| TRIM22 is a member of the tripartite motif (TRIM) family of proteins, which are involved in diverse cellular processes, including cell proliferation, differentiation, apoptosis, and transcriptional regulation etc. In recent years, some TRIM proteins are found to play important roles in antiviral processes. Of which, TRIM5a is the most intensively studied, which plays a crucial role in the antiviral activity against HIV-1. Notably, many TRIM proteins can be upregulated by interferons, supporting their potential role as effectors in the anti-viral cellular response. Thus, it has been speculated that the TRIM proteins might represent a new and widespread class of antiviral molecules involved in innate immunity.In physiological condition, TRIM22 is mainly expressed in lymphoid tissues, such as thymus and spleen. It has been reported that TRIM22 may be involved in hematopoietic differentiation. Although the basal expression level of TRIM22 in many other tissues is pretty low, TRIM22 expression could be strongly induced upon the stimulation of IFN, LPS or p53. Our previous data demonstrated that TRIM22, but not TRIM5a, was one of the most strongly induced TRIMs in response to IFNs treatment in human hepatocytes and could inhibit Hepatitis B virus (HBV) gene expression and replication efficiently. Intrahepatic gene expression analysis of acutely infected chimpanzees has shown that the TRIM22 is associated with the IFN-y-mediated noncytopathic inhibition of HBV replication. Additionally, it was reported that TRIM22, but not TRIM5a, was implicated in IFN-mediated anti-HIV activity. Based on these observations, we previously investigated the molecular mechanisms of TRIM22 induction by IFN-y. We identified a special cis-element named 5'extended IFN-stimulating response element (5'eISRE) that was crucial for IFN-y-induced TRIM22 expression. Furthermore, we demonstrated that interferon regulatory factor (IRF)-1 played a key role in the expression of TRIM22 induced by IFN-y via binding to this cis-element.Histone deacetylases (HDACs) play an important role in the epigenetic transcriptional regulation of many genes. Histone deacetylation enzymes have often been associated with the suppression of eukaryotic gene transcription. However, recent studies show that the effect of HDACs on gene transcriptional regulation is much more complicated than previously expected. First, although HDACs often act as suppressors for gene expression, they can also be crucial for the transcriptional activation of some genes. Second, HDACs can also regulate the acelytion of non-histones, such as transcription factors. Third, HDACs can also play an import role in the ubiquitination of some proteins. Interestingly, some studies demonstrated that HDACs inhibitors, such as TSA, played a crucial role in the transcriptional regulation of some genes by IFNs, although the underlying mechanisms remained unclear.Accumulating data show that the low acelyted level of histons correlates closely with pathogenesis of malignant tumours. HDACs inhibitors, such as TSA, NaB and VPA, are therefore received much attention in anti-tumor therapy for their role in regulation of protein acelytion. However, during the course of HDACs inhibitors-mediated anti-tumor therapy, there are some reports that HDACs inhibitors treatment can lead to the higher replication of HCV or HBV, indicating HDACs inhibitors may interrupt the expression of some ISGs. As we previously demonstrated that TRIM22 played an important role in the innate immunity against HBV and it was a key effector in IFN-mediated antiviral activity, we hypothesize that the enhancement of viral replication by HDACs inhibitors may be due to their inhibitory effect on IFN-y induction of TRIM22. To address this question, we have studied on the effect of HDACs inhibitors on IFN-y induction of TRIM22 and its molecular mechanisms. This study may help to further decipher the molecular mechanisms of TRIM22 induction by IFN-y, and might also be useful for designing TRIM22-based antiviral strategy.Part I TSA blocked the IFN-y-induced TRIM22 expressionOur previous data showed that the expression level of TRIM22 in quicient hepatocytes was pretty low, but could be strongly induced in response to IFN-y stimulation. To examine the role of HDACs inhibitors in the IFN-y-induced TRIM22 expression, we treated HepG2 cells with TSA plus IFN-y. After 24 hours, the protein expression of TRIM22 was determined. Results showed that TSA could efficiently inhibit the TRIM22 induction by IFN-y at both mRNA and protein levels in a dose-and time-dependent manner. To determine at which level TSA exerted its effect on IFN-y induction of TRIM22, we examined TRIM22 mRNA stability in DMSO-or TSA-treated cells using an actinomycin D inhibition assay. Results showed the half-life of TRIM22 mRNA induced by IFN-y appeared to be similar between DMSO-and TSA-treated cells for the indicated time period, indicating TSA didn't affect TRIM22 mRNA stability. The data agreed with the notion that the inhibitory effect of TSA on TRIM22 induction by IFN-y was likely at the transcriptional level. Then, we tansfected TRIM22 promoter dependent luciferase reporter plasmid, pLuc-160, into HepG2 cells, and treated these cells with different concentrations of TSA in the presence of IFN-y.24 h later, luciferase activity in the cell Iysate was examined. Results showed that TSA could effectively inhibit the IFN-y-induced promoter activity of TRIM22 in a dose-and time-dependent manner, indicating TSA inhibited the IFN-y-induced TRIM22 expression at the transcriptional level. To further verify the role of TSA on IFN-y induction of TRIM22 is due to its specific inhibitory effect on HDACs, we examined the effect of another HDACs inhibitor NaB on IFN-y-induced TRIM22 transcriptional activity. It was found that both TSA and NaB could enhance the acelytion of histone H4 significantly, indicating both of them could inhibit the HDACs activity in HepG2 cells dramatically. Further study showed that NaB could also inhibit the IFN-y-induced promoter activity of TRIM22, indicating it was the blockage of HDACs activity that led to the inability of IFN-y to induce TRIM22 transcription.Part II TSA blocked the IFN-y-induced TRIM22 expression via enhancing the degradation of IRF-1 through ubiquitin-proteasome pathwayOur previous work found that IRF-1 played an important role in IFNs-induced TRIM22 expression via binding with 5'eISRE. As the above-mentioned data indicated that TSA inhibited IFN-y-induced TRIM22 expression at the transcriptional level, we therefore investigated whether TSA could have effect on the IFN-y-induced expression of IRF-1, which is the key transcription factor for TRIM22 induction. The ELISA-based transcription factor assay showed that the binding of nuclear IRF-1 from TSA-treated cells with 5'eISRE was decreased significantly. Western blot results also showed that upon TSA treatment, the expression level of nuclear IRF-1 was reduced dramatically. Next, we examined the IFN-y-induced IRF-1 protein expression at different time points (0.5,1,2,6,12,24 h) after TSA treatment. Results showed that TSA could inhibit IFN-y-induced IRF-1 protein expression at each time point, further confirming the effect of TSA on IRF-1 protein expression. To determine whether the effect of TSA on IRF-1 protein expression is due to its effect on IRF-1 transcription, we examined the effect of TSA on IRF-1 mRNA expression. Results showed that TSA did not affect the IFN-y-induced IRF-1 mRNA expression. It was well known that IRF-1 transcription was under control of STAT1, we therefore investigated the effect of TSA on IFN-y-induced STAT1 phosphorylation. It was found there was no difference between DMSO-or TSA-treated cells in the context of IFN-y-induced STAT1 phosphorylation. These data indicated that TSA did not affect IRF-1 transcription. We then tested the stability of IRF-1 protein in the presence of TSA, and found that TSA treatment decreased the stability of IFN-y-induced IRF-1 protein significantly. We also tested the effect of TSA on the stability of ectopic IRF-1 protein and found that TSA could also accelerate the degradation of ectopic IRF-1 protein. Taken together, these data indicated that TSA could decrease the protein level of IRF-1 via enhancing the degradation of IRF-1 protein.Studies have reported that, half-life of IRF-1 protein is very short, and its degradation occurs mainly through the ubiquitin-proteasome pathway. We therefore tested the effect of the proteasome inhibitor MG132 on TSA-mediated degradation of IRF-1 protein. We found that MG132 could inhibit TSA-mediated degradation of IRF-1 protein. Meanwhile, we also tested the effect of lysosome inhibitor Chloroquine on TSA-mediated degradation of IRF-1 protein. Results showed that Chloroquine failed to block the effect of TSA. These data indicated that TSA might enhance the degradation of IRF-1 via the ubiquitin-proteasome pathway. To further verify this phenomenon, we transfected pIRF-1-Myc and Ub-HA plasmids into HepG2 cells, then conducted Co-IP with anti-Myc for pull-down and with Ub or HA antibodies for detection. Results showed that TSA could increase the ubiquitination level of IRF-1 protein significantly.HDAC1 is the target gene of TSA. Under physiological conditions, the main function of HDAC1 is to reduce the acetylation of histone. However, recent study showed that HDAC1 may function as a deubiquintin ligase. We found that both IRF-1 and HDAC1 could bind to the 5'eISRE of TRIM22 promoter as determined by avidin gel precipitation test, which might act as a platform to facilitate the deubiquitination of IRF-1. Further study showed that overexpression HDAC1 could decrease the level of IRF-1 ubiquitination significantly.In summary, this study demonstrated that the histone deacetylase inhibitor TSA could block the transcription of IFN-y-induced TRIM22 expression through enhancing the degradation of IRF-1 protein via ubiquitin-proteasome pathway. It provides new data for explanation of the regulatory and functionary mechanism of the anti-HBV innate molecule-TRIM22.
|
PDF Full Text Request