Insight into the physiology of skeletal development – role of BCL-2-associated athanogene-1 (BAG-1)

Abstract

Endochondral ossification, the process by which majority of the skeleton is formed, relies on the precise development of cartilaginous templates of the future bones that serve as scaffolds for deposition of bone matrix. Disruption of the fine balance coordinating chondrocyte differentiation within the cartilaginous templates and the transition from chondrogenesis to osteogenesis at the vascular front often results in a heterogeneous group of congenital anomalies, referred to as osteochondral dysplasias, which are characterised by dwarfism, skeletal malformations and, in some cases, prenatal death. Moreover, dysregulation of early skeletal development is directly linked to diseases of the adult skeleton such as osteoporosis and osteoarthritis. Improved appreciation of skeletal development, therefore, will not only enhance our understanding of musculoskeletal diseases, but also augment the development of novel strategies for replacement/regeneration of skeletal tissues. The role of molecular chaperones and their co-chaperones in health and disease has attracted much attention in recent years. The co-chaperone, BCL-2-associated athanogene-1 (BAG-1), is a multifunctional protein, which by its ability to bind multiple partners, predominantly the heat shock chaperone proteins (HSC70/HSP70), can regulate gene transcription and signalling crucial for cell proliferation, differentiation and apoptosis. In prenatal murine development, highest expression of Bag-1 mRNA is detected in cartilaginous tissues. Both isoforms of BAG-1 (50 kDa and 32 kDa) are expressed ubiquitously in long bones of adult mice by chondrocytes and osteoblast-lineage cells. BAG-1 protects mammalian chondrocytes against apoptosis and plays an important role in the regulation of expression of chondrogenic markers. Interestingly, expression of BAG-1 is increased in human osteoarthritic cartilage, most likely in response to the dysregulation of homeostasis in osteoarthritic chondrocytes. Comprehensive characterisation of the role of BAG-1 and the significance of its interaction with HSC70 in the regulation of skeletal development would, therefore, assist in improved understanding of the molecular regulation of skeletogenesis. The present study aimed to elucidate the role of Bag-1 in endochondral ossification, using Bag-1 null (Bag-1-/-) and Bag-1 heterozygous (Bag-1+/-) mice, in conjunction with ex vivo models of chondrocyte and osteoblast development. Micromass cultures of embryonic limb bud cells of Bag-1-/- mice were characterised by significantly high number of mineralised cartilage nodules and increased expression of genes crucial for chondrocyte hypertrophy, mineralisation and maintenance of hypertrophic chondrocyte morphology. Bone marrow stromal cells (BMSCs) of skeletally mature Bag-1+/- female mice exhibited increased proliferation rate, decreased BMP-2-directed osteogenic differentiation and negligible apoptosis. Moreover, genes crucial for osteogenic differentiation, matrix biosynthesis, mineralisation and maintenance of the mature osteoblast phenotype were expressed at significantly lower levels in cultures of Bag-1+/- BMSCs supplemented with BMP-2, while the expression levels of genes with roles in inhibition of BMP-2-directed osteoblast differentiation were significantly upregulated. Since BAG-1, via its interaction with HSC70, can assist in the establishment of functional estrogen receptors (ERs) and modulate cellular responses to estrogen (E2), the significance of BAG-1-regulated modulation of ER function by HSC70 in E2- facilitated, BMP-directed osteogenic differentiation of BMSCs was investigated. Inhibition of the binding of BAG-1 to HSC70 by the small-molecule chemical inhibitor, Thioflavin-S, and a short peptide containing the 8 amino acid binding core of helix 2 of the BAG domain, resulted in significant downregulation of E2/ER-facilitated, BMP-directed osteogenic response of BMSCs of both wild-type and Bag-1+/- mice. Thus, the present study has demonstrated an important role for BAG-1 in skeletal development, specifically in the terminal differentiation of chondrocytes, mineralisation of the hypertrophic cartilage and osteogenic differentiation of BMSCs. Furthermore, downregulation of E2/ER-facilitated, BMP-directed osteogenic differentiation of BMSCs due to disruption of binding between BAG-1 and HSC70 highlighted the significance of BAG-1-mediated protein-protein interactions in osteogenic differentiation of BMSCs.

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ORCID for Rahul Tare: orcid.org/0000-0001-8274-8837

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