INTEGRIN-EXTRACELLULAR MATRIX INTERACTIONS AND OSTEOBLAST DIFFERENTIATION

该提案的总体目标是了解信号的作用 细胞与细胞外基质 (ECM) 相互作用的结果 成骨细胞的分化。 大量数据支持这一观点 成骨细胞谱系源自多能间充质干 也具有形成其他结缔组织细胞能力的细胞 类型。 最近建立了几种多能细胞系 可以在体外分化成具有成骨细胞的细胞 特征，给予适当的刺激。 最近的 表征的大鼠 C26 成骨细胞系表达水平非常低 成骨细胞分化的早期标志物，例如碱性 磷酸酶，并在汇合处形成肌管。 然而，C26 细胞可以 被刺激表现出分化的成骨细胞表型 通过在 I 型胶原凝胶中培养或接触 BMP-2（一种 TGF-β 样物质） 生长因子。 根据这些数据，要检验的假设是 由整合素成员介导的特定细胞-ECM 相互作用 ECM 受体家族，在促进成骨细胞形成中发挥关键作用 差异化。 可能发生这种情况的一种机制预测 ECM 配体通过其整合素受体调节 TGF-β 的表达 β样生长因子。 或者整合素-ECM 相互作用和 TGF-β样生长因子可能发挥协同或协同作用 在成骨细胞分化中。 目前，人们对此知之甚少 临界表达的特定细胞表面 ECM 受体的身份 OB 分化的各个阶段，或者关于它们的目标 矩阵。 此外，这种细胞-ECM相互作用的机制 可能向间充质前体发出信号，使其沿着 成骨细胞途径和维持稳定的成骨细胞表型是 不太了解。 本提案旨在增进了解 这些重要领域。 具体来说，我们将： 1）表征 C26 细胞对选定 ECM 成分的成骨反应， 优化 ECM 诱导的 C26 细胞成骨细胞分化的目标； 2）检验ECM与特定物质之间相互作用的假设 成骨细胞分化需要整合素受体； 3） 确定特定的整合素-ECM 相互作用是否调节 成骨细胞过程中TGF-β样生长因子的表达 差异化； 4) 确定是否选定TGF-β成员 超家族和 ECM 成分协同作用，促进 C26细胞的成骨细胞分化； 5）检验假设 特征性成骨细胞 ECM 的积累和稳定 涉及基质金属蛋白酶及其抑制剂的调节 特定的整合素-ECM 相互作用。 更加深入地了解 成骨细胞分化的调节机制应该导致 开发更有效的材料来恢复 由于衰老、关节炎、手术、创伤导致的骨骼缺陷 和先天性缺陷。

The overall goal of this proposal is to understand the role of signals resulting from cell-extracellular matrix (ECM) interactions in the differentiation of osteoblasts. Considerable data support the idea that the osteoblast lineage is derived from a multipotential mesenchymal stem cell that also has the capacity to form other connective tissue cell types. Several multipotential cell lines have recently been established that can differentiate in vitro into cells with osteoblastic characteristics, given the appropriate stimulus. The recently characterized rat C26 osteoblastic cell line expresses very low levels of early markers of osteoblastic differentiation, such as alkaline phosphatase, and forms myotubes at confluence. However, C26 cells can be stimulated to exhibit a differentiated osteoblastic phenotype either by culture in type I collagen gels or exposure to BMP-2, a TGF-beta-like growth factor. Based on these data, the hypothesis to be tested is the specific cell-ECM interactions, mediated by members of the integrin family of ECM receptors, play a critical role in promoting osteoblastic differentiation. One mechanism by which this might occur predicts that ECM ligands, via their integrin receptors, regulate expression of TGF- beta-like growth factors. Alternatively integrin-ECM interactions and TGF-beta-like growth factors might play a cooperative or synergistic role in osteoblastic differentiation. At present, little is known about the identities of specific cell surface ECM receptors expressed at critical stages in the differentiation of OB, or about their targets in the matrix. In addition, the mechanisms by which such cell-ECM interactions might signal mesenchymal precursors to differentiate along the osteoblastic pathway and to maintain a stable osteoblastic phenotype are poorly understood. This proposal is designed to increase understanding of these important areas. Specifically, we will: 1) Characterize the osteogenic response of C26 cells to selected ECM constituents, with the goal of optimizing ECM-induced osteoblastic differentiation of C26 cells; 2) test the hypothesis that interactions between ECM and specific integrin receptors are required for osteoblastic differentiation; 3) determine whether particular integrin-ECM interactions regulate expression of TGF-beta-like growth factors during osteoblastic differentiation; 4) determine whether selected members of the TGF-beta superfamily and ECM constituents act synergistically to promote osteoblastic differentiation of C26 cells; 5) test the hypothesis that accumulation and stabilization of the characteristic osteoblastic ECM involves regulation of matrix-metalloproteinases and their inhibitors by specific integrin-ECM interactions. Greater understanding of the mechanisms by which osteoblast differentiation is regulated should lead to development of more effective materials for the restoration of skeletal deficiencies resulting from aging, arthritis, surgery, trauma and congenital defects.