RE1-silencing transcription factor (Rest) has been identified as a master negative regulator of neuronal differentiation. Nothing is known about Rest function in bone cells. In this study, we examined the Rest expression levels and role during osteoblast differentiation. We found that Rest is abundantly expressed in bone marrow stromal cells, calvarial osteoblasts, and MC3T3-E1 osteoblasts. Treatment of primary osteoblasts with ascorbic acid (AA) down regulated Rest mRNA expression at an early stage, but not in later stages of differentiation. Consistent with treatment of primary cultures, AA treatment of MC3T3-E1 cells significantly reduced Rest protein expression at day 3 and at day 8 after initiation of osteoblast differentiation. Treatment of bone marrow stromal cells with BMP-2 and dexamethasone, but not IGF-I for 3 days greatly decreased Rest mRNA expression. To test the function of Rest during osteoblast differentiation, Rest expression was knocked down in MC3T3-E1 cell subclones segregated on the basis of ALP activity (differentiation status) using lentivirus expressing shRNA against Rest. An 80% knockdown of Rest expression decreased Osterix (Osx) expression by 52-57% and as a result, increased both basal and AA induced ALP expression and activity in the subclone that expresses low basal level of ALP (undifferentiated). By contrast, a 98% knockdown of Rest expression in cells that express high basal levels of ALP (differentiated cells) caused a significant reduction in Osx expression, basal and AA induced ALP expression and activity. These data suggest that Rest regulates early osteoblast differentiation via modulating Rest expression that is independent of Osx expression.
Keywords: ALP; OSTEOBLAST; OSTERIX; REST; SHRNA KNOCKDOWN.
© 2015 Wiley Periodicals, Inc.