Extracellular Matrix Regulation of Fibroblast Viability

描述（由申请人提供）：IPF是一种致命性疾病，其特征为肺泡壁和肺泡腔内成纤维细胞进行性积聚，随后胶原蛋白沉积在肺泡腔内。在正常肺修复期间，成纤维细胞通过细胞凋亡被消除，从而恢复正常的解剖模式。相比之下，IPF的特征在于纤维化病灶中成纤维细胞的异常持续存在，在纤维化病灶中成纤维细胞存款胶原蛋白，扭曲正常的肺结构，导致器官功能障碍。鉴定在富含胶原蛋白的环境中调节肺成纤维细胞活力的关键信号分子将有助于深入了解IPF中成纤维细胞的异常持久性。我们已经发现，B1整合素PI-3-激酶/Akt活力途径调节肺成纤维细胞在胶原基质中的存活。当成纤维细胞通过B1粘附到胶原蛋白上时，整合素Akt被磷酸化，提供存活信号。然而，响应于胶原基质收缩，Akt变得去磷酸化并且成纤维细胞经历凋亡。B1整联蛋白活性途径的强制激活可挽救成纤维细胞免于凋亡。在这个修订后的提案中，我们将评估与B1整联蛋白相关的整联蛋白a亚基的身份，以确定哪种整联蛋白负责调节胶原基质中Akt活性和成纤维细胞活力。此外，我们已经开发的新的初步数据表明，胶原基质收缩促进细胞凋亡的分子机制是通过增加PTEN磷酸酶活性导致Akt去磷酸化。我们的研究表明，PTEN活性增加的机制涉及SHP 1介导的细胞骨架解体。重要的是，我们提出了新的发现，表明IPF成纤维细胞具有独特的表型，其特征在于这种整合素活力信号通路的功能障碍。这些观察结果使我们假设：1）B1整联蛋白通过胶原基质中的PI-3-激酶/Akt信号传导途径调节肺成纤维细胞活力涉及激活Akt的FAK和ILK以及抑制Akt活性的PTEN磷酸酶。2)响应于胶原收缩，SHP 1介导的细胞骨架分解上调抑制B1整联蛋白活力途径的PTEN活性。3)IPF成纤维细胞中B1整联蛋白活力途径和PTEN活性的疾病特异性改变是其在胶原基质中存活改变的基础。为了解决我们的假设，我们将：目标1。鉴定在调节Akt活力信号中与B1整联蛋白相关的整联蛋白a亚基。目的2探讨胶原收缩促进PTEN激活、Akt去磷酸化及成纤维细胞凋亡的分子机制。检查PTEN在调节B1整合素/PI-3-激酶/Akt存活信号和成纤维细胞活力中的作用。目标3：探索富含胶原蛋白（纤维化）微环境中IPF成纤维细胞中整合素活力信号传导和PTEN活性的疾病特异性改变。

DESCRIPTION (provided by applicant): IPF is a deadly disease characterized by the progressive accumulation of fibroblasts within the alveolar wall and airspace with subsequent deposition of collagen in the airspace. During normal lung repair fibroblasts are eliminated by apoptosis permitting restoration of normal anatomic patterns. In contrast, IPF is characterized by the abnormal persistence of fibroblasts in fibrotic foci where they deposit collagen distorting normal lung architecture resulting in organ dysfunction. Identifying key signaling molecules that regulate lung fibroblast viability in a collagen-rich environment will provide insight into the abnormal persistence of fibroblasts in IPF. We have discovered that a B1 integrin PI-3-kinase/Akt viability pathway regulates lung fibroblast survival in collagen matrices. When fibroblasts attach to collagen via B1 integrin Akt is phosphorylated providing a survival signal. However, in response to collagen matrix contraction, Akt becomes dephosphorylated and fibroblasts undergo apoptosis. Enforced activation of the B1 integrin viability pathway rescues fibroblasts from apoptosis. In this revised proposal we will evaluate the identity of the integrin a subunit(s) associated with B1 integrin to determine which integrin is responsible for regulating Akt activity and fibroblast viability in collagen matrices. Furthermore, novel preliminary data we have developed suggest that the molecular mechanism by which collagen matrix contraction promotes apoptosis is by increases in PTEN phosphatase activity resulting in Akt dephosphorylation. Our studies suggest that the mechanism for the increase in PTEN activity involves SHP1-mediated cytoskeletal disassembly. Importantly, we present novel findings indicating that IPF fibroblasts have a distinct phenotype that is characterized by dysfunction of this integrin viability signaling pathway. These observations lead us to hypothesize that: 1) B1 integrin regulation of lung fibroblast viability through the PI-3-kinase/Akt signaling pathway in collagen matrices involves FAK and ILK which activate Akt and PTEN phosphatase which inhibits Akt activity. 2) In response to collagen contraction, SHPl-mediated cytoskeletal disassembly up-regulates PTEN activity inhibiting the B1 integrin viability pathway. 3) Disease specific alterations in the B1 integrin viability pathway and PTEN activity in IPF fibroblasts underlie their altered survival in collagen matrices. To address our hypotheses we will: Aim 1. Identify the integrin a subunit(s) associated with B1 integrin in regulating the Akt viability signal. Aim 2 Investigate the molecular mechanism by which collagen contraction promotes activation of PTEN, Akt dephosphorylation, and fibroblast apoptosis. Examine the role of PTEN in regulating the B1 integrin/PI-3-kinase/Akt survival signal and fibroblast viability. Aim 3. Explore disease-specific alterations in integrin viability signaling and PTEN activity in IPF fibroblasts in a collagen-rich (fibrotic) microenvironment.