A Dystroglycan/Plectin Scaffold Mediates Mechanical Pathway Bifurcation in Lung Epithelial Cells

Abstract

In alveolar epithelial cells (AECs), the membrane-anchored proteoglycan dystroglycan (DG) is a mechanoreceptor that transmits mechanical stretch forces to activate independently the 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 cross-linker plectin, known to bind both DG and AMPK in muscle cells, acts as a scaffold to regulate DG-mediated mechanical stimulation and pathway bifurcation. We demonstrate that plectin and DG form a complex in AECs and that this complex interacts with ERK1/2 and AMPK. Plectin knockdown reduces DG interaction with AMPK but not with ERK1/2. Despite this, mechanoactivation of both signaling pathways is significantly attenuated in AECs 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. We conclude that the DG-plectin complex plays a central role in transmitting mechanical stress from the extracellular matrix to the cytoplasm.In the lung, alveolar epithelial cells (AECs) 3 not only mediate the exchange of gases between the circulation system of the host and its external environment but are also highly responsive to a number of mechanical forces (1). These forces include deformation and strain that occur during lung expansion and relaxation from breathing movements, and shear stress during the distension of the airway walls and blood vessels from bulk air and blood flow (2). Over the past few years, there has been increasing interest in identifying molecules that "sense" physical forces on the cell surface and in defining the signaling pathways activated by mechanical stimulation (1, 3-6). Stretch-activated ion channels, integrins, cell-cell adhesion molecules, cytoskeleton elements, and the extracellular matrix (ECM) have all been implicated in transducing mechanical signals in a manner that is detectable as chemical signals (e.g. protein phosphorylation) in the cytoplasm of the stimulated cell (1, 7).We are interested in investigating the molecular underpinnings of cellular responses to physical force in rat AECs. In particular, we have previously tested the hypothesis that matrix molecules secreted by cultured AECs and transmembrane matrix receptors on the substratum surface of these cells are crucial molecular links in the process of "converting" a mechanical stimulus in the form of cyclic stretching into a cytoplasmic signal (8). Specifically, in prior studies we demonstrated that rat AECs assemble an ECM rich in fibers composed of the ␣3, ␤1, and ␥1 subunits of laminin (laminin-311), complexed with perlecan and nidogen (8). This complex transmits mechanosignals in the form of stretch, via the matrix receptor dystroglycan (DG), to activate ERK1/2 (8). Moreover, we have also shown that DG is required for stretch-induced activation of the adenosine 5Ј-monophosphate-activated protein kinase (AMPK) sign...

Networking and anchoring through plectin: a key to IF functionality and mechanotransduction

“…Conversely, there is evidence that cage-bound plectin acts as a platform for proteins shuttling between the nucleus and the cytoplasm, such as FUS [13]. Similarly, plectin has been shown to recruit and regulate the activity of the non-receptor tyrosine kinase Fer [14] in fibroblasts, and of AMP-activated protein kinase AMPK in differentiated myofibers [15] and alveolar epithelial cells [16 ]. In compartmentalizing IF networks, plectin is likely to affect many of the signaling functions ascribed to them [17,18].…”

Plectin-containing, centrally-localized focal adhesions exert traction forces in primary lung epithelial cells

“…Additionally, there is precedence for FAs being important for IF organization (Bhattacharya et al, 2009;Burgstaller et al, 2010;Windoffer et al, 2006). Thus, we next determined if the plectincontaining cFAs influence the distribution of the IF keratin cytoskeleton of rAEC (Eisenberg et al, 2011;Takawira et al, 2011). In rAEC exhibiting rings of cFAs and pFAs, keratin bundles are restricted to the central cell area and closely associate with cFAs (Fig.…”

“…For certain experiments, following reattachment, rat cells were infected with adenoviruses encoding short hairpin RNAs (shRNAs) against rat DG, plectin and human lamin A/C (control) as previously described (Jones et al, 2005;Takawira et al, 2011). AEC were infected with the adenoviruses on day 3, and maximal knockdown of both proteins was obtained 48 hours thereafter, so knock-down assays were conducted on day 5 (about 72 hours after replating) as done previously .…”

“…The latter was created using a paxillin/GFP-N plasmid (obtained from Dr Christopher Turner at State University of New York) following standard protocols (Jones et al, 2005;Takawira et al, 2011).…”

Signalling through mechanical inputs – a coordinated process

“…3A9). Interestingly, VAB-10A, the core component of C. elegans hemidesmosomes, is homologous to vertebrate plectin, which, following its binding to the dystroglycan complex in alveolar epithelial cells, can relay mechanical inputs into the cell to activate protein kinases (Takawira et al, 2011). Thus, the involvement of cytoskeletal crosslinkers such as VAB-10A and plectin in mechanotransduction seems to be a common feature, although the cellular outputs vary depending on the cell types.…”