Regulation of the Type II Hemidesmosomal Plaque Assembly in Intestinal Epithelial Cells

Hemidesmosomes (HDs) are cellular junctions that anchor epithelial cells to the extracellular matrix (ECM) and are associated morphologically with the cytoskeleton. Hemidesmosomal molecular components include two proteins involved in linking intermediate filaments, HD1/plectin and BP230, and two transmembrane proteins, BP180 and the alpha6beta4 integrin, a laminin receptor. In cells lacking BP230 and BP180, HD1/plectin still associates with alpha6beta4 integrin, forming HD-like structures, called type II HDs. In the present study, we used an intestinal epithelial cell line that expresses HD1/plectin and the alpha6beta4 integrin to investigate the regulation of assembly of these proteins in type II HDs. These compounds were found to be clustered at sites of cell-ECM contact and their polarized localization was influenced by either cell confluency or extracellular matrix deposition. Conventional and immunoelectron microscopy showed that HD1/plectin and the beta4 integrin subunit are colocalized in an adhesion structure. Using cytoskeleton-disrupting drugs and confocal microscopy, we demonstrated that type II HDs are made up of numerous individual plaques whose assembly into a cluster requires actin filaments, but not microtubules.     

Human bronchial epithelial cells secrete laminin 5, express hemidesmosomal proteins, and assemble hemidesmosomes.

Epithelial cells attach to the basement membrane through adhesive contacts between the basal cells of the epithelium and the proteins of the extracellular matrix (ECM). The hemidesmosome (HD) is a specialized cell-ECM contact, that mediates the attachment of the epithelial cell basal surface to the ECM. In bronchial epithelial cells, the protein components that constitute the HD have not been demonstrated. Using immunohistochemical techniques, we determined that normal human bronchial epithelial (NHBE) cells express the HD cell surface integrin alpha6beta4 and produce laminin 5, the ECM protein associated with HDs. Furthermore, expression of the HD-associated structural proteins, bullous pemphigoid antigens 1 (BPAG 1) and 2 (BPAG 2), was demonstrated in NHBE cells by immunofluorescence microscopy and immunoblot analyses. In addition, we confirmed the presence of laminin 5 in the basement membrane (BM) of bronchial epithelial biopsy specimens and of BP230, BP180, and the alpha6beta4 integrin heterodimer at the site of bronchial epithelial cell-ECM interaction in vivo. Finally, using electron microscopy, we were able to demonstrate intact HDs in a glutaraldehyde-fixed NHBE cell monolayer. These findings suggest that bronchial epithelium forms HDs and that the laminin 5-alpha6beta4 integrin interaction may be important in stabilizing epithelial cell adhesion to the BM in the lung.     

Phosphorylation of a Novel Site on the β4 Integrin at the Trailing Edge of Migrating Cells Promotes Hemidesmosome Disassembly

Hemidesmosomes (HDs) are multiprotein structures that anchor epithelial cells to the basement membrane. HD components include the α6β4 integrin, plectin, and BPAGs (bullous pemphigoid antigens). HD disassembly in keratinocytes is necessary for cells to migrate and can be induced by EGF through β4 integrin phosphorylation. We have identified a novel phosphorylation site on the β4 integrin: S₁₄₂₄. Preventing phosphorylation by mutating S→A₁₄₂₄ results in increased incorporation of β4 into HDs and resistance to EGF-induced disassembly. In contrast, mutating S→D₁₄₂₄ (mimicking phosphorylation) partially mobilizes β4 from HDs and potentiates the disassembly effects of other phosphorylation sites. In contrast to previously described sites that are phosphorylated upon growth factor stimulation, S₁₄₂₄ already exhibits high constitutive phosphorylation, suggesting additional functions. Constitutive phosphorylation of S₁₄₂₄ is distinctively enriched at the trailing edge of migrating keratinocytes where HDs are disassembled. Although most of this S₁₄₂₄-phosphorylated β4 is found dissociated from HDs, a substantial amount can be associated with HDs near the cell margins, colocalizing with plectin but always excluding BPAGs, suggesting that phospho-S₁₄₂₄ might be a mechanism to dissociate β4 from BPAGs. S₁₄₂₄ phosphorylation is PKC dependent. These data suggest an important role for S₁₄₂₄ in the gradual disassembly of HDs induced by cell retraction.     

EGF-induced MAPK Signaling Inhibits Hemidesmosome Formation through Phosphorylation of the Integrin β4

Migration of keratinocytes requires a regulated and dynamic turnover of hemidesmosomes (HDs). We and others have previously identified three serine residues on the integrin β4 cytoplasmic domain that play a critical role in the regulation of HD disassembly. In this study we show that only two of these residues (Ser-1356 and Ser-1364) are phosphorylated in keratinocytes after stimulation with either PMA or EGF. Furthermore, in direct contrast to previous studies performed in vitro, we found that the PMA- and EGF-stimulated phosphorylation of β4 is not mediated by PKC, but by ERK1/2 and its downstream effector kinase p90RSK1/2. EGF-stimulated phosphorylation of β4 increased keratinocyte migration, and reduced the number of stable HDs. Furthermore, mutation of the two serines in β4 to phospho-mimicking aspartic acid decreased its interaction with the cytoskeletal linker protein plectin, as well as the strength of α6β4-mediated adhesion to laminin-332. During mitotic cell rounding, when the overall cell-substrate area is decreased and the number of HDs is reduced, β4 was only phosphorylated on Ser-1356 by a distinct, yet unidentified, kinase. Collectively, these data demonstrate an important role of β4 phosphorylation on residues Ser-1356 and Ser-1364 in the formation and/or stability of HDs.     

Hemidesmosome Formation Is Initiated by the β4 Integrin Subunit,Requires Complex Formation of β4 and HD1/Plectin,and Involves a Direct Interaction between β4 and the Bullous Pemphigoid Antigen 180

Hemidesmosomes (HDs) are stable anchoring structures that mediate the link between the intermediate filament cytoskeleton and the cell substratum. We investigated the contribution of various segments of the β4 integrin cytoplasmic domain in the formation of HDs in transient transfection studies using immortalized keratinocytes derived from an epidermolysis bullosa patient deficient in β4 expression. We found that the expression of wild-type β4 restored the ability of the β4-deficient cells to form HDs and that distinct domains in the NH₂- and COOH-terminal regions of the β4 cytoplasmic domain are required for the localization of HD1/plectin and the bullous pemphigoid antigens 180 (BP180) and 230 (BP230) in these HDs. The tyrosine activation motif located in the connecting segment (CS) of the β4 cytoplasmic domain was dispensable for HD formation, although it may be involved in the efficient localization of BP180. Using the yeast two-hybrid system, we could demonstrate a direct interaction between β4 and BP180 which involves sequences within the COOH-terminal part of the CS and the third fibronectin type III (FNIII) repeat. Immunoprecipitation studies using COS-7 cells transfected with cDNAs for α6 and β4 and a mutant BP180 which lacks the collagenous extracellular domain confirmed the interaction of β4 with BP180. Nevertheless, β4 mutants which contained the BP180-binding region, but lacked sequences required for the localization of HD1/plectin, failed to localize BP180 in HDs. Additional yeast two- hybrid assays indicated that the 85 COOH-terminal residues of β4 can interact with the first NH₂-terminal pair of FNIII repeats and the CS, suggesting that the cytoplasmic domain of β4 is folded back upon itself. Unfolding of the cytoplasmic domain may be part of a mechanism by which the interaction of β4 with other hemidesmosomal components, e.g., BP180, is regulated.     

Activated Rac1 Selectively Up-regulates the Expression of Integrin α6β4 and Induces Cell Adhesion and Membrane Ruffles of Nonadherent Colon Cancer Colo201 Cells

Functions of small GTPases in integrin expression were investigated when the interaction of nonadherent human colon carcinoma 201 cells with the extracellular matrix (ECM) was examined. By transfection of the constitutively active form of a small GTPase Rac1, Rac V12, adhesion of cells to the ECM increased with concomitant cell spreading and formation of membrane ruffles. Activated Cdc42 and Cdc42 V12, but not wild-type Rac1, Cdc42, or RhoA, also induced the adhesion and spreading of Colo201 cells. This adhesion is integrin β4 dependent since an antibody for integrin β4 inhibited the RacV12-dependent cell adhesion and numbers of adhesive cells on laminin-coated plates exceeded those on collagen- and fibronectin-coated plates. By immunofluorescence, in addition to clustering of integrin molecules, expression of integrin α6β4 on the cell surface of Rac V12- and Cdc42 V12-expressing cells was selectively up-regulated without an increase in biosynthesis of α6β4 integrin. Treatment of Rac V12-expressing cells with wortmannin or LY294002, specific inhibitors of phosphoinositide 3-OH kinase, decreased the up-regulated α6β4 and cell adhesion. In light of this evidence, we propose that the regulation of integrin α6β4 expression induced by Rac1 and Cdc42 may play an important role in cell adhesion and tumorigenesis of colon carcinoma cells.     

Advances and perspectives of the architecture of hemidesmosomes: Lessons from structural biology

Hemidesmosomes (HD) are adhesive protein complexes that mediate stable attachment of basal epithelial cells to the underlying basement membrane. The organization of HDs relies on a complex network of protein-protein interactions, in which integrin α6β4 and plectin play an essential role. Here we summarize the current knowledge of the structure of hemidesmosomal proteins, which includes the structures of the first and second fibronectin type III (FnIII) domains and the calx-β domain of the integrin β4 subunit, the actin binding domain of plectin, and two non-overlapping pairs of spectrin repeats of plectin and BPAG1e. Binding of plectin to the β4 subunit is critical for the formation and the stability of HDs. The recent 3D structure of the primary complex between the integrin β4 subunit and plectin has provided a first insight into the macromolecular recognition mechanisms responsible for HD assembly. Two missense mutations in β4 linked to non lethal forms of epidermolysis bullosa map on the plectin-binding surface. Finally, the formation of the β4-plectin complex induces conformational changes in β4 and plectin, suggesting that their interaction may be subject to allosteric regulation.     

Regulation of hemidesmosome disassembly by growth factor receptors

Hemidesmosomes (HDs) promote the stable adhesion of basal epithelial cells to the underlying basement membrane (BM). Critical for the mechanical stability of the HD is the interaction between integrin alpha6beta4 and plectin, which is destabilized when HD disassembly is required, for instance, to allow keratinocyte migration during wound healing. Growth factors such as epidermal growth factor (EGF) can trigger HD disassembly and induce phosphorylation of the beta4 intracellular domain. Whereas tyrosine phosphorylation appears to mediate cooperation with growth factor signaling pathways and invasion in carcinoma cells, serine phosphorylation seems the predominant mechanism for regulating HD destabilization. Here, we discuss recent advances that shed light on the residues involved, the identity of the kinases that phosphorylate them, and the interactions that become disrupted by these phosphorylations.     

Linking Integrin α6β4-based Cell Adhesion to the Intermediate Filament Cytoskeleton: Direct Interaction between the β4 Subunit and Plectin at Multiple Molecular Sites

Recent studies with patients suffering from epidermolysis bullosa simplex associated with muscular dystrophy and the targeted gene disruption in mice suggested that plectin, a versatile cytoskeletal linker and intermediate filament-binding protein, may play an essential role in hemidesmosome integrity and stabilization. To define plectin's interactions with hemidesmosomal proteins on the molecular level, we studied its interaction with the uniquely long cytoplasmic tail domain of the β₄ subunit of the basement membrane laminin receptor integrin α₆β₄ that has been implicated in connecting the transmembrane integrin complex with hemidesmosome-anchored cytokeratin filaments. In vitro binding and in vivo cotransfection assays, using recombinant mutant forms of both proteins, revealed their direct interaction via multiple molecular domains. Furthermore, we show in vitro self-interaction of integrin β₄ cytoplasmic domains, as well as disruption of intermediate filament network arrays and dislocation of hemidesmosome-associated endogenous plectin upon ectopic overexpression of this domain in PtK2 and/or 804G cells. The close association of plectin molecules with hemidesmosomal structures and their apparent random orientation was indicated by gold immunoelectron microscopy using domain-specific antibodies. Our data support a model in which plectin stabilizes hemidesmosomes, via directly interlinking integrin β₄ subunits and cytokeratin filaments.     

A Cell Signal Pathway Involving Laminin-5, α3β1 Integrin, and Mitogen-activated Protein Kinase Can Regulate Epithelial Cell Proliferation

Laminin-5 (LN5) is a matrix component of epithelial tissue basement membranes and plays an important role in the initiation and maintenance of epithelial cell anchorage to the underlying connective tissue. Here we show that two distinct LN5 function-inhibitory antibodies, both of which bind the globular domain of the α3 subunit, inhibit proliferation of epithelial cells. These same antibodies also induce a decrease in mitogen-activated protein kinase activity. Inhibition of proliferation by the function-perturbing LN5 antibodies is reversed upon removal of the antibodies and can be overcome by providing the antibody-treated cells with exogenous LN5 and rat tail collagen. Because epithelial cells use the integrin receptor α3β1 to interact with both LN5 and rat tail collagen, we next investigated the possibility that integrin α3β1 is involved in mediating the proliferative impact of LN5. Proliferation of human epithelial cells is significantly inhibited by a function-perturbing α3 integrin antibody. In addition, antibody activation of β1 integrin restores the proliferation of epithelial cells treated with LN5 function-perturbing antibodies. These data indicate that a complex comprising LN5 and α3β1 integrin is multifunctional and contributes not only to epithelial cell adhesion but also to the regulation of cell growth via a signaling pathway involving mitogen-activated protein kinase. We discuss our study in light of recent evidence that LN5 expression is up-regulated at the leading tips of tumors, where it may play a role in tumor cell proliferation.     

Laminin-based cell adhesion anchors microtubule plus ends to the epithelial cell basal cortex through LL5α/β

LL5β has been identified as a microtubule-anchoring factor that attaches EB1/CLIP-associating protein (CLASP)–bound microtubule plus ends to the cell cortex. In this study, we show that LL5β and its homologue LL5α (LL5s) colocalize with autocrine laminin-5 and its receptors, integrins α3β1 and α6β4, at the basal side of fully polarized epithelial sheets. Depletion of both laminin receptor integrins abolishes the cortical localization of LL5s, whereas LL5 depletion reduces the amount of integrin α3 at the basal cell cortex. Activation of integrin α3 is sufficient to initiate LL5 accumulation at the cell cortex. LL5s form a complex with the cytoplasmic tails of these integrins, but their interaction might be indirect. Analysis of the three-dimensional distribution of microtubule growth by visualizing EB1-GFP in epithelial sheets in combination with RNA interference reveals that LL5s are required to maintain the density of growing microtubules selectively at the basal cortex. These findings reveal that signaling from laminin–integrin associations attaches microtubule plus ends to the epithelial basal cell cortex.     

Hemidesmosome formation in vitro

Intact, viable sheets of adult rabbit corneal epithelium, 9 mm in diameter, were prepared by the Dispase II method (Gipson, I. K., and S. M. Grill, 1982, Invest. Ophthalmol. Vis. Sci. 23:269-273). The sheets, freed of the basal lamina, retained their desmosomes and stratified epithelial characteristics, but lacked hemidesmosomes (HD). Epithelial sheets were placed on fresh segments of corneal stroma with denuded basal laminae and incubated in serum-free media for 1, 3, 6, 18, or 24 h. Tissue was processed for electron microscopy, and the number of HD/micron membrane, the number of HDs with anchoring fibrils directly across the lamina densa from them, and the number of anchoring fibrils not associated with HDs were counted. After 6 h in culture, the number of newly formed HD was 82% of controls (normal rabbit corneas), and by 24 h the number had reached 95% of controls. At all time periods studied, 80-86% of HDs had anchoring fibrils directly across the lamina densa from them. Anchoring fibrils not associated with HDs decreased with culture time. These data indicate that the sites where anchoring fibrils insert into the lamina densa may be nucleation sites for new HD formation. Corneal epithelial sheets placed on two other ocular basal laminae, Descemet's membrane and lens capsule, had not formed HDs after 24 h in culture. These two laminae do not have anchoring fibrils associated with them. Rabbit epithelial sheets placed on the denuded epithelial basal lamina of rat and human corneas formed new HDs. Thus, at least in these mammalian species, HD formation may involve some of the same molecular components.     

Both type-I hemidesmosomes and adherens-type junctions contribute to the cell-substratum adhesion system in myoepithelial cells

Myoepithelial cells present in exocrine glands cause secretion from the glands by contraction. They have mixed characteristics with regard to cytoskeletal elements, containing both epithelial-type intermediate filaments and smooth muscle-type myofilaments. For further characterization, myoepithelial cells from bovine apocrine sweat glands and tracheal glands were here examined with special attention to the cell-substratum adhesion system. Immunofluorescence microscopy using a panel of antibodies against adherens-type junctional and hemidesmosomal proteins demonstrated two types of cell-substratum junctions in myoepithelial cells from both glands. Type-I hemidesmosomes (HDs) consisting of plectin, BP230, integrin alpha6beta4, and BP180 were thus observed as punctate arrays longitudinally arranged along myoepithelial cell surfaces, while adherens-type junctions were similarly evident as linear rib-like structures. Double-label immunofluoresence revealed the two junctions to be distributed in a mutually exclusive or independent manner. Electron microscopy further demonstrated that apocrine myoepithelial cells surround secretory epithelial cells completely, without any gaps, HDs being abundant along the basement membrane, but with no distinct structures in the inter-hemidesmosomal regions. Immunoelectron microscopy, however, revealed an interhemidesmosomal localization of vinculin, pointing to the existence of adherens-type junctions. Secretory epithelial cells in tracheal glands were found not to be completely covered with myoepithelial cells, so that more than half of them are directly attached to the basement membrane, where they form type II-HDs lacking BP230 and BP180, but no detectable adherens junctions, like epidermal basal cells and sebaceous gland cells. These observations demonstrate that, in addition to their cytoskeleton, myoepithelial cells have both epithelial- and smooth muscle-type cell-substratum adhesion structures, i.e. HDs and dense plaque-like adherens junctions.     

Positive role of IQGAP1, an effector of Rac1, in actin-meshwork formation at sites of cell-cell contact

The small guanosine triphosphatase Rac1 is activated by E-cadherin-mediated cell-cell adhesion and is required for the accumulation of actin filaments, E-cadherin, and β-catenin at sites of cell-cell contact. However, the modes of activation and action of Rac1 remain to be clarified. We here found that suppression of IQGAP1, an actin-binding protein and an effector of Rac1, by small interfering RNA apparently reduced the accumulation of actin filaments, E-cadherin, and β-catenin at sites of cell-cell contact in Madin-Darby canine kidney II epithelial cells under the conditions in which knockdown of Rac1 reduced them. Knockdown of Rac1 did not affect the localization of these junctional components in cells expressing a constitutively active IQGAP1 mutant defective in Rac1/Cdc42 binding. Knockdown of either Rac1 or IQGAP1 accelerated the 12-O-tetradecanoylphorbol-13-acetate-induced cell-cell dissociation. The basal Rac1 activity, which was maintained by E-cadherin-mediated cell-cell adhesion, was inhibited in the IQGAP1-knocked down cells, whereas the Rac1 activity was increased in the cells overexpressing IQGAP1. Together, these results indicate that Rac1 enhances the accumulation of actin filaments, E-cadherin, and β-catenin by acting on IQGAP1 and suggest that there exists a positive feedback loop comprised of “E-cadherin-mediated cell-cell adhesion→Rac1 activation→actin-meshwork formation by IQGAP1→increasing E-cadherin-mediated cell-cell adhesion.”     

A functional analysis of Wnt inducible signalling pathway protein ?1 (WISP-1/CCN4)

Wnt-1 inducible signalling pathway protein 1 (WISP-1/CCN4) is an extracellular matrix protein that belongs to the Cyr61 (cysteine-rich protein 61), CTGF (connective tissue growth factor) and NOV (CCN) family and plays a role in multiple cellular processes. No specific WISP-1 receptors have been identified but emerging evidence suggests WISP-1 mediates its downstream effects by binding to integrins. Here we describe a functional analysis of integrin receptor usage by WISP-1. Truncated WISP-1 proteins were produced using a baculovirus expression system. Full length WISP-1 and truncated proteins were evaluated for their ability to induce adhesion in A549 epithelial cells and β-catenin activation and CXCL3 secretion in fibroblasts (NRK49-F cells). Subsequent inhibition of these responses by neutralising integrin antibodies was evaluated. A549 cells demonstrated adhesion to full-length WISP-1 whilst truncated proteins containing VWC, TSP or CT domains also induced adhesion, with highest activity observed with proteins containing the C-terminal TSP and CT domains. Likewise the ability to induce β-catenin activation and CXCL3 secretion was retained in truncations containing C-terminal domains. Pre-treatment of A549s with either integrin αVβ5, αVβ3 or β1 neutralising antibodies partially inhibited full length WISP-1 induced adhesion whilst combining integrin αVβ5 and β1 antibodies increased the potency of this effect. Incubation of NRK49-F cells with integrin neutralising antibodies failed to effect β-catenin translocation or CXCL3 secretion. Analysis of natural WISP-1 derived from human lung tissue showed the native protein is a high order oligomer. Our data suggest that WISP-1 mediated adhesion of A549 cells is an integrin-driven event regulated by the C-terminal domains of the protein. Activation of β-catenin signalling and CXCL3 secretion also resides within the C-terminal domains of WISP-1 but are not regulated by integrins. The oligomeric nature of native WISP-1 may drive a high avidity interaction with these receptors in vivo.     

αvβ3 Integrin Boosts the Innate Immune Response Elicited in Epithelial Cells through Plasma Membrane and Endosomal Toll-Like Receptors

We report that αvβ3 integrin strongly affects the innate immune response in epithelial cells. αvβ3 integrin greatly increased the response elicited via plasma membrane Toll-like receptors (TLRs) by herpes simplex virus or bacterial ligands. The endosomal TLR3, not the cytosolic sensor interferon gamma-inducible protein 16 (IFI16), was also boosted by αvβ3 integrin. The boosting was exerted specifically by αvβ3 integrin but not by αvβ6 or αvβ8 integrin. Current and previous work indicates that integrin-TLR cooperation occurs in epithelial and monocytic cells. The TLR response should be considered an integrin-TLR response.     

Impact of Laminitis on the Canonical Wnt Signaling Pathway in Basal Epithelial Cells of the Equine Digital Laminae

The digital laminae is a two layer tissue that attaches the distal phalanx to the inner hoof wall, thus suspending the horse''s axial skeleton in the hoof capsule. This tissue fails at the epidermal:dermal junction in laminitic horses, causing crippling disease. Basal epithelial cells line the laminar epidermal:dermal junction, undergo physiological change in laminitic horses, and lose versican gene expression. Versican gene expression is purportedly under control of the canonical Wnt signaling pathway and is a trigger for mesenchymal-to-epithelial transition; thus, its repression in laminar epithelial cells of laminitic horses may be associated with suppression of the canonical Wnt signaling pathway and loss of the epithelial cell phenotype. In support of the former contention, we show, using laminae from healthy horses and horses with carbohydrate overload-induced laminitis, quantitative real-time polymerase chain reaction, Western blotting after sodium dodecylsulfate polyacrylamide gel electrophoresis, and immunofluorescent tissue staining, that positive and negative regulatory components of the canonical Wnt signaling pathway are expressed in laminar basal epithelial cells of healthy horses. Furthermore, expression of positive regulators is suppressed and negative regulators elevated in laminae of laminitic compared to healthy horses. We also show that versican gene expression in the epithelial cells correlates positively with that of β-catenin and T-cell Factor 4, consistent with regulation by the canonical Wnt signaling pathway. In addition, gene and protein expression of β-catenin correlates positively with that of integrin β4 and both are strongly suppressed in laminar basal epithelial cells of laminitic horses, which remain E-cadherin⁺/vimentin⁻, excluding mesenchymal transition as contributing to loss of the adherens junction and hemidesmosome components. We propose that suppression of the canonical Wnt signaling pathway, and accompanying reduced expression of β catenin and integrin β4 in laminar basal epithelial cells reduces cell:cell and cell:basement membrane attachment, thus, destabilizing the laminar epidermal:dermal junction.