Hemidesmosomes modulate force generation via focal adhesions.

Hemidesmosomes are specialized cell-matrix adhesion structures that are associated with the keratin cytoskeleton. Although the adhesion function of hemidesmosomes has been extensively studied, their role in mechanosignaling and transduction remains largely unexplored. Here, we show that keratinocytes lacking hemidesmosomal integrin α6ß4 exhibit increased focal adhesion formation, cell spreading, and traction-force generation. Moreover, disruption of the interaction between α6ß4 and intermediate filaments or laminin-332 results in similar phenotypical changes. We further demonstrate that integrin α6ß4 regulates the activity of the mechanosensitive transcriptional regulator YAP through inhibition of Rho-ROCK-MLC- and FAK-PI3K-dependent signaling pathways. Additionally, increased tension caused by impaired hemidesmosome assembly leads to a redistribution of integrin αVß5 from clathrin lattices to focal adhesions. Our results reveal a novel role for hemidesmosomes as regulators of cellular mechanical forces and establish the existence of a mechanical coupling between adhesion complexes.

Integrin α6ß4 Recognition of a Linear Motif of Bullous Pemphigoid Antigen BP230 Controls Its Recruitment to Hemidesmosomes.

Mechanical stability of epithelia requires firm attachment to the basement membrane via hemidesmosomes. Dysfunction of hemidesmosomal proteins causes severe skin-blistering diseases. Two plakins, plectin and BP230 (BPAG1e), link the integrin α6ß4 to intermediate filaments in epidermal hemidesmosomes. Here, we show that a linear sequence within the isoform-specific N-terminal region of BP230 binds to the third and fourth FnIII domains of ß4. The crystal structure of the complex and mutagenesis analysis revealed that BP230 binds between the two domains of ß4. BP230 induces closing of the two FnIII domains that are locked in place by an interdomain ionic clasp required for binding. Disruption of BP230-ß4 binding prevents recruitment of BP230 to hemidesmosomes in human keratinocytes, revealing a key role of this interaction for hemidesmosome assembly. Phosphomimetic substitutions in ß4 and BP230 destabilize the complex. Thus, our study provides insights into the architecture of hemidesmosomes and potential mechanisms of regulation.

CCAR-1 affects hemidesmosome biogenesis by regulating unc-52/perlecan alternative splicing in the C. elegans epidermis.

Hemidesmosomes are epithelial-specific attachment structures that maintain tissue integrity and resist tension. Despite their importance, how hemidesmosomes are regulated at the post-transcriptional level is poorly understood. Caenorhabditiselegans hemidesmosomes (CeHDs) have a similar structure and composition to their mammalian counterparts, making C. elegans an ideal model for studying hemidesmosomes. Here, we focus on the transcription regulator CCAR-1, identified in a previous genetic screen searching for enhancers of mutations in the conserved hemidesmosome component VAB-10A (known as plectin in mammals). Loss of CCAR-1 function in a vab-10(e698) background results in CeHD disruption and muscle detachment from the epidermis. CCAR-1 regulates CeHD biogenesis, not by controlling the transcription of CeHD-related genes, but by affecting the alternative splicing of unc-52 (known as perlecan or HSPG2 in mammals), the predicted basement extracellular matrix (ECM) ligand of CeHDs. CCAR-1 physically interacts with HRP-2 (hnRNPR in mammals), a splicing factor known to mediate unc-52 alternative splicing to control the proportions of different UNC-52 isoforms and stabilize CeHDs. Our discovery underlines the importance of post-transcriptional regulation in hemidesmosome reorganization. It also uncovers previously unappreciated roles of CCAR-1 in alternative splicing and hemidesmosome biogenesis, shedding new light on the mechanisms through which mammalian CCAR1 functions in tumorigenesis.

Hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity in oral squamous cell carcinoma derived cells.

BPAG1e and Plectin are hemidesmosomal linker proteins which anchor intermediate filament proteins to the cell surface through ß4 integrin. Recent reports indicate that these proteins play a role in various cellular processes apart from their known anchoring function. However, the available literature is inconsistent. Further, the previous study from our laboratory suggested that Keratin8/18 pair promotes cell motility and tumor progression by deregulating ß4 integrin signaling in oral squamous cell carcinoma (OSCC) derived cells. Based on these findings, we hypothesized that linker proteins may have a role in neoplastic progression of OSCC. Downregulation of hemidesmosomal linker proteins in OSCC derived cells resulted in reduced cell migration accompanied by alterations in actin organization. Further, decreased MMP9 activity led to reduced cell invasion in linker proteins knockdown cells. Moreover, loss of these proteins resulted in reduced tumorigenic potential. SWATH analysis demonstrated upregulation of N-Myc downstream regulated gene 1 (NDRG1) in linker proteins downregulated cells as compared to vector control cells. Further, the defects in phenotype upon linker proteins ablation were rescued upon loss of NDRG1 in linker proteins knockdown background. These data together indicate that hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity possibly through NDRG1 in OSCC derived cells.

Hemidesmosome integrity protects the colon against colitis and colorectal cancer.

OBJECTIVE: Epidemiological and clinical data indicate that patients suffering from IBD with long-standing colitis display a higher risk to develop colorectal high-grade dysplasia. Whereas carcinoma invasion and metastasis rely on basement membrane (BM) disruption, experimental evidence is lacking regarding the potential contribution of epithelial cell/BM anchorage on inflammation onset and subsequent neoplastic transformation of inflammatory lesions. Herein, we analyse the role of the α6ß4 integrin receptor found in hemidesmosomes that attach intestinal epithelial cells (IECs) to the laminin-containing BM. DESIGN: We developed new mouse models inducing IEC-specific ablation of α6 integrin either during development (α6ΔIEC) or in adults (α6ΔIEC-TAM). RESULTS: Strikingly, all α6ΔIEC mutant mice spontaneously developed long-standing colitis, which degenerated overtime into infiltrating adenocarcinoma. The sequence of events leading to disease onset entails hemidesmosome disruption, BM detachment, IL-18 overproduction by IECs, hyperplasia and enhanced intestinal permeability. Likewise, IEC-specific ablation of α6 integrin induced in adult mice (α6ΔIEC-TAM) resulted in fully penetrant colitis and tumour progression. Whereas broad-spectrum antibiotic treatment lowered tissue pathology and IL-1ß secretion from infiltrating myeloid cells, it failed to reduce Th1 and Th17 response. Interestingly, while the initial intestinal inflammation occurred independently of the adaptive immune system, tumourigenesis required B and T lymphocyte activation. CONCLUSIONS: We provide for the first time evidence that loss of IECs/BM interactions triggered by hemidesmosome disruption initiates the development of inflammatory lesions that progress into high-grade dysplasia and carcinoma. Colorectal neoplasia in our mouse models resemble that seen in patients with IBD, making them highly attractive for discovering more efficient therapies.

The molecular architecture of hemidesmosomes, as revealed with super-resolution microscopy.

Hemidesmosomes have been extensively studied with immunofluorescence microscopy, but owing to its limited resolution, the precise organization of hemidesmosomes remains poorly understood. We studied hemidesmosome organization in cultured keratinocytes with two- and three-color super-resolution microscopy. We observed that, in the cell periphery, nascent hemidesmosomes are associated with individual keratin filaments and that ß4 integrin (also known as ITGB4) is distributed along, rather than under, keratin filaments. By applying innovative methods to quantify molecular distances, we demonstrate that the hemidesmosomal plaque protein plectin interacts simultaneously and asymmetrically with ß4 integrin and keratin. Furthermore, we show that BP180 (BPAG2, also known as collagen XVII) and BP230 (BPAG1e, an epithelial splice variant of dystonin) are characteristically arranged within hemidesmosomes with BP180 surrounding a central core of BP230 molecules. In skin cross-sections, hemidesmosomes of variable sizes could be distinguished with BP230 and plectin occupying a position in between ß4 integrin and BP180, and the intermediate filament system. In conclusion, our data provide a detailed view of the molecular architecture of hemidesmosomes in cultured keratinocytes and skin.

The calcium/calcineurin pathway promotes hemidesmosome stability through inhibition of ß4 integrin phosphorylation.

Cell migration depends on cells being able to create and disassemble adhesive contacts. Hemidesmosomes are multiprotein structures that attach epithelia to basal lamina and disassemble during migration and carcinoma invasion. Phosphorylation of the ß4 integrin, a hemidesmosome component, induces disassembly. Although kinases involved in ß4 phosphorylation have been identified, little is known about phosphatases countering kinase action. Here we report that calcineurin, a serine-threonine protein phosphatase, regulates ß4 phosphorylation. Calcineurin inhibitor cyclosporin A (CsA) and calcineurin-siRNA increase ß4 phosphorylation, induce hemidesmosome disassembly, and increase migration in HaCat keratinocytes, suggesting that calcineurin negatively regulates ß4 phosphorylation. We found no direct dephosphorylation of ß4 by calcineurin or association between ß4 and calcineurin, suggesting indirect regulation of ß4 phosphorylation. We therefore assessed calcineurin influence on MAPK and PKC, known to phosphorylate ß4. CsA increased MAPK activity, whereas MAPK inhibitors reduced CsA-induced ß4 phosphorylation, suggesting that calcineurin restricts ß4 phosphorylation by MAPK. Calcineurin is activated by calcium. Increased [Ca(2+)](i) reduces ß4 phosphorylation and stabilizes hemidesmosomes, effects that are reversed by CsA, indicating that calcineurin mediates calcium effects on ß4. However, MAPK activation is increased when [Ca(2+)](i) is increased, suggesting that calcineurin activates an additional mechanism that counteracts MAPK-induced ß4 phosphorylation. Interestingly, in some squamous cell carcinoma cells, which have reduced hemidesmosomes and increased ß4 phosphorylation, an increase in [Ca(2+)](i) using thapsigargin, bradykinin, or acetylcholine can increase hemidesmosomes and reduce ß4 phosphorylation in a calcineurin-dependent manner. These findings have implications in calcineurin-inhibitor induced carcinoma, a complication of immunosuppressive therapy.

Targeted proteolysis of plectin isoform 1a accounts for hemidesmosome dysfunction in mice mimicking the dominant skin blistering disease EBS-Ogna.

Autosomal recessive mutations in the cytolinker protein plectin account for the multisystem disorders epidermolysis bullosa simplex (EBS) associated with muscular dystrophy (EBS-MD), pyloric atresia (EBS-PA), and congenital myasthenia (EBS-CMS). In contrast, a dominant missense mutation leads to the disease EBS-Ogna, manifesting exclusively as skin fragility. We have exploited this trait to study the molecular basis of hemidesmosome failure in EBS-Ogna and to reveal the contribution of plectin to hemidesmosome homeostasis. We generated EBS-Ogna knock-in mice mimicking the human phenotype and show that blistering reflects insufficient protein levels of the hemidesmosome-associated plectin isoform 1a. We found that plectin 1a, in contrast to plectin 1c, the major isoform expressed in epidermal keratinocytes, is proteolytically degraded, supporting the notion that degradation of hemidesmosome-anchored plectin is spatially controlled. Using recombinant proteins, we show that the mutation renders plectin's 190-nm-long coiled-coil rod domain more vulnerable to cleavage by calpains and other proteases activated in the epidermis but not in skeletal muscle. Accordingly, treatment of cultured EBS-Ogna keratinocytes as well as of EBS-Ogna mouse skin with calpain inhibitors resulted in increased plectin 1a protein expression levels. Moreover, we report that plectin's rod domain forms dimeric structures that can further associate laterally into remarkably stable (paracrystalline) polymers. We propose focal self-association of plectin molecules as a novel mechanism contributing to hemidesmosome homeostasis and stabilization.

Dual targets for mouse mast cell protease-4 in mediating tissue damage in experimental bullous pemphigoid.

Mouse mast cell protease-4 (mMCP-4) has been linked to autoimmune and inflammatory diseases, although the exact mechanisms underlying its role in these pathological conditions remain unclear. Here, we have found that mMCP-4 is critical in a mouse model of the autoimmune skin blistering disease bullous pemphigoid (BP). Mice lacking mMCP-4 were resistant to experimental BP. Complement activation, mast cell (MC) degranulation, and the early phase of neutrophil (PMN) recruitment occurred comparably in mMCP-4(-/-) and WT mice. However, without mMCP-4, activation of matrix metalloproteinase (MMP)-9 was impaired in cultured mMCP-4(-/-) MCs and in the skin of pathogenic IgG-injected mMCP-4(-/-) mice. MMP-9 activation was not fully restored by local reconstitution with WT or mMCP-4(-/-) PMNs. Local reconstitution with mMCP-4(+/+) MCs, but not with mMCP-4(-/-) MCs, restored blistering, MMP-9 activation, and PMN recruitment in mMCP-4(-/-) mice. mMCP-4 also degraded the hemidesmosomal transmembrane protein BP180 both in the skin and in vitro. These results demonstrate that mMCP-4 plays two different roles in the pathogenesis of experimental BP, by both activating MMP-9 and by cleaving BP180, leading to injury of the hemidesmosomes and extracellular matrix of the basement membrane zone.

Interaction of nectin-like molecule 2 with integrin alpha6beta4 and inhibition of disassembly of integrin alpha6beta4 from hemidesmosomes.

In normal epithelial cells, integrin α(6)ß(4) is abundantly expressed and forms hemidesmosomes, which is a cellular structure that mediates cell-extracellular matrix binding. In many types of cancer cells, integrin α(6)ß(4) is up-regulated, laminin is cleaved, and hemidesmosomes are disrupted, eventually causing an enhancement of cancer cell movement and facilitation of their invasion. We previously showed that the immunoglobulin-like cell adhesion molecule Necl-2 (Nectin-like molecule 2), known as a tumor suppressor, inhibits cancer cell movement by suppressing the ErbB3/ErbB2 signaling. We show here that Necl-2 interacts in cis with integrin α(6)ß(4). The binding of Necl-2 with integrin ß(4) was mediated by its extracellular region. In human colorectal adenocarcinoma Caco-2 cells, integrin α(6)ß(4) was localized at hemidesmosomes. Small interfering RNA-mediated suppression of Necl-2 expression enhanced the phorbol ester-induced disruption of the integrin α(6)ß(4) complex at hemidesmosomes, whereas expression of Necl-2 suppressed the disruption of this structure. These results indicate that tumor-suppressive functions of Necl-2 are mediated by the stabilization of the hemidesmosome structure in addition to the inhibition of the ErbB3/ErbB2 signaling.

Interaction of syndecan and alpha6beta4 integrin cytoplasmic domains: regulation of ErbB2-mediated integrin activation.

The alpha6beta4 integrin is a laminin 332 (LN332) receptor central to the formation of hemidesmosomes in epithelial layers. However, the integrin becomes phosphorylated by keratinocytes responding to epidermal growth factor in skin wounds or by squamous cell carcinomas that overexpress/hyperactivate the tyrosine kinase ErbB2, epidermal growth factor receptor, or c-Met. We show here that the beta4-dependent signaling in A431 human squamous carcinoma cells is dependent on the syndecan family of matrix receptors. Yeast two-hybrid analysis identifies an interaction within the distal third (amino acids 1473-1752) of the beta4 cytoplasmic domain and the conserved C2 region of the syndecan cytoplasmic domain. Via its C2 region, Sdc1 forms a complex with the alpha6beta4 integrin along with the receptor tyrosine kinase ErbB2 and the cytoplasmic kinase Fyn in A431 cells. Engagement of LN332 or clustering of the alpha6beta4 integrin with integrin-specific antibodies causes phosphorylation of ErbB2, Fyn, and the beta4 subunit as well as activation of phosphatidylinositol 3-kinase and Akt and their assimilation into this complex. This leads to phosphatidylinositol 3-kinase-dependent cell spreading and Akt-dependent protection from apoptosis. This is disrupted by RNA interference silencing of Sdc1 but can be rescued by mouse Sdc1 or Sdc4 but not by syndecan mutants lacking their C-terminal C2 region. This disruption does not prevent the phosphorylation of ErbB2 or Fyn but blocks the Fyn-mediated phosphorylation of the beta4 tail. We propose that syndecans engage the distal region of the beta4 cytoplasmic domain and bring it to the plasma membrane, where it can be acted upon by Src family kinases.

Lgl2 and E-cadherin act antagonistically to regulate hemidesmosome formation during epidermal development in zebrafish.

The integrity and homeostasis of the vertebrate epidermis depend on various cellular junctions. How these junctions are assembled during development and how their number is regulated remain largely unclear. Here, we address these issues by analysing the function of Lgl2, E-cadherin and atypical Protein kinase C (aPKC) in the formation of hemidesmosomes in the developing basal epidermis of zebrafish larvae. Previously, we have shown that a mutation in lgl2 (penner) prevents the formation of hemidesmosomes. Here we show that Lgl2 function is essential for mediating the targeting of Integrin alpha 6 (Itga6), a hemidesmosomal component, to the plasma membrane of basal epidermal cells. In addition, we show that whereas aPKClambda seems dispensable for the localisation of Itga6 during hemidesmosome formation, knockdown of E-cadherin function leads to an Lgl2-dependent increase in the localisation of Itga6. Thus, Lgl2 and E-cadherin act antagonistically to control the localisation of Itga6 during the formation of hemidesmosomes in the developing epidermis.

Target proteins in inherited and acquired blistering skin disorders.

Maintenance of an intact epidermis depends on secure adhesion between adjacent keratinocytes, and between basal keratinocytes and the underlying epidermal basement membrane. The major adhesion units that achieve this are the hemidesmosomes and desmosomes, but when these structures are disrupted, e.g., by gene mutations or autoantibodies, the resilience of the epidermis is lost and blisters develop. Recently, there have been considerable advances in our knowledge of the proteins and glycoproteins that contribute to maintaining keratinocyte adhesion via hemidesmosomes and desmosomes, as well as new insights into the molecular pathogenesis of several inherited and autoimmune blistering skin diseases. These new basic scientific data are clinically relevant, helping to improve patient management and to provide a rationale for developing better and more specific treatments for patients with inherited or acquired blistering skin diseases. In addition, there have also been improvements in our understanding of the organization and assembly of these adhesion structures, and their involvement in signalling pathways, intricately linked to skin development, wound healing and tumour invasion. This review provides an update on the structure and organization of hemidesmosomes and desmosomes, and on the molecular pathology of their various components that result in bullous skin diseases.

Epidermolysis bullosa. I. Molecular genetics of the junctional and hemidesmosomal variants.

INTRODUCTION: Epidermolysis bullosa (EB), a group of autosomal heritable blistering diseases, is characterised by extensive phenotypic variability with considerable morbidity and mortality. EB is classified into distinct subtypes depending on the location of blistering within the cutaneous dermoepidermal basement membrane zone. Ten genes are known to harbour mutations in the major types of EB, and the level of expression of these genes within the cutaneous basement membrane zone and in extracutaneous tissues, as well as the types and combinations of the mutations, explain in general terms the phenotypic variability. METHODS: The DebRA Molecular Diagnostics Laboratory, established in 1996 and supported in part by the patient advocacy organisation DebRA of America, has analysed over 1000 families with different forms of EB. RESULTS: In total, 265 cases were submitted with the preliminary diagnosis of junctional or hemidesmosomal forms of EB. We found 393 mutant alleles in seven different genes, with 173 of the mutations being distinct and 71 previously unpublished. DISCUSSION: These findings attest to the clinical and molecular heterogeneity of the junctional and hemidesmosomal subtypes of EB. The results also reveal exceptions to the general rules on genotype-phenotype correlations, unusual phenotypes, and surprising genetics. Collectively, mutation analysis in different forms of EB provides the basis for improved classification with prognostic implications and for prenatal and preimplantation diagnosis in families at risk for recurrence of EB.

Downregulation of hemidesmosomal proteins in nasopharyngeal carcinoma cells.

Hemidesmosome (HD) is a transmembrane complex that mediates attachment of epithelial cells to the basement membrane. Abnormal expression of HD components has been reported in several types of human cancers and is believed to play a role in tumor invasion and metastasis. Using differential gene display, we have identified downregulation of BPAG1 expression in nasopharyngeal carcinoma cells. BPAG1 is a major component of hemidesmosome. In the present study, we have extended our work to investigate the expression pattern of other components in the HD complex, namely, BPAG2, ITGalpha6 and ITGbeta4 in three distinct biological groups of nasopharyngeal epithelial cells: (a) non-malignant nasopharyngeal epithelial cells established from primary culture of nasopharyngeal explants, (b) non-malignant nasopharyngeal epithelial cells immortalized by viral oncogenes, SV40 or HPV16E6E7, and (c) nasopharyngeal carcinoma (NPC) cells. Both non-malignant primary cultured nasopharyngeal epithelial cells and immortalized nasopharyngeal epithelial cell lines expressed all the HD components examined, although the immortalized cells expressed a lower level of HD components compared with the non-malignant nasopharyngeal cells established from primary culture. In contrast, downregulation of HD components is commonly observed in nasopharyngeal carcinoma cells. Loss of HD expression in NPC may be associated with the undifferentiated properties of NPC cells and may have prognostic significance.