Novel Roles of Plectin in the Mechanobiology of Cultured Alveolar Epithelial Cells

In cultured alveolar epithelial cells (cAECs) the membrane-anchored proteoglycan dystroglycan (DG) is a mechanoreceptor that transmits mechanical stretch forces to activate independently the extracellular signal-regulated kinases 1 and 2 (ERK1/2) and the adenosine 5'-monophosphate-activated protein kinase (AMPK) signaling cascades in a process called pathway bifurcation. We tested the hypothesis that the cytoskeleton-crosslinker plectin, known to bind both DG and AMPK in muscle cells, acts as a scaffold to regulate DG-mediated mechanical stimulation and pathway bifurcation. Using immunofluorescence and immunoprecipitation assays we demonstrate that plectin and DG form a complex in cAECs and that this complex interacts with ERK1/2 and AMPK. Plectin knockdown by adenoviral shRNA reduces DG interaction with AMPK but not with ERK1/2. Despite this, mechanoactivation of both signaling pathways is significantly attenuated in cAECs deficient in plectin. Thus, DG has the dual role of mechanical receptor and scaffold for ERK1/2, whereas plectin acts as a scaffold for AMPK signaling but is also required for DG-mediated ERK1/2 activation. From these data we conclude that the DG/plectin complex plays a central role in transmitting mechanical stress from the extracellular matrix (ECM) to the cytoplasm. Since the cAECs are not amenable to complex genetic manipulation, we assayed immortalized cell lines as substitute models for probing DG/plectin mechanosignaling complexes in detail. A fibroblast line (3T3) and a murine lung epithelial carcinoma line (MLE-12) were found to activate ERK1/2 on the ECM secreted by cAECs but not on their own matrix. However, this ERK1/2 mechanoactivation was independent of DG in both cell lines, suggesting that these cells are not suitable substitute model systems. Going back to the cAECs, we reasoned that the established role of plectin as a cytoskeletal crosslinker implies a possible mechanosignaling role for the cytoarchitecture. Disruption of cytoskeletal elements with drugs did not attenuate mechanoactivation of both ERK1/2 and AMPK. Additionally, analysis of plectin knockdown cells revealed reorganized keratin networks and disrupted focal adhesions in the perinuclear zone without obvious effects on adhesion, cell size, or cell shape. Thus, our results suggest that plectin has roles in mechanical signaling as well as in maintaining adhesion structures in a specific area of cAECs.