Immunochemical characterization of three components of the hemidesmosome and their expression in cultured epithelial cells.

Klatte, David H.; Kurpakus, Michelle A.; Grelling, Kent A.; Jones, Jonathan

doi:10.1083/jcb.109.6.3377

Cited by 134 publications

(95 citation statements)

References 48 publications

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“…These extracts and immunoprecipitates were separated by SDS-PAGE, transferred to nitrocellulose, and processed for immunoblotting as described previously (35)(36)(37). Immunoreactive antibodies against DG (8D5), AMPK, plectin, phospho-ERK1/2, phospho-JNK, or phospho-ACC (a downstream target of activated AMPK) were used and detected by enhanced chemiluminescence (Thermo Scientific, Rockford, IL).…”

Section: Methodsmentioning

confidence: 99%

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

Takawira¹,

Budinger

Hopkinson³

et al. 2011

Journal of Biological Chemistry

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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...

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Section: Methodsmentioning

confidence: 99%

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

Takawira¹,

Budinger

Hopkinson³

et al. 2011

Journal of Biological Chemistry

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“…Matrix proteins and integrins were separated on 7.5-12% SDS-polyacrylamide gels following standard procedures (21). Gels were stained or separated proteins were transferred to nitrocellulose, which was subsequently processed for Western blotting as previously described (21)(22)(23).…”

Section: Methodsmentioning

confidence: 99%

Complex interactions between the laminin α4 subunit and integrins regulate endothelial cell behavior in vitro and angiogenesis in vivo

Gonzalez

Gonzales

Herron

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

114

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The ␣4 laminin subunit is a component of the basement membrane of blood vessels where it codistributes with the integrins ␣v␤3, ␣3␤1, and ␣6␤1. An antibody against the G domain (residues 919-1207; G 919 -1207 ) of the ␣4 laminin subunit inhibits angiogenesis in a mouse-human chimeric model, indicating the functional importance of this domain. Additional support for the latter derives from the ability of recombinant G 919 -1207 to support endothelial cell adhesion. In particular, endothelial cell adhesion to G 919 -1207 is half-maximal at 1.4 nM, whereas residues 919-1018 and 1016 -1207 of the G domain are poor cellular ligands. Function blocking antibodies against integrins ␣v␤3 and ␤1 and a combination of antibodies against ␣3 and ␣6 integrin subunits inhibit endothelial cell attachment to G 919 -1207 . Moreover, both ␣v␤3 and ␣3␤1 integrin bind with high affinity to G 919 -1207 . Together, our studies demonstrate that the G domain of laminin ␣4 chain is a specific, high affinity ligand for the ␣v␤3 and ␣3␤1 integrin heterodimers and that these integrins, together with ␣6␤1, function cooperatively to mediate endothelial cell-␣4 laminin interaction and hence blood vessel development. We propose a model based on these data that reconcile apparent discrepancies in the recent literature with regard to the role of the ␣v␤3 integrin in angiogenesis. matrix ͉ matrix receptor ͉ blood vessels

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“…12, January 2001for immunoblotting as previously described (Laemmli 1970;Zackroff et al, 1984;Harlow and Lane, 1988;Klatte et al, 1989).…”

Section: Matrix Interaction Of Endothelial Cellsmentioning

confidence: 99%

Structure and Function of a Vimentin-associated Matrix Adhesion in Endothelial Cells

Gonzales

Weksler

Tsuruta

et al. 2001

MBoC

148

134

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The ␣4 laminin subunit is a component of endothelial cell basement membranes. An antibody (2A3) against the ␣4 laminin G domain stains focal contact-like structures in transformed and primary microvascular endothelial cells (TrHBMECs and HMVECs, respectively), provided the latter cells are activated with growth factors. The 2A3 antibody staining colocalizes with that generated by ␣v and ␤3 integrin antibodies and, consistent with this localization, TrHBMECs and HMVECs adhere to the ␣4 laminin subunit G domain in an ␣v␤3-integrin-dependent manner. The ␣v␤3 integrin/2A3 antibody positively stained focal contacts are recognized by vinculin antibodies as well as by antibodies against plectin. Unusually, vimentin intermediate filaments, in addition to microfilament bundles, interact with many of the ␣v␤3 integrin-positive focal contacts. We have investigated the function of ␣4-laminin and ␣v␤3-integrin, which are at the core of these focal contacts, in cultured endothelial cells. Antibodies against these proteins inhibit branching morphogenesis of TrHBMECs and HMVECs in vitro, as well as their ability to repopulate in vitro wounds. Thus, we have characterized an endothelial cell matrix adhesion, which shows complex cytoskeletal interactions and whose assembly is regulated by growth factors. Our data indicate that this adhesion structure may play a role in angiogenesis.

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Immunochemical characterization of three components of the hemidesmosome and their expression in cultured epithelial cells.

Cited by 134 publications

References 48 publications

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

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

Complex interactions between the laminin α4 subunit and integrins regulate endothelial cell behavior in vitro and angiogenesis in vivo

Structure and Function of a Vimentin-associated Matrix Adhesion in Endothelial Cells

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