| Runx2 is a transcription factor which plays pivotal roles in osteoblast differentiation. In the process of Runx2 induced stem cell osteoblastic differentiation, Runx2 transactivation activity relies on changes of chromatin structure and facilitative epigenetic factors, which regulate Runx2's ability of binding to its target DNA and its activities. For example, inhibiting the enzymatic activity of a histone deacetylase, HDAC3, can significantly enhance Osteocalcin expression and accelerate mineralization.On the basis of the studies on HDACs, Runx2 and their interactions by others, we further studied the roles of HDAC1 in regulating Runx2 induced Osteopontin expression. First, we used dual luciferase assay to investigate nine HDACs (HDAC1,-3,-4,-5,-6,-7,-8,-10,Sirt2) and three HATs (P300,GCN5,CBP) in their abilities of repressing or activating Runx2 stimulated Osteopontin promoter activity. Our results indicated that HDAC1 and HDAC3 strongly repressed Runx2 stimulated Osteopontin promoter activity; and TSA, the specific HDAC inhibitor, diminished HDAC1's repression capability and enhanced Runx2 stimulated Osteopontin promoter activity. We also subcloned the Osteopontin promoter into a promoter-less, luciferase reporter vector, which can form nucleosome structure in the cell, and we co-transfected this construct together with Runx2 and HDAC1 into the cells. Our results showed that nucleosomal structure did not affect HDAC1 capability and TSA treatment's efficacy in repressing and enhancing Runx2 stimulated Osteopontin promoter activity, respectively. Therefore we supposed that the HDAC regulation of Runx2 transactivation activity is nucleosome structure independent.By using immuno-fluorescence and co-immuno-precipitation techniques, we next found that HDAC1 and Runx2 co-localized in the nucleus, suggesting that they may form a complex. Using chromatin immuno-precipitation technique, we further identified that both HDAC1 and Runx2 occupied on the Osteopontin promoter. Through transiently transfecting HDAC1 expression vectors into the Runx2 overexpressing cells, we found that overexpression of HDAC1 significantly repressed Runx2 induced Osteopontin mRNA levels but interestingly did not repressed Runx2 induced Alkaline phosphatase mRNA levels. If HDAC enzyme activities were inhibited by TSA, Runx2 induced Osteopontin mRNA levels were significantly elevated but Runx2 induced Alkaline phosphatase mRNA levels were not affected. These results were similar to those obtained from dual luciferase assays and indicated that Runx2 induced Osteopontin expression is specifically regulated by HDAC1, as compared to that of Alkaline phosphatase, although both of them are induced by Runx2 during early osteoblast differentiation. Our immuno-blotting results indicated that OPN mRNA levels increased after six days of osteoblast differentiation, and in the meantime HDAC1 protein levels decreased. In addition, we also analyzed different cell lines representing different differentiation statuses which range from mesenchymal stem cell, pre-osteoblast, osteoblast to osteocyte. As differentiation status progresses, HDAC1 protein levels also decreased. Taken together, our results suggested that HDAC1 may act as a repressing barrier for Runx2 to induce Osteopontin gene expression and even osteoblast differentiation. Removal of this barrier will facilitate Runx2. Further we noticed that the amount of Runx2 proteins was not changed by overexpression of HDAC1 but accumulated after differentiation. This phenomenon indicates that there may be HDAC1 independent epigenetic mechanisms governing the amount of Runx2 proteins during osteoblast differentiation. In summary, our study revealed a molecular mechanism by which HDAC1 represses Runx2 induced OPN gene expression and provided useful data which may help therapy of bone diseases in the future. |
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