Bone Abstracts

The co-chaperone, Bcl-2-associated athanogene-1 (BAG-1), is expressed by chondrocytes and osteogenic cells, and interacts via heat shock chaperones (HSC70/HSP70) with diverse proteins such as nuclear hormone receptors to regulate cell proliferation, differentiation and apoptosis. Early embryonic lethality in Bag-1 null mice has limited the investigation of the function of BAG-1 in skeletal development. The present study aimed to elucidate the role of BAG-1 in skeletal development by using Bag-1 null and heterozygous mice.

Micromass cultures of limb bud mesenchymal cells of E11.5 Bag-1^−/− mice demonstrated significantly high number of mineralised cartilage nodules compared to micromass cultures of limb bud mesenchymal cells of Bag-1^+/− and wild-type mice. Significantly increased expression of hypertrophic genes, in combination with robust Alkaline phosphatase and Alizarin red staining of the cartilage nodules, indicated marked upregulation of chondrocyte hypertrophy and matrix mineralisation in the cartilage nodules generated by limb bud mesenchymal cells of Bag-1^−/− mice. Deletion of one functional Bag-1 allele significantly decreased the osteogenic differentiation potential of bone marrow stromal cells (BMSCs) of Bag-1^+/− female mice in response to BMP-2. Addition of 17-β-estradiol (E2) enhanced responsiveness of BMSCs of 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 assisting the establishment of functional estrogen receptors (ERs), crucially, via its interaction with HSC70/HSP70. The interaction between BAG-1 and HSC70 in BMSCs was inhibited by the small-molecule chemical inhibitor, Thioflavin-S, and the C-terminal BAG domain-derived short peptide, and resulted in significant downregulation of E2/ER-facilitated BMP-2-directed osteogenic differentiation of BMSCs.

Thus, by regulating terminal differentiation of chondrocytes and hypertrophic cartilage mineralisation, BAG-1 plays an important role in the transition from chondrogenesis to osteogenesis during endochondral ossification. The study has also demonstrated the significance of BAG-1-mediated protein-protein interactions, specifically, BAG-1-regulated activation of ER by HSC70, in the regulation of osteoblast development.