Regulatory Mechanisms Involved In Ubiquitin-Proteasome Proteolytic Pathway Mediated Degradation Of Retinoic Acid Receptor α(RARα)

IntroductionDifferentiation-inducing means the malignant cells are induced differentiation to normal or almost normal state in the treatment with differentiation-inducing agents. It emphasizes the reversals of malignant phenotypes and immature states in tumor cells which generally does not cause destruction of either tumor cells or normal cells, therefore the side effects of traditional chemotherapies are avoided. As a treatment different from the traditional cancer treatments, differentiation-inducing therapy causes more and more attentions. At present, the differentiation-inducing agents commonly used include the retinoids, polar compounds, cytokines, vitamin D3and its analogues, some anti-cancer drugs and herbs, etc., wherein the retinoids is the most important and efficacious clinical used agent. With the treatment success of all-trans retinoic acid (ATRA) in remission of acute promyelocytic leukemia (APL), the retinoids are also increasingly being used for prevention and treatment of many other cancers including breast cancer, skinmalignancies, cervical cancer and central nervous system tumors. The wide applications and striking clinical benefits of the retinoids arouse the enthusiasm of researchers in clarifying the mechanisms of their actions.Studies have shown that the physiological actions of retinoic acid (RA) are mediated through two distinct nuclear receptor families, the retinoic acid receptors and the retinoid X receptors, each of which has α, β,γ three subtypes. RARs and RXRs bind as homodimers or heterodimers to a specific DNA response element (RARE) found in target genes, thereby promoting gene transcription. In-depth studies showed that RA directs ubiquitin-proteasome pathway mediated degradation of RARα in company with cell differentiation. Our previous studies have demonstrated that ATRA triggers degradation of RARα which might limit the sensitivity of cells to the cytodifferentiating and antiproliferative activities of ATRA. Furthermore, protecting RARa from degradation by proteasome inhibitors such as MG132or PS341has synergistic differentiation effect of ATRA. Therefore researches related to the mechanisms by which ATRA promotes ubiquitination mediated degradation of RARα may provide new breakthroughs for efficacy elevation of ATRA.Our study will start with the observations of related molecular phenomenons during ATRA-induced cell differentiation and aims to find some potential regulators involved in ubiquitination mediated degradation of RARa. The current study wants to solve the following three problems:1, How sumo-1modification may influence the ubiquitination mediated degradation of RARa? Existing literature suggested that RARα can be modified by sumo-1. Furthermore, sumo-1was confirmed involved in ubiquitinaiton of target proteins by complicated regulatory mechanisms. Therefore investigating whether and how sumo-1modification regulates ubiquitination mediated degradation of RARα is the main purpose of this section.2, What’s role of E2F1in regulating ubiquitination mediated RARα degradation? Transcriptional factor E2F1is involved in regulation of many intracellular molecular events by expression inductions of its downstream proteins. However, our previous studies have shown that the protein level but not mRNA level of RARa is regulated by E2F1, suggesting a probable transcription-independent role of E2F1in RARa regulation. We will study its role in regulating ubiquitination mediated degradation of RARa in this section.3, Is MDM2likely to be the ubiquitin E3ligase of RARa? Protein ubiquitination is an orderly process that a variety of enzymes are involved in. The ubiquitin E3ligase is a crucial factor in the whole process responsible for specifically recognizing target proteins. Our previous studies showed that ubiquitin E3ligase MDM2may promote ubiquitination mediated degradation of RARa. Therefore we will further explore if there are some possibilities that MDM2is the ubiquitin E3ligase of RARα in this section based on preliminary findings.Section1The role of sumo-1modification in modulating ubiquitination mediated degradation of RARaObjective:RARa has been demonstrated to be one of the most important retinoic acid receptors and functions through mutual interactions with other pathways by regulating gene networks that control cell growth, differentiation, survival and death. Previous studies demonstrated that RARa undergoes ubiquitination mediated degradation during its ligand, RA treatment, but very little is known about the mechanism by which RA regulates RARa’s stability. The small ubiquitin-related modifier (sumo) has been identified within the past few decades and shown to covalently modify a large number of proteins with important roles in many cellular processes. Distinct from ubiquitination, which is involved in processing proteins for degradation via the proteasome pathway, sumo modification has been implicated in regulating protein stability and in some cases, has been demonstrated to be antagonistic to ubiquitination. In this study, we will systematically investigate the role of sumo-1modification in regulating ubiquitination mediated degradation of RARa and cell differentiation by using ATRA as a tool drug.Methods and results:Firstly, to establish relationships between sumoylation and ubiquitination of RARa, we mutated a possible sumo acceptor lysine within RARa, lysine399to arginine, generating RARa-K399R mutant. Immunoprecipitation and Western blotting results showed that RARa-K399R is almost totally incapable of sumoylated by sumo-1. Therefore, RARa-K399R was used in the following experiments to investigate how sumo-1modification may modulate the ubiquitination of RARa. Interestingly, sumo-1and sumo-1modified-RARa were both found downregulated in accompany with ubiquitination and degradation of RARα triggered by ATRA, suggesting a positive role for sumo-1modification in maintaining the stability of RARα. The comparison of half-life of the wild type and the lysine399mutant forms of RARa and their abilities to conjugate to ubiquitin demonstrated that lysine399is a critical amino acid residue in protecting RARα from ubiquitin-dependent degradation. Similarly, sumo-1dificiency was found significantly shorten the half-life of RARa protein. Based on these findings, we further demonstrated that functional lysine399and normal sumo-1expression are both required for full transactivation of RARa and efficient ATRA-triggered RARa-mediated differentiation.Conclusion:These results, thus, briefly suggested sumo-1modification may potentially play an antagonistic role to ubiquitination of RARα, and consequently has a positive effect in promoting transactivation of RARa and cell differentiation induced by ATRA.Section2The role of E2F1in regulating ubiquitination mediated degradation of RARαObjective:E2F1is a critical transcriptional factor that regulates various target genes with important roles in cell growth, apoptosis and differentiation. Recently, in-depth insights of E2F1revealed its transcription-independent capacity. Our preliminary study demonstrated a probable role of E2F1in regulating ubiquitination mediated degradation of RARα. Based on this finding, we will further investigate how E2F1modulates ubiquitination mediated degradation of RARα in this study, which aims to discover a new regulator involved in RARa degradation signaling as well as a target gene regulated by E2F1in a transcription-independent manner.Methods and results:First of all, we studied the immunohistochemical expressions of RARα and E2F1in three dozen cases of osteosarcomas. Results showed that approximately71.4%of osteosarcoma samples demonstrate low level of RARα expression while86.5%demonstrate high level of E2F1expression. Interestingly, there is a significant negative correlation between E2F1and RARα expression (R=-0.402, P<0.01), suggesting a certain regulation might exist between the two proteins. Furthermore, we provided evidences that overexpression of E2F1contributes to transcription-independent downregulation of RARa which can be significantly reversed by proteasome inhibitor MG132. Meanwhile, RARα was found decrease much more rapidly in E2F1overexpression-U2OS cells, both suggesting that E2F1may be involved in regulating ubiquitination mediated degradation of RARα. This deduce was further confirmed by the experiment that suppression of E2F1by siRNA upregulates RARa and inhibits its polyubiquitination induced by ATRA. The former data were consistent with results from the luciferase reporter assay and Real Time RT-PCR experiment that E2F1inhibits ligand-dependent transactivation of RARa. In addition, immunofluorescence, immunoprecipitation and GST-pull down experiments all demonstrated that E2F1colocalizes and interacts with RARa. Amino acid residues191-379within E2F1were demonstrated critical for E2F1-RARα interaction and once deleted, RARa would not be downregulated by E2F1any more. Additionally, full length E2F1as well as E2F1-N but not E2F1-N-Δ191-379mutant were found decrease sensitivities of U2OS cells to cytodifferentiating activity of ATRA. The expression level of E2F1was further demonstrated determine the responses of primary osteosarcoma cells after stimulation with ATRA to some extent.Conclusion:To summarize, our findings suggested that E2F1can promote the ubiquitination of RARα via physical interaction and thus inhibit its transcriptional activity, which plays an important regulatory role in ATRA-induced U2OS differentiation.Section3Study on the mechanism by which MDM2promotes ubiquitination mediated degradation of RARaObjective:According to the previous two sections, we have found two possible regulatory factors regulating RARa degradation via ubiquitin proteasome pathway. However, ubiquitination is a complicated and well-organized procedure, which is mediated by various enzymes. Among these, ubiquitin E3ligase is the most important as it specifically identifies the target proteins, thus keeping the accuracy of the whole ubiquitination process. So finding out the ubiquitin E3ligase of RARa protein will promote our understanding of the regulation of RARa ubiquitination and degradation. In screen of the proteins which interact with RARa, we got an important ubiquitin E3ligase, MDM2. Here, we will find out whether MDM2is an ubiquitin E3ligase of RARa.Methods and results:As literatures have reported, we found RARa is downregulated with ATRA treatment in U2OS cells. While LMB, a nuclear export inhibitor, significantly reverses ATRA-induced downregulation of RARα. It indicated that ATRA induces the degradation of RARa in the cytosols. According to the results of Western blotting and immunofluorescence assay that MDM2increases and accumulates in the cytosols of U2OS cells after treatment with ATRA for a short period of time, we speculated a potential correlation between MDM2and RARa. In order to further understand this procedure, we overexpressed MDM2protein in U2OS cells. Western blotting results showed that RARa is downregulated by wild type but not ubiquitin E3ligase activity-dificiency MDM2protein. Meanwhile the protein level of RARa was also detected in a stable transfection U2OS cell line with low expression level of MDM2. Results showed that repression of MDM2upregulates RARa and increases its stability. Furthermore, MDM2was found promote ubiquitination of RARα either by in vivo or in vitro ubiquitination assay. On the other hand, by immunofluorescence and immunoprecipitation experiments, we confirmed a physical interaction exist between MDM2and RARa. In addition, domain containing1-109amino acids within MDM2was demonstrated the binding site of RARα and once deletion, MDM2cannot promote RARa degradation any more. Nutlin-3, a well-known p53-MDM2binding inhibitor which was reported occupy this domain as well significantly interferes the interaction of MDM2with RARa and reverses the downregulation of RARa induced by ATRA. Consistent with the negative regulatory role on RARa, MDM2was found decrease the sensitivities of U2OS cells to the cytodifferentiating and antiproliferative activities of ATRA. What’s more, it demonstrated that MDM2ubiquitin E3ligase activity inhibitor HLI373can inhibit ubiquitination mediated degradation of RARα and facilitate the differentiation-inducing activity of ATRA.Conclusion:MDM2promotes RARa degradation via ubiquitination proteasome pathway which relies on its ubiquitin E3ligase activity and interaction with RARa. Thus, our results represented a possibility that MDM2is an ubiquitin E3ligase of RARa.