Dexamethasone-induced PPARG expression in osteogenic differentiation in vitro: impact on SOX9 and RUNX2 levels

ABSTRACT Background The effects of dexamethasone during in vitro human osteogenesis present a complex picture. On one side, dexamethasone promotes the osteogenic differentiation of human bone marrow mesenchymal stromal cells (BMSCs) by downregulating SOX9 . On the other side, it simultaneously promotes adipogenesis through the upregulation of PPARG . The regulation of SOX9 and PPARG levels appears to be mediated by the transactivation function of the glucocorticoid receptor (GR), suggesting an indirect effect of dexamethasone on SOX9 downregulation. This study aims to determine whether PPAR-γ affects the expression levels of SOX9 , as suggested by several studies. Methods Human BMSCs were isolated from bone marrow and cultured in different osteogenic induction media containing 10 or 100 nM dexamethasone. Undifferentiated cells were used as control. Cells were treated either with a pharmacological PPAR-γ inhibitor (T0070907) or with a PPARG -targeting siRNA. Differentiation markers or PPAR-γ target genes were analysed by RT-qPCR. Mineral deposition was assessed by Alizarin Red staining. Two-way ANOVA followed by a Sidak multiple comparison test was used to compare the effects of treatments. Results Pharmacological inhibition of PPAR-γ had a mild effect on the expression of PPAR-γ target genes but hindered adipocyte formation. Neither RUNX2 nor SOX9 expression were affected by T0070907. siRNA treatment successfully downregulated PPARG expression, as well as that of PPAR-γ target genes LPL, LPAR1 , and ADIPOQ . Contrary to expectations, RUNX2 was significantly downregulated by the PPARG -siRNA treatment during osteogenic differentiation both in the absence and presence of dexamethasone, while SOX9 levels were downregulated in undifferentiated cells. Overall, Alizarin Red staining analysis showed no change in mineralization levels when PPARG expression or activity was inhibited. Conclusions Understanding how dexamethasone regulates human BMSC differentiation is crucial to refine current in vitro models. These results suggest that PPAR-γ is not involved in SOX9 or RUNX2 repression during in vitro osteogenic differentiation of human cells.