Effect of lentiviral silencing of Rcor2 in MC3T3-E1 cells during osteoblast differentiation
Epigenetic regulation of bone formation is a complex network of active proteins and their cofactors. Recent developments in calcified tissue research have revealed several important pathways of epigenetic regulation leading to transcriptional changes in bone cells. Rest Corepressor 2 (Rcor2) is a coregulator of Lysine-specific histone demethylase 1 (Lsd1), a demethylase linked to osteoblast activity, hematopoietic stem cell differentiation and malignancy of different neoplasms. However, the role of Rcor2 in osteoblast differentiation has not yet been examined in detail. We have previously shown that Rcor2 is highly expressed in mesenchymal stromal cells (MSC) and particularly in the osteoblastic lineage. The role of Rcor2 in osteoblastic differentiation in vitro was further characterized and we demonstrate here that lentiviral silencing of Rcor2 in MC3T3-E1 cells led to a decrease in osteoblast differentiation, as shown by decreased alkaline phosphatase and von Kossa stainings as well as by decreased expression of several osteoblast-related marker genes. RNA-sequencing of the Rcor2-downregulated MC3T3-E1 cells revealed decreased repression of Rcor2 target genes, as well as significant upregulation of majority of the differentially expressed genes. While the heterozygous, global loss of Rcor2 in vivo did not lead to a detectable bone phenotype, conditional deletion of Rcor2 in limb-bud mesenchymal cells led to a moderate decrease in cortical bone volume, which was not accentuated by challenging bone formation by ovariectomy or tibial fracture. Furthermore, a global deletion of Rcor2 led to decreased amount of white adipose tissue in vivo and decreased the capacity of primary cells to differentiate into adipocytes in vitro, while the conditional deletion led to decreased adiposity in fracture calluses. Taken together, these results suggest that epigenetic regulation of mesenchymal stromal cell differentiation is mediated by Rcor2, which could thus play an important role in defining the MSC fate. Overall design: To study the relevance of Rcor2 upregulation during osteoblast differentiation, we silenced Rcor2 expression in MC3T3-E1 cells by stable shRNA expression.