HOPX counteracts Twist-1/ EZH2 regulation of BMSC cell fate determination via suppression of adipogenic gene pathways

Previous studies of global binding patterns identified the histone 3 lysine 27 methyltransferase, EZH2, as a regulator of the homeodomain-only protein homeobox (HOPX) gene during bone marrow stem/stromal cell (BMSC) osteogenic differentiation, suggesting a potential role for HOPX in regulating BMSC lineage specification. In the present study we explored the function of HOPX in BMSC proliferation and differentiation. Human BMSC were found to express low levels of HOPX transcripts, which were dramatically upregulated during both BMSC osteogenic and adipogenic induction. Interestingly, HOPX gene expression was negatively correlated with TWIST-1, a basic helix-loop-helix transcription factor, previously reported to increase expression of the epigenetic repressor, EZH2, where both molecules inhibit BMSC osteogenic differentiation but promote adipogenesis. Functional siRNA mediated HOPX knockdown studies resulted in a decreased proliferative capacity by human BMSC, as assessed by BrdU incorporation. Moreover, knockdown of HOPX resulted in an enhanced adipogenic capacity by human BMSC in vitro. However, functional studies failed to observe any affect by HOPX on BMSC osteogenic differentiation. Conversely, retroviral mediated HOPX enforced expression by BMSC resulted in an increased capacity for proliferation and decreased capacity to undergo adipogenesis. RNA-seq analysis of HOPX overexpressing and vector control BMSC during adipogenesis suggested that HOPX function may be acting through suppression of adipogenic pathways associated genes such as ADIPOQ, FABP4, PLIN1 and PLIN4. Collectively, these data suggest that HOPX is a promoter of BMSC growth and self-renewal, and an inhibitor of adipogenesis by suppressing adipogenic associated genes.