Osteocyte Interactome in mechanotransduction and bone osteogenesis - Project Summary Osteocytes, the most abundant bone cell type, serve as the primary mechanosensory cells, orchestrating bone remodeling by regulating bone formation and bone resorption from their embedded locations within the mineralized matrix. These osteocytes are interconnected through gap junction channels and communicate with the extracellular environments via hemichannels, both primarily formed by connexin (Cx) 43. Our previous studies have shown that impaired Cx43 hemichannels, and the integrins that regulate these channels, suppress osteogenic responses to mechanical loading, in part through the inhibition of prostaglandin E2 released via hemichannels. Preliminary data also indicate that the Piezo1 mechanosensitive ion channel regulates Cx43 hemichannel opening. Furthermore, osteocyte hemichannels may influence the senescence and differentiation of bone marrow mesenchymal stem progenitor cell (BM-MSPC). Together, these three proteins, Piezo1, integrins, and Cx43, appear to form a mechanosensitive interactome in osteocyte. To explore these mechanisms, we have developed two transgenic mouse models expressing dominant negative Cx43 mutants that selectively impair gap junctions or hemichannels in osteocytes, as well as osteocyte-specific integrin α5 conditional knockout mice. We are also generating an osteocyte-specific Piezo1 gene knockout mouse model. In addition, we have produced monoclonal antibodies that specifically inhibit or activate Cx43 hemichannels without affecting gap junctions. Notably, we have developed a hemichannel-activating antibody, Cx43(M2) antibody, which enhances osteocyte mechanosensitivity and protects bone loss in models of aging and disuse. The objective of this application is to elucidate the regulatory role of the osteocyte interactome and Cx43 HCs during mechanical loading, and to determine their functional impact on BM-MSPC differentiation into osteogenic commitment, and bone homeostasis, with the goal of developing targeted therapeutics for osteoporosis and bone loss. Our central hypothesis is that the activation of Cx43 hemichannels via Piezo1 and integrin signaling in osteocytes enhances BM-MSPC osteogenic differentiation and mediates the anabolic effects of mechanical loading on bone. A hemichannel-activating agent holds strong therapeutic potential for preventing or reversing osteoporosis and bone loss. Three specific aims are proposed: 1) To determine the mechanistic roles of the osteocytic interactome, Piezo1, integrin, and Cx43 HCs in bone anabolism and remodeling under mechanical loading. 2) To determine how the Piezo1, integrin α5β1, and Cx43 HC interactome in osteocytes regulates BM-MSPC cellular senescence, differentiation, and osteogenic commitment. 3) To Evaluate whether a Cx43-HC activating antibody, Cx43(M2), mitigates bone loss and enhances BM-MSPC osteogenesis in osteoporotic models. The proposed studies are expected to uncover novel mechanotransduction pathways critical for osteogenesis and mechanical responsiveness in bone and provide new therapeutic targets and candidates for treating bone diseases like osteoporosis and osteopenia.
