Abstract
Alveolar type II epithelial cells (ATII) are instrumental in early wound healing in response to lung injury, restoring epithelial integrity through spreading and migration. We previously reported in separate studies that focal adhesion kinase-1 (FAK) and the chemokine receptor CXCR4 promote epithelial repair mechanisms. However, potential interactions between these two pathways were not previously considered. In the present study, we found that wounding of rat ATII cells promoted increased association between FAK and CXCR4. In addition, protein phosphatase-5 (PP5) increased its association with this heteromeric complex, while apoptosis signal regulating kinase-1 (ASK1) dissociated from the complex. Cell migration following wounding was decreased when PP5 expression was decreased using shRNA, but migration was increased in ATII cells isolated from ASK1 knockout mice. Interactions between FAK and CXCR4 were increased upon depletion of ASK1 using shRNA in MLE-12 cells, but unaffected when PP5 was depleted. Furthermore, we found that wounded rat ATII cells exhibited decreased ASK1 phosphorylation at Serine-966, decreased serine phosphorylation of FAK, and decreased association of phosphorylated ASK1 with FAK. These changes in phosphorylation were dependent upon expression of PP5. These results demonstrate a unique molecular complex comprising CXCR4, FAK, ASK1, and PP5 in ATII cells during wound healing.
Highlights
Epithelial repair mechanisms are initiated immediately following lung injury and involve an acute inflammatory response, immune cell recruitment, and activation of the coagulation cascade
To determine whether CXCR4 interacts with focal adhesion kinase-1 (FAK) in Alveolar type II epithelial cells (ATII) cells, we performed immunoprecipitation (IP) studies in unwounded rat ATII cells and in cells 24 hr after multiple scratch wounds were applied to enrich the population of migrating cells
Using an approach in which we enriched the population of migrating cells in a multiple scratch wound model, we identified for the first time that CXCR4 and PP5 increased their interactions with FAK in migrating cells, while apoptosis signal regulating kinase-1 (ASK1) dissociated from this complex
Summary
Epithelial repair mechanisms are initiated immediately following lung injury and involve an acute inflammatory response, immune cell recruitment, and activation of the coagulation cascade (reviewed in ref. 1). We previously found that cell migration in a scratch wound model was dependent upon FAK interactions with c-jun N-terminal kinase (JNK) mediated via JNK-interacting protein-3 (JIP3)[17] Through these complexes, FAK promotes several elements of cell migration including membrane protrusion and focal adhesion turnover. Knockdown or inhibition of ASK1 has been shown to either promote[26] or diminish[27, 28] cell migration in tumor cells, but this has not previously been investigated in ATII cells Since these interactions may be dependent upon changes in phosphorylation of ASK1, we investigated the role of protein phosphatase-5 (PP5), a key regulator of ASK1 activity[29, 30]. We identified a molecular complex of FAK, CXCR4, PP5, and ASK1 that changed in composition in cells following wounding and that was dependent upon changes in phosphorylation of both FAK and ASK1
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