HDAC5 Controls MEF2C-Driven Sclerostin Expression in Osteocytes

Abstract

Osteocytes secrete paracrine factors that regulate the balance between bone formation and destruction. Among these molecules, sclerostin (encoded by the gene SOST) inhibits osteoblastic bone formation, and is an osteoporosis drug target. The molecular mechanisms underlying SOST expression remain largely unexplored. Here we report that histone deacetylase 5 (HDAC5) negatively regulates sclerostin levels in osteocytes in vitro and in vivo. HDAC5 shRNA increases, whereas HDAC5 overexpression decreases SOST expression in the novel murine Ocy454 osteocytic cell line. HDAC5 knockout mice show increased levels of SOST mRNA, more sclerostin-positive osteocytes, decreased Wnt activity, low trabecular bone density, and reduced bone formation by osteoblasts. In osteocytes, HDAC5 binds and inhibits the function of MEF2C, a crucial transcription factor for SOST expression. Using chromatin immunoprecipitation, we have mapped endogenous MEF2C binding in the SOST gene to a distal intergenic enhancer 45 kB downstream from the transcription start site. HDAC5 deficiency increases SOST enhancer MEF2C chromatin association and H3K27 acetylation and decreases recruitment of co-repressors NCoR and HDAC3. HDAC5 associates with and regulates the transcriptional activity of this enhancer, suggesting direct regulation of SOST gene expression by HDAC5 in osteocytes. Finally, increased sclerostin production achieved by HDAC5 shRNA is abrogated by simultaneous knockdown of MEF2C, indicating that MEF2C is a major target of HDAC5 in osteocytes.

The Wnt Inhibitor Sclerostin Is Up-regulated by Mechanical Unloading in Osteocytes in Vitro

“…Ocy454 cells show rapid, high level expression of SOST/sclerostin that is responsive to hormonal (PTH), cytokine (PGE 2 ), and mechanical stimuli. Furthermore, G s ␣ knockdown in Ocy454 led to significant increases in SOST expression matching known osteocyte in vivo regulation (35), demonstrating the broad utility of this new osteocytic cell line for studying SOST/sclerostin regulation as we have recently reported (36). Ocy454 also showed an enhanced osteocytic phenotype when cultured on a three-dimensional biomaterial by increasing FGF23 expression upon PTH stimulation, highlighting the importance of optimizing in vitro culture conditions for studying certain aspects of osteocyte biology.…”

“…To confirm these in vivo results in Ocy454 cells, we used shRNA to knock down G s ␣ in Ocy454 as was done previously for HDAC5 (36). The range of sclerostin secretion (normalized to cell number) was determined in each experiment using 10 separate control lentiviruses expressing shRNAs against non-expressed genes (LacZ, luciferase, GFP, and red fluorescent protein).…”

“…In an effort to establish a more homogeneous osteocytic population, we also performed FACS on Ocy454 to isolate single cell subclones. Ocy454 and several single cell clones (36) have the same osteocyte marker expression and hormonal (PTH, PGE 2 , and shear stress) response.…”

Histone deacetylases (HDAC) in physiological and pathological bone remodelling

“…shRNA to HDAC 4 or 7 in these cells did not increase SOST expression [74]. In subsequent studies, HDAC 5 global knock out mice were shown to have higher numbers of sclerostin secreting osteocytes particularly the periendosteal osteocytes.…”

“…These studies are consistent with HDAC 5 suppressing bone formation and selective HDAC 5 inhibitors elevating bone formation, although this has yet to be investigated. In contrast, a recent study revealed that HDAC 5 global knockout mice have reduced numbers of osteoblasts and hence low bone density [74]. This study focused on the fact that HDAC 5 knockout increases SOST expression by osteocytes.…”