Role of IGF-I/integrin signaling in the periosteal response to load

 描述(申请人提供)：骨骼卸载导致骨形成停止和骨吸收的初始增加。伴随这些变化的是对胰岛素样生长因子1(IGF1)对骨骼的合成代谢作用的抵抗。另一方面，骨骼负荷增加了IGF1的产生，并增强了IGF1R的IGF1活性，这是其促进骨形成的机制的一部分。在寻找负荷诱导的IGF1信号调节机制的过程中，我们发现骨骼卸载与整合素的表达减少有关，特别是带有b1(IGTB1)和b3(ITGB3)亚基的整合素的表达减少。我们发现，在成骨细胞中，IGF1增加了ITGB3与IGF1R的结合，如果ITGB3下调，IGF1就不能再激活IGF1R。粘着斑激酶(FAK)和/或其相关家族成员蛋白酪氨酸激酶2β(PTK2B)在整合素和生长因子受体通路之间提供了联系，包括在ITGB3和IGF1R之间。IGF1R的IGF1激活导致FAK的磷酸化(假定激活)，而抑制FAK则阻止IGF1或LOAD激活IGF1R。为了直接在体内测试IGF1信号的作用，我们建立了在成熟成骨细胞中IGF1R缺失的小鼠，并检测了这只小鼠对骨骼卸载或重新加载是否有反应。缺乏IGF1R的小鼠在卸载到对照组的过程中表现出同样的骨形成减少，但在重新加载过程中未能增加骨形成。惊人的结果是， 然而，只有在骨膜骨形成中才发现这种对重新加载的反应失败；骨内膜和骨小梁形成的反应与对照组相当。这些结果将我们的注意力集中在骨膜上，在骨膜上，骨祖细胞不仅受到来自骨细胞和肌肉的负荷诱导的IGF1，而且还受到整合素配体Perostin的作用，我们假设这种结合将使其在机械负荷下最大限度地增殖、分化和形成新骨。在这个项目中，我们将验证这样的假设，即骨骼负荷刺激骨细胞和肌肉产生IGF1，并在骨膜成骨细胞(POP)中形成IGF1R/整合素复合体，这是IGF1介导的细胞对负荷的合成代谢反应所必需的。这将通过以下三个目标实现：目标1--确定体外骨膜细胞对负荷和/或IGF1反应形成的IGF1受体复合体的成分，并评估它们在促进这一反应中的作用；目标2--确定体内从POP中删除IGF1和ITGB3对它们调节骨骼对负荷的反应的能力的影响；目标3--确定IGF1的来源(S)--促进负荷诱导的骨膜形成，特别注意骨细胞和肌肉。我们将利用新的动物模型和最先进的技术来实现这些目标。结果将提供 对IGF1/整合素信号相互作用如何调节骨骼对机械负荷的反应有了新的理解，为进一步研究开辟了新的途径，并为防止固定和衰老造成的骨丢失提供了潜在的靶点。

 DESCRIPTION (provided by applicant): Skeletal unloading results in a cessation in bone formation and an initial increase in bone resorption. These changes are accompanied by resistance to the anabolic actions of insulin like growth factor 1 (IGF1) on bone. On the other hand skeletal loading increases IGF1 production, and enhances IGF1 activation of IGF1R as part of the mechanism by which it increases bone formation. In seeking a mechanism for the load induced regulation of IGF1 signaling we discovered that skeletal unloading was associated with a decrease in the expression of integrins, in particular integrins with b1 (IGTB1) and b3 (ITGB3) subunits. We discovered that in osteoblasts, IGF1 increased the binding of ITGB3 to IGF1R, and that if ITGB3 were downregulated, IGF1 could no longer activate IGF1R. Focal adhesion kinase (FAK) and/or its related family member protein tyrosine kinase 2 beta (PTK2B) provide a link between the integrin and growth factor receptor pathways including between ITGB3 and IGF1R. IGF1 activation of IGF1R results in phosphorylation (presumed activation) of FAK, whereas inhibition of FAK blocks activation of IGF1R either by IGF1 or by load. To test the role of IGF1 signaling directly in vivo we developed mice in which the IGF1R was deleted in mature osteoblasts and examined whether this mouse would respond to skeletal unloading or reloading. Mice lacking IGF1R showed an equivalent decrease in bone formation during unloading to controls, but failed to increase bone formation during reloading. The striking result, however, was that this failure to respond to reloading was found only in periosteal bone formation; endosteal and trabecular bone formation responded comparable to controls. These results focused our attention on the periosteum where the osteoprogenitors are exposed not only to load induced IGF1 emanating from both osteocytes and muscle, but to the integrin ligand periostin, a combination we hypothesize will maximize their proliferation, differentiation, and formation of new bone in response to mechanical load. In this project we will test the hypothesis that skeletal loading stimulates IGF1 production in osteocytes and muscle and formation of the IGF1R/integrin complex in periosteal osteoprogenitors (pOP) required for the IGF1 mediated anabolic response of these cells to load. This will be achieved in the following three aims: Aim1--Determine the components of the IGF1R complex that forms in response to load and/or IGF1 in periosteal cells in vitro, and assess their role in contributing to that response; Aim 2-- Determin the impact of deleting Igf1r and Itgb3 from pOP in vivo with respect to their ability to mediate th skeletal response to load; Aim 3--Determine the source(s) of IGF1 facilitating load induced periosteal bone formation with particular attention to osteocytes and muscle. We will be utilizing novel animal models and state of the art techniques to fulfill these aims. The results will provide new understanding of how IGF1/integrin signaling interactions regulate the skeletal response to mechanical load, open up new avenues for further investigation, and provide potential targets for preventing the bone loss of immobilization and aging.