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

整合素α2β1 对破骨细胞在分化和骨溶解中的作用和氧化还原调节

• 批准号: 448288075
• 负责人: Professor Dr. Johannes Andreas Eble
• 金额: --
• 依托单位: Institut für Physiologische Chemie und Pathobiochemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/448288075?language=en
• 关键词: Role; redox; regulation; integrins; osteoclasts

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.

破骨细胞(OCs)是由单核细胞血液干细胞分化而来的，是维持骨稳态和骨重建所必需的。特定的细胞因子促进合胞体的形成和OCS与骨的细胞外基质(ECM)的黏附，以及细胞外溶酶体样陷窝的形成。它们由α2β1和αVβ3富含整合素的密封区密封，其中OCS牢固地附着在细胞外基质上。在分化状态依赖的方式下，OCs产生活性氧物种(ROS)，提示通过半胱氨酸对之间的硫醇开关，可能位于构象和活性决定铰链区域内，对黏附介导的整合素进行氧化还原调节。尽管骨细胞外基质中的胶原含量很高，但与αVβ3相比，胶原结合的α2β1整合素在OCS中的作用及其氧化还原调节作用在很大程度上尚不清楚。因此，我们的目标是(1)确定α2β1整合素中氧化还原调节的硫醇开关；(2)研究胶原依赖的α2β1和OSCAR介导的OC分化和骨溶解；(3)分析OCS形成ROS和α2β1介导的氧化还原调节其分化和骨溶解；(4)阐明ROS调控的整合素α2β1和OSCAR介导的信号转导事件。为了在分子水平上表征α2β1整合素胞外结构域中的硫醇开关，我们将鉴定氧化还原敏感的半胱氨酸，并将其突变为氧化还原不敏感的残基，然后与野生型进行蛋白质化学特征比较(结合和构象测试，力测量)。对于α2β1整合素的体外鉴定，在从ER-HoxB8细胞分化而来的OCs中，αV将被关闭或抑制。此外，小鼠ITGA2基因将分别被编码野生型和半胱氨酸α2β1突变体的同源人cDNA取代。α-2、β-1介导的与胶原和整合素特异的迷你胶原的黏附所致的OC分化将通过定量逆转录-聚合酶链式反应和流式细胞术进行监测。骨溶解将通过荧光显微镜(整合素和肌动蛋白细胞骨架在封闭区的定位)和陷窝的功能测试(荧光显微镜检测酸化、羟基磷灰石再吸收、胶原酶和产物)来测量。利用受体特异性抗体、迷你胶原蛋白和毒素，选择性地测试α2β1或OSCAR介导的细胞-胶原接触的效果。除了定量OCs产生的ROS外，表达氧化还原可调节或半胱氨酸突变的α2β1整合素的OCs将被过氧化氢处理，以分析α2β1整合素的氧化还原调节对OC分化和骨溶解的影响。通过α-2、β-1和OSCAR信号转导蛋白的磷酸化模式来分析胶原诱导的信号通路。了解α-2-β-1整合素的作用及其对OC分化和骨溶解的氧化还原调节作用有助于了解骨质疏松等溶骨性骨病的分子和细胞过程。