Role and redox-regulation of integrins α2β1 on osteoclasts in differentiation and osteolysis

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Osteoclasts (OCs) differentiating from monocytic blood stem cell are essential for bone homeostasis and remodeling. Specific cytokines promote syncytia formation and adhesion of OCs to the extracellular matrix (ECM) of the bone as well as the formation of extracellular lysosome-like lacunae. These are sealed off by an α2β1 and αVβ3 integrin-rich sealing zone, in which OCs adhere firmly to the ECM. In a differentiation state-dependent manner, OCs produce reactive oxygen species (ROS), suggesting redox-regulation of adhesion-mediating integrins via thiol-switches between cysteine pairs, likely located within the conformation-and activity-determining hinge region. Despite the high collagen content of bone ECM, the role of collagen-binding α2β1 integrin in OCs, in contrast to αVβ3, and its redox-regulatory effects are largely unknown.Our goals are therefore (1) to identify redox-regulated thiol switches in α2β1 integrin; (2) to characterize collagen-dependent α2β1- and OSCAR-mediated OC differentiation and osteolysis; (3) to analyze the formation of ROS by OCs and the α2β1-mediated redox regulation of their differentiation and osteolysis; and (4) to elucidate ROS-regulated integrin α2β1 and OSCAR-mediated signaling events.To characterize thiol switches in α2β1 integrin ectodomains at the molecular level, redox-sensitive cysteines will be identified and mutated to redox-insensitive residues and then characterized protein-chemically in comparison to the wild type (binding and conformational tests, force measurements). For in vitro characterization of α2β1 integrin, αV will be turned off or inhibited in OCs differentiated from ER-HoxB8 cells. In addition, the murine ITGA2 gene will be replaced by homologous human cDNAs encoding wild type and cysteine α2β1 mutants, respectively. OC differentiation due to its α2β1-mediated adhesion to collagen and integrin-specific mini-collagens will be monitored by qRT-PCR and flow cytometry. Osteolysis will be measured by fluorescence microscopy (localization of integrins and actin cytoskeleton in the sealing zone) and by functional tests of the lacunae (fluorescence microscopic detection of acidification, hydroxyapatite resorption, collagenolytic enzymes and products). With receptor-specific antibodies, mini-collagens and toxins, the effect of α2β1- or OSCAR-mediated cell-collagen contact is selectively tested. In addition to quantifying ROS production by OCs, OCs expressing redox-regulatable or cysteine-mutated α2β1 integrin will be treated with hydrogen peroxide to analyze the effects of redox regulation of α2β1 integrin on OC differentiation and osteolysis. Collagen-induced signaling pathways through α2β1 and OSCAR will be analyzed by the phosphorylation patterns of signal transduction proteins in OCs.Elucidating the role of α2β1 integrin and its redox regulatory effect on OC differentiation and osteolysis helps to understand the molecular and cellular processes in osteolytic bone diseases such as osteoporosis.