Extracellular Matrix Regulation of Fibroblast Viability

描述（由申请人提供）：IPF是一种致命疾病，其特征是成纤维细胞在肺泡壁和肺泡腔内进行性积累，随后在肺泡腔内沉积胶原。在正常的肺修复过程中，成纤维细胞被细胞凋亡消除，从而恢复正常的解剖形态。相反，IPF的特征是成纤维细胞在纤维化灶中异常持续存在，在那里它们沉积的胶原扭曲了正常的肺结构，导致器官功能障碍。确定在富含胶原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成纤维细胞在IPF中的异常持久性。我们发现B1整合素pi -3-激酶/Akt活性通路调节胶原基质中肺成纤维细胞的存活。当成纤维细胞通过B1整合素附着于胶原蛋白时，Akt被磷酸化，提供生存信号。然而，在胶原基质收缩的反应中，Akt发生去磷酸化，成纤维细胞发生凋亡。强制激活B1整合素生存途径可使成纤维细胞免于凋亡。在本修订提案中，我们将评估与B1整合素相关的整合素亚基的身份，以确定哪种整合素负责调节Akt活性和胶原基质中成纤维细胞的活力。此外，我们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PTEN磷酸酶活性的增加导致Akt去磷酸化。我们的研究表明，PTEN活性增加的机制涉及shp1介导的细胞骨架分解。重要的是，我们提出的新发现表明，IPF成纤维细胞具有独特的表型，其特征是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整合素通过胶原基质中pi -3激酶/Akt信号通路调节肺成纤维细胞活力，涉及激活Akt和抑制Akt活性的FAK和ILK。2)在胶原收缩的反应中，shpl介导的细胞骨架分解上调PTEN活性，抑制B1整合素活力途径。3) IPF成纤维细胞中B1整合素活力通路和PTEN活性的疾病特异性改变是其在胶原基质中生存改变的基础。为了解决我们的假设，我们将：目标1。鉴定与B1整合素相关的整合素a亚基调控Akt活力信号。目的2探讨胶原收缩促进PTEN活化、Akt去磷酸化和成纤维细胞凋亡的分子机制。研究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。探讨富胶原微环境中IPF成纤维细胞中整合素活力信号和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.