Molecular consequences of deletion of the cytoplasmic domain of bullous pemphigoid 180 in a patient with predominant features of epidermolysis bullosa simplex.
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Bullous pemphigoid antigen 2 (BP180; COL17A1) collagen gene mutations typically result in nonlethal junctional epidermolysis bullosa. We have identified a patient, who had phenotypic features of mainly epidermolysis bullosa simplex and evidence for both intraepidermal and junctional blister formation. Mutation analysis disclosed compound heterozygous mutations in the COL17A1 gene, leading to deletion of Ile-18 to Asn-407 from the intracellular domain of BP180, BP180 Delta 18-407. To gain insight into the mechanisms underlying the phenotype, we have investigated the functional consequences of this truncation in BP180. The results demonstrate that: (1) in cultured keratinocytes of the patient, the assembly of hemidesmosomes, and their linkage with intermediate filaments are impaired; (2) BP180 Delta 18-407 is not capable of binding to the hemidesmosomal components BP230, plectin, and the beta 4 subunit of the alpha 6 beta 4 integrin in yeast two-hybrid assays; (3) BP180 Delta 18-407 is recruited into hemidesmosome-like structures in both normal and BP180-deficient transfected keratinocytes when ectopically expressed, suggesting that the extracellular domain of BP180 Delta 18-407 determines its topogenic fate; and, finally (4) the proteolytic shedding of the extracellular domain of BP180 Delta 18-407 is not impaired in transfected COS-7 cells. Collectively, the data demonstrate that the truncation of the intracellular domain of BP180 impairs the organization of hemidesmosomes, affecting both the mechanical stability of basal keratinocytes and dermoepidermal cohesion. (+info)
Characterization of the anti-BP180 autoantibody reactivity profile and epitope mapping in bullous pemphigoid patients.
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Bullous pemphigoid is a subepidermal bullous disease of skin and mucosae associated with autoantibodies to BP180. To characterize the humoral response to BP180, we generated a random BP180 epitope library displayed on lambda bacteriophage. After validation of the library by epitope mapping of three BP180-specific monoclonal antibodies, 15 novel or known BP180 epitopes were identified using 10 bullous pemphigoid serum samples. Fifty-seven bullous pemphigoid and 81 control sera were then assayed against the selected epitopes. Thirty-one out of 57 (54%) bullous pemphigoid sera reacted with at least an additional antigenic site other than the NC16A, within the extracellular (37%) and intracellular (28%) domains of BP180. In addition, the reactivity with extracellular epitopes of BP180 contained within the residue stretches 508-541 and 1331-1404 appeared to be related to the presence of both skin and mucosal involvement. Finally, a preliminary analysis of the epitope pattern in the disease course indicated that bullous pemphigoid patients exhibit a specific reactivity pattern, and that binding to intracellular epitopes of BP180, in addition to NC16A, may be detectable at an early clinical stage. Our findings provide novel insights into the pathophysiology of bullous pemphigoid and show the potential of the utilized approach as a tool for a rapid diagnosis of bullous pemphigoid patients and their management. (+info)
An experimental study on the features of peri-implant epithelium: immunohistochemical and electron-microscopic observations.
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The purpose of this study was to investigate the immunohistochemical and the ultrastructural features of the implant circumference epithelium of the beagle dog using various types of antibodies. The peri-implant epithelium was at an acute-angle from the gingival epithelium and was arranged in parallel to the implant surface. With immunohistochemical staining, the peri-implant epithelium was strongly positive for KL-1, and weakly positive for CK4, CK8 and CK19. These positive reactions for keratins and also for PCNA and BM-1 were similar to those seen in the oral mucosa. In the peri-implant epithelium, a plentitude of microvilli were observed at the periphery of cells at the implant sites, and bacteria were observed between the implant and the peri-implant epithelium without the formation of half desmosomes. There were many lipid-like vacuoles or lysosome-like granules. The intercellular space was wider than the junctional epithelium, and random migrations of large numbers of neutrophils could be seen. Taken together, the peri-implant epithelium is similar to that seen in the oral mucosa, and it is structurally different from the junctional epithelium. (+info)
Protein kinase C-alpha phosphorylation of specific serines in the connecting segment of the beta 4 integrin regulates the dynamics of type II hemidesmosomes.
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Although the regulation of hemidesmosome dynamics during processes such as epithelial migration, wound healing, and carcinoma invasion is important, the mechanisms involved are poorly understood. The integrin alpha 6 beta 4 is an essential component of the hemidesmosome and a target of such regulation. Epidermal growth factor (EGF) can induce hemidesmosome disassembly by a mechanism that involves serine phosphorylation of the beta 4 integrin subunit. Using a combination of biochemical and mutational analyses, we demonstrate that EGF induces the phosphorylation of three specific serine residues (S(1356), S(1360), and S(1364)) located within the connecting segment of the beta 4 subunit and that phosphorylation on these residues accounts for the bulk of beta 4 phosphorylation stimulated by EGF. Importantly, phosphorylation of these serines is critical for the ability of EGF to disrupt hemidesmosomes. Using COS-7 cells, which assemble hemidesmosomes type II upon exogenous expression of the alpha 6 beta 4 integrin, we observed that expression of a beta 4 construct containing Ser-->Ala mutations of S(1356), S(1360), and S(1364) reduced the ability of EGF to disrupt hemidesmosomes and that this effect appears to involve cooperation among these phosphorylation sites. Moreover, expression of Ser-->Asp mutants that mimic constitutive phosphorylation reduced hemidesmosome formation. Protein kinase C-alpha (PKC-alpha) is the kinase responsible for phosphorylating at least two of these serines, based on in vitro kinase assays, peptide mapping, and mutational analysis. Together, these results highlight the importance of serine phosphorylation in regulating type II hemidesmosome disassembly, implicate a cluster of serine residues within the connecting segment of beta 4, and argue for a key role for PKC-alpha in regulating these structures. (+info)
Inhibition of basement membrane formation by a nidogen-binding laminin gamma1-chain fragment in human skin-organotypic cocultures.
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Basement membranes generally determine different tissue compartments in complex organs, such as skin, playing not only an important structural but also a regulatory role. We have previously demonstrated the formation of a regular basement membrane in organotypic three-dimensional (3D)-cocultures of human skin keratinocytes and fibroblasts by indirect immunofluorescence and transmission electron microscopy. In this assembly process, cross-linking of type IV collagen and the laminin gamma1 chain by nidogen is considered a crucial step. For a functional proof, we have now competitively inhibited nidogen binding to laminin in 3D-cocultures with a recombinant laminin gamma1 fragment (gamma1III3-5 module) spanning this binding site. Repeated treatment abolished the deposition of nidogen at the epithelial-matrix interface but also greatly perturbed the presence of other matrix constituents such as laminin and perlecan. This effect persisted over the entire observation period of 10 to 21 days. In contrast, some components of the basement membrane zone were only moderately affected, with the laminin-5 isoform (gamma2 chain), type IV collagen and integrin alpha6ss4 still showing a distinct staining at their regular position, when seen by light microscopy. Furthermore, epidermal morphology and differentiation remained largely normal as indicated by the regular location of keratins K1/K10 and also of late differentiation markers. Ultrastructural examination demonstrated that the gamma1 fragment completely suppressed any formation of basement membrane structures (lamina densa) and also of hemidesmosomal adhesion complexes. As a consequence of hemidesmosome deficiency, keratin filament bundles were not attached to the ventral basal cell aspect. These findings were further substantiated by immuno-electron microscopy, revealing either loss or drastic reduction and dislocation of basement membrane and hemidesmosomal components. Taken together, in this simplified human skin model (representing a 'closed system') a functional link has been demonstrated between compound structures of the extra- and intracellular space at the junctional zone providing a basis to interfere at distinct points and in a controlled fashion. (+info)
Keratinocytes display normal proliferation, survival and differentiation in conditional beta4-integrin knockout mice.
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The alpha6beta4 integrin is located at the basal surface of keratinocytes, in hemidesmosomal structures that mediate stable adhesion of epidermal cells to the underlying basement membrane component laminin-5. The absence of alpha6beta4 integrin causes junctional epidermolysis bullosa, a severe blistering disease of the skin leading to perinatal death, confirming its essential role in mediating strong keratinocyte adhesion. Several studies have suggested that alpha6beta4 integrin can also regulate signaling cascades that control cell proliferation, survival and migration through a mechanism independent of its adhesive function. We have generated a conditional knockout mouse strain, in which the gene encoding the beta4 integrin subunit (Itgb4) was inactivated only in small stretches of the skin. These mice were viable and permitted an accurate analysis of the consequences of the loss of beta4 on various biological processes by comparing beta4-positive and -negative parts of the skin in the same animal. Despite the complete loss of hemidesmosomes in regions lacking alpha6beta4 integrin, the distribution of a range of adhesion receptors and basement membrane proteins was unaltered. Moreover, loss of alpha6beta4 did not affect squamous differentiation, proliferation or survival, except for areas in which keratinocytes had detached from the basement membrane. These in vivo observations were confirmed in vitro by using immortalized keratinocytes - derived from beta4-subunit conditional knockout mice - from which the gene encoding beta4 had been deleted by Cre-mediated recombination. Consistent with the established role of alpha6beta4 in adhesion strengthening, its loss from cells was found to increase their motility. Our findings clearly demonstrate that, after birth, epidermal differentiation, proliferation and survival all proceed normally in the absence of alpha6beta4, provided that cell adhesion is not compromised. (+info)
Zebrafish penner/lethal giant larvae 2 functions in hemidesmosome formation, maintenance of cellular morphology and growth regulation in the developing basal epidermis.
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Epithelial cells are equipped with junctional complexes that are involved in maintaining tissue architecture, providing mechanical integrity and suppressing tumour formation as well as invasiveness. A strict spatial segregation of these junctional complexes leads to the polarisation of epithelial cells. In vertebrate epithelia, basally localised hemidesmosomes mediate stable adhesion between epithelial cells and the underlying basement membrane. Although components of hemidesmosomes are relatively well known, the molecular machinery involved in governing the formation of these robust junctions, remains elusive. Here, we have identified the first component of this machinery using a forward genetic approach in zebrafish as we show that the function of penner (pen)/lethal giant larvae 2 (lgl2) is necessary for hemidesmosome formation and maintenance of the tissue integrity in the developing basal epidermis. Moreover, in pen/lgl2 mutant, basal epidermal cells hyper-proliferate and migrate to ectopic positions. Of the two vertebrate orthologues of the Drosophila tumour suppressor gene lethal giant larvae, the function of lgl2 in vertebrate development and organogenesis remained unclear so far. Here, we have unravelled an essential function of lgl2 during development of the epidermis in vertebrates. (+info)
Differential roles for beta2 integrins in experimental autoimmune bullous pemphigoid.
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Bullous pemphigoid (BP) is an autoimmune disease associated with autoantibodies directed against the hemidesmosomal antigens anti-BP230 and anti-B180. Neonatal mice injected with rabbit anti-mouse BP180 (mBP10) IgG develop a BP-like disease. Complement, immune complexes, mast cells, and neutrophils play a key role in subepidermal blistering in this animal model. In this study we investigated the role of beta2 integrins in experimental BP. Wild-type (WT) mice pretreated with neutralizing antibody against CD11a (LFA-1), CD11b (Mac-1), CD11a plus CD11b, or CD18 alone failed to develop BP when injected with pathogenic anti-mBP180 IgG. This was associated with a significant reduction in neutrophil accumulation in neutralizing antibody-treated mice. Mac-1-deficient (Mac-1 knockout [KO]) mice were resistant to experimental BP despite normal complement deposition and mast cell and neutrophil degranulation. Neutrophil infiltration in Mac-1 KO mice was severely impaired at 24 hours. However, more neutrophils accumulated in the skin of Mac-1 KO mice compared with WT mice at early time points (2-4 hours), which was associated with an increase in their survival as determined by apoptosis markers. These data suggest that beta2 integrins play differential roles in experimental BP: LFA-1 is required for neutrophil recruitment, while Mac-1 mediates late neutrophil accumulation and apoptosis of infiltrating neutrophils. (+info)