Centre for Human Development, Stem Cells and Regeneration, Institute of Developmental Sciences, University of Southampton, Southampton SO16 6YD, United Kingdom.
Cancer Research UK Centre Cancer Sciences Unit, Somers Building, University of Southampton, Southampton SO16 6YD, United Kingdom.
Sci Rep. 2016 Sep 16;6:33504. doi: 10.1038/srep33504.
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.
B 细胞淋巴瘤 2 相关抗凋亡基因 1（BAG-1）在成骨细胞系细胞中表达；然而，Bag-1 基因敲除小鼠的早期胚胎致死限制了对 Bag-1 在成骨细胞发育中功能的研究。在本研究中，骨形态发生蛋白 2/BMP-2 指导的骨髓基质细胞（BMSCs）成骨分化中，Bag-1（+/）（杂合子）雌性小鼠的分化显著降低。在补充 BMP-2 的 Bag-1（+/）BMSCs 培养物中，与成骨分化、骨基质形成和矿化有关的关键基因的表达水平显著降低，而抑制 BMP-2 指导的成骨细胞生成的基因则显著上调。17-β-雌二醇（E2）增强了野生型和 Bag-1（+/）小鼠 BMSCs 对 BMP-2 的反应性，并促进了 BMSCs 对 BMP-2 的强有力的成骨分化。BAG-1 可以通过调节功能性雌激素受体（ER）的建立来调节细胞对 E2 的反应，这一点至关重要，通过与热休克蛋白（HSC70/HSP70）相互作用。小分子化学抑制剂硫黄素-S 抑制 BAG-1 与 HSC70 的结合，以及源自 C 端 BAG 结构域的短肽，该肽介导与 HSC70 的 ATP 酶结构域的结合，导致 E2/ER 促进的 BMP-2 指导的 BMSCs 成骨分化显著下调。这些研究首次证明了 BAG-1 介导的蛋白-蛋白相互作用的重要性，特别是 BAG-1 调节 HSC70 激活 ER，在调节 E2 促进的 BMP-2 指导的成骨细胞发育中的作用。
