COL1A1 and COL1A2 are genes for Collagen 1 and 2
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- Official Symbol
- COL1A1provided by HGNC
- Official Full Name
- collagen, type I, alpha 1provided by HGNC
- Primary source
- HGNC:HGNC:2197
- See related
- Ensembl:ENSG00000108821; HPRD:00362; MIM:120150; Vega:OTTHUMG00000148674
- Gene type
- protein coding
- RefSeq status
- REVIEWED
- Organism
- Homo sapiens
- Lineage
- Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; Haplorrhini; Catarrhini; Hominidae; Homo
- Also known as
- OI4
- Summary
- This gene encodes the pro-alpha1 chains of type I collagen whose triple helix comprises two alpha1 chains and one alpha2 chain. Type I is a fibril-forming collagen found in most connective tissues and is abundant in bone, cornea, dermis and tendon. Mutations in this gene are associated with osteogenesis imperfecta types I-IV, Ehlers-Danlos syndrome type VIIA, Ehlers-Danlos syndrome Classical type, Caffey Disease and idiopathic osteoporosis. Reciprocal translocations between chromosomes 17 and 22, where this gene and the gene for platelet-derived growth factor beta are located, are associated with a particular type of skin tumor called dermatofibrosarcoma protuberans, resulting from unregulated expression of the growth factor. Two transcripts, resulting from the use of alternate polyadenylation signals, have been identified for this gene. [provided by R. Dalgleish, Feb 2008]
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- In all, 38 miRNAs were identified to be upregulated and 31 downregulated, in TGFβ1-stimulated HTFs. Among those, the miR-29b, downregulated in TGFβ1-treated HTFs, targeted a cadre of mRNAs that encode proteins involved in fibrosis, including PI3Kp85α, Sp1, and collagen type I alpha1 (Col1A1). Treatment of HTFs with TGFβ1 activated the PI3K/Akt/Sp1 pathway and, consequently, induced an increase in the expression of type I collagen. Overexpression of miR-29b inhibited the PI3K/Akt/Sp1 pathway, and attenuated the expression of Col1A1. CONCLUSIONS. miR-29b acted as a suppressor of type I collagen gene by repressing the PI3K/Akt/Sp1 pathway in HTFs. Overexpression of miR-29b protected subconjunctival tissues against collagen production and fibrosis. These findings provided a novel rationale for the development of miRNA-based strategies for attenuating scar formation after glaucoma filtering surgery.
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Type I collagen is the major constituent of the skin and the reduction of dermal type I collagen content is closely associated with the intrinsic skin aging. We here found that esculetin, 6,7-dihydroxycoumarin, strongly induces type I procollagen expression in human dermal fibroblasts. Esculetin not only increased protein levels of type I procollagen but also increased mRNA levels of COL1A1 but not COL1A2. Esculetin activated the MAPKs (ERK1/2, p38, JNK) and PI3K/Akt pathways, through which it promoted the type I procollagen expression. We also demonstrated that the binding motifs for transcription factor Sp1 occur with the highest frequency in the COL1A1 promoter and that esculetin increases the Sp1 expression through the MAPK and PI3K/Akt pathways. These results suggest that esculetin promotes type I procollagen expression through the MAPK and PI3K/Akt pathways and that Sp1 might be involved in the esculetin-induced type I procollagen expression via activation of the COL1A1 transcription.
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The present study was designed to assess the potential inhibitory activity of curcumin on the α-melanocyte stimulating hormone (α-MSH)-stimulated melanogenesis signal pathway in B16F10 melanoma cells. The molecular mechanism of curcumin-induced inhibitory activity on the α-MSH-stimulated melanogenesis signal pathway, including expression of melanogenesis-related proteins and activation of melanogenesis-regulating proteins, was examined in B16F10 cells. Curcumin suppressed the cellular melanin contents and the tyrosinase activity in α-MSH-stimulated B16F10 cells. In addition, the expression of melanogenesis-related proteins such as microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related protein 1 and 2 was suppressed by curcumin in the α-MSH-stimulated B16F10 cells. Notably, a melanogenesis-regulating signal such as mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) or phosphatidylinositol 3-kinase (PI3K)/Akt was activated by curcumin in the B16F10 cells treated with or without α-MSH. The suppressive activity of curcumin on α-MSH-induced melanogenesis was down-regulated by PD98059 and by LY294002. Our results suggest that the suppressive activity of curcumin on α-MSH-stimulated melanogenesis may involve the down-regulation of MITF and its downstream signal pathway through the activation of MEK/ERK or PI3K/Akt.
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Now HMS researchers have discovered that MC1R-RHC, the mutation responsible for the red-hair phenotype, also triggers an important cancer-promoting pathway. The findings, reported in the August 22 issue of Molecular Cell, help explain the molecular mechanisms that underlie redheads’ well-known risk of developing melanoma and may provide new insights for preventing and treating this dangerous type of skin cancer.
Melanoma is the least common but the most lethal of skin cancers. Accounting for 75 percent of all skin-cancer deaths, it originates in pigment-producing skin cells called melanocytes. Two types of UV radiation—UVA and UVB—can mutate DNA in these skin cells and lead to melanoma.
“In this current study, we have demonstrated that the mutation MC1R-RHC promotes the PI3K/Akt signaling pathway when a red-haired individual is exposed to UV radiation,” says co-senior author Wenyi Wei, an HMS associate professor of pathology at Beth Israel Deaconess Medical Center. PI3K/Akt is a well-known cancer-promoting pathway implicated in breast, ovarian, and lung cancers.
The team also found that in the MC1R-RHC pigment cells, elevated PI3K/Akt activity boosted cell proliferation and synchronized with another well-known cancer mutation in the BRAF gene to accelerate cancer development.
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Table of contents
COL1A1 - collagen, type I, alpha 1
Homo sapiens
Disease relevance of COL1A1
- Deregulation of the platelet-derived growth factor B-chain gene via fusion with collagen gene COL1A1 in dermatofibrosarcoma protuberans and giant-cell fibroblastoma [1].
- Amplified cDNAs prepared from lymphoblastoid cells were used to identify previously characterized heterozygous mutations in the COL1A1 and COL1A2 genes from two patients with osteogenesis imperfecta and in the COL3A1 gene from a patient with the Ehlers-Danlos syndrome type IV [2].
- Lack of association between osteoarthritis of the hip and gene polymorphisms of VDR, COL1A1, and COL2A1 in postmenopausal women [3].
- The data obtained exclude linkage of Marfan syndrome to the two major fibrillar collagen (COL1A1, COL1A2, and COL2A1) genes [4].
- The allele frequency of the G-->T mutation of COL1A1 gene (collagen type I alpha 1 gene, GenBank accession n. AF017178) was analyzed by a newARMS-PCR method in 240 osteoporotic subjects bearing a femoral neck fracture [5].
High impact information on COL1A1
- COL1A1 is a major constituent of the connective tissue matrix [1].
- Here we describe efficient and reproducible gene targeting in fetal fibroblasts to place a therapeutic transgene at the ovine alpha1(I) procollagen (COL1A1) locus and the production of live sheep by nuclear transfer [6].
- The authors found a novel missense mutation in COL1A1, the gene encoding the alpha1 chain of type I collagen, in all affected individuals in 3 discrete pedigrees [7].
- Affected individuals and obligate carriers were heterozygous for a missense mutation (3040Ctwo head right arrowT) in exon 41 of the gene encoding the alpha1(I) chain of type I collagen (COL1A1), altering residue 836 (R836C) in the triple-helical domain of this chain [8].
- A novel COL1A1 mutation in infantile cortical hyperostosis (Caffey disease) expands the spectrum of collagen-related disorders [8].
Chemical compound and disease context of COL1A1
- We have previously shown that hypoxia (2% O2), when compared to standard oxygen tension (20% O2), upregulates the mRNA levels of the human alpha1(I) (COL1A1) procollagen gene and transforming growth factor-beta1 (TGF-beta1) in human dermal fibroblasts [9].
- TNF-alpha inhibited the CAT activity of fibroblasts transfected with plasmids containing 2.3 kb of 5' flanking sequences of COL1A1, whereas the activity of control plasmids containing the herpes simplex thymidine kinase promoter gene (pBLCAT) was unaltered [10].
- OBJECTIVE: To evaluate the effects of mithramycin on COL1A1 expression in systemic sclerosis fibroblasts [11].
- Detection of COL1A1-PDGFB fusion transcripts in dermatofibrosarcoma protuberans by reverse transcription-polymerase chain reaction using archival formalin-fixed, paraffin-embedded tissues [12].
Biological context of COL1A1
- Frameshift mutation near the 3' end of the COL1A1 gene of type I collagen predicts an elongated Pro alpha 1(I) chain and results in osteogenesis imperfecta type I [13].
- Thus, in contrast to mutations in genes that encode the dominant protein of a tissue (e.g., COL1A1 and COL2A1), in which "null" mutations result in phenotypes milder than those caused by mutations that alter protein sequence, the phenotypes produced by these mutations in COL3A1 overlap with those of the vascular form of EDS [14].
- The two genes of type I collagen, COL1A1 and COL2A1, have been mapped previously to chromosomes 17 and 7, respectively [15].
- Variable expression of osteogenesis imperfecta in a nuclear family is explained by somatic mosaicism for a lethal point mutation in the alpha 1(I) gene (COL1A1) of type I collagen in a parent [16].
- To assay the genomic DNA, we established a consensus sequence for the first 12 kb of the COL1A1 gene and for 30 kb of new sequences of the 38-kb COL1A2 gene [17].
Anatomical context of COL1A1
- By allele-specific oligonucleotide hybridization to amplified genomic sequences from paternal tissues we determined that the mutant allele accounted for approximately 50% of the COL1A1 alleles in fibroblasts, 27% of those in blood, and 37% of those in sperm [16].
- Collagen produced from osteoblasts cultured from "Ss" heterozygotes had an increased ratio of alpha 1(I) protein relative to alpha 2(I), and this was accompanied by an increased ratio of COL1A1 mRNA relative to COL1A2 [18].
- NIH-3T3 cells were simultaneously transfected with a Tax expressor plasmid and a chimeric construct containing regulatory sequences (-804 to +42 bp) of the alpha 1(I) procollagen gene (COL1A1) promoter [19].
- Here we show that the hammerhead ribozyme Rzpol1a1, targeting a common polymorphism within transcripts from the COL1A1 gene, downregulates COL1A1 transcript in human mesenchymal progenitor cells at a ribozyme to transcript ratio of only 1:1 [20].
- These results suggest that cis sequences found in ColCAT3.6 mediate high levels of COL1A1 expression in bone and tendon, but not in vascular smooth muscle cells (VSMC), whereas sequences located within the minigene, but not found in ColCAT3.6, mediate VSMC-specific expression [21].
Associations of COL1A1 with chemical compounds
- Subsequent amplification of the cDNA by the PCR and nucleotide sequencing revealed a single-base mutation that substituted an aspartate codon for glycine at position alpha 1-541 in the COL1A1 gene [22].
- The nucleotide sequence of a fragment amplified from genomic DNA confirmed the location of the cysteine residue and showed that the mutation was a single nucleotide change in one COL1A1 allele [23].
- Inhibition of basal and transforming growth factor-beta-induced stimulation of COL1A1 transcription by the DNA intercalators, mitoxantrone and WP631, in cultured human dermal fibroblasts [24].
- The individual was heterozygous for a G to A transition in the COL1A1 gene that resulted in the substitution of serine for glycine 883 in one or both of the pro alpha 1 (I) chains [25].
- Cyanogen bromide cleavage and subsequent sequencing revealed a G-to-T base substitution at nucleotide 2420 of COL1A1, resulting in a Gly586Val substitution [26].
Physical interactions of COL1A1
- CONCLUSION: Some cases of otosclerosis and osteoporosis could share a functionally significant polymorphism in the Sp1 transcription factor binding site in the first intron of the COL1A1 gene [27].
- Association of otosclerosis with Sp1 binding site polymorphism in COL1A1 gene: evidence for a shared genetic etiology with osteoporosis [27].
Regulatory relationships of COL1A1
- Competition of the drugs for Sp1 binding and their effect on TGF-beta-induced stimulation of COL1A1 transcription was also examined [24].
- In addition, by employing full-length or deleted B-Myb cDNA construct, we found that B-Myb down-regulates the COL1A1 proximal promoter through its C-terminal domain [28].
- The results indicate that IFN-gamma inhibits COL1A1 expression in fibroblasts principally at the level of gene transcription [29].
- A 1.9-Kb 5' fragment from the human COL1A1 gene drives inappropriate expression of the human COL2A1 gene in tissues of transgenic mice that normally express only the COL1A1 gene [30].
- METHODS: Nuclear extracts from dermal fibroblasts from 4 patients with SSc and 4 age- and sex-matched control individuals were examined by electrophoresis mobility shift assays with a COL1A1 promoter fragment encompassing nucleotides -174 to -50 bp [31].
Other interactions of COL1A1
- It is interesting that such glycine substitutions inside the COL1A1 or COL1A2 genes have been associated with many cases of osteogenesis imperfecta [32].
- In this study, we analyzed the regulation of the alpha1(I) procollagen (COL1A1) promoter and the alpha2(I) procollagen (COL1A2) promoter by IL-4 in normal human lung fibroblasts [33].
- Sequence analysis revealed that the ends of exons 42, 29 and 38 in the COL1A1 gene were fused with the start of exon 2 in the PDGFB gene in case 1, 2 and 3, respectively [34].
- Polymorphisms were detected by digestion with Bsm I for VDR, Acc B7I for COL1A1, and Pvu II for COL2A1 [3].
- The Sp1 transcription factor plays a crucial role in COL1A1 transcriptional regulation under normal and pathologic conditions and under the effects of transforming growth factor-beta (TGF-beta) [24].
Analytical, diagnostic and therapeutic context of COL1A1
- Collagen production was examined by enzyme-linked immunosorbent assay and metabolic labeling, COL1A1 steady-state mRNA levels, and stability were assessed by Northern hybridizations, and COL1A1 transcription by in vitro nuclear transcription assays and transient transfections [24].
- We describe a dominant point mutation in the COL1A1 gene causing extremely severe osteogenesis imperfecta (OI type II/III) which was detected in the dermal fibroblasts of a proband, diagnosed by ultrasonography at 24 weeks of gestation [35].
- Meta-analysis of COL1A1 Sp1 polymorphism in relation to bone mineral density and osteoporotic fracture [36].
- Northern-blot analysis showed an inverse relationship between COL1A1 mRNA expression and that of B-Myb during exponential cell growth and during quiescence in human SSc fibroblasts [28].
- The DNA binding activity of nuclear proteins recognizing the regulatory regions in the COL1A1 promoter was examined by gel mobility shift assays [37].
References
- Deregulation of the platelet-derived growth factor B-chain gene via fusion with collagen gene COL1A1 in dermatofibrosarcoma protuberans and giant-cell fibroblastoma. Simon, M.P., Pedeutour, F., Sirvent, N., Grosgeorge, J., Minoletti, F., Coindre, J.M., Terrier-Lacombe, M.J., Mandahl, N., Craver, R.D., Blin, N., Sozzi, G., Turc-Carel, C., O'Brien, K.P., Kedra, D., Fransson, I., Guilbaud, C., Dumanski, J.P. Nat. Genet. (1997) [Pubmed]
- Low basal transcription of genes for tissue-specific collagens by fibroblasts and lymphoblastoid cells. Application to the characterization of a glycine 997 to serine substitution in alpha 1(II) collagen chains of a patient with spondyloepiphyseal dysplasia. Chan, D., Cole, W.G. J. Biol. Chem. (1991) [Pubmed]
- Lack of association between osteoarthritis of the hip and gene polymorphisms of VDR, COL1A1, and COL2A1 in postmenopausal women. Aerssens, J., Dequeker, J., Peeters, J., Breemans, S., Boonen, S. Arthritis Rheum. (1998) [Pubmed]
- Linkage analysis of five fibrillar collagen loci in a large French Marfan syndrome family. Boileau, C., Jondeau, G., Bonaiti, C., Coulon, M., Delorme, G., Dubourg, O., Bourdarias, J.P., Junien, C. J. Med. Genet. (1990) [Pubmed]
- Allele frequency of the G-->T mutation of the col1A1 gene analyzed by an ARMS-PCR in osteoporotic subjects with femoral neck fractures. Montanaro, L., Arciola, C.R. Clin. Chem. Lab. Med. (2002) [Pubmed]
- Production of gene-targeted sheep by nuclear transfer from cultured somatic cells. McCreath, K.J., Howcroft, J., Campbell, K.H., Colman, A., Schnieke, A.E., Kind, A.J. Nature (2000) [Pubmed]
- Caffey disease: an unlikely collagenopathy. Glorieux, F.H. J. Clin. Invest. (2005) [Pubmed]
- A novel COL1A1 mutation in infantile cortical hyperostosis (Caffey disease) expands the spectrum of collagen-related disorders. Gensure, R.C., Mäkitie, O., Barclay, C., Chan, C., Depalma, S.R., Bastepe, M., Abuzahra, H., Couper, R., Mundlos, S., Sillence, D., Ala Kokko, L., Seidman, J.G., Cole, W.G., Jüppner, H. J. Clin. Invest. (2005) [Pubmed]
- Low oxygen tension stimulates collagen synthesis and COL1A1 transcription through the action of TGF-beta1. Falanga, V., Zhou, L., Yufit, T. J. Cell. Physiol. (2002) [Pubmed]
- The transcription of human alpha 1(I) procollagen gene (COL1A1) is suppressed by tumour necrosis factor-alpha through proximal short promoter elements: evidence for suppression mechanisms mediated by two nuclear-factorbinding sites. Mori, K., Hatamochi, A., Ueki, H., Olsen, A., Jimenez, S.A. Biochem. J. (1996) [Pubmed]
- Inhibition of collagen gene expression in systemic sclerosis dermal fibroblasts by mithramycin. Sandorfi, N., Louneva, N., Hitraya, E., Hajnoczky, G., Saitta, B., Jimenez, S.A. Ann. Rheum. Dis. (2005) [Pubmed]
- Detection of COL1A1-PDGFB fusion transcripts in dermatofibrosarcoma protuberans by reverse transcription-polymerase chain reaction using archival formalin-fixed, paraffin-embedded tissues. Wang, J., Hisaoka, M., Shimajiri, S., Morimitsu, Y., Hashimoto, H. Diagn. Mol. Pathol. (1999) [Pubmed]
- Frameshift mutation near the 3' end of the COL1A1 gene of type I collagen predicts an elongated Pro alpha 1(I) chain and results in osteogenesis imperfecta type I. Willing, M.C., Cohn, D.H., Byers, P.H. J. Clin. Invest. (1990) [Pubmed]
- Haploinsufficiency for one COL3A1 allele of type III procollagen results in a phenotype similar to the vascular form of Ehlers-Danlos syndrome, Ehlers-Danlos syndrome type IV. Schwarze, U., Schievink, W.I., Petty, E., Jaff, M.R., Babovic-Vuksanovic, D., Cherry, K.J., Pepin, M., Byers, P.H. Am. J. Hum. Genet. (2001) [Pubmed]
- Chromosomal assignments of the genes coding for human types II, III, and IV collagen: a dispersed gene family. Solomon, E., Hiorns, L.R., Spurr, N., Kurkinen, M., Barlow, D., Hogan, B.L., Dalgleish, R. Proc. Natl. Acad. Sci. U.S.A. (1985) [Pubmed]
- Variable expression of osteogenesis imperfecta in a nuclear family is explained by somatic mosaicism for a lethal point mutation in the alpha 1(I) gene (COL1A1) of type I collagen in a parent. Wallis, G.A., Starman, B.J., Zinn, A.B., Byers, P.H. Am. J. Hum. Genet. (1990) [Pubmed]
- Analysis of the COL1A1 and COL1A2 genes by PCR amplification and scanning by conformation-sensitive gel electrophoresis identifies only COL1A1 mutations in 15 patients with osteogenesis imperfecta type I: identification of common sequences of null-allele mutations. Körkkö, J., Ala-Kokko, L., De Paepe, A., Nuytinck, L., Earley, J., Prockop, D.J. Am. J. Hum. Genet. (1998) [Pubmed]
- A COL1A1 Sp1 binding site polymorphism predisposes to osteoporotic fracture by affecting bone density and quality. Mann, V., Hobson, E.E., Li, B., Stewart, T.L., Grant, S.F., Robins, S.P., Aspden, R.M., Ralston, S.H. J. Clin. Invest. (2001) [Pubmed]
- Stimulation of alpha 1 (I) procollagen gene expression in NIH-3T3 cells by the human T cell leukemia virus type 1 (HTLV-1) Tax gene. Muñoz, E., Suri, D., Amini, S., Khalili, K., Jiménez, S.A. J. Clin. Invest. (1995) [Pubmed]
- Validation in mesenchymal progenitor cells of a mutation-independent ex vivo approach to gene therapy for osteogenesis imperfecta. Millington-Ward, S., Allers, C., Tuohy, G., Conget, P., Allen, D., McMahon, H.P., Kenna, P.F., Humphries, P., Farrar, G.J. Hum. Mol. Genet. (2002) [Pubmed]
- Regulation of the alpha 1(I) collagen promoter in vascular smooth muscle cells. Comparison with other alpha 1(I) collagen-producing cells in transgenic animals and cultured cells. Bedalov, A., Breault, D.T., Sokolov, B.P., Lichtler, A.C., Bedalov, I., Clark, S.H., Mack, K., Khillan, J.S., Woody, C.O., Kream, B.E. J. Biol. Chem. (1994) [Pubmed]
- A single base mutation in type I procollagen (COL1A1) that converts glycine alpha 1-541 to aspartate in a lethal variant of osteogenesis imperfecta: detection of the mutation with a carbodiimide reaction of DNA heteroduplexes and direct sequencing of products of the PCR. Zhuang, J.P., Constantinou, C.D., Ganguly, A., Prockop, D.J. Am. J. Hum. Genet. (1991) [Pubmed]
- Substitution of cysteine for glycine within the carboxyl-terminal telopeptide of the alpha 1 chain of type I collagen produces mild osteogenesis imperfecta. Cohn, D.H., Apone, S., Eyre, D.R., Starman, B.J., Andreassen, P., Charbonneau, H., Nicholls, A.C., Pope, F.M., Byers, P.H. J. Biol. Chem. (1988) [Pubmed]
- Inhibition of basal and transforming growth factor-beta-induced stimulation of COL1A1 transcription by the DNA intercalators, mitoxantrone and WP631, in cultured human dermal fibroblasts. Gaidarova, S., Jiménez, S.A. J. Biol. Chem. (2002) [Pubmed]
- Substitution of serine for glycine 883 in the triple helix of the pro alpha 1 (I) chain of type I procollagen produces osteogenesis imperfecta type IV and introduces a structural change in the triple helix that does not alter cleavage of the molecule by procollagen N-proteinase. Lightfoot, S.J., Atkinson, M.S., Murphy, G., Byers, P.H., Kadler, K.E. J. Biol. Chem. (1994) [Pubmed]
- (G586V) substitutions in the alpha 1 and alpha 2 chains of collagen I: effect of alpha-chain stoichiometry on the phenotype of osteogenesis imperfecta? Lund, A.M., Skovby, F., Schwartz, M. Hum. Mutat. (1997) [Pubmed]
- Association of otosclerosis with Sp1 binding site polymorphism in COL1A1 gene: evidence for a shared genetic etiology with osteoporosis. McKenna, M.J., Nguyen-Huynh, A.T., Kristiansen, A.G. Otol. Neurotol. (2004) [Pubmed]
- B-Myb acts as a repressor of human COL1A1 collagen gene expression by interacting with Sp1 and CBF factors in scleroderma fibroblasts. Cicchillitti, L., Jimenez, S.A., Sala, A., Saitta, B. Biochem. J. (2004) [Pubmed]
- Negative modulation of alpha1(I) procollagen gene expression in human skin fibroblasts: transcriptional inhibition by interferon-gamma. Yuan, W., Yufit, T., Li, L., Mori, Y., Chen, S.J., Varga, J. J. Cell. Physiol. (1999) [Pubmed]
- A 1.9-Kb 5' fragment from the human COL1A1 gene drives inappropriate expression of the human COL2A1 gene in tissues of transgenic mice that normally express only the COL1A1 gene. Ala-Kokko, L., Yuan, C.M., Le Guellec, D., Franc, S., Fertala, A., Khillan, J.S., Sokolov, B.P., Prockop, D.J. Ann. N. Y. Acad. Sci. (1996) [Pubmed]
- CCAAT binding transcription factor binds and regulates human COL1A1 promoter activity in human dermal fibroblasts: demonstration of increased binding in systemic sclerosis fibroblasts. Saitta, B., Gaidarova, S., Cicchillitti, L., Jimenez, S.A. Arthritis Rheum. (2000) [Pubmed]
- Substitution of arginine for glycine 325 in the collagen alpha 5 (IV) chain associated with X-linked Alport syndrome: characterization of the mutation by direct sequencing of PCR-amplified lymphoblast cDNA fragments. Knebelmann, B., Deschenes, G., Gros, F., Hors, M.C., Grünfeld, J.P., Zhou, J., Tryggvason, K., Gubler, M.C., Antignac, C. Am. J. Hum. Genet. (1992) [Pubmed]
- Transcriptional activation of the type I collagen genes COL1A1 and COL1A2 in fibroblasts by interleukin-4: analysis of the functional collagen promoter sequences. Büttner, C., Skupin, A., Rieber, E.P. J. Cell. Physiol. (2004) [Pubmed]
- Analysis of gene mutations in three cases of dermatofibrosarcoma protuberans (DFSP): ordinary DFSP, DFSP with fibrosarcomatous lesion (DFSP-FS) and lung metastasis of DFSP-FS. Saeki, H., Hoashi, T., Tada, Y., Ashida, R., Kuwano, Y., Le Pavoux, A., Tsunemi, Y., Shikada, J., Torii, H., Kawabata, Y., Kikuchi, K., Tamada, Y., Matsumoto, Y., Tamaki, K. J. Dermatol. Sci. (2003) [Pubmed]
- Paternal mosaicism for a COL1A1 dominant mutation (alpha 1 Ser-415) causes recurrent osteogenesis imperfecta. Mottes, M., Gomez Lira, M.M., Valli, M., Scarano, G., Lonardo, F., Forlino, A., Cetta, G., Pignatti, P.F. Hum. Mutat. (1993) [Pubmed]
- Meta-analysis of COL1A1 Sp1 polymorphism in relation to bone mineral density and osteoporotic fracture. Mann, V., Ralston, S.H. Bone (2003) [Pubmed]
- Identification of elements in the promoter region of the alpha1(I) procollagen gene involved in its up-regulated expression in systemic sclerosis. Hitraya, E.G., Varga, J., Artlett, C.M., Jiménez, S.A. Arthritis Rheum. (1998) [Pubmed]
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" α-MSH reduced scar area and improved the organization of the collagen fibers indicating that it may direct the healing into a more-regenerative/less-scarring pathway. "
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Abstract
Intermittent application of parathyroid hormone (PTH) has well established anabolic effects on bone mass in rodents and humans. Although transcriptional mechanisms responsible for these effects are not fully understood, it is recognized that transcriptional factor cAMP response element binding protein (CREB) mediates PTH signaling in osteoblasts, and that there is a communication between the PTH-CREB pathway and the BMP2 signaling pathway, which is important for osteoblast differentiation and bone formations. These findings, in conjunction with putative cAMP response elements (CREs) in the BMP2 promoter, led us to hypothesize that the PTH-CREB pathway could be a positive regulator of BMP2 transcription in osteoblasts. To test this hypothesis, we first demonstrated that PTH signaling activated CREB by phosphorylation in osteoblasts, and that both PTH and CREB were capable of promoting osteoblastic differentiation of primary mouse osteoblast cells and multiple rodent osteoblast cell lines. Importantly, we found that the PTH-CREB signaling pathway functioned as an effective activator of BMP2 expression, as pharmacologic and genetic modulation of PTH-CREB activity significantly affected BMP2 expression levels in these cells. Lastly, through multiple promoter assays, including promoter reporter deletion, mutation, chromatin immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA), we identified a specific CRE in the BMP2 promoter which is responsible for CREB transactivation of the BMP2 gene in osteoblasts. Together, these results demonstrate that the anabolic function of PTH signaling in bone is mediated, at least in part, by CREB transactivation of BMP2 expression in osteoblasts.
Figures
Citation: Zhang R, Edwards JR, Ko S-Y, Dong S, Liu H, et al. (2011) Transcriptional Regulation of BMP2 Expression by the PTH-CREB Signaling Pathway in Osteoblasts. PLoS ONE 6(6): e20780. doi:10.1371/journal.pone.0020780
Editor: Jean-Marc A. Lobaccaro, Clermont Université, France
Received: March 4, 2011; Accepted: May 9, 2011; Published: June 9, 2011
Copyright: © 2011 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors have no support or funding to report.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Parathyroid hormone (PTH) plays an important role in skeletal metabolism. In mice [1]–[4] and rats [5], [6], intermittent administration of PTH has anabolic effects on bone mass and bone formation. Similarly, when PTH is administered intermittently to humans, it has been proven to increase bone mass and decrease fracture risk in post-menopausal women, elderly men, and women with glucocorticoid-induced osteoporosis [7]–[10]. Despite its clinical efficacy, the precise molecular mechanisms responsible for the anabolic effects of PTH on bone, particularly at the transcriptional level, need to be further elucidated.
Although multiple intracellular signaling mechanisms are involved in mediating PTH function, the major downstream signaling pathway of PTH in bone cells involves 5′-cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), and cAMP response element binding protein (CREB). The anabolic function of this cAMP-PKA-CREB pathway in bone has been characterized in vivo and in vitro [11]–[30]. Increasing activity of this pathway promotes osteoblast differentiation [11]–[26] and stimulates bone formation [27]–[30]. In osteoblasts, PTH binds to a PTH receptor and induces formation of cAMP, leading to activation of PKA, which in turn phosphorylates and activates CREB, a member of a large family of basic leucine zipper (bZIP) domain DNA-binding proteins [11]–[14]. Activated CREB, in association with the other co-activators, binds to target genes through a cAMP response element (CRE) and activates their transcription. Therefore, in this pathway, CREB plays a pivotal role by converting the PTH signal to activation of gene expression. In various cell systems, CREB induces the expression of some osteoblast-related genes, such as bone sialoprotein (BSP) and osteocalcin (OCN), by directly binding to these promoters [13]–[22]. However, the major downstream targets of CREB transactivation, which have a predominant role in initiating osteoblast differentiation and stimulating bone formation, are unknown.
Bone morphogenetic protein 2 (BMP2) is an important growth factor that stimulates osteoblast differentiation and bone formation [31]–[33]. The PTH-cAMP-CREB signaling pathway synergizes the anabolic signaling of BMP2 for osteoblast differentiation and bone formation [23], [25], [26], [28], [29], [34], indicating that there is a communication between the PTH-cAMP-CREB and BMP2 signaling pathways. Recently, we have reported that osteoblast-specific deficiency of CREB in mice causes reduction of postnatal bone mass and decreases BMP2 expression in osteoblasts [35], [36], suggesting that BMP2 could be a critical transcriptional target of CREB in osteoblasts. This is consistent with the findings of a genome-wide study, based on a chromatin immunoprecipitation (ChIP) assay, in which the cAMP-CREB signaling pathway was identified as a positive transcriptional regulator of BMP2 mRNA expression in PC12 cells [37]. Furthermore, we have identified multiple CREs in the BMP2 promoter by DNA sequence analysis. Based on the collective findings reviewed above, it is reasonable to hypothesize that the transcription factor CREB is an activator of BMP2 expression in osteoblasts, and that CREB mediates the anabolic function of PTH-cAMP signaling in bone, at least in part, by this mechanism. This hypothesis was examined in the current investigation through pharmacological and genetic experiments, and promoter analyses.
Results
PTH signaling activates CREB in osteoblasts
CREB activity is controlled by phosphorylation through multiple signaling pathways, including the PTH signaling pathway [11]–[14]. Here, we determined the effects of manipulating the signaling activity of the PTH-cAMP-signaling cascade on CREB phosphorylation in osteoblasts. First, we determined the effect of PTH on CREB phosphorylation in osteoblasts. Pluripotent mesenchymal precursor C2C12 cells, which are capable of differentiating into an osteoblast lineage, were treated with PTH, and phosphorylation levels of CREB were detected by Western blot, with non-phosphorylated CREB and β-actin as controls. The results showed that treatment with PTH at 0–500 nM substantially, and dose-dependently, increased CREB phosphorylation (Fig. 1A). The minimum dose with a discernable effect was approximately 50 nM (Fig. 1A). The PTH-induced CREB phosphorylation in osteoblasts was also time-dependent between 5 and 60 minutes (Fig. 1B). As a second messenger, cAMP is known to mediate PTH signaling. Thus, we determined the effects of the cAMP enhancer 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cAMP phosphodiesterase, on CREB phosphorylation. Western blot showed that incubation of C2C12 cells with IBMX at doses of 10–300 µM increased intracellular levels of phosphorylated CREB protein (Fig. 1C), and that this stimulation was induced in a time-dependent manner up to 4 hours (Fig. 1D). As a direct downstream target of cAMP, PKA phosphorylates CREB. Next, we examined the effects of inhibition of PKA activity on CREB phosphorylation using KT5720, a PKA inhibitor. We found that the KT5720-induced inhibition of PKA activity markedly reduced levels of phosphorylated CREB in C2C12 cells, in both dose- and time-dependent manners (Fig. 1E, 1F). The levels in the Western blot of phosphor-CREB treated by IBMX and KT5720 were quantitated. The quantitative results confirmed that these cAMP and PKA drugs dose- and time-dependently regulated CREB phosphorylation in osteoblasts (see the quantitative data in Figure S1).
Figure 1. PTH signaling activates CREB phosphorylation in osteoblasts.
(A–F) CREB phosphorylation levels in C2C12 cells, treated with PTH at 0 to 500 nM for 1 hour (A); PTH at 50 nM for 5 to 60 min (B); or IBMX at 0 to 300 µM for 1 hour (C); IBMX at 50 µM for 0 to 4 hours (D); or KT5720 at 0 to 10 µM for 1 hour (E); KT5720 at 10 µM for 0 to 2 hours (F), were detected by Western blot with anti-phosphorylated CREB antibody, with normalization by non-phosphorylated CREB and β-actin. (G) C2C12 cells were co-transfected with CRE-Luc reporter and CREB expression vector, and treated with PTH at 50 nM for 36 hours. The reporter luciferase activity was measured with normalization by β-gal activity. # p<0.01 (CREB vs vector; mean±SE, n = 6); * p<0.05 (PTH vs vehicle; mean±SE, n = 6).
doi:10.1371/journal.pone.0020780.g001Phosphorylated CREB migrates into nuclei and transactivates target genes through CRE binding sites. Consequently, we evaluated the effects of PTH, which induces CREB phosphorylation, on transcriptional activity of CREB. The luciferase assay using a CREB-specific reporter CRE-Luc showed that overexpression of CREB in C2C12 cells dose-dependently increased the activity of the CRE reporter, and that treatment with PTH at 50 nM further elevated the CREB-induced reporter activity (Fig. 1G).
PTH-CREB pathway promotes osteoblast differentiation
It has been postulated that the anabolic effect of PTH on bone is mediated through osteoblasts. In this series of experiments, we characterized the role of the PTH-CREB signaling pathway in osteoblast differentiation. First, we determined the effects of PTH on alkaline phosphatase (ALP) activity of C2C12 cells by incubating cells with PTH for 24 hours. Measurement of ALP activity in cell lysates showed that treatment with PTH at 0–200 nM stimulated ALP activity in a pattern that approached dose-dependence, with a maximum effect (2-fold increase) at PTH concentration 100 nM (Fig. 2A). Real time PCR showed that the mRNA levels of osteoblast maturation genes, Runx2 and type I collagen 1a1 (Col1a1), were significantly enhanced by treatment with PTH at doses of 10–100 nM (Fig. 2B).
Figure 2. PTH-CREB pathway promotes osteoblast differentiation.
(A) ALP activity in C2C12 cells, treated with PTH at 0 to 200 nM for 24 hours, was measured with normalization by cell proteins. * p<0.05 (vs vehicle; mean±SE, n = 8). (B) mRNA levels of Runx2 and Col1a1 in C2C12 cells, treated with PTH for 12 hours, were quantitated by real time PCR, normalized by 18S rRNA. * p<0.01 (vs vehicle; mean±SE, n = 6). (C) ALP activity in C2C12 and 2T3 cells was measured after transfection with CREB expression plasmid and treated with noggin at 500 nm/ml for 48 hours. * p<0.01 (CREB vs vector); # p<0.05 (noggin vs vehicle; mean±SE, n = 8). (D) mRNA levels of Runx2, Col1a1 and OCN in C2C12 cells, transfected with CREB for 24 hours, were measured by real time PCR, normalized by 18S rRNA. * p<0.05 (vs vector; mean±SE, n = 6). (E) 2T3 cells were transfected with vector (top) or CREB plasmid (bottom) and cultured under osteogenic conditions for 14 and 21 days. Von Kossa staining was performed to visualize the mineralized matrix formation. (F) Isolated calvarial osteoblastic cells from newborn conditional CREB knockout mice (CREB cKO) and their CREBfloxed littermate controls were cultured for 24 hours and ALP activity was measured. * p<0.05 (cKO vs control); # p<0.05 (PTH on cKO vs PTH on control); ** p<0.05 (PTH va vehicle on control mice; mean±SE, n = 6); NS: not significant.
doi:10.1371/journal.pone.0020780.g002Then, we determined the effects of overexpression of CREB on osteoblast differentiation. Osteoblast precursor C2C12 and 2T3 cells [38]–[40] were transfected with CREB expression plasmid or its empty vector for 48 hours. Enzymatic quantitation of ALP activity showed that CREB overexpression significantly increased ALP activity in both C2C12 and 2T3 cells, compared with vehicle controls (Fig. 2C). Interestingly, CREB-stimulation of ALP activity was completely reversed in these cells by adding noggin (500 ng/ml), a natural BMP antagonist, to the culture medium (Fig. 2C). This finding is critical evidence for our hypothesis that the stimulatory effect of CREB on ALP depends on BMP signaling. mRNA measurement of C2C12 cells also showed that CREB transfection significantly increased expression of osteoblast-specific marker genes, including Runx2, Col1a1 and osteocalcin (OCN) (Fig. 2D). Next, we determined whether overexpression of CREB affects bone matrix formation in osteoblast precursor 2T3 cells, which can form mineralized matrices in culture under osteogenic conditions [33], [37]–[40]. Von Kossa staining showed that mineralized bone nodule formation was greatly enhanced in 2T3 cell cultures 14 and 21 days after CREB transfection, compared with control 2T3 cells transfected with empty vector (Fig. 2E).
In order to confirm the role of CREB in promoting osteoblast differentiation, we performed a loss-of-function study with primary CREB-deficient calvarial osteoblast cells. These cells were isolated from conditional CREB knockout mice (CREB cKO), that were generated by crossing CREB floxed mice with Col1a1-Cre mice. We have recently reported that these osteoblast-specific CREB knockout mice exhibit low bone mass [35], [36]. In the present study, we found that CREB deficiency markedly decreased both basal and PTH-induced ALP activity in calvarial osteoblast cells, compared with floxed control cells (Fig. 2F). We also noticed that, despite at a much lower level than PTH treatment in the control group, PTH appeared still capable of increasing ALP activity in the absence of CREB, compared with vehicle treatment. However, the statistical analysis did not show a significant difference between the treatment groups in the CREB deficient cells. This suggests that PTH stimulation of ALP activity was attenuated by removal of the CREB gene in the calvairal osteoblast cells, (Fig. 2F). Together, these results suggest that the PTH-CREB pathway is an activator of osteoblast differentiation.
PTH up-regulates BMP2 expression through CREB in osteoblasts
The finding that noggin blocked CREB-induced ALP activity in osteoblasts (Fig. 2C) suggested an involvement of the BMP pathway in CREB function. A previous genome-wide study has found that BMP2, a prototype member of the BMP family, is a transcriptional target of the CREB pathway in other cell systems [37]. Given these facts, and the presence of potential CREB binding elements in the BMP2 promoter (vide infra), we hypothesized that PTH-CREB signaling may function in bone, at least in part, by up-regulating BMP2 transcription in osteoblasts. To test this hypothesis, we first examined the effects of PTH on BMP2 expression. C2C12 cells were incubated with PTH at 0–200 nM for 12 hours, and real time PCR was performed to quantitate BMP2 mRNA levels. The results showed that PTH treatment increased BMP2 mRNA expression in a dose-dependent manner (Fig. 3A). We also found that stimulation of BMP2 mRNA expression by PTH (50 nM) was time-dependent from 0 to 12 hours (Fig. 3B). In BMP2 promoter assays, C2C12 cells were transfected with a mouse BMP2 promoter reporter, −2712/+165-Luc [38]–[40], and cultured in the presence or absence of PTH. The promoter reporter luciferase assays showed that treatment with PTH for 24 hours increased BMP2 promoter activity (Fig. 3C), further demonstrating that PTH is an activator of BMP2 transcription in osteoblasts.
Figure 3. PTH up-regulates BMP2 expression through CREB.
(A, B) BMP2 mRNA levels in C2C12 cells, treated with PTH at 0 to 200 nM for 12 hours (A); or PTH at 50 nM for 0 to 12 hours (B), were measured by real time PCR with GAPDH normalization. * p<0.01 (vs vehicle; mean±SE, n = 6). (C) BMP2 promoter reporter activity in C2C12 cells, transfected with −2712/+165-Luc and treated with PTH at 0 to 200 nM for 24 hours, was measured with β-gal normalization. * p<0.01 (vs vehicle; mean±SE, n = 8). (D) BMP2 promoter reporter activity in C2C12 cells, co-transfected with CREB plasmid or vector and reporter −2712/+165-Luc, and treated with IBMX at 50 µM or KT5720 at 5 µM for 24 hours, was measured. * p<0.01 (CREB vs vector); # p<0.01 (CREB+IBMX vs CREB+vehicle); ** p<0.05 (CREB+KT5720 vs CREB+vehicle; mean±SE, n = 8). (E) BMP2 mRNA levels in calvrial cells, isolated from CREB cKO and control mice, and treated with PTH at 50 nM for 24 hours, were measured by real time PCR. * p<0.01 (cKO vs control); # p<0.01 (PTH on cKO vs PTH on control); ** p<0.05 (PTH vs vehicle on control mice; mean±SE, n = 6); NS: not significant.
doi:10.1371/journal.pone.0020780.g003Next, we determined the effects of drugs, IBMX and KT5720 that regulate PTH signaling activity, on BMP2 promoter activity. C2C12 cells that carry the BMP2 promoter reporter were transfected with CREB expression vector, and then treated with these drugs. We found that CREB transfection substantially increased BMP2 promoter activity, and that treatment with the cAMP enhancer IBMX further increased CREB stimulation. In contrast, treatment with the PKA inhibitor KT5720 significantly decreased CREB-induced BMP2 promoter activity (Fig. 3D).
Furthermore, we examined the effects of PTH on BMP2 transcription in the absence of CREB using the CREB-deficient calvarial cells described above. We found that CREB deficiency significantly reduced BMP2 mRNA levels compared with the control cells (Fig. 3E, black bars) and that the stimulation of BMP2 expression by treatment with PTH (50 nM) for 24 hours was largely abolished by inactivation of CREB in the CREB-deficient cells, compared with the control cells (Fig. 3E, gray bars). These data indicate that PTH signaling acts as an upstream stimulator of BMP2 gene expression in osteoblasts, and that this function is mediated through CREB.
CREB activates BMP2 transcription in osteoblasts
We have shown that overexpression of CREB enhanced BMP2 promoter activity (Fig. 3D). To fully characterize the activating function of CREB on the BMP2 gene, we performed multiple gain- and loss-of-function experiments to determine the effects of manipulating CREB levels on BMP2 gene expression in osteoblasts. We found that overexpression of CREB in osteoblastic cell lines, including C2C12, 2T3 and UMR106 cells, substantially increased BMP2 mRNA levels in these cells, compared with vector controls (Fig. 4A). Next, we evaluated the effects of CREB transfection on BMP2 promoter activity. The luciferase assay with −2712/+165-Luc reporter showed that CREB markedly increased transcriptional activity of the BMP2 promoter in these osteoblastic cells, with a maximal stimulation of approximately 15-fold in C2C12 cells (Fig. 4B). In contrast, small interfering RNA (siRNA) knockdown of endogenous CREB in these osteoblast cell lines had significant inhibitory effects on BMP2 mRNA transcription, measured by real time PCR, compared with siRNA controls (Fig. 4C). The loss-of-function by siRNA was further confirmed by CREB knockout. mRNA quantitation demonstrated that an osteoblast-specific CREB deletion markedly reduced BMP2 mRNA levels in primary calvarial osteoblastic cells, compared with cells isolated from control mice (Fig. 4D).
Figure 4. CREB activates BMP2 expression.
(A) BMP2 mRNA levels in C2C12, 2T3 and UMR106 cells, transfected with CREB plasmid or vector for 24 hours, were measured by real time PCR with GAPDH106 cells. * p<0.01 (vs vector; mean±SE, n = 6). (B) BMP2 promoter activity in C2C12, 2T3 and UMR106 cells, co-transfected with −2712/+165-Luc and CREB plasmid for 24 hours, was measured by luciferase activity with β-gal normalization. * p<0.01 (vs vector; mean±SE, n = 8). (C) BMP2 mRNA levels in C2C12, 2T3 and UMR106 cells, transfected with CREB siRNA or control siRNA for 36 hours, were measured by real time PCR. * p<0.05 (vs control siRNA; mean±SE, n = 6). (D) BMP2 mRNA levels in calvarial cells of CREB cKO and control mice were measured by real time PCR. * p<0.01 (vs control; mean±SE, n = 6). (E) BMP2 promoter reporter activity in C2C12 cells, co-transfected with −2712/+165-Luc and CREB or ATF4 plasmid for 24 hours, was measured. * p<0.01 (vs vector; mean±SE, n = 8). (F) BMP2 promoter reporter activity in C2C12 cells, co-transfected with −2712/+165-Luc or BMP4-Luc and CREB plasmid for 24 hours, was measured. * p<0.01 (vs vector; mean±SE, n = 8).
doi:10.1371/journal.pone.0020780.g004In addition to CREB and BMP2, other members of the CREB and BMP families also are involved in osteoblast function. The closest members in these respective families are activating transcription factor 4 (ATF4) and BMP4. In order to test the specificity of the effects of CREB on BMP2 expression, we examined the effects of ATF4 on BMP2 expression and the effects of CREB on BMP4 expression. The factor ATF4 belongs to the same large bZIP family of transcription factors as CREB, and has proven to be important for osteoblast differentiation [41], [42]. We found that, compared with CREB stimulation, ATF4 transfection did not affect BMP2 promoter activity in C2C12 cells (Fig. 4E). In the BMP family, BMP4 is structurally closest to BMP2. Using a BMP4 promoter reporter, BMP4-Luc, we found that, CREB failed to enhance BMP4 promoter activity, in contrast to CREB enhancement of BMP2 promoter activity (Fig. 4F). Collectively, these results strongly support the conclusion that the transcriptional factor CREB is a specific and potent enhancer of BMP2 gene expression in osteoblasts.
CREB transactivates BMP2 through CRE in the BMP2 promoter
CREB is known to transactivate target genes through a specific CRE consensus sequence in their promoters [17]–[22]. Sequence analysis revealed multiple CREs throughout the mouse BMP2 promoter from −2712 to +165 (data not shown). To identify the functional CRE(s) responsible for CREB transactivation of the BMP2 promoter, we first performed a promoter deletion study. C2C12 cells were transiently co-transfected with CREB expression plasmid or empty vector plus a series of truncated BMP2 promoter reporters representing −2712/+165, −2457/+165, −199/+165, −1803/+165, −968/+165, −838/+165, −310/+165, and −150/+165 of the 5′ promoter region of the BMP2 gene. Interestingly, we found that none of these progressively truncated BMP2 promoter reporters lost or reduced their responsiveness to CREB transactivation of luciferase activity (Fig. 5A). These results suggest that the potential CREs may be located within the −150/+165 region of the BMP2 promoter. Next, we analyzed the sequence of this narrow region in the promoter, and identified three putative CREs (CRE1, CRE2 and CRE3) in this area (Fig. 5B). To determine the role of these putative CREs in CREB transactivation, we performed site-directed mutagenesis. Each of these putative CREs located within the BMP2 promoter-luciferase construct (−150/+165-Luc) was mutated by deleting or replacing core nucleotides as shown in figure 5C. The results of luciferase reporter assays showed that levels of BMP2 transcriptional activity induced by CREB were comparable for mutant CRE1 and CRE3 reporters (mCRE1 and mCRE3) and the wild-type −150/+165-Luc reporter (Fig. 5D). Mutation of CRE2 (mCRE2), however, resulted in complete abrogation of the response to CREB stimulation (Fig. 5D), indicating that CRE2, in region −150/+165 of the BMP2 promoter, is a functional CRE responsible for CREB's action on the BMP2 gene. Furthermore, we also examined the effects of CRE2 mutation on PTH stimulation of BMP2 promoter activity. As shown in Figure 5E, we found that PTH lost its stimulatory effect on BMP2 promoter activity when the CRE2 is mutated, compared with wild-type CRE2 control.
Figure 5. CREB transactivates BMP2 through CRE in the BMP2 promoter.
(A) BMP2 promoter reporter activity in C2C12 cells, co-transfected with CREB plasmid and a series of truncated reporter constructs for 24 hours, was measured with β-gal normalization. (B) Sequence of −150/+165 of mouse BMP2 promoter. Putative sequences for CRE1, CRE2 and CRE3 are underlined and bolded. Sequences of ChIP primers are underlined only. (C) Sequences of mutant CRE1, CRE2 and CRE3 (mCRE1, mCRE2 and mCRE3). (D) BMP2 promoter reporter activity in C2C12 cells, co-transfected with wild-type −150/+165-Luc (WT) and mutant −150/+165-Luc in which CREs were mutated (mCRE1, mCRE2 and mCRE2), and with CREB plasmid for 24 hours, was measured. * p<0.01 (CREB transfection on mCRE2 vs WT or mCRE1 or mCRE3 reporters; mean±SE, n = 8). (E) Effects of PTH on BMP2 promoter activity. C2C12 cells were co-transfected with wild-type −150/+165-Luc (WT) and mutant −150/+165-Luc in which CRE2 was mutated (mCRE2) and treated with PTH at 50 nM for 24 hours. * p<0.01 (PTH mCRE2 vs PTH on WT reporter; mean±SE, n = 8).F) ChIP assay. Nuclear DNA-protein complexes were extracted from C2C12 cells treated with PTH at 50 nM for 6 hours and precipitated with anti-CREB antibody. PCR was performed to amplify the region of the BMP2 promoter that contained CRE2, using PCR primers indicated in (B). Input: BMP2 promoter DNA. IgG: Goat IgG as a negative control. (G) EMSA assay. Nuclear extracts of C2C12, 2T3 and MC3T3-E1 cells were incubated with a biotin-labeled DNA probe containing the CRE2 binding site sequence in the BMP2 promoter, in the absence or presence of anti-CREB antibody. The shift and super shift bands were analyzed using a 5% polyacrylamide gel.
doi:10.1371/journal.pone.0020780.g005We subsequently performed ChIP and EMSA assays to verify the interaction of CREB with CRE2 in the BMP2 promoter. In the ChIP assay, a 230 bp fragment of the BMP2 promoter containing the specific CRE2 sequence was amplified after the addition of anti-CREB antibody to nuclear DNA-protein complexes extracted from C2C12 cells, compared with non-specific IgG control (Fig. 5F, lane 3 vs lane 2). This suggests that CREB binds directly to CRE2 in the BMP2 promoter. Importantly, treatment of these cells with PTH (50 nM) for 6 hours further increased CREB binding ability to CRE2, evidenced by increased PCR product in the gel (Fig. 5F, lane 4 vs lane 3). Furthermore, EMSA was performed to obtain direct evidence of physical binding of CREB with CRE2. Nuclear proteins were extracted from C2C12, 2T3 and MC3T3-E1 osteoblast cells and reacted with a biotin-labeled DNA probe containing the CRE2 sequence (−13 to +20) of the BMP2 promoter. The addition of the CRE2 probe to these cell extracts induced a shift in mobility of the probe in the gel, (Fig. 5G, lane 2–4 vs lane 1), indicating a direct interaction between the CRE2 probe and nuclear proteins. Further, addition of anti-CREB antibody to the reaction mixture of C2C12 cells produced a super shift in mobility of the band (Fig. 5G, lane 7 vs lane 6), indicating that CREB binds directly to this CRE2 complex. Based on the combined results of luciferase reporter, ChIP and EMSA assays, we propose that the transcriptional factor CREB transactivates the BMP2 gene through a specific CRE2 site in the BMP2 promoter.
Discussion
These collective results provide evidence that the PTH signaling pathway is an effective activator of BMP2 gene expression in osteoblasts, and this function is mediated by CREB transactivation of the BMP2 promoter. This transcriptional mechanism, at least in part, accounts for the anabolic function of the PTH-CREB pathway in osteoblast differentiation and bone formation.
It is well documented that intermittent dosing with PTH has an anabolic effect on bone [1]–[10], and that the PTH signaling pathway is a physiological regulator of osteoblast function in bone [11]–[30]. As a major mediator of PTH signaling, the transcriptional factor CREB has been found to play a role in osteoblast differentiation by regulating expression of osteoblast-specific genes [13]–[22], by which CREB may function as an anabolic regulator for bone mass. Several previous in vivo studies have provided evidence for the anabolic function of CREB in bone [30], [35], [36], [43], [44]. A recent report showed that small molecules with potent osteogenic activity induce osteoblast differentiation by activating intracellular CREB activity [43]. In studies with Lrp 5 knockout mice that exhibit low bone mass, Yadav et al reported that Lrp5 deficiency increases duodenal production of serotonin, which inhibits osteoblast function by suppressing CREB activity in osteoblasts [44]. Furthermore, they found that Lrp5 and CREB double knockout mice have a significantly lower bone mass than Lrp5 single null mutants [44]. Recently, we have reported that osteoblast-specific knockout of CREB in mice produces a skeletal phenotype with low bone mineral density and low bone volume [35], [36]. This is consistent with a previously reported similar osteopenic phenotype of the osteoblast-specific ICER (inducible cAMP early repressor) transgenic mice [30]. ICER belongs to the same bZIP family of transcription factors as CREB, and acts as a dominant negative regulator of gene transcription through CRE. Thus, it appears that CREB plays an important role in postnatal bone formation through its effect on osteoblast cells. To explore the mechanisms by which CREB exerts its effects on bone, in the current gain- and loss-of-function studies employing primary osteoblast cells and multiple osteoblast cell lines, we found that manipulation of activity and expression levels of PTH and CREB affects osteoblast function. We also demonstrated that the PTH-CREB signaling pathway is a positive regulator of osteoblast differentiation. These findings further support the hypothesis that promoting osteoblast differentiation is one of cellular mechanisms by which the PTH-CREB signaling pathway exerts its anabolic function in bone.
The cAMP-PKA-CREB axis represents a major signaling pathway that mediates PTH signaling [11]–[30]. In the current study, we demonstrated that PTH induces CREB phosphorylation in osteoblasts and that this function is regulated by pharmacological manipulation of the PTH signaling cascade in the upstream of CREB. The results of experiments with the cAMP-enhancer and the PKA-inhibitor suggest that the capacity of CREB to enhance BMP2 transcription in osteoblasts is phosphorylation-dependent. In these studies, however, we found that the PKA-inhibitor (KT5720) failed to completely abolish the capacity of CREB to increase BMP2 transcription in osteoblasts, suggesting that other protein kinases, in addition to PKA (such as protein kinase C), also may be involved in this process. Similar results have been reported with other cell systems [45], [46]. In a separate experiment, we found that the cAMP-enhancer IBMX had biphasic effects on the BMP2 promoter, depending on the duration of treatment. In contrast to the stimulatory effect on BMP2 expression observed in this study when cells were exposed to IBMX for only 24 hours, extending exposure up to 48 hours resulted in inhibition of BMP2 promoter activity (data not shown). This latter effect of IBMX on the BMP2 gene may be due to the degradation of transcription factor Gli2, which we previously identified as a stimulator of BMP2 expression in osteoblasts [39], [40].
CREB transactivates target genes through a consensus cAMP response element (CRE), by which CREB has been shown to transactivate multiple osteoblast maturation related marker genes, such as BSP and OCN [13]–[22]. The rationale in this study to focus on the BMP2 gene as a critical transcriptional target of CREB in osteoblasts includes the following: (1) BMP2 is the prototypic member of the BMP family responsible for osteoblast differentiation; (2) cAMP, an upstream activator of CREB, up-regulates BMP2 gene expression in other cells [37], and; (3) multiple putative CRE sites can be found throughout the BMP2 promoter. Therefore, we mapped out the molecular interaction between CREB protein and the BMP2 promoter. Utilizing promoter deletion, mutation, ChIP and EMSA studies, we defined a specific CRE (CRE2) located within the basal promoter loci from −150 to +165, as a functional binding element responsible for CREB transactivation of the BMP2 gene. We recognize, however, that regulation of the BMP2 gene is complex, and that multiple additional mechanisms, involving Gli proteins [39], [40], [47], β-catenin/TCF [48] and ERα [49], have been reported to contribute to the regulation of BMP2 transcription. Thus, we do not exclude the possibility that additional untested CRE sequences in the upstream of the BMP2 promoter may also possess potential cis-functions in BMP2 gene transcription in response to other nuclear transcription factors such as those identified above. Interestingly, in this study, we found that ATF4 (a member of the CREB/ATF family) failed to stimulate BMP2 expression. In other cell types, ATF4 and CREB have been shown to share the same DNA binding element (TGACGTCA) in transactivating the promoter activities of target genes [49]. We presume, therefore, that CREB transactivation of the BMP2 gene in osteoblasts may require other co-activators associated within the CREB transcriptional machinery that are structurally and functionally distinct from those associated with ATF4 action. However, more comparison studies about the transcriptional mechanisms of these factors on the BMP2 gene are needed to examine this possibility.
The experiments in which noggin blocked CREB-induced ALP activity support the concept that the function of CREB in osteoblasts is likely BMP-dependent, because noggin blocks BMP signaling by hindering BMP ligands from binding to BMP receptors [50], [51]. The present data showed that CREB, as a powerful stimulator of BMP2 transcription, lacks the capability to induce expression of BMP4, the closest member of BMP2 in the BMP family. However, it is possible that CREB may have a positive regulating function for transcription of other BMP family members. This possibility warrants further investigation.
In addition to the capability of increasing BMP2 gene expression in osteoblasts demonstrated in the current study, the PTH-cAMP-CREB pathway also has been shown to enhance BMP2 activity. For example, it has been demonstrated that this pathway is capable of enhancing BMP2-stimulated Smad signaling [34], and synergizing the BMP2-induced anabolic effects on differentiation of osteoblasts [23], [25], [26] and chondrocytes [23], [52] in vitro, and new bone formation in vivo [28], [29]. In contrast, BMP2 is also known to have a positive feedback effect on CREB function. In osteoblast precursor C2C12 cells, BMP2 synergizes with PKA-CREB to enhance ALP activity [25]. Therefore, the crosstalk between the PTH-CREB and BMP2 signaling pathways is complex, and could occur at multiple steps in these signaling cascades. In order to further test the role of BMP2 in mediating the function of PTH-CREB signaling in osteoblast differentiation and bone formation, a BMP2 invalidated mouse model is needed. We are now proposing a study using osteoblast-specific BMP2 knockout mice to determine the effects of BMP2 deficiency on the anabolic effects of intermittent PTH on bone mass. The present finding that PTH-CREB up-regulates of BMP2 gene expression in osteoblasts provides new insights into the relation between these important anabolic signaling pathways in bone.
In summary, the functional and mechanistic studies in this study demonstrate that PTH signaling is a positive regulator of BMP2 gene expression in osteoblasts, mediated directly by CREB transactivation of BMP2 promoter activity. This transcriptional mechanism contributes to osteoblast differentiation. The collective results of these studies support the hypothesis that the anabolic effects of PTH on bone mass are mediated through the cAMP-PKA-CREB-BMP2 pathways.
Materials and Methods
DNA constructs and compounds
The CREB expression plasmid pRC/RSV-CREB, its empty vector pRC/RSV, and CREB responsive reporter (CRE-Luc) containing multiple CREs were obtained from Dr. Richard Goodman [53]. The ATF4 expression vector was a gift of Dr. Xiangli Yang [42]. Mouse BMP2 promoter luciferase reporter (−2712/+165-Luc) was constructed by linking the 5′ promoter region (−2712/+165) of mouse the BMP2 gene to firefly luciferase in a pGL3 vector [39]–[40]. The promoter was truncated from the distal end by enzymatic digestion to create a series of deletion constructs, including −2457/+165-Luc, −199/+165-Luc, −1803/+165-Luc, −968/+165-Luc, −838/+165-Luc, −310/+165-Luc, and −150/+165-Luc. Mouse BMP4 promoter reporter (BMP4-Luc) was obtained from Dr. Stephen Harris. The CREB siRNA (CREB ShortCut® siRNA Mix), and negative control siRNA (New England Biolabs, Beverly, MA), IBMX and KT5720 (Calbiochem, San Diego, CA), and
On Thursday, December 4, 2014 11:20:46 PM UTC-8, Uhohinc wrote:
Farma Zutical
But I wonder if Clinuvel will ever consider a license scenario where big pharma gets to use Scenesse for some of all the other indications this implant seems to
treat or cure. To an extend, I would say it is close to unethical not to persue this considering that patients would benefit greatly from the implant. Take for instance
HHD. If Clinuvel shelves HDD many patients won't get the relief they are in my view entitled to. If ther eis a medicine and if it works one should have the right
to access it. If Clinuvel won't persue this I hope others will. this is not only about money. This is about a drug that can eventually change the World.
Uhohinc
Matrix metalloproteinase and Collagen 1
Uhohinc
Thesis research indicating stimulation of mcr 1 and mcr 3 will downreg the same negative interlukines including 1 and 6 and upreg the good interlukeins including 10 as well as negating bad cytokines. Including tnf.
Uhohinc
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Although α-MSH has been shown to decrease TNF-α induced MMP expression, the parent hormone ACTH can cause terminal differentiation of chondrocytes and subsequent cartilage degeneration (Evans et al., 2004). Rodent chondrocytes and chondrocyte cell-lines express MC3, matrix deposition was concentration dependently elevated in the presence of ACTH. These data highlight that the melanocortin system promotes chondrocyte phenotype development and their differentiation into mature chondrocytes, leading to an elevation in intracellular free calcium (Evans et al., 2005).
1.4.2 Rheumatoid arthritis.
RA is a complex pathology affecting many systems outside of the joints, with 40 % of RA deaths due to cardiovascular disease. Therapeutic intervention in RA is a leading cause of complications, for example NSAIDs may cause elevated blood pressure (Getting et al., 2009) and glucocorticoids increase cardiovascular problems by accelerating the rate of pathologies such as arterial thickening and narrowing (Getting et al., 2009). Methotrexate on the other hand may also promote heart disease by increasing levels of homocysteine. As a result of these complications and the fact that NSAIDs are contraindicated in the elderly due to high possibility of kidney failure, the progress of novel endogenous therapeutics is indispensable.
The role of melanocortin peptides in rheumatoid arthritis has not been fully ascertained yet. Catania et al. detected increased concentration of α-MSH in the synovial fluid of patient suffering from RA, juvenile arthritis, but not OA, that were greater than that detected in the plasma (Catania et al., 1994; Grässel et al., 2009) suggesting local production of the peptide, i.e. that the anti-cytokine molecule α-MSH is produced within a site of inflammation. This increase in αMSH within synovial fluid also suggests the possibility of an endogenous antiinflammatory loop maintaining a homeostatic balance within the joint and controlling the host inflammatory response. Joint concentrations of α-MSH in these patients were directly proportional to the degree of inflammation, whilst systemic (plasma) levels remained at physiological concentrations (Catania et al., 1994).
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Utilising the adjuvant-induced arthritis model, α-MSH was shown to modulate weight loss, arthritic score, joint damage, and swelling, characteristic features of the disease. This effect on preventing weight loss was in contrast to that observed by the glucocorticoid prednisolone, which caused significant weight loss in these animals (Ceriani et al., 1994). The inflamed joint is characterised not only by infiltrating leukocytes, but also by activated resident bone/cartilage cells, such as osteoclasts and chondrocytes. Some evidence exists to suggest melanocortin receptor expression on these cells (Yoon et al., 2008). In situ hybridization shows that all MCs are expressed on chondrocytes in the mouse femoral bone and mRNA signals for all MCs, except MC1, were detected on primary rat osteoclasts, which also expressed the POMC gene (Zhong et al., 2005), thereby suggesting the possibility that POMC peptides generated by these cells could act in an autocrine/paracrine manner through their corresponding MCs. α-MSH has been shown to inhibit TNF-α-induced MMP13 by modulating p38 and NF-κB signaling in human chondrosarcoma cell-line HTB-94 (Yoon et al., 2008). These findings indicate that α-MSH may be beneficial in arthritic conditions.
1.4.3 Gouty arthritis.
On Monday, January 19, 2015 at 1:17:03 AM UTC-8, Uhohinc wrote:
Uhohinc
In order to investigate whether the melanocortin peptides have chondroprotective properties, cell viability was quantified as described earlier. Briefly, cells were treated for 6 h with 3.0 µg/ml α-MSH or D[TRP]8-γ-MSH, or 1.0 µM dexamethasone and then incubated with the fluorescent indicators Calcein-AM (5.0 µM) and Propidium Iodide (1.0 µM) for 30 min prior to CLSM, permitting the measurement of cell viability.
Figure 3.54 demonstrates that α-MSH caused a reduction in chondrocyte death compared to basal levels (2.95 %). Cell death decreased to 0.97 %, when cartilage explants were incubated for 6 h with α-MSH (3.0 µg/ml) prior to CLSM imaging, representing 67 % reduction in cell death, compared to untreated controls (p≤ 0.05). However, neither D[TRP]8-γ-MSH (3.0 µg/ml) nor dexamethasone (1.0 µM) was able to show any significant effect on this parameter.
Uhohinc
In healthy articular cartilage, chondrocytes are actively maintaining the expression and ratio of collagens and proteoglycans (Hall, 1998). Chondrocytes are very sensitive to pro-inflammatory cytokines, an observation supported by this study. Studies have reported that pro-inflammatory cytokines either reduce or enhance the production of type II collagen, a marker of normal function of chodrocytes (Ho et al., 2006). Therefore to determine if the cell-line responded in a similar fashion to primary cells, the direct effect of TNF-α on the expression of the cartilage specific collagens was determined by RT-PCR. TNF-α stimulation caused COL1A1 and COL2A1 levels to decline significantly, therefore inhibiting the chondrocyte compensatory synthesis pathways, required to restore integrity of the degraded matrix (Goldring and Goldring, 2004). It is important to point out, that TNF-α did not influence the differentiation indicator ratio of COL2A1: COL1A1 detected in unstimulated C-20/A4 cells, which is an important observation as dedifferentiated cells would be undesirable for the purposes of this study.
To ensure that our in vitro cell-line system responded in a similar fashion to primary cells the effect of the glucocorticoid dexamethasone and the NSAID Indomethacin was evaluated given the role they play in the treatment of inflammatory pathologies. Glucocorticoids are powerful anti-inflammatory molecules shown to be able to repress transcriptional activation of genes including IL1, IL-6, IL-8, TNFα , γ-interferon, colony stimulating factor (CSF)1/macrophage, granulocyte macrophage (GM)-CSF (Taniguchi, 1988). Most of these genes are activated by the transcription factors NF-κB and AP-1, and their down-regulation confirms that glucocorticoids are interfering with these pathways
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(Vayssiere et al., 1997). NSAIDs exert their effects by inhibiting COX enzyme (part of the arachidonic acid cascade) and reducing prostaglandins leading to a diminished inflammation and pain (Vane, 1971, 1976). At present only COX-1 and COX-2 are clinically relevant, with COX-1 regarded as housekeeping enzyme responsible (via prostaglandins and thromboxane A2) for physiological functions including protection of gut mucosal integrity and vascular homeostasis (Chen et al., 2008), whilst COX-2 appears to be a more important mediator of inflammation and thus a key factor in arthritic pain (Chen et al., 2008).
Dexamethasone and Indomethacin were administered 30 min prior to TNF-α stimulation of C-20/A4 chondrocytes. Dexamethasone and indomethacin have previously been shown to inhibit cytokine production in other cell systems (Mukaida et al., 1991). Glucocorticoids activate intracellular receptors that then bind to glucocorticoid-responsive elements in the promoters of various genes, or inhibit NF-κB transocation in the nucleus (Vayssiere et al., 1997). Furthermore, they inhibit AP-1 DNA binding ability and therefore block the respective gene expression (Vayssiere et al., 1997). In this study they were shown to inhibit TNFα (60.0 pg/ml) induced expression of IL-6 and IL-8 mRNA and protein, an effect accompanied by a abrogation of the expression of MMP1, MMP3 and MMP13 over the time-course. This ability to modulate inflammatory pathways in this celltype was in agreement with previous studies utilizing primary chondrocytes (Richardson and Dodge, 2003). Both the glucocorticoid dexamethasone and the NSAID indomethacin completely abrogated the production of IL-6 and IL-8 at all time points tested, and in doing so they negatively surpassed even the basal levels of production of these cytokines detected in unstimulated cells. However, although this effect may be desireable in management of acute inflammation, in chronic inflammatory diseases such as OA, this could lead to supression of the HPA axis and lead to impaired wound healing, Cushings syndrome and opportunistic infections (Gupta et al., 2000; Alekseev et al., 2001; Dorscheid et al., 2006).
Data generated here looking at exempler cytokines, shows that TNF-α and LPS alone trigger a cascade of cytokines in this in vitro model. The results obtained from the ELISA and the PCR revealed a minimum 2-fold increase in inflammatory marker genes in these activated C-20/A4 chondrocytes. Of importance was the
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observation that classical anti-inflammatory drugs (dexamethasone and indomethacin) were able to modulate these pathways. These data sugests that its possible to use this cell-line as a surrogate in vitro model for investigating inflammatory pathways within these cells and to evaluate the effects of the melanocortin peptides.
Melanocortin receptor expression in C-20/A4 chondrocyte and the role of melanocortins in modulation of pro-inflammatory cytokine production
Melanocortin peptides have potent antipyretic and anti-inflammatory effects (Grabbe, 1996; Getting et al., 1999, 2001, 2008, 2009; Getting 2002, 2006, Luger, 2000), which they deliver via activation of a family of 7TM-GPCRs (Catania et al., 2004). These are termed melanocortin receptors and to date five subtypes have been identified (Getting et al., 2009). Previous research within the field of inflammation has highlighted that these peptides can modulate the effect of several pro-inflammatory cytokines and chemokines such as TNF-α, IL-1β, IL6, and IL-8 (Catania et al., 1999; Grassel et al., 2009) and they are inducers of the anti-inflammatory cytokine IL-10 (Redondo et al., 1998; Lam et al., 2005) . This study tested the hypothesis that targeting melanocortin receptors may provide a novel therapeutic approach to treatment of chondrocyte inflammation, such as that observed in OA.
For this purpose, pharmacological and molecular techniques were used employing the melanocortin receptor pan-agonist α-MSH (Catania et al., 2006; Getting, 2002, 2006; Getting et al., 2009; Rajora et al., 1996, 1997) and the selective MC3 agonist D[TRP]8-γ-MSH (Grieco, 2000; Getting, 2006; Getting et al., 2008, 2009). To date, two receptors have been identified to mediate the antiinflammatory effects of melanocortin peptides, namely the MC1 and MC3 (Getting, 2002, 2006, Getting et al., 2009). However, some evidence points towards a role played by MC5 in inflammation, since its expression has been detected on B-lymphocytes (Buggy, 1998) and T-lymphocytes (Taylor and Namba, 2001), suggesting a potential role in immuno-modulation.
Initially, expression of MC1, MC3 and MC5 was determined in C-20/A4 chondrocytes, reported here for the first time with a strong signal MC1 and MC3, and a very faint signal corresponding to MC5. Recently, gene expression of MC1
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was identified in the human chondrosarcoma cell line HTB-94 (Yoon et al., 2008), and MC1, MC3 and MC5 transcripts have been detected in primary articular chondrocytes (Grässel et al., 2009). Whilst the detection of MC1 is in agreement with previous studies, to our knowledge the detection of MC3 expression in human chondrocytes is novel. Given the apparent disparity in the results observed and those generated previously in primary cells, western blotting was used to determine if mRNA for MC1 and MC3 was translated into protein.
Following the identification of mRNA and protein for MC1,3 and mRNA for MC5, the functionality of the receptors was determined by evaluating a panel of melanocortin peptides (displaying different receptor selectivity) on cAMP accumulation as detected by EIA. Melanocortin receptors are positively coupled to adenylate cyclase, which upon activation causes increase in intracellular cAMP formation (Catania et al., 2006 ; Gantz et al., 2003; Getting et al., 2009). In order to determine the functionality of the melanocortin receptors, at first we tested whether the MC1 receptor agonist α-MSH, the MC3 selective agonist D[TRP]8-γ-MSH, and the MC5 selective agonists SHU9119, PG901 and PG911 could induce cAMP accumulation in the human C-20/A4 cells. Our functional studies showed that both α-MSH and D[TRP]8-γ-MSH were able to ellicit a significant and concentration-dependent increase in cAMP formation, this effect being observed in C-20/A4 chondrocytes for the first time. Whilst α-MSH has previously been shown to induce cAMP accumulation in chondrocytes (Grassel et al., 2009) the ability of the MC3 agonist D[TRP]8-γ-MSH to incude increases in cAMP has not been previously demostrated in chondrocytes. This increase in cAMP occurred in a bell-shaped manner and is in agreement with previous studies utilizing these peptides in other cell-types (Getting et al., 2006).
Given that MC5 mRNA was observed in C-20/A4 chondrocytes and has previously been shown to be epxressed on primary articular chondrocytes (Grassel et al., 2009), the effect of selective MC5 agonists PG901, PG911 (Grieco et al., 2002) and the mixed agonist/antagonist SHU9119 (Hruby et al., 1995) was evaluated. Treatment of cells with these peptides at all the concentrations evaluated did not cause an increase in cAMP accumulation. This lack of effect of the selective MC5 agonists PG901 and PG911, was perhaps due
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to the weak mRNA expression of this receptor, not translated to protein. Whilst we cannot completely rule out the expression of a functionally active MC5 in the C-20/A4 cell model, given the fact that more selective compounds maybe developed in the future, which would allow further investigation of this receptor. At present, it is highly unlikely that this receptor plays a role in modulating the effects of the melanocortin peptides given the low expression level and lack of functionality displayed here in this model.
Given that α-MSH and D[TRP]8-γ-MSH significantly elevated cAMP levels, the peptides were evaluated in the presence of the MC3/4 anatagonist SHU9119 (Fan et al., 1997) used at a concentration previously shown to inhibit the cAMP accumulation elicited by these peptides (Getting et al., 2006), thus allowing identification of whether MC1 or MC3 was involved. Incubation of the C-20/A4 chondrocytes with SHU9119 was able to inhibit D[TRP]8-γ-MSH confirming previous findings in other cell types (Getting et al., 2006) and in models of inflammation (Getting et al., 2008, Leoni et al., 2008, Patel et al., 2010), whereby D[TRP]8-γ-MSH mediates its effects via MC3. Not surprisingly, SHU9119 (10.0 µg/ml) failed to block α-MSH at all concentrations, except 3.0 µg/ml, which caused an extremely modest reduction in cAMP levels. At MC3, the peptides ACTH1-39, α-MSH, β-MSH and γ-MSH are equipotent (Getting, 2006) and even though α-MSH preferentially activates MC1, it cannot be excluded that, at this concentration, some of the increase in cAMP observed with α-MSH may be inpart due to activation of the MC3.
Numerous studies have highlighted the ability of melanocortin peptides to inhibit cytokine release in vitro (Lam et al., 2005, 2006) and also in vivo models of inflammation (Ceriani et al., 1994, Getting, 2002, Getting et al., 2003, 2006, 2008). However, to date only one study has investigated the effect of melanocortin peptides on chondrocytes (Grässel et al., 2009) and none using the selective MC3 agonist D[TRP]8-γ-MSH. C-20/A4 chondrocytes were treated with α-MSH (3.0 µg/ml), D[TRP]8-γ-MSH (3.0 µg/ml), and SHU9119 (10.0 µg/ml) to ascertain their effect on basal release of cytokines from these cells. In order to determine the effect of the petides in the absence of detectible inflammation, IL
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1β, IL-6, IL-8 and MCP-1 levels were determind by ELISA and showed that none of the peptides caused an elevation in basal cytokine release from these cells.
The effect of melanocortin peptides on TNF-α induced inflammatory markers, was then evaluated since its has been shown to be involved in activation of chondrocytes leading to the degradation of cartillage within the knee joint. α-MSH has long been known to suppress inflammation by down-regulating the expression of pro-inflammatory cytokines, and to have anti-inflammatory and immuno-modulatory actions in rodent models of inflammation in a corticosteroneindependent manner (Getting et al., 1999). Here it inhibited TNF-α-induced release of IL-1β, IL-6, IL-8 and MCP-1 from C-20/A4 chondrocytes in a concentration-dependent manner. The peptide displayed potent anti-cytokine effects at both 2, and 6 h, a similar observation that was noted in primary murine peritoneal macrophages (Getting et al., 1999) and macrophage cell-lines (Lam et al., 2006). The peptide did not elicit any effect following 24 h incubation. The suppression of IL-1β, IL-6 and IL-8 by α-MSH in C-20/A4 chondrocytes is in accordance with the overall anti-inflammatory and protective capacity of the peptide (Catania et al., 2004).
Given the elevation in cAMP observed with the MC3 agonist D[TRP8]-γ-MSH, it was evaluated in this model. Here the effect of D[TRP]8-γ-MSH (0.1 – 30.0 µg/ml) on modulation of pro-inflammatory cytokine release from human C-20/A4 chondrocytes activated by TNF-α was determined. C-20/A4 chondrocytes were treated with the selective melanocortin peptide for 30 min prior to stimulation with TNF-α, and subsequently incubated for 2-24 h, when supernatants were collected and analysed for IL-1β, IL-6 and IL-8 release. D[TRP]8-γ-MSH showed a bell-shaped inhibition of IL-1β release with 3.0 µg/ml and 10.0 µg/ml being consistently the most potent concentrations, causing ~ 70-80 % inhibition. Similarly, a bell-shaped response in IL-6 was observed following 2 and 6 h of incubation, peaking at 3.0 µg/ml D[TRP]8-γ-MSH, however, when the treatment was continued for 24 h, the peptide showed a concentration dependent inhibition of IL-6. IL-8 production was inhibited by D[TRP]8-γ-MSH in a concentrationdependent manner at all time points, with 10.0 and 30.0 µg/ml being the most potent with a 70-80% reduction in this chemokine following TNF-α stimulation.
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These data highlight for the first time the ability of this peptide to inhibit cytokine release from chondrocytes. The peptide displayed efficacy at all time-points evaluated and was still active at 24 h post treatment, correlating with the findings of this peptide in urate crystal-induced inflammatory cytokine release from macrophages (Lam et al., 2005) The maximal anti-inflammatory effect at 2 and 6 h post stimulation was reached by 3 and 10.0 µg/ml of D[TRP]8-γ-MSH concentrations previously shown to be effective (Getting et al., 2006, 2008) with a 70-80 % inhibition of IL-1β release.
This ability of the peptide to supress cytokine release was recently confirmed in a model of LPS-induced lung inflammation (Getting et al., 2008). With respect to models of arthritis α-MSH has been shown to repress experimental adjuvantinduced arthritis in rats (Ceriani et al., 1994), whilst a recent study by the Perretti group has highlighted the importance of the MC3 agonist D[TRP]8-γ-MSH in a model of serum transfer arthritis where the peptide was effective in wild type mice but not in MC3-/- null mice (Patel et al., 2010). However, neither of the selective MC5 agonists (MC3/4 antagonists) PG901 and PG911 was able to cause significant decrease of TNF-α-induced pro-inflammatory cytokines, which together with the inability of the peptides to elicit cAMP increases, suggesting that this receptor is not functionally active in C-20/A4 chondrocytes.
The anti-inflammatory effects of α-MSH and D[TRP]8-γ-MSH were evaluated on IL-6 and IL-8 transcription levels in the presence or absence of the MC3/4 antagonist SHU9119, at a dose previously shown to abrogate the inhibitory effects of γ2-MSH on chemokine release (10.0 µg/ml) (Getting and Perretti, 2000). α-MSH has been shown previously shown to suppress an array of inflammatory cytokines including TNF-α (Rajora, 1997 ; Delgado Hernandez, 1999) and IL-1β (Getting et al., 2003). α-MSH has tremendous effect on chemotaxis, further supported by the finding of Luger’s group that this melanocortin peptide inhibits the production and release of IL-8 (Brzoska et al., 1999). Our study demonstrates and confirms the anti-inflammatory effect of not only α-MSH, but also D[TRP]8-γ-MSH in human C-20/A4 chondrocytes. The pretreatment of C-20/A4 chondrocytes with α-MSH (3.0 µg/ml) prior to TNF-α stimulation caused an ~30 % inhibition in the expression of IL-6 (35 %) and IL-8
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(25 %), therefore supporting the collected evidence that this peptide and its putative receptor have marked impact on IL-6 and IL-8 regulation. Additionally, αMSH not only inhibited the transcription of these genes, but also the protein release of IL-6, IL-8 and MCP-1 from the cells. Cytokine ELISAs showed that there was a 70 %, 60 % and 21 % reduction in IL-6, IL-8 and MCP-1 protein levels released from C-20/A4 chondrocytes. The ability of α-MSH to exert antiinflammatory actions has been well documented (Martin, 1991 ;Lipton, 1997, 1999; Catania, 1999), whereas the use of the mixed MC3/4 antagonist SHU9119 (Hruby et al., 1995; Fan et al., 1997; Getting et al., 2006) did not affect the observed anti-cytokine effects of α-MSH, suggesting that the latter must be preferentially activating MC1 in order to exert its effect.
D[TRP]8-γ-MSH was also able to diminish pro-inflammatory cytokines expression causing 27% and 36% inhibition of TNF-α-stimulated IL-6 and IL-8 transcription, respectively. Furthermore, similarly to the action of α-MSH, D[TRP]8-γ-MSH led to 68 %, 45 % and 26 % reduction in IL-6, IL-8 and MCP-1 production, respectively, an observation in accordance with other studies exemplifying the anti-inflammatory and anti-migratory effects of D[TRP]8-γ-MSH on cultured Mø both in vitro and in vivo (Getting et al., 2006). An important finding was that SHU9119 completely obliterated the effect of D[TRP]8-γ-MSH not only on cytokine transcription levels, but also on the synthesis and release of IL-6, IL-8 and MCP-1 from C-20/A4 cells compared to the effect of D[TRP]8-γ-MSH alone. These data, together with the functional studies of MC1 and MC3 receptor activation, highlight not only the anti-inflammatory properties of α-MSH and D[TRP]8-γ-MSH, but also confirms that both MC1 and MC3 might be the main targets for inflammation modulation in C-20/A4 chondrocytic system. Of interest is the fact that unlike dexamethasone and indomethacin, neither of the melanocortin peptides caused a complete abrogation of pro-inflammatory cytokine production. In contrast, they modulated the production of the tested cytokines, therefore allowing for some level of synthesis from the chondrocytes.
A novel finding of this study is the expression of functionally active MC3 on C20/A4 chondrocytes and that agonism of this receptor modulates the inflammatory response of TNF-α-activated human C-20/A4 chondrocytes.
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However in this cell-line no one has ever investigated the effect of melanocortin peptides or their receptors on matrix metalloproteinases expression following TNF-α stimulation. We have shown that TNF-α potently up-regulates the expression of MMP1 and 13 and that there is an upregulation in pro-inflammatory cytokines confirming previous findings (Fernandes et al., 2002; Martel-Pelletier et al., 1999). α-MSH pre-treatment of TNF-α-activated C-20/A4 chondrocytes led to a marked 48 % reduction in transcription of MMP1, one of the interstitial collagenases, significantly up-regulated in human osteoarthritic cartilage compared to healthy tissue (Reboul et al., 1996; Kevorkian et al., 2004).
Collagenase 3, or MMP13, is highly up-regulated in chondrocytes isolated from human osteoarthritic chondrocytes (Shlopov et al., 1997) and following TNF-α stimulation (Rai et al., 2008), and was significantly down regulated by α-MSH (3.0 µg/ml) in our model of TNF-α-activated C-20/A4 chondrocytes. The melanocortin peptide down-regulated the expression of MMP13 by 67 %, compared to TNF-α-stimulated levels. These findings are in agreement with a recent study showing that α-MSH can inhibit TNFα-induced MMP13 expression in the chondrosarcoma cell line HTB-94 (Yoon et al., 2008).
In order to additionally confirm the involvement of MC1 and MC3 in the transmission of these effects, SHU9119 was added in conjunction with α-MSH, but no significant effect was observed on the expression of either MMP1 or MMP3. However, an interesting observation was made when analysing the effect of this combination on MMP13 expression. RT-PCR showed that SHU9119 and α-MSH synergistically inhibited the expression of this collagenase, leading to 87 % reduction compared to TNF-α-stimulated levels, thereby suggesting other mechanism by which this combination might affect the expression of this particular collagenase.
Similar results were obtained following pre-treatment of chondrocytes with the selective MC3 agonist. D[TRP]8-γ-MSH was more potent than α-MSH in reducing MMPs expression, with reductions of 89 %, 76 % and 92 % in MMP1, MMP3 and MMP13 expression, respectively, compared to levels detected following TNF-α treatment. However, when cells were treated with SHU9119 and D[TRP]8-γ-MSH,
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the effect was completely attenuated in all cases, confirming the involvement of MC3 in the modulation of degradative matrix metalloproteinases. All these actions contribute to a local attenuation of the host’s inflammatory response. Given the wealth of knowledge generated so far, few studies have looked at the potential of the melanocortins in inducing anti-inflammatory mediators in chondrocytes (Iannone et al., 2001; Fernandes et al., 2002).
In contrast to the suppressive effects of endogenous melanocortin peptides on pro-inflammatory cytokines production and release, they have been shown to elicit significant elevations in the production of the anti-inflammatory cytokine with potent immuno-suppressive properties, IL-10 (Bhardwaj et al., 1996; Redondo et al., 1998; Lam et al., 2006). The importance of IL-10 in melanocortin receptor biology was first demonstrated in a model of contact hypersensitivity, where an antibody against this cytokine abrogated the protective action attained by α-MSH application (Grabbe et al., 1996). The anti-inflammatory effect of α-MSH observed here and in other models, could be dependent on IL-10 induction, given that α-MSH was inactive in IL-10 knock out mice in a murine model of allergic airway inflammation (Raap et al., 2003). This study sought to reveal, whether αMSH and D[TRP]8-γ-MSH could stimulate the production of IL-10 in C-20/A4 chondrocytes in the presence and absence of TNF-α. Additionally, the ability of SHU9119, PG901 and PG911 was tested in order to investigate whether these peptides could stimulate their cognate receptor (MC5) to induce IL-10 synthesis. Human chondrocytes from healthy and osteoarthritic cartilage have been shown to express the anti-inflammatory cytokine IL-10 and its putative receptor IL10R (Iannone et al., 2001), which upon interacting down-regulate TNF-α-induced MMP1 and MMP13 (Shlopov et al., 2000). Here, we have demonstrated, that treatment with α-MSH and D[TRP]8-γ-MSH (3.0 µg/ml), but not SHU9119 (10.0 µg/ml), PG901 and PG911 (3.0 pg/ml) leads to significant increase in basal IL-10 release from C-20/A4 chondrocytes. In addition, it was apparent that the response of the chondrocytes was time-dependent, with both α-MSH and D[TRP]8-γ-MSH causing maximal induction of IL-10 at 6 h post-stimulation. αMSH was significantly more potent than D[TRP]8-γ-MSH at inducingIL-10 although both peptides elevated basal levels of IL-10 as early as 2 h postadministration, and maintained them for 24 h. These results demonstrate that in
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part melanocortin peptides can exert a homeostatic control over chondrocyte physiology with the ability to induce anti-inflammatory cytokines. This therefore suggests a possible role in modulating basal levels of pro-inflammatory synthesis in this cell type even when no apparent inflammation is occurring.
Given the induction of basal IL-10 by the melanocortin peptides, their effect were investigated over a concentration range in the presence of TNF-α-induced chondrocyte inflammation over a time-course. α-MSH caused a concentrationdependent bell shaped response with maximal release of IL-10 caused by 1.0 µg/ml at early time-points and 10.0 µg/ml at later time points (24 h), whilst D[TRP]8-γ-MSH (30.0 µg/ml) caused a maximal release of IL-10 at 2 h poststimulation. The ability of α-MSH and D[TRP]8-γ-MSH to trigger the production of IL-10 clearly suggests that activated melanocortin receptors may have crucial anti-inflammatory properties, conducted through activation of this cytokine.
The anti-inflammatory protein HO-1 has been implicated in the protection against tissue injury and is modulated by cytokines such as TNF-α (Fernandes et al., 2003). It has been shown to be expressed and functionally active in human osteoarthritic chondrocytes from OA tissue with IL-10 shown to modulate its production (Lee and Chau, 2002; Fernandes et al., 2003). A potential link between melanocortin receptor-dependent cAMP formation and HO-1 induction has previously been identified (Lam et al., 2005). This idea stemmed from the fact that cAMP analogues have been shown to induce HO-1 in rat hepatocyte culture (Immenschuh et al., 1998). To address this, the human C-20/A4 chondrocytes were employed to monitor alteration in HO-1 protein production, following incubation with α-MSH and D[TRP]8-γ-MSH at 3.0 µg/ml in the presence and absence of TNF-α. C-20/A4 chondrocytes produced detectable basal levels of HO-1, and melanocortin peptides were able to provoke a marked up-regulation of HO-1 evident at 6 h post-incubation with the melanocortin receptor pan-agonist α-MSH and the synthetic MC3/4 agonist D[TRP]8-γ-MSH. Interestingly, when chondrocytes were pre-treated with the melanocortin peptides prior to stimulation with TNF-α, there was notable elevation in HO-1 production. Together with the fact that TNF-α moderately, but significantly induced the production of HO-1, these results are in accordance with published data
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(Wagener et al., 2003). The downstream sequence of events currently remains unclear, and further investigation would be needed to elucidate the action of melanocortin receptor signalling in chondrocytes.
Chondrocyte apoptosis and the protective effect of melanocortin peptides
Chondrocytes are the only cell type present within articular cartilage and thus chondrocyte apoptosis plays an important part during the processes of cartilage development, aging and in cartilage pathologies (Blanco et al., 1998). Chondrocytes have been shown to be susceptible to endogenous degradative stimuli, such as TNF-α and IL-1β by up-regulating the synthesis of proinflammatory cytokines and matrix metalloproteinases, inhibiting collagen and proteoglycan synthesis, therefore causing loss of cartilage (Ismail et al., 1992; Martel-Pelletier et al., 1999; Fernandes et al., 2002; Kapoor et al., 2011). The importance of apoptosis has been identified with an increase in the number of apoptotic chondrocytes in osteoarthritic lesional than in non-lesional cartilage (Kim et al., 1999; Kim et al., 2000; Kouri et al., 2000; Hashimoto et al., 1998; Kirsch et al., 2000). In addition, chondrocyte apoptosis and the reduction of tissue cellularity represent an important step in the pathogenesis of cartilage degradation (Blanco et al., 1998; Maneiro et al., 2003).
Currently, it remains unclear which pathways induce apoptosis and are responsible for the loss of chondrocytes and subsequent cartilage degradation. DeWolf and colleagues observed that TNF-α (30.0 ng/ml) stimulated caspase-3 driven apoptosis in human chondrocytic cell line (Nuttal et al., 2000) and that TNF-α induced apoptosis of bovine chondrocytes in vitro (Schuerwegh et al., 2003). In our in vitro study, we demonstrate that TNF-α potently induces chondrocyte apoptosis, suggesting that this is part of the mechanism of cartilage destruction, thereby substantiating those existing data. TNF-α concentrations (60.0 – 80.0 pg/ml) caused approximately 28% of the C-20/A4 chondrocytes to die, whereas lower concentrations caused around 10-15 % rate. Clinical studies have demonstrated that pathophysiological concentrations of TNF-α detected in OA synovium of patients with severe disease progression are in the range of 1.0 – 10.0 ng/ml (Westacott et al., 1990), much higher than the concentrations used
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in this study. One reason for using lower concentrations is that chondrocytes in vivo die in the context of extracellular matrix, which may physically limit the levels of pro-inflammatory cytokines reaching the chondrocytes as opposed to in vitro experiments, where the chondrocytes are cultured in monolayer, allowing for fast and equal distribution of TNF-α to all chondrocytes, thereby increasing susceptibility of the chondrocyte to undergo apoptosis in response to TNF-α.
Moreover, the expression of p55 TNF-α receptor has also been localized in areas of osteoarthritic lesions of human cartilage (Webb et al., 1997), and given that the pro-inflammatory cytokine TNF-α is particularly important in the pathophysiology of cartilage disease, I aimed to further investigate the precise role it plays in chondrocyte apoptosis. It was confirmed that TNF-α modulates the activation of apoptotic pathways in human C-20/A4 chondrocytes and may be partially dependent on the activation of caspase-3 and -7. Western blot analysis showed that there was a significant 24 % increase in the protein levels of the activated executioner caspase-3 (Asp-175; 17, 19 kDa) following treatment of the C-20/A4 chondrocytes with TNF-α for 6 h. In addition, TNF-α treatment (60.0 pg/ml; 6 h) led to marked 5.7-fold increase in caspase-3/7 activities, which was confirmed by Caspase-Glo® 3/7 assay analysis. These results are an important finding and are not in accordance with the work by the Blanco group, who detected increased mRNA and protein levels of both caspase-3 and -7 in cultured human OA chondrocytes stimulated with TNF-α, but protein analysis detected only the intermediate, inactive forms of these enzymes (Lopez-Armada et al., 2006). Contrary to those reports, recent studies have demonstrated that TNF-α causes enhanced chondrocyte apoptosis by increasing capsase-3/7 activities (Nuttal et al., 2000; John et al., 2007; Kayal et al., 2010).
Role of melanocortins in prevention of pro-inflammatory cytokine-induced apoptosis.
Following the identification of α-MSH’s and D[TRP]8-γ-MSH’s ability to markedly reduce the synthesis and production of pro-inflammatory cytokines, as well as down-regulating degradative matrix metalloproteinases expression, their effect on TNF-α induced cellular toxicity and cell-death inducing signals was evaluated. Both peptides inhibited TNF-α-induced cell death and significantly down
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regulated the production and activity of caspase-3 and -7. Both peptides at all concentrations tested failed to cause any damage to the treated C-20/A4 chondrocytes and additionally caused 40 - 50 % reduction in cleaved caspase-3 protein expression as determined by western blot. These results were confirmed by testing the activity of the executioner caspases 3 and 7 and measuring mitochondrial functionality following pre-treatment with α-MSH and D[TRP]8-γMSH on TNF-α-activated C-20/A4 chondrocytes. The peptides exhibited strong concentration-dependent protective effect against TNF-α-induced cell death, whereby 49 % and 55 % reduction in chondrocyte apoptosis was observed following treatment with α-MSH (3.0 µg/ml) and D[TRP]8-γ-MSH respectively.
Other studies support the molecular mechanism by which melanocortin peptides prevent apoptosis in chondrocytes, with one particular study on neuronal cell-line GT1-I demonstrating the inhibitory effect of the melanocortin peptide NDP-MSH on caspase-3 activation as readout of apoptosis (Windebank et al., 1994) whilst α-MSH prevents LPS/INF-γ-induced astrocyte apoptosis via activation of MC4 (Caruso et al., 2007).
To our knowledge, this study is the first to show the inhibitory effect of α-MSH and D[TRP]8-γ-MSH in a model of TNF-α-induced chondrocyte apoptosis in vitro. Through antagonism of MC3/4, demonstrates that MC1 and MC3 are involved in the transmission of the anti-apoptotic effects of α-MSH and D[TRP]8-γ-MSH respectively in the human C-20/A4 chondrocytic cell line. SHU9119 (10.0 µg/ml) antagonized the effects of D[TRP]8-γ-MSH, but not α-MSH on down-regulating the production of cleaved caspase-3 as a marker of apoptosis and consistently reversed the protective effect of the selective MC3 agonist on mitochondrial function in the model of TNF-α-induced chondrocyte death. The combination led to 27 % up-regulation of cleaved caspase-3 production and cell death was observed at TNF-α-produced levels regardless of the concentration used.
Combined, these results demonstrate, that MC3 is particularly important in transmitting the anti-inflammatory, cyto-protective, anti-apoptotic and immunomodulatory effects of melanocortin peptides. In addition, the role of MC1 has been also confirmed, given the fact that MC5 is non-functional in this C-20/A4 chondrocyte model and MC2 is solely activated by ACTH1-39 (Getting, 2006).
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Effect of hypotonic solution on chondrocyte function.
To further understand the potential role that melanocortin peptides play in modulating chondrocyte activity, their effect was investigated on the function of osmotically challenged chondrocytes. Articular cartilage is highly hydrated tissue whereby approximately 30 % of the water in the cartilage is found within the collagen intrafibrillar space (Hall, 1998). The amount of water in the cartilage depends on the fixed charge density of the proteoglycans, which bear strong negative electrical charges (Maroudas et al., 1979), neutralised by positive ions in the surrounding fluid. The high concentration of ions in the extracellular matrix, compared to the outside the tissue, has been shown to increase osmotic pressure (Maroudas, 1979; Maroudas and Evans, 1972). In OA and upon matrix degradation, the water content of cartilage increases and leads to over-hydration of the negatively charged proteoglycans, which alters the chondrocyte extracellular physio-chemical environment by reducing the osmolality causing an increase in cell volume; an early event during osteoarthritis (Gardner, 1992; Bush & Hall, 2004).
C-20/A4 chondrocytes were subjected to hypotonic conditions (280 to 140 mOsm), and a significant time-dependent up-regulation of pro-inflammatory cytokines IL-6 and IL-8 from C-20/A4 chondrocytes was observed, with a 37-fold increase in IL-6 and 8-fold up-regulation of IL-8 expression at 24 h and 42-fold and 10 fold, respectively at 72 h. These results were further substantiated by ELISA detection of these cytokines, which confirmed that the intensification on cytokine transcription, in response to chronic hypo-osmotic challenge, was translated into protein released from the C-20/A4 chondrocytes.
This study, to our knowledge, is the first to investigate the effect of lowered media osmolarity on C-20/A4 chondrocyte inflammatory profile. A novel finding, demonstrated by this work, is that upon reduction of extracellular osmolarity, C20/A4 chondrocytes respond by increasing not only pro-inflammatory cytokines, but also the expression of MMP1 and MMP13, which increased in a timedependent manner. MMP1 mRNA was up-regulated 2.3-fold following 24 h incubation in the 140 mOsm DMEM and this increased to 4-fold, compared to cells incubated in normal 280 mOsm culture media. Interestingly, MMP13 mRNA
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(which is not present in non-treated C-20/A4 or healthy human cartilage) was elevated following stimulation with 140 mOsm media, and the detected amounts were significantly higher compared to TNF-α-stimulated levels. Hypo-tonicity did not seem to affect MMP3 expression in the first 24 h of incubation, but caused increased transcription when the chondrocytes were incubated for 72 h. This finding is supported by work on other cell type showing that cellular stresses such as pro-inflammatory cytokines and osmotic stress activate MAPK pathways (JNK, ERK1/2 and p38) (Lewis, 1998; Garrington, 1999). It has been shown that JNK and ERKs phosphorylate AP-1 family member c-Jun (Karin, 1995, Leppa et al., 1998), which then dimerizes with c-Fos and initiates the transcription of various MMP genes. Other groups have shown that the ERK1/2 pathway mediates the activation of the MMP1 promoter via an AP-1 element (Frost, 1994; Rutter, 1995; Korzus, 1997).
However, whether the altered osmolality of the C-20/A4 chondrocytes media is directly triggering the expression of these MMPs cannot be concluded by this work, as 140 mOsm DMEM also affects the synthesis of various cytokines, such as IL-6, which in turn can directly alter both MMP1 and MMP13 gene expression in a concentration-dependent manner. This is an important finding, since osteoarthritic cartilage is also defined by increased expression and synthesis of pro-inflammatory cytokines, matrix metalloproteinases and increased tissue hydration.
Since the events of OA, which we attempted to mimic here in the C-20/A4 chondrocyte model (increased production and release of pro-inflammatory cytokines and catabolic matrix metalloproteinases, increased degradation and reduced collagen type II production, chondrocyte apoptosis and cartilage hydration), it seems likely that a perpetuation of self-inducible and pathological events could lead to the chronic profile of this joint disorder, thus novel avenues for pharmacological intervention could look at targeting these processes.
The effect of α-MSH and D[TRP]8-γ-MSH on hypo-tonicity induced overexpression of matrix metalloproteinases was determined. D[TRP]8-γ-MSH downregulated both MMP1 and MMP13 expression even at 24 h post stimulation with 140 mOsm DMEM. However, α-MSH only caused a modest non-significant
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decrease in the transcription of MMP1 and MMP13, its effect was not statistically different from the levels caused by the hypotonic media alone. None of the peptides altered the expression of MMP3 by C-20/A4 chondrocytes.
These results suggest, that α-MSH probably due to its short half-life (6 h) is unable to down-regulate MMPs expression at 24 h, especially, since the hypoosmotic medium, surrounding the chondrocytes seems to be exerting strong proinflammatory effects, which is unlike the effect of the pro-inflammatory stimuli used for various cell stimulation. The fact that IL-6 and IL-8 levels are increasing even at 72 h post incubation of C-20/A4 chondrocytes with 140 mOsm DMEM, suggested a possible role for these cytokines in the synergistic induction of MMPs expression at time-points as late as 24 and 72 h.
Primary bovine chondrocyte (P0) activation by various pro-inflammatory stimuli.
Following the identification of the protective role that melanocortin peptides could play on pro-inflammatory cytokines and MMP expression following TNF−α stimulation in C-20/A4 chondrocytes, it was decided to determine whether these effects translated to primary cells and cartillage. C-20/A4 chondrocytes have been shown throughout this thesis to respond to various stimuli by secreting significant amounts of pro-inflammatory cytokines, chemokines and other noncytokine pro-inflammatory mediators, such as NO and matrix metalloproteinases. Even though immortalized cell lines are a suitable method for studying the function and the response of chondrocytes to various stimuli, primary cultures o
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Bleomycin-induced fibrosis in MC1 signalling-deficient C57BL/6J-Mc1r(e/e) mice further supports a modulating role for melanocortins in collagen synthesis of the skin.
Abstract
The melanocortin-1 receptor (MC1 ) binds α-melanocyte-stimulating hormone (α-MSH) with high affinity and has a physiological role in cutaneous melanin pigmentation. Previously, we reported that human dermal fibroblasts also express functional MC1 . α-MSH suppressed transforming growth factor-β1 - and bleomycin (BLM)-induced collagen synthesis in vitro and in vivo. Using MC1 signalling-deficient C57BL/6J-Mc1r(e/e) mice, we tested as to whether MC1 has a regulatory role on dermal collagen synthesis in the BLM model of scleroderma. Notably, mice with a C57BL/6J genetic background were previously shown to be BLM-non-susceptible. Interestingly, treatment of C57BL/6J-Mc1r(e/e) but not of C57BL/6J-wild-type mice with BLM increased cutaneous collagen type I content at RNA and protein level along with development of skin fibrosis. Cutaneous levels of connective tissue growth factor and monocyte chemotactic protein-1 were also increased in BLM-treated C57BL/6J-Mc1r(e/e) mice. Primary dermal fibroblasts from C57BL/6J-wt mice further expressed MC1 , suggesting that these cells are directly targeted by melanocortins to affect collagen production of the skin. Our findings support the concept that MC1 has an endogenous regulatory function in collagen synthesis and controls the extent of fibrotic stress responses of the skin.
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Related Articles
If further studies prove the variation to cause fibrosis (scarring) in the kidneys of transplant recipients, researchers may be able to use it to better screen potential donors and improve transplant outcomes. Furthermore, uncovering the protein pathways that trigger kidney fibrosis may help researchers design drugs that prevent this disease process in kidney transplant recipients, and perhaps in all patients with chronic kidney disease.
"It is critically important that we identify new therapeutic targets to prevent scarring within transplanted kidneys, and our study has linked a genetic marker, and related protein pathways, to poor outcomes in kidney transplantation," said Barbara Murphy, MD, Chair, Department of Medicine, Murray M. Rosenberg Professor of Medicine (Nephrology) and Dean for Clinical Integration and Population Health at the Icahn School of Medicine at Mount Sinai. "Drug designers may soon be able to target these mechanisms."
A commonly used study type in years, the genome-wide association study (GWAS) looks at differences at many points in the genetic code to see if, across a population, any given variation in the genetic code is found more often in those with a given trait; in the case of the current study, with increased fibrosis in recipients of donated kidneys.
Even the smallest genetic variations, called single nucleotide polymorphisms (SNPs), can have a major impact on a trait by swapping just one of 3.2 billion "letters" making up the human DNA code. The current study found a statistically significant association between SNP identified as rs17319721 in the gene SHROOM3 and progressive kidney scarring (fibrosis) and function loss in a group of kidney donors, mostly of European descent. In many cases, certain SNPs will be more common in families or ethnic groups.
The kidneys filter the blood to remove extra blood sugar and waste products that trickle down the kidney tubes to become urine, while re-absorbing key nutrients. The build-up of scar tissue in these delicate structures over time interferes with proper renal function.
Chronic kidney disease already affects 10 percent of US adults and its prevalence is increasing. Along with leading to kidney failure in many cases, chronic kidney disease increases the risk of cardiovascular disease. Fibrosis in kidney tubules is a common pathogenic process for many types of chronic kidney disease, and a central part of chronic disease in donated kidneys (chronic allograft nephropathy, or CAN).
CAN comes with a steady, gradual loss of function in the donated kidneys. A significant percentage of patients with chronic kidney disease and fibrosis in their kidney tubules will eventually progress to renal failure that requires dialysis or transplantation of kidneys, with demand far higher that supply. To date, there is no effective therapy to prevent the progression of kidney disease.
Researchers and clinicians have made great gains in preventing transplant rejection during the first few years by selectively suppressing the immune system, but long term damage and disease remain a major challenge. The eventual development of an assay to predict whether a donor's kidney, once transplanted, would be more susceptible to inflammation or scarring may help overcome this challenge.
Newfound Pathways Reveal Drug Targets
The Journal of Clinical Investigation study found that the SNP rs17319721 in the gene SHROOM3, when present in the donor of kidney, correlates with increased expression of the SHROOM3 genes, and a greater quantity of SHROOM3 protein in the organ once transplanted. More SHROOM3 turns on more transcription factor 7-like 2 (TCF7L2). This, in turn, turns on several genes with many functions in cells. TCF7L2 is a member of the Wnt signaling pathway, and ultimately results in increased signaling by transforming growth factor beta 1 (TGF-β1) and increased COL1A1 expression.
TGF-β1 signals for the building of connective tissue (scar tissue), which normally restores tissue architecture as part of healing, but may also drive fibrosis in the wrong context. COL1A1 (Collagen, type I, alpha 1) is the gene that codes for the major component in type I collagen, the major protein component of connective tissues (e.g. bone. cartilage) and of scar tissue that forms as wounds heal. Together, these factors contribute to excess tissue fibrosis.
While SHROOM3 had been associated with chronic kidney disease by earlier work, the specific role of and mechanisms by which SHROOM3 contributed to transplant injury and kidney fibrosis was unknown going into this study.
The current study results proceed from an ongoing NIH-sponsored study in kidney transplant recipients [Genomics of Chronic Allograft Rejection (GOCAR) study]. The research team performed biopsies of transplanted kidneys at pre-specified time points after transplantation and matched gene activation (expression) levels in the transplanted kidneys 3 months after transplantation to indices of transplant dysfunction at 12 months.
The link between the SHROOM3 gene, related protein pathways and fibrosis detected in the GWAS was confirmed in studies of mice engineered to be models of human kidney disease.
"Further work is needed before a clinical application of the study can be introduced," said Dr. Murphy. "However, our results are a crucial and optimistic step towards improving treatment of chronic kidney disease."
Story Source:
The above story is based on materials provided by Mount Sinai Medical Center. Note: Materials may be edited for content and length.
Journal Reference:
- Madhav C. Menon, Peter Y. Chuang, Zhengzhe Li, Chengguo Wei, Weijia Zhang, Yi Luan, Zhengzi Yi, Huabao Xiong, Christopher Woytovich, Ilana Greene, Jessica Overbey, Ivy Rosales, Emilia Bagiella, Rong Chen, Meng Ma, Li Li, Wei Ding, Arjang Djamali, Millagros Saminego, Philip J. O’Connell, Lorenzo Gallon, Robert Colvin, Bernd Schroppel, John Cijiang He, Barbara Murphy. Intronic locus determines SHROOM3 expression and potentiates renal allograft fibrosis. Journal of Clinical Investigation, 2014; DOI: 10
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1600-0625/asset/bannerforeground.gif?v=1&s=286b1e3eed08e680174fd346cc9fdf2ece9544a4)
Original Article
Improved cutaneous wound healing after intraperitoneal injection of alpha-melanocyte-stimulating hormone
- Kênia Soares de Souza1,
- Thiago Anselmo Cantaruti1,
- Geraldo Magela Azevedo Junior1,2,
- Daniel Antero de Almeida Galdino1,
- Claudiney Melquíades Rodrigues1,
- Raquel Alves Costa1,3,
- Nelson Monteiro Vaz4 and
- Cláudia Rocha Carvalho1,*
Article first published online: 12 JAN 2015
DOI: 10.1111/exd.12609
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Issue
Experimental Dermatology
Early View (Online Version of Record published before inclusion in an issue)
Additional Information(Show All)
How to CiteAuthor InformationPublication HistoryFunding Information
How to Cite
de Souza, K. S., Cantaruti, T. A., Azevedo, G. M., Galdino, D. A. d. A., Rodrigues, C. M., Costa, R. A., Vaz, N. M. and Carvalho, C. R. (2015), Improved cutaneous wound healing after intraperitoneal injection of alpha-melanocyte-stimulating hormone. Experimental Dermatology. doi: 10.1111/exd.12609
Author Information
- 1
Departamento de Morfologia, Universidade Federal de Minas Gerais, Belo Horizonte, Brasil
- 2
Hospital Público Regional de Betim, Minas Gerais, Brasil
- 3
Universidade Federal de São João del Rey, Minas Gerais, Brasil
- 4
Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, Brasil
*Correspondence: Cláudia Rocha Carvalho, Departamento de Morfologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, CEP: 31270-901, Brasil, Tel.: +55-31-34092797, Fax: +55-31-34092810, e-mail: cro...@icb.ufmg.br
Publication History
- Article first published online: 12 JAN 2015
- Accepted manuscript online: 28 NOV 2014 06:56AM EST
- Manuscript Accepted: 23 NOV 2014
Funded by
- Fundação de Amparo à Pesquisa de Minas Gerais, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
- Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil
- Abstract
- Article
- References
- Cited By
Keywords:
- alpha-melanocyte-stimulating hormone and scarless healing;
- inflammation;
- scars;
- wound healing
Abstract
Skin wound healing is a complex process involving many types of cells and molecules and often results in scar tissue formation in adult mammals. However, scarless healing occurs in foetal skin and minimal scars may occur after cutaneous healing in the adult with reduced inflammation. Alpha-melanocyte-stimulating hormone (α-MSH) is widely distributed within the central nervous system and in other body regions, such as the skin, and has strong anti-inflammatory activity. The aim in the present experiments was to learn whether intraperitoneal (i.p) injection of α-MSH just before skin wounds antagonize inflammation and improves skin wound healing in adult mice. C57BL/6 young adult mice received an i.p. injection of 1 mg/kg of α-MSH and, 30 min later, two circular through-and-through holes (6.5 mm diameter) were made in their dorsal skin under anaesthesia. Control mice were wounded after vehicle injection. The wound healing process was analysed macroscopically and microscopically at 3, 7, 40 and 60 days. Skin samples were fixed in formalin, embedded in paraffin, sectioned at 5 μm, stained with H&E or toluidine blue for cell analysis or Gomori's trichrome for extracellular matrix (ECM) analysis. Other samples were fixed in DMSO+methanol, embedded in paraplast and incubated with anti-CD45, antismooth muscle actin, anticollagen-I and anticollagen-III for immunofluorescence analysis. Alpha-MSH significantly reduced the number of leucocytes, mast cells and fibroblasts at 3 and 7 days after injury. On days 40 and 60, α-MSH reduced scar area and improved the organization of the collagen fibres indicating that it may direct the healing into a more-regenerative/less-scarring pathway.
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The just above abstract indicated that an injection of alpha melanocyte stimulating hormone resulted in a more successful healing with a affect on the collagen that infers some level of less scarring. Unfortunately no data on the numbers of how much difference over the control mice.
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In the discussion, Melanocyte stimulating hormone induces collagen I and II as well as Sox9. It looks as if Scenesse will have a homeostacis not just in the skin, but in the skin structure with the extracellular matrix, the collagens, and the bones and cartilage.
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Scenesse will regulate Sox9, Sox9 will regulate Runx2. The bones in adults are like the GoldenGate Bridge. Since 1939, everyday there is a guy sanding and scraping the paint. Then there is a guy repainting. It never ends because of the moist salt air. As soon as they finish one end of the bridge they start over. If the oxidation is not stopped with electrode galvanic rods, or paint, it will break down the steel.
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B-Catenin in this article relative to endometriosis, but implications to SOX9, and Collagen 1, and stem cell regulation
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Review Article | Open Access
Malaria remains a significant global health problem, but the development of effective antimalarial drugs is challenging due to the parasite’s complex life cycle and lack of knowledge about the critical specific stages. Medicinal plants have been investigated as adjuvant therapy for malaria, so this systematic review summarizes 46 primary articles published until December 2020 that discuss curcumin and piperine as antimalarial agents. The selected articles discussed their antioxidant, anti-inflammatory, and antiapoptosis properties, as well as their mechanism of action against Plasmodium species. Curcumin is a potent antioxidant, damages parasite DNA, and may promote an immune response against Plasmodium by increasing reactive oxygen species (ROS), while piperine is also a potent antioxidant that potentiates the effects of curcumin. Hence, combining these compounds is likely to have the same effect as chloroquine, that is, attenuate and restrict parasite development, thereby reducing parasitemia and increasing host survival. This systematic review presents new information regarding the development of a curcumin-piperine combination for future malaria therapy.
1. IntroductionMalaria is still a significant health problem, with more than 220 million people affected and millions of deaths annually worldwide, particularly children and pregnant women [1]. The current availability of antimalarial drugs in reducing malaria morbidity and mortality in endemic areas does not positively impact. Still, it creates new problems, such as the emergence of drug-resistant parasites [2, 3]. This is a significant challenge to human health; consequently, new antimalarial drugs or treatment strategies are urgently needed. However, developing an effective antimalarial drug is challenging due to the complex life cycle of parasite. Plasmodia infection begins with an asymptomatic liver-stage, followed by symptomatic blood-stage infection [2, 4]. Most studies have shown that protective immunity will automatically develop against blood-stage infections, after repeated exposure to the parasites, but it remains unclear at the liver stage [2, 5, 6]. Furthermore, it becomes more challenging to fortify the host when the parasites enter the blood-stage without being interfered at the liver-stage; consequently, the load of the parasite in the blood could be unruly high [2].
The use of medicinal plants can modulate the immune response, which significantly impacts health [2–7]. For example, in India, mostly Indians consume foods containing spices/herbs, such as garlic, ginger, turmeric, and black pepper, which are known to have antimalarial activity [8–11]. Turmeric (Curcuma longa) is an ancient spice from Southeast Asia, used as a dye and a condiment [12]. It is one of the cheapest spices globally and has been used for 4,000 years to treat various ailments [12, 13]. It contains an active substance, curcumin (bis-α, β-unsaturated β-diketone), commonly known as diferuloylmethane, which has a broad spectrum of biological and pharmacological activities, including antioxidant [14], anti-inflammatory [15], antimicrobial, and anticarcinogenic [14] properties. Additionally, the hepato- and nephroprotective [16, 17], thrombosis suppression [18], myocardial infarction protective [19], hypoglycemic [20], and antirheumatic [21] effects of curcumin are also well established. Curcumin exhibits potent activity against other parasites including Leishmania [22], Cryptosporidium parvum [23], Schistosoma mansoni [24], Giardia lamblia [25], and Trypanosoma cruzi [26]. Moreover, it has been shown to possess antimalarial activity against various Plasmodium species in vitro [27–31]. Similar to turmeric, black pepper (Piper nigrum) is also used as a traditional antimalarial medicine in Calabria (South Italy) and India, especially for treating malaria with symptoms of periodic fever and hepatomegaly [32, 33]. It is also an ancient spice from the coast of Malabar in India, which contains an active substance called piperine (chemically, piperoylpiperidine), which has been used to treat cholera, flatulence, arthritis, digestive disorders, asthma, and cancer [34–37].
Many studies (in vitro, in vivo, as well as clinical trials) have described the use of curcumin and piperine as antimalarial drugs, either alone or combined with current antimalarial drugs [10, 28, 30, 38–40]. However, no studies have discussed the potential use of curcumin-piperine combinations and their possible mechanisms of action. This systematic review summarizes the use of curcumin and piperine, identifies their possible antimalarial mechanisms, and determines the role of curcumin-piperine in the management of malaria. For the future, this study can be used as a reference to produce a potential antimalarial agent.
2. Materials and Methods2.1. Literature Search StrategyTwo electronic databases, i.e., Google Scholar and PubMed, were searched for relevant studies published between 1995 and December 2020. The search terms used for this systematic review included “curcumin, curcuma, malaria” or “piperine, piper nigrum, malaria.” The language was restricted to English.
2.2. Eligibility Criteria2.2.1. Inclusion CriteriaAll articles published in English language between 1995 and December 2020 in any setting with an aim of finding the potential use of curcumin or piperine for malaria regardless of the Plasmodium species whether P. falciparum, P. vivax, P. berghei, P. chabaudi, or P. yoelii.
2.2.2. Exclusion CriteriaStudies of curcumin or piperine in malaria do not provide complete data or unclear outcome indicator. Review articles, case reports, letter to the editor, conference papers, and articles published in languages other than in English. Full texts are not accessible/irretrievable. The systematic review was guided by the PRISMA guidelines. The PRISMA diagram detailing the selection process is shown in Figure 1.
2.3. Study Selection and Data ExtractionFor this systematic review, two researchers independently read the title and abstract for screening, continued by reading the full text of the research study and performing literature screening and data extraction according to inclusion and exclusion criteria. Disagreements of two researchers will be resolved by involving the third researcher to make final decision. The following data were extracted: year of publication, first author, type of study, subject, intervention characteristics (i.e., dosage and compound`s activities), and outcome measures.
2.4. Data AnalysisDue to the heterogeneity of the included studies, a meta-analysis was not conducted. Data analysis was performed descriptively using Microsoft Excel 2019. Data analysis was presented in a narrative form.
3. Results and Discussion3.1. Selection StudiesA total of 352 articles were obtained according to the search strategy. We acquired the remaining 253 articles after removing duplicates and were subsequently filtered by title and abstract so that 165 studies were excluded. The remaining 88 articles were further screened by reading the full-text articles, and 42 articles were excluded. Finally, this review includes 46 qualitative studies.
3.2. Curcumin as an AntiplasmodiumIn total, 46 primary articles were identified and 41 articles discussed curcumin (Table 1), reporting that curcumin exerts antiplasmodium effects through various activities/mechanisms. The pathogenesis of malaria is multifactorial involving the complex life cycle of the parasites. During a blood meal, a malaria-infected mosquito inoculates sporozoites (SPZ) into the human skin, enter the liver via bloodstream, and infect hepatocytes. At the liver-stage (exoerythrocytic), SPZ produce thousands of infective merozoites, enter the bloodstream, and infect the red blood cells (RBCs) to recruit the erythrocytic cycle that is responsible for clinical sign of the disease [75]. The infection level correlates with the parasite burden that elicits a defense mechanism to prevent the parasite from multiplying [30]. Curcumin (turmeric) acts as a prooxidant and antioxidant to modulate the innate immune response through the production of intracellular reactive oxygen species (ROS) for the clearance of parasites. ROS enhances the scavenger expression of the CD36 receptor on monocytes or macrophages, which mediates phagocytosis of the nonopsonization parasite-infected erythrocyte by macrophages [30, 42]. Also, curcumin promotes the immune response through induction of ROS production, resulting in the activation of PPARɣ/Nrf2 and upregulation of CD36 expression in monocytes/macrophages that recital the parasiticidal activity on the blood-stage parasite when administered in mice [30, 72]. ROS production can also be caused by the accumulation of large amounts of free heme, known as ferriprotoporphyirin [13], inducing oxidative stress which leads to parasite death. In this case, the parasite requires a free heme detoxification process by changing it to a nontoxic, inert, insoluble, crystal pigment, and blackish-brown form called hemozoin or β-hematin [76]. The formation of β-hematin is considered a key mechanism for heme detoxification in Plasmodium [76, 77]. The study conducted by Padmanaban et al. demonstrated that the curcumin-artemisinin combination inhibited hemozoin formation and increased ROS production in mice infected with P. berghei. The result suggests that the combination of these compounds is synergistic and results in optimal efficacy. Furthermore, in vitro, curcumin 0.4 mM exhibits an inhibitory effect on the formation of β-hematin, with an efficiency of 78.8% compared to amodiaquine (91.8%) and DMSO (10.7%) [13]. Similar findings were also obtained by Akhtar et al. [29], who reported that curcumin bound to chitosan nanoparticles cured rats of P. yoelii infection and inhibited the synthesis of β-hematin in vitro at IC50 (122 μM ± 2.7). Curcumin bound to chitosan nanoparticles could increase bioavailability and metabolic stability. Some antimalarial drugs, i.e., chloroquine and amodiaquine, inhibit hemozoin formation in food vacuoles, preventing the detoxification of the released heme, thereby killing the parasites. Curcuminoid isolate has a similar role to chloroquine, so the interaction between ferriheme and curcumin is likely to allow the interaction of the Fe3+ metal center with one of the carbonyl groups on curcumin. Furthermore, the side-chain carboxyl group of heme will interact with one of the hydroxyl groups of curcumin [3]. The capability of curcumin as a prooxidant is also known to trigger the production of ROS, resulting in mitochondrial and core DNA damage and triggering pH changes in organelles that cause parasite death [42]. Furthermore, curcumin-induced hypoacetylation occurs on H3 in K9 and K14; nevertheless, not on H4 in K5, K8, K12, and K16. The result prompts us to think that curcumin can cause inhibition of the HAT PfGCN5 involved in parasite chromatin modifications [42]. Chromatin is a pivotal component of various cellular processes such as DNA transcription, replication, and repair [78]. It is composed of a nucleosome containing two copies of histones H2A, H2B, H3, and H4 which play a role in the epigenetic regulation of gene expression. Histone lysine acetylation is catalyzed by histone acetyltransferases (HATs), and it is eliminated by histone deacetylases (HDACs). Previous studies revealed that histone acetylation has great potential as a new therapeutic target. To date, several HDAC inhibitors have also been clinically tested for anticancer therapy [79]. P. falciparum general control nondepressed 5 (PfGCN5) is a HAT that acetylates K9 and K14 from H3 histone. Drugs that impact on HDACs and impede histone acetylation in parasites have powerful antiparasitic actions. Curcumin serves as a HAT p300/CREB-binding protein (GST) inhibitor, but its inhibitory effect is selective because curcumin does not suppress the P300-associated factor of GNAT (GEN5-related acetyltransferase), a member of the HAT superfamily. Cui et al. [78] revealed that curcumin specifically inhibits PfGCN5 in vitro and has a cytotoxic effect against the parasite. Curcumin (5 μM) is also reported to disrupt cellular microtubules of Plasmodium through depolarization of the microtubules during mitosis due to elevated ROS and is more prominent in the second cycle [31], similar to the effect of the microtubule-destabilizing drug vinblastine on P. falciparum. Molecular docking predicts that curcumin might bind to the alpha-beta tubulin heterodimer interface leading to altered microtubule morphology. This is supported by drug combination trials with antagonistic interactions between curcumin and colchicine which show competition for the same binding site. Alternatively, it is possible that curcumin does not bind directly to tubulin but is involved in global cell damage or due to the targeted effect of curcumin. Impaired microtubules inhibit cellular functionality, including apicoplast morphology [31, 80]. Previous studies regarding the effect of curcumin on Plasmodium viability have also been reported. Reddy et al. [27] revealed that curcumin (IC50 of 5 mM) inhibits the development of P. falciparum via PfATP6, the orthologue parasite on the SERCA (sarcoplasmic-endoplasmic reticulum Ca2+- ATPase) mammalian cells. Curcumin inhibits Ca2+-ATPase by stimulating a conformational change, which impedes the ATP from attachment. In this case, curcumin has the same activity as artemisinin [27]; thus, it is hypothesized that curcumin can decrease Plasmodium viability and reduce blood parasitemia, significantly increasing the survival rate.
Malaria is a highly inflammatory disease that requires drugs that can suppress the inflammatory response. Curcumin (therapeutic and prophylactic) can reduce TNF-α and IFN-γ (proinflammatory cytokines), increase IL-10 and IL-4 (anti-inflammatory cytokines), as well as modulate inflammatory cytokines mediated by inhibition of GSK3β (serine/threonine kinase which functions in glycogen metabolism and is the target of malaria therapy) [73]. Furthermore, sequestration is a pathological hallmark of P. falciparum infection, where erythrocytes can attach to the endothelium of vital organs in an attempt by the malaria parasite to evade the immune system [81]. The sequestration process can occur in both infected and uninfected erythrocytes due to lack of microvascular flow, causing damage to the blood-brain barrier, cerebral edema, and tissue hypoxia [30]. Sequestration is also recognized as a consequence of the expression of adhesion molecules (mostly ICAM1, VCAM1, and E-selectin) in brain endothelial cells induced by excessive production of inflammatory cytokines or by direct attachment of P. falciparum [82]. In vitro studies show that inflammation through the expression of ICAM1 results from P. falciparum adhesion to brain endothelial cells [30]. Curcumin can effectively control the inflammatory cascade due to the host immune response in cerebral malaria via the modulation of NF-κB (nuclear factor κ beta), which plays an essential role in malaria. Furthermore, Pf-IRBC has been shown to induce the NF-κB-regulated inflammatory pathway in human cerebral endothelium [83]. Also, curcumin has been shown to reduce the production of proinflammatory cytokines (TNF, IL-12, and IL-6) in vitro, and inhibition of iNOS by curcumin suppresses the production of IFN-γ and IL-12. iNOS has been shown to mediate ROS production, which is cytotoxic against Plasmodium [84]. Furthermore, curcumin can upregulate heme oxygenase-1 (HO-1) gene and protein expression by protecting brain endothelial cells from peroxide-mediated toxicity and toxicity due to free heme [85]. Another study reported that curcumin suppresses activation of C-Jun N-terminal kinases (JNK), which belongs to the mitogen active kinase family (MAP kinase) and is activated in response to inflammatory cytokines and stress conditions [2, 86]. Its activation induces a transcription-dependent apoptotic signaling pathway, resulting in cell death during experimental cerebral malaria (CM) [39, 86]. In a murine model of CM, curcumin administration resulted in a partial reduction of CM and delayed death [66]. Interestingly, curcumin has been shown to suppress proinflammatory cytokine responses and provide protection to endothelial cells.
The pathogenesis of malaria is determined by the interaction between P. falciparum and human host cells. P. falciparum infection can develop into severe malaria, even CM, associated with sequestration of P. falciparum-infected erythrocytes blood cells (Pf-IRBC) in the brain, causing coma [87]. Pf-IRBC is known to play a role in the apoptosis of bEnd. Three cells are amplified by parasitemia levels and incubation period [39]. The increase in the apoptosis of bEnd.3 cells depends on the synergy between parasitemia, host cells, platelets, and peripheral blood mononuclear cells (PBMC) [39]. The apoptotic mechanism of brain endothelial cells induced by Pf-IRBC is associated with the cytoadherence of Pf-IRBC. Pino et al. [84] revealed that the cytoadherence of Pf-IBRC modulated brain endothelial expression of the TNF-α superfamily genes, apoptosis-related genes (Bad, Bax, caspases, and iNOS) and activated the Rho-kinase signaling pathway that induces the production of ROS by endothelial cells that cause cell death. Several possible mechanisms cause endothelial cell dysfunction, including sequestration and adhesion-independent mechanisms [39]. Curcumin (IC50:10 μM) inhibited the growth of P. falciparum and was able to protect endothelially, by reducing apoptosis of bEnd.3 cells, with Pf-IRBC, platelets, and PBMC. These findings suggest that curcumin is a potential adjunctive therapy for treating CM in the future.
3.3. Piperine as an AntiplasmodiumOnly five articles (Table 2) discussed piperine as antiplasmodium despite black pepper (Piper nigrum) being long used as a traditional medicine for malaria. However, the number of publications is likely to increase as research trends develop. Piperine is a potent antioxidant and has been reported in many experimental models of cancer [89]. Piperine exhibits a higher antioxidant potential compared to vitamin E, attributed to its strong hydrogen-donating ability, metal chelating capacity, and effectiveness to scavenge free radicals, mainly ROS [90]. During malaria infection, both the host and parasites are under oxidative stress, with ROS (e.g., superoxide anions and hydroxyl radicals) produced by activated neutrophils in the host and during hemoglobin degradation in parasites. The effects of ROS on malaria can be both beneficial and pathological, depending on the amount and location of production. Piper nigrum has been used by South Indian traditional healers to treat fevers in general, malaria, asthma, cold, intermittent fever, cholera, colic pain, and diarrhea [91, 92]. Kamaraj et al. [38] reported that the ethyl acetate seeds extract of Piper nigrum showed promising in vitro antiplasmodial activity against P. falciparum 3D7 and INDO strains with IC50 values of 12.5 and 12.0 μg/mL, respectively, with low cytotoxicity (TC50 = 87.0 g/mL). Furthermore, the significant therapeutic index of 7.0 in alkaloids piperine, guineensine, piperidine, N-feruloyltyramine, and N-isobutyl-2E, and 4E-dodecadienamide have been isolated from Piper nigrum, and piperine has been reported as a stimulator of in vitro melanocyte proliferation [93]. Also, an ethnobotanical survey of twenty traditional healers in India reported that Piper nigrum was used in decoction form for malaria chemoprophylaxis [33]. In 2013, Thiengsusuk et al. researched 27 medicinal plants in Thailand, including Piper chaba Hunt (the active compound is piperine), showing that the extract Piper chaba Hunt showed potent antimalarial activity IC50: <10 μg/ml [88]. Furthermore, piperine IC50: 111.5 μM and IC90: 329 μM change parasite (3D7 P. falciparum) morphology after 48 hours of exposure. Specifically, morphological changes (cytoplasm condenses) start at 8 hours, but effects were observed after 12 hours of piperine exposure compared to untreated cells, slowing the growth of some surviving parasites. At IC90, almost all parasites died after 8 hours of exposure to piperine, suggesting that the window of activity is likely to be the late ring to trophozoite stages (8–12 h) [40]. However, there were no effects of piperine observed on modulating (inducing or inhibiting) the expression of all P. falciparum resistance genes under investigation including Plasmodium falciparum multidrug resistance 1 (pfmrp1), Plasmodium falciparum multidrug resistance protein 1 (pfmdr1), and Plasmodium falciparum chloroquine resistance transporter (pfcrt) [40], implying a low risk of development of resistance development to piperine of P. falciparum. In a recent study, Piper nigrum (IC50: 16.25 and 20.26 μg/mL) showed promising antimalarial activity against insensitive and resistant P. falciparum (FCK2 and INDO) strains in inhibiting Plasmodium lactate dehydrogenase (PfLDH) [10]. However, the mechanism of action of piperine at molecular and cellular level remains unclear.
3.4. The Potential Use of a Curcumin-Piperine Combination as an Antimalarial AgentBased on our understanding from various studies, curcumin has already shown great potential against Plasmodium spp, both in vitro and in vivo [28, 41]. Nevertheless, its poor bioavailability and also rapid metabolism are issues to overcome to exploit the full benefits of this plant-derived compound [8]. Bioenhancers such as piperine, extract from black pepper (Piper nigrum) can improve the bioavailability of curcumin by 2000-fold [8, 94]. Martinelli et al. [28] evaluated the effect of curcumin-artemisinin combination against an artemisinin-resistant clone of P. chabaudi. Also, they tested the efficacy of piperine in increasing the bioavailability of curcumin, thus increasing its efficacy [95]. The study showed that oral administration of 300 mg/kg BW of curcumin combined with 20 mg/kg BW of piperine and 150 mg/kg of artemisinin had no conclusive effect on the course of infection. However, the peak parasitemia and antimalarial activity reached by the curcumin and curcumin/piperine treatment groups were significantly lower than the control untreated group [28].
Furthermore, Neto et al. [68] evaluated the efficacy and the drug interactions between curcumin/piperine/chloroquine with curcumin/piperine/artemisinin in P. chabaudi parasites resistant to chloroquine (AS-3CQ) and artemisinin (AS-ART). Also, they verified the effects of curcumin, chloroquine, and artemisinin drug treatment on the UPS (ubiquitin/proteasome system), showing that the interaction between curcumin/piperine/chloroquine was additive, reducing the parasite load seven days after treatment. Interestingly, although both drugs have different structures and modes of action, they both have anti-inflammatory properties which possibly contribute to the reduction in parasitemia [70]. Curcumin is known for its immunomodulatory properties, including activation of TLR2, increased IL-10, and production of antiparasite antibodies [70]. Chloroquine is well known for its antimalarial schizonticidal activity and its anti-inflammatory properties such as inhibition of TNF-α, IL-1β, and IL-6, making both drug combinations attractive for the treatment of other diseases where an excess of proinflammatory cytokines is produced. It is believed that curcumin is a potential compound for adjunctive treatment of CM, which is often treated with quinine (chloroquine derives) [30]. However, the curcumin/piperine/artemisinin combination did not show a favorable drug interaction in this murine model of malaria [68]. Based on the mechanism of action of curcumin and piperine that has been discussed previously, it is likely that most parasite development is impaired at the blood stage. Meanwhile, at the liver-stage, plasmodia infection becomes very limited to trigger an immune response to the liver stage. Although curcumin and piperine are known to act at different phases, it is hypothesized that the combination of curcumin and piperine can attenuate plasmodia in the early stages of the blood stage (Figure 2), increasing the immune response to malaria liver-stage infection, which implies increased protection (Figure 3). This phenomenon prompts us to think that the combination of curcumin and piperine significantly reduces the likelihood of developing severe clinical manifestations of malaria (i.e., inflammation, hepatosplenomegaly, and anemia) (Figure 4). The combination of curcumin and piperine is expected to be a potential candidate in the development of future antimalarial drugs.
The data presented in this review demonstrates the potential combination of curcumin and piperine (therapeutic and prophylactic) as a candidate antimalarial drug. Curcumin has many pharmacological activities, with antioxidant, anti-inflammatory, and antiapoptotic properties. Piperine is a potent antioxidant and a bioenhancer that may potentiate the effect of curcumin, especially by producing ROS which is cytotoxic against malaria parasites. Combining these compounds is likely to have the same effect as chloroquine that attenuate and restrict the development of parasites. A comprehensive approach is also needed to evaluate the specific mechanism of action of these compounds as antimalarial agents. For further large-scale development, research related to evaluating the potential for the combination of curcumin and piperine is urgently needed [96].
Data AvailabilityThe data supporting this review article are from previously reported studies, which have been cited.
DisclosureAll figures in this systematic review were created with BioRender.com.