Identification of the role of a novel conserved leucine Zipper - containing protein in osteogenic differentiation of mesenchymal stem cells

ABSTRACT Adult bone marrow mesenchymal stem cells have the ability to differentiate into different cell types such as osteoblasts, adipocytes, chondrocytes, myoblasts, neural cells etc. The multipotency of mesenchymal stem cells, its easy isolation and culture as wells as their high ex vivo expansion potential make these cells to be exploited for therapeutic purpose. Despite enormous data available at molecular and cellular levels, it is evident that number of fundamental queries regarding molecular and cellular mechanisms needs to be elucidated. The present study is aimed at understanding differentiation of osteoblasts from mesenchymal stem cells (MSC) by certain candidate genes. shRNA based library screening was performed in our laboratory to silence specific mRNA that interfere with osteoblasts differentiation from MSC. shRNA library led to the identification of several candidate genes of known as well as unknown function. We focused our studies on candidate genes whose function is unknown and termed as osteoblasts inducer (ObI/OB). Current study is limited to understand a particular gene termed as ࢠOB3ࢠin succession to our previously identified and characterized genes. OB3 gene identified during shRNA library screening was validated in both bone marrow stromal cell lines as well as murine primary mesenchymal stem cells. Its expression during osteoblasts differentiation has to been elucidated and its silencing effects on bone markers has been shown. We have by microarray few targets that are repressed upon OB3 silencing and involved in osteoblasts differentiation. Moreover, OB3 protein undergoes proteasome degradation and we have attempted if not completely to show that OB3 degradation is mediated through ubiquitin-proteasome. We have identified Creb3 as a partner of OB3 and have attempted to show creb3 silencing during osteoblasts differentiation. Furthermore overexpression of both OB3 and Creb3 independently delay osteoblasts differentiation. Attempts to identify the molecular mechanisms through which these two candidate genes are involved in osteoblasts differentiation is being explored.