Integrin Regulation of IGF-1 Responsiveness in Bone During Mechanical Loading

描述（由申请人提供）：大量证据支持IGF-I信号传导在介导骨骼对负荷的反应中的作用。骨细胞和成熟的成骨细胞是机械载荷最初传递成新骨形成信号的可能候选者。机械负荷后这些细胞中IGF-I的产生增加，并且这种IGF-I可能是产生新成骨细胞的信号。此外，机械负荷增强骨骼对IGF-I的反应，正如骨骼卸载导致骨形成和骨祖细胞增殖对IGF-I的抵抗一样。尽管IGF-IR水平和IGF-I与其受体的结合是正常的，但这种在体内或体外对IGF-I无应答的卸载是由于IGF-I未能激活IGF-I受体（IGF-IR）。通过体内骨重负荷或体外成骨细胞重负荷，可以逆转骨骼去负荷引起的对IGF-I的抵抗。整合素在这一过程中起着重要作用。骨骼卸载减少整合素的表达，这种表达的抑制可以通过与IGF-I反应性恢复相称的骨骼重新加载来恢复。免疫共沉淀和共聚焦研究证实了整合素1和3亚基与IGF-IR的直接结合，IGF-I增强了这种结合。在特定的整联蛋白底物上生长的细胞显示IGF-IR通过IGF-I和负载的增强的活化，以及IGF-I对β 3整联蛋白亚基的增强的活化。击倒要么是1或？3整合素亚单位阻断IGF-I激活其受体的能力，并通过负载伴随下游IGF-I信号传导的破坏来防止其激活。因此，IGF-I信号传导对于骨骼对骨的反应是重要的，并且该信号传导由整合素调节。在这个项目中，我们将测试的假设，骨合成代谢的反应，机械负荷需要整合素和IGF-I信号之间的协同作用。提出了三个目标：1。确定在机械负荷过程中整合素调节IGF-I信号的机制，以及成骨细胞成熟改变这种调节的程度。这些研究将通过脉动流体流（PFF）机械加载成骨细胞和骨细胞细胞系进行，分析整合素/IGF-IR复合物的组分和作用。2.确定骨骼对机械负荷的反应对IGF-I和IGF-IR的需求。这些研究将使用从骨祖细胞和成骨细胞/骨细胞中删除IGF-I和IGF-IR的小鼠进行，然后确定体内和体外对负荷的反应。3.确定骨骼对机械负荷的反应中对整合素亚基<$1和<$3的需求。这些研究将通过从骨祖细胞和成骨细胞/骨细胞中删除<$1和<$3整联蛋白，然后确定它们对负荷和IGF-I的反应来进行。因此，使用体内和体外方法，我们将建立整合素/IGF-I信号传导在骨骼对负荷的反应中的作用和重要性，这些结果对于我们理解骨骼固定期间骨丢失的各种原因（包括瘫痪和受伤或疾病后的长期固定）至关重要。 公共卫生相关性：骨骼卸载导致骨丢失，这部分是由于骨不能响应IGF-1的合成代谢作用。这种失败与整合素表达的丧失有关。本项目将评估整合素和IGF-1信号在骨骼对机械负荷的反应中的相互作用。

DESCRIPTION (provided by applicant): Substantial evidence supports the role for IGF-I signaling in mediating the skeletal response to load. Osteocytes and mature osteoblasts are the likely candidates by which mechanical load is initially transmitted into signals for new bone formation. IGF-I production is increased in these cells following mechanical load, and this IGF-I may be the signal for the production of new osteoblasts. Furthermore, mechanical load enhances the skeletal response to IGF-I just as skeletal unloading leads to resistance to IGF-I with respect to bone formation and osteoprogenitor proliferation. This failure to respond to IGF-I in vivo or in vitro with unloading is due to a failure of IGF-I to activate the IGF-I receptor (IGF-IR), although IGF-IR levels and binding of IGF-I to its receptor are normal. The resistance to IGF-I caused by skeletal unloading can be reversed by reloading the bones in vivo or the osteoblasts in vitro. Integrins play an important role in this process. Skeletal unloading reduces integrin expression, and this inhibition of expression can be restored by reloading the bones commensurate with restoration of IGF-I responsiveness. Co-immunoprecipitation and confocal studies demonstrate direct binding of ¿1 and ¿3 integrin subunits to the IGF-IR, binding which is enhanced by IGF-I. Cells grown on specific integrin substrates demonstrate enhanced activation of the IGF-IR by both IGF-I and loading, and enhanced IGF-I activation of the ¿3 integrin subunit. Knockdown of either the ¿1 or ¿3 integrin subunits in osteoblasts blocks the ability of IGF-I to activate its receptor and prevents its activation by loading concomitant with a disruption of downstream IGF-I signaling. Thus, IGF-I signaling is important for the skeletal response to bone, and this signaling is regulated by integrins. In this project we will test the hypothesis that the anabolic response of bone to mechanical load requires the synergistic interaction between integrin and IGF-I signaling. Three aims are proposed: 1. Determine the mechanism for regulation of IGF-I signaling by integrins during mechanical loading, and the extent to which the maturation of the osteoblast modifies this regulation. These studies will be done with osteoblast and osteocyte cell lines mechanically loaded by pulsatile fluid flow (PFF), analyzing the components and roles of the integrin/IGF-IR complex. 2. Determine the requirement for IGF-I and IGF-IR in the skeletal response to mechanical load. These studies will be done with mice in which IGF-I and IGF-IR are deleted from osteoprogenitors and osteoblasts/osteocytes then determining the response to load in vivo and in vitro. 3. Determine the requirement for the ¿1 and ¿3 integrin subunits in the skeletal response to mechanical load. These studies will be done by deleting ¿1 and ¿3 integrin from osteoprogenitors and osteoblasts/osteocytes then determining their response to load and to IGF-I. Thus using both in vivo and in vitro approaches we will establish the role and importance of integrin/IGF-I signaling in the skeletal response to load, results critical to our understanding of bone loss during skeletal immobilization from a variety of causes including paralysis and prolonged immobilization following injury or sickness. PUBLIC HEALTH RELEVANCE: Skeletal unloading results in the loss of bone which is due in part to failure of bone to respond to the anabolic actions of IGF-1. This failure is associated with loss of integrin expression. This project will evaluate the interaction between integrin and IGF-1 signaling in the skeletal response to mechanical load.