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.
