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Sharif Garmon
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Acute promyelocytic leukemia (APL) is characterized by a specific t(15;17) translocation that fuses the retinoic acid receptor alpha (RAR alpha) to a novel gene product, PML. The involvement of RAR alpha is particularly intriguing in view of the efficient therapeutic effect of retinoic acid (RA) in this disease. In this report, we show that PML is specifically localized within a discrete subnuclear compartment corresponding to nuclear bodies recognized by patient autoimmune sera. In APL cells, the PML-RAR alpha hybrid displays an abnormal localization and directs RXR and other nuclear antigens into aberrant structures that are tightly bound to chromatin. This suggests that the hybrid could exert a dominant negative effect by diverting a subset of proteins from their natural sites of action. Interestingly, treatment of APL cells with RA induces a complete relocalization of each of these proteins. We propose that the beneficial role of RA in promoting myeloid differentiation in APL might be related to its ability to restore a normal subnuclear organization.
Nuclear retinoic acid receptors (RARs) are transcriptional regulators controlling the expression of specific subsets of genes in a ligand-dependent manner. The basic mechanism for switching on transcription of cognate target genes involves RAR binding at specific response elements and a network of interactions with coregulatory protein complexes, the assembly of which is directed by the C-terminal ligand-binding domain of RARs. In addition to this scenario, new roles for the N-terminal domain and the ubiquitin-proteasome system recently emerged. Moreover, the functions of RARs are not limited to the regulation of cognate target genes, as they can transrepress other gene pathways. Finally, RARs are also involved in nongenomic biological activities such as the activation of translation and of kinase cascades. Here we will review these mechanisms, focusing on how kinase signaling and the proteasome pathway cooperate to influence the dynamics of RAR transcriptional activity.
In the classical pathway of retinoic acid (RA) mediated gene transcription, RA binds to a nuclear hormone receptor dimer composed of retinoic acid receptor (RAR) and retinoid X receptor (RXR), to induce the expression of its downstream target genes. In addition to nuclear receptors, there are other intracellular RA binding proteins such as cellular retinoic acid binding proteins (CRABP1 and CRABP2) and cytochrome P450 (CYP) enzymes, whose contributions to the RA signaling pathway have not been fully understood. The objective of this study was to compare the significance of various RA binding receptors, i.e. CRABP1, CRABP2, CYP and RAR in the RA signaling pathway. In this regard, we developed a mathematical model of the RA pathway, which is one of the few models, if not the only one, that includes all main intracellular RA binding receptors. We then performed a global sensitivity analysis (GSA) to investigate the contribution of the RA receptors to RA-induced mRNA production, when the cells were treated with a wide range of RA levels, from physiological to pharmacological concentrations.
Our simulations indicate that the significance of the RA binding proteins in the RA pathway of gene expression strongly depends on intracellular concentration of RA. This study not only can explain why various cell types respond to RA therapy differently, but also can potentially help develop pharmacological methods to increase the efficacy of the drug.
Retinoic acid (RA), a biologically active form of vitamin A, plays essential roles in the growth and development of various cell types. RA has also been widely used as an anticancer drug due to its ability to inhibit cancer cell growth and induce cell differentiation. It is believed that RA mainly exerts its effects by regulating gene expression. The classical pathway of RA-induced gene transcription involves binding of RA to retinoic acid receptor (RAR), a member of the nuclear hormone family. The liganded RAR binds as a heterodimer (RA:RAR:RXR) to DNA and regulates gene expression. RAR:RXR heterodimer is the main transcription factor in the classical RA signaling pathway. The formation rate of RA:RAR:RXR complex, is highly affected by other intracellular RA binding receptors such as cellular retinoic acid binding proteins (CRABPs) and cytochrome P450 (CYP) enzymes. CRABPs are high affinity cytosolic receptors for RA that can potentially limit the access of RA to the RARs; CRABP1 and CRABP2 are the main members of the CRABP family. It has been reported that CRABP1 is responsible for sequestering RA in the cytosol, and thus controlling the level of free intracellular RA available for binding to RARs [1]. CRABP1 can also facilitate RA degradation by directing RA molecules to RA-degrading enzymes, cytochrome P450 (CYP) [2]. However, other in vitro studies have indicated that CRABP1 is dispensable in the RA signaling pathway [3, 4]. CRABP2, whose expression pattern is different from CRABP1 [5], delivers RA to both nuclear hormone receptors and CYP enzymes [6, 7]. CRABPs are bound to CYPs prior to adding RA to the cell [6, 8].
The main advantage of the current study over previous in vitro studies is that our results were obtained using wide ranges of RA receptor concentrations for any given RA concentration, thus our results are applicable to most cell types or to a population of cells of the same type. Furthermore, our study is able to reveal the synergistic effects of a combination of parameters across a broad range of parameter values. In contrast, the obtained results from previous experimental studies [3, 11] reveal the sensitivity of the system with respect to one parameter when the rest of the parameters remain unchanged.
The model included gene transcription, protein translation, and degradation of mRNA and protein. The model involved the mechanisms by which RA is degraded. The core set of reactions describing the RA metabolism process were taken directly from [6]. We simulated RA-induced gene transcription through the interactions between liganded transcription factor and DNA (Additional file 1). The model also describes how RA binding receptors interact with each other in the absence or presence of RA (Fig. 1). In the absence of RA, CRABPs complex with CYP enzymes, while RARs are not bound to CRABPs or CYPs [6, 8]. Once RA diffuses into the cell, it binds to different RA binding receptors with various binding affinities. CRABP1, which has the highest binding affinity for RA compared to the other RA receptors, regulates the metabolic fate of RA by directing RA molecules to CYP enzymes. In theory, CRABP1 can also transport RA to RAR. This process involves dissociation of RA from CRABP1, followed by association of RA with RAR. CRABP2 is the second high-affinity receptor for RA [5] and can deliver RA to RAR and CYP. RA is transported from CRABP2 to RAR by a mechanism that involves direct interactions between CRABP2 and RAR [5].
Simplified schematic of RA signaling pathway. CRABP1 is shown in green, while CRABP2 is shown in blue. Red circles, gray ellipsoids and yellow hexagons represent RA molecules, CYP enzymes and RAR molecules, respectively
RA is transferred to CYP enzymes either freely or bound to CRABPs. RA-induced gene transcription depends on the rate of transfer of RA to RAR. Free RA molecules can interact with RARs directly. CRABP1 and CRABP2 can also deliver RA to RAR by different mechanisms. Liganded transcription factors can enhance the transcriptional activation of CYP, RAR and CRABP2 genes after binding to DNA at a retinoic acid response element (RARE). We also assumed that RA was degraded only by CYP, while RA binding receptors, i.e. RAR, CRABP1, CRABP2, CYP were degraded by first-order reactions.
Analysis of the model behavior required the initial concentrations of the species and the kinetic parameters. Our model had 44 parameters, which included total concentrations of the RA binding receptors, the kinetic rate constants for binding/unbinding reactions, transcription and translation rate constants and mRNA and protein degradation rates. We assumed that total concentrations of CRABP1, CRABP2, CYP and RAR were unknown, which implies that these proteins are expressed at various levels across different tissues and across a population of cells of the same type. In the absence of RA, total concentrations of RA receptors were given by
where [] indicates molar concentration, while subscripts t and f stand for total and free receptors. RAR does not have any interaction with the remainder of the RA binding receptors, i.e. CRABP1, CRABP2 and CYP, before RA treatment. However, RAR can homodimerize, and heterodimerize with other proteins such as RXR in the absence of RA. In this study, we assumed that RA molecules can bind to free RARs, and to RARs bound to other proteins, with the same binding affinity. Thus, all RARs are receptive to RA binding.
We used in vitro values for 30 model parameters (see Additional file 1), while the remaining 14 parameters were unknown for which we considered some physiological bounds (Table 3). We also assumed that for a given gene the values of transcription rate constants, translation rate constants, forward and reverse rate constants of the binding reactions and the elimination rates of proteins and mRNAs can vary within the in vitro values by a factor of two. This is because not only can these parameters vary across cell type and across cells of the same type, but also all in vitro parameters are subject to error.
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