Histone deacetylase 8 suppresses osteogenic differentiation of bone marrow stromal cells by inhibiting histone H3K9 acetylation and RUNX2 activity

https://doi.org/10.1016/j.biocel.2014.07.003Get rights and content

Abstract

Bone marrow stromal cells (BMSCs) are multipotent progenitor cells with capacities to differentiate into the various cell types and hold great promise in regenerative medicine. The regulatory roles of histone deacetylases (HDACs) in osteoblast differentiation process have been increasingly recognized; however, little is known about the precise roles of HDAC8 in the osteogenic differentiation of BMSCs. Herein we aimed to investigate the roles of HDAC8 in the osteogenic differentiation of rat BMSCs by pharmacological and genetic manipulations in vitro. During osteogenic differentiation of BMSCs, pharmacological inhibition of HDAC8 by HDAC inhibitor valproic acid (VPA) promoted the level of histone H3 lysine 9 acetylation (H3K9Ac) and significantly enhanced the expression of osteogenesis-related genes Runx2, Osterix, osteocalcin (OCN), osteopontin (OPN) and alkaline phosphatase (ALP). Similarly, knockdown of HDAC8 using short interfering RNA triggered H3K9Ac and enhanced osteogenic differentiation of BMSCs, largely phenocopied the effects of VPA-mediated HDAC8 depletion. However, enforced expression of HDAC8 significantly reduced the level of H3K9Ac and inhibited osteogenic differentiation of BMSCs, which can be attenuated by VPA addition. Mechanistically, HDAC8 suppressed osteogenesis-related genes expression by removing the acetylation of histone H3K9, thus leading to transcriptional inhibition during osteogenic differentiation of BMSCs. Importantly, we found that HDAC8 physically associated with Runx2 to repress its transcriptional activity and this association decreased when BMSCs underwent osteogenic differentiation. Taken together, these results indicate that epigenetic regulation of Runx2 by HDAC8-mediated histone H3K9 acetylation is required for the proper osteogenic differentiation of BMSCs.

Introduction

Bone marrow stromal cells (BMSCs) are progenitor cells as defined by their self-renewal capabilities and multilineage differentiation potentials that give rise to various tissues and organs both in vitro and in vivo (Undale et al., 2009). Under certain conditions, BMSCs are able to differentiate into osteoblasts and hold significant promise for clinical applications, especially for bone regeneration in skeletal defects, largely due to easy harvest, accessibility and lack of immunogenicity (Caplan and Bruder, 2001, Kon et al., 2012, Wagers and Weissman, 2004). Previous studies have been focused on endeavoring to promote the capability of BMSCs to undergo osteogenic differentiation via epigenetic modifications, such as histone acetylation, which may contribute to differentiation of BMSCs into osteoblasts lineage (Hu et al., 2013, McGee-Lawrence and Westendorf, 2011, Teven et al., 2011). However, the detailed molecular mechanisms underlying these processes remain incompletely recognized.

Histone acetylation is a reversible epigenetic process modulated by the combined and delicate activities of histone acetylases (HATs) and histone deacetylases (HDACs). Hyperacetylation contributes to relaxing the chromatin structure and allows the binding of DNA sequences by transcription factors, thereby activating transcription, whereas histone deacetylation results in transcriptional silencing (Cheung et al., 2000, Choudhary et al., 2009, Teven et al., 2011). HDACs regulate transcriptional process by affecting the structure of chromatin and activities of relevant transcription factors under diverse biological contexts. Upon removal of negatively charged acetyl groups (CH3CO-) from the ɛ-amino groups of lysine residues by HDACs, such histone changes result in chromatin condensation, thus decreasing the accessibility of DNA-binding transcription factors (Choudhary et al., 2009, Glozak et al., 2005). During the differentiation of stem cells, the relative abundance of epigenetic marks usually reflect the activation or repression of genes which further guide cells development toward a particular cell lineage. Modified histone domains such as acetylated histone H3 lysine 9 (H3K9) are considered as key epigenetic signatures as indicative of transcriptional activation (Zheng et al., 2013). Although histone modifications play crucial roles in the transcriptional regulation, the relationship between the differentiation of specific cell types and histone modifications is not completely understood. Therefore, it is of great interest and importance to investigate the effects of chromatin modifications by individual HDACs on the osteogenic differentiation of BMSCs, which may advance our understanding of stem cell fate decision and facilitate the clinical translation of BMSCs-mediated bone repair.

The HDACs are grouped into four subclasses based on structural and functional similarities (Hewitson et al., 2013). A line of evidence has indicated the inhibitory roles of HDAC1ā€“7 in osteoblast differentiation by regulating histone acetylation or interacting with RUNX2, a master transcription factor for osteoblast differentiation (Jensen et al., 2007, Jensen et al., 2009Lee et al., 2006, Li et al., 2009, Schroeder, 2004, Shimizu et al., 2010, Westendorf et al., 2002). HDAC inhibitors (HDIs), such as valproic acid (VPA) and sodium butyrate, are often utilized to promote osteoblast differentiation and maturation (Cho et al., 2005, Iwami and Moriyama, 1993, Lee et al., 2006). HDAC8 is a unique member of class I HDACs, as it exhibits a special crystal structure (Gantt et al., 2010) and lacks the conserved C-terminal domain (Somoza et al., 2004), suggesting a distinct and specific biological function for HDAC8 in various pathophysiological processes. Interestingly, knockdown of HDAC8 in cultured neuroblastoma cells has no effect on histone acetylation (Oehme et al., 2009), whereas the class I HDACs 1ā€“3 are potent regulators of histone acetylation in osteoblasts. Moreover, HDAC8 specifically controls craniofacial skeletal patterning by repressing a subset of transcription factors in cranial neural crest cells upon conditional HDAC8 deletion, whereby HDAC8 is shown to regulate these genes at the chromatin level (Haberland et al., 2009). Therefore, an interesting question arises whether HDAC8 has some uncharacterized key roles during osteogenic differentiation of BMSCs.

In this study, we aimed to investigate the biological roles of HDAC8 on the osteogenesis of BMSCs and the associated regulatory mechanisms. Our findings indicated that HDAC8 functioned as a transcriptional repressor via regulating the level of H3K9Ac and interacting with RUNX2 during the specification of BMSCs to the osteoblasts lineage in vitro, suggesting that therapeutic inhibition of HDAC8 might be beneficial for bone repair and regeneration.

Section snippets

Cell culture and differentiation

All experiments and animal protocols were performed with the approval of the Ethics Committee and Animal Care Committee of Nanjing Medical University. Primary BMSCs were obtained from the tibia bone marrow of three-week-old male Sprague Dawley rats as we described before (Zhang et al., 2012), and used between passages 3 and 5. To induce osteogenic differentiation of BMSCs in vitro, cells were cultured in osteogenic medium consisting of DMEM supplemented with 50Ā Ī¼M ascorbic acid (Sigma, St.

Histone H3K9 is hyperacetylated and associated with reduced expression of HDAC8 in osteogenic differentiation of BMSCs

To investigate the potential roles of HDAC8 in osteogenic differentiation of BMSCs, we initially determined the temporospatial expression pattern of HDAC8 in rat BMSCs cultured with osteogenic medium. The formation of mineralized nodules was readily observed on day 7 and increased significantly on day 14 as assessed by Alizarin Red staining (Fig. 1A). The level of RUNX2 protein increased gradually from day 3 to day 7 and then declined on day 14. At the same time, two representative osteogenic

Discussion

The fate decision and lineage differentiation of BMSCs is a complex biological process orchestrated by multiple layers of regulation including epigenetic, transcriptional and post-transcriptional ways. It is becoming increasingly clear that the chromatin modifications play crucial roles during these processes (Teven et al., 2011). Among them, histone acetylation is a widely studied type of chromatin modifications and correlates with transcriptional activation, and histone deacetylases (HDACs)

Acknowledgements

This work was supported by National Natural Science Foundation of China grant (81070810) and a project funded by the Priority Academic Program for the Development of Jiangsu Higher Education Institutions (2014-37).

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