Regulation of osteoblast development by Bcl-2-associated athanogene-1 (BAG-1)

Joanna Greenhough,Paul A Townsend,Richard O C Oreffo,Rahul S Tare,Emmanouil S Papadakis,Ramsey I Cutress

doi:10.1038/srep33504

Abstract

BCL-2-associated athanogene-1 (BAG-1) is expressed by osteoblast-lineage cells; early embryonic lethality in Bag-1 null mice, however, has limited the investigation of BAG-1 function in osteoblast development. In the present study, bone morphogenetic protein-2/BMP-2-directed osteogenic differentiation of bone marrow stromal cells (BMSCs) of Bag-1+/− (heterozygous) female mice was decreased significantly. Genes crucial for osteogenic differentiation, bone matrix formation and mineralisation were expressed at significantly lower levels in cultures of Bag-1+/− BMSCs supplemented with BMP-2, while genes with roles in inhibition of BMP-2-directed osteoblastogenesis were significantly upregulated. 17-β-estradiol (E2) enhanced responsiveness of BMSCs of wild-type and Bag-1+/− mice to BMP-2, and promoted robust BMP-2-stimulated osteogenic differentiation of BMSCs. BAG-1 can modulate cellular responses to E2 by regulating the establishment of functional estrogen receptors (ERs), crucially, via its interaction with heat shock proteins (HSC70/HSP70). Inhibition of BAG-1 binding to HSC70 by the small-molecule chemical inhibitor, Thioflavin-S, and a short peptide derived from the C-terminal BAG domain, which mediates binding with the ATPase domain of HSC70, resulted in significant downregulation of E2/ER-facilitated BMP-2-directed osteogenic differentiation of BMSCs. These studies demonstrate for the first time the significance of BAG-1-mediated protein-protein interactions, specifically, BAG-1-regulated activation of ER by HSC70, in modulation of E2-facilitated BMP-2-directed osteoblast development.

Highlights

Endochondral ossification, the process responsible for formation of majority of the bones that make up the skeleton, involves deposition of bone matrix by osteoblasts on intermediary cartilaginous primordia, which serve as scaffolds on which bones are subsequently built
bone marrow stromal cells (BMSCs) cultures of 14-week-old wild-type and Bag-1 heterozygous mice were utilised for analyses of cell proliferation, osteogenic differentiation and apoptosis
The present study has highlighted the significance of Bcl-2-associated athanogene-1 (BAG-1)-regulated activation of estrogen receptors (ERs) by HSC70 in the modulation of E2-facilitated BMP-2-stimulated osteogenic differentiation of murine BMSCs

Summary

Introduction

Endochondral ossification, the process responsible for formation of majority of the bones that make up the skeleton, involves deposition of bone matrix by osteoblasts on intermediary cartilaginous primordia, which serve as scaffolds on which bones are subsequently built. Embryonic lethality in Bag-1 null mice has limited the investigation of the role of BAG-1 in bone development. This is primarily because vascular invasion of the calcified hypertrophic cartilage, resulting in the recruitment of osteoclasts and osteoblasts for the gradual replacement of the cartilaginous matrix with bone, occurs between E14.5 and E15.513. BAG-1 interacts with a diverse array of molecular targets, namely the 70-kDa heat shock chaperone proteins (HSC70/HSP70), RAF-1 kinase, components of the ubiquitylation/proteasome machinery and nuclear hormone receptors (NHRs), to regulate gene transcription and molecular signalling crucial for cell proliferation, differentiation and apoptosis[15]. A very small region comprising of 8 amino acid residues in helix 2 of the BAG domain has been shown to be vital for binding of BAG-1 to HSC7020

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Conclusion