Dr. Daniel Polley, a leading expert in the physiology and plasticity of auditory processing areas of the brain, is using cutting edge technologies for chronic imaging at a cellular scale to assess changes in the patterns of neural activity that represent the underlying signature of tinnitus in mouse models. His work is also investigating new strategies for direct brain stimulation that might reverse pathological patterns of activity and restore more normal sound perception.
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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].
We used large ranges for unknown initial concentrations of CRABP1, CRABP2, CYP and RAR [13]. This is because the cellular levels of these proteins can vary significantly across cell types, or in a particular cell type as a consequence of cancer and cancer therapy. We then performed a global sensitivity analysis to identify the influential unknown parameters in the RA signaling pathway.
One serious drawback of the clinical use of RA is that RA has a rapid and variable degradation rate [23, 24]. Thus, a relatively high concentration of RA is required to induce the expression of target genes in various cell types. The pattern of RA degradation is important since it can directly influence cell differentiation and gene expression by RA. In this section, we simulated the variations in total concentration of RA within 24 h after RA treatment. For this purpose, we sampled several sets of parameters within their ranges of variability, which characterized various cell types or various cells of the same type. We then added 0.1 μM of RA to each model and obtained the changes in total RA concentration over time. Our results showed that RA exhibited different elimination patterns depending on intracellular concentrations of the RA binding proteins, i.e. CRABP1, CRABP2, CYP and RAR (Fig. 10). Furthermore, RA can both down- and up-regulate its own degradation.
Retinoic acid, a metabolite of vitamin A, modulates a wide variety of biological processes such as cell growth, cell differentiation and cell proliferation. RA has also been known to be effective in treatment of various types of cancer. Even though a vast number of studies have focused on exploring the regulatory target genes for RA, the significance and roles of various intracellular RA receptors in transduction of the RA signal have not been fully understood. CRABP1, CRABP2, CYP enzymes and RARs are the main intracellular proteins which can bind to RA as receptors. Few previous studies have attempted to investigate the effects of overexpression of CRABPs on the RA signaling pathway, and in some cases somewhat contradictory results have been reported for different cell lines [2,3,4]. In this study, we developed a mathematical model to analyze the importance of CRABP1, CRABP2, CYP and RAR in production of mRNA and RA metabolites. In this regard, after proposing a well-mixed model of the RA signaling pathway, we performed a global sensitivity analysis to investigate the relative importance of RA binding receptors in total mRNA production via the RA pathway. Our results indicate that CRABP2 is the most important RA receptor at physiological levels of RA, while RAR concentration has the least importance among all four RA receptors. At pharmacological levels of RA, the total mRNA production was more sensitive to variations in RAR and CYP levels than CRABP1 and CRABP2 levels. It is important to note that all RA binding receptors could influence RA-induced mRNA production within the entire region of parameter space where the concentrations of RA binding proteins change considerably. They are all important since their sensitivity indices were of the same order of magnitude. These results can explain the conflict between previous experimental results regarding the effects of CRABP1 on transcriptional activity of target genes [1, 3, 4]. Our results were obtained using GSA, which quantifies the effects of the model inputs on the model output by perturbing the inputs within large ranges. Therefore, our results indicate that in a broader region of parameter space, which represents various cells with various levels of RA receptors, all of the RA binding receptors are influential. However, there is a possibility that for a certain parameter set which specifies a specific tissue or cell, CRABP1 is unimportant in the RA pathway. Thus, for a given cell type, an accurate parameter set is necessary to determine whether a parameter has a substantial control on the system performance.
This study has some limitations. First, we assumed that RA influences gene expression through the classical pathway, which involves binding of RA to a nuclear hormone receptor heterodimer (RAR:RXR). The liganded heterodimer can initiate the transcription of target genes after binding to a DNA response element. However, there may be other intermediate transcription factors or nonclassical pathways that can transduce RA signal, thus our results can only be applied to the genes which are direct targets of the classical RA signaling pathway. Second, we assumed that all RA binding proteins undergo first-order degradation processes. This may not be the case for all types of tissues with various expression levels of degradation enzymes. The mechanisms mediating the elimination of RA binding receptors have not been fully understood, thus the model can be improved once more information regarding these mechanisms is available. Third, we used the kinetic rate constants of CYP26B1 in the model. CYP26B1 is a member of the 26 family (CYP26s) of the CYP enzymes which is mainly responsible for metabolism of RA during adult life [6, 31,32,33]. However, RA can also be degraded by other families of CYP which are different from CYP26B1 in terms of rate constants and binding affinities. In the current model, we assumed that the kinetic rate constants of degrading enzymes can vary by a factor of two around the in vitro values for CYP26B1. This assumption increases the applicability of our results to other cell types with different types of CYP. Thus, our results are applicable to those cell lines that express higher levels of CYP26B1 compared to other CYP families and to those cell types which have CYP enzymes with kinetic rate constants within the specified ranges in this study. The current simulation can be run using the kinetic rate constants of any arbitrary CYP enzyme. In that case, this model can be expanded to include the effects of RA metabolites on RA-induced gene expression if the CYP of interest forms high levels of active RA metabolites. The current model is applicable to those cell types whose main degrading enzyme is CYP26B1. The primary metabolite formed by CYP26B1 from RA is 4-OH-RA [34, 35]. CYP26B1 forms non-bioactive dehydroxylated products from 4-OH-RA [35]. Thus, we believe that the endogenous levels of RA metabolites formed by CYP26B1 do not play significant roles in the RA signaling pathway. However, there are other active RA metabolites such as 4-oxo-RA which can potentially compete with RA for binding to RAR and activating the transcription of target genes [22]. Fourth, we neglected the possible effects of RA treatment on the model parameters such as translation rate constants, transcription rate constants, and degradation rate constants of proteins and mRNAs. Fifth, for simplicity, we proposed a well-mixed model, thus our model is not able to capture the dynamics of protein diffusion through the nuclear membrane. RARs are located inside the cell nucleus. RA must diffuse across the nucleus membrane to be able to bind to RARs. In reality, RA binds to CRABPs after diffusing across the cellular membrane. RA can diffuse across the nuclear membrane alone or bound to CRABPs. We believe that our well-mixed model can approximate this process due to the rank order of binding affinity of RA for various RA receptors. RA binds to CRABP1 and CRABP2 with higher affinity than to RAR, which implies that RA is primarily available for CRABPs. The remaining RA molecules can bind to RARs and CYP enzymes. Finally, we assumed that the ratio of total transcription factor concentration to total RAR concentration (f) remains constant after adding RA to the cell. However, this depends on the gene- and cell-type. It is believed that RARs and RXRs each have three isotypes, namely RARα, RARβ, RARγ, RXRα, RXRβ, RXRγ, which can form nine different heterodimers. Depending on the gene-type, one or some of these heterodimers can initiate the transcription of the target gene after binding to RA. Little is known about the expression levels of the nuclear hormone receptors in various cell types, and their interactions with each other. The model presented in this paper can be expanded once there is more information about the nuclear hormone receptor expression levels and functions. 2ff7e9595c
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