BAG-1 interacts with diverse proteins including heat shock proteins (HSC70/ HSP70) and nuclear hormone receptors to modulate cell proliferation, differentiation and apoptosis. We have shown that bone marrow stromal cells (BMSCs) of Bag-1+/− female mice display reduced BMP-2-directed osteogenic differentiation potential. Estradiol binds to estrogen receptors (ERs) and is functionally involved in BMP-directed osteogenic differentiation of osteoprogenitor cells by upregulating Bmpr2 transcription and Smad 1/5/8 signalling. BAG-1 enhances estradiol-dependent transcription and HSC70 is a likely mediator of BAG-1 function, since BAG-1 binds HSC70 and regulates refolding/ activation of ERs by HSC70 when transitioning from ligand-bound to ligand-free conformations. thioflavin-S inhibits BAG-1:HSC70 binding and serves as a small-molecule chemical inhibitor to investigate the functional significance of BAG-1-mediated protein interactions.
The present study analysed differences in expression of 84 genes crucial for skeletal development between BMSCs of WT and Bag-1+/− female mice following BMP-2-directed osteogenic differentiation, and investigated the effect of inhibition of BAG-1-mediated protein interactions by thioflavin-S on BMP-2-directed osteogenic differentiation of WT BMSCs in presence of 17-β-estradiol.
In response to BMP-2, 30 genes were differentially expressed between BMSCs of WT and Bag-1+/− female mice. Notably, genes crucial for osteogenic differentiation (Bmp-2, Bmp-4, Bmp-7, Pdgfa, Vegfa, Osterix/Sp-7), matrix biosynthesis (Col1a1, Col5a1, Serpinh-1) and mineralisation (Alp, Osteopontin, Osteocalcin, Phex, Sclerostin) were expressed at significantly lower levels in Bag-1+/− BMSC cultures. 17-β-estradiol enhanced BMP-2-induced osteogenic gene expression by upregulating Bmpr2 expression in WT BMSCs, which expressed both ERs. Addition of thioflavin-S to BMSC cultures supplemented with BMP-2 and 17-β-estradiol resulted in significant downregulation of Bmpr2 and osteogenic gene expression. No differences in DNA content/ cell number between the two culture conditions confirmed that thioflavin-S was not cytotoxic.
Thus, BAG-1 has an important role in osteoblastogenesis and inhibition of BAG-1 function by thioflavin-S reduces osteogenic differentiation of BMSCs in response to 17-β-estradiol and BMP-2.
17 May 2014 - 20 May 2014