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PURPOSE: To identify specific site(s) on human ss4 molecule to which sera from ocular cicatricial pemphigoid (OCP) patients bind and to determine its role in the process of blister formation. METHODS: Clone the fragments representing the extracellular and intracellular domain of ss4 molecule from normal human conjunctival mRNA into an expression vector; map the region to which sera from OCP patients bind by Western blot analysis. Determine the role of the immunodominant region in pathogenesis by demonstrating the ability of the rabbit antibody to the immunodominant region to produce separation of basement membrane zone (BMZ) from the basal epithelial layer when incubated with normal human conjunctiva in an in vitro organ culture model. RESULTS: Majority of the OCP sera tested bound to the C-terminal end of the intracellular domain (IC3.0) of the human ss4 integrin. Further subcloning of IC3.0 demonstrated that a smaller fragment extending from 1489 aa to 1572 aa (IC3.4) was responsible for this binding. This region may have multiple antibody binding sites. Antibody to human IC3.0 and IC3.4 produced in rabbit, resulted in BMZ separation, histologically identical with that observed when normal human conjunctiva was cultured with OCP sera in an human conjunctival organ culture model. CONCLUSIONS: These observations identify IC3.4 as the antibody binding site for sera of OCP patients and suggest a possible role for it in blister formation. Indirectly it highlights certain important aspects of the structural and functional dynamics of the biology of the hemidesmosomes and basement membranes.
Integrin-mediated adhesion of cells to extracellular matrix proteins has been shown to activate various intracellular signaling events. In the present study, we demonstrate that the addition of a monoclonal antibody raised against the beta4 integrin subunit in the culture medium of a clone derived from the colon adenocarcinoma cell line LoVo specifically results in stimulation of cell migration and invasion through reconstituted basement membrane matrices. Moreover, an increase in MMP-2 activity is observed. Conversely, monoclonal anti-alpha6 and anti-beta1 have no effect on MMP-2 expression. The s. c. co-injection of adenocarcinoma cells with antibodies raised against the beta4 integrin subunit to immunosuppressed newborn rats gives rise to tumors displaying altered and disorganized peri-tumoral basement membranes compared with tumors obtained when cells are injected with adenocarcinoma cells alone. Higher metastatic capacity of cells results when they are co-injected with antibodies to the beta4 integrin subunit. Our results suggest that the beta4 subunit of alpha6beta4 integrin, a laminin receptor in colon adenocarcinoma, may be responsible for the specific signals which stimulate cell motility, expression of MMP-2 and tumor invasion.
BACKGROUND: The aim of this work was to study the expression of alpha, beta1, and beta4 integrin subunits in meningiomas. METHODS: Seventeen atypical or anaplastic meningiomas were retrieved from the files of Hôpital de Bellevue, Saint-Etienne, France. They were compared with 17 benign meningiomas consecutively examined in 1997 and 6 schwannomas. The tumors were classified according to standard histologic criteria. Frozen sections were immunostained for alpha1, alpha2, alpha3, alpha4, alpha5, alpha6, beta1, and beta4 integrin subunits; collagen; laminin and fibronectin; cytokeratin; vimentin; neural cell adhesion molecule (NCAM); and MIB-1. RESULTS: The study included 7 fibrous meningiomas, 6 transitional meningiomas, 19 syncytial meningiomas, and 2 secretory meningiomas. The expression of alpha1, alpha3, alpha5, alpha6, and beta1 was constant. The expression of alpha1 was higher in fibrous meningiomas than in syncytial meningiomas. Only in transitional, syncytial, and secretory meningiomas was the expression of alpha2 detected. The expression of alpha2 and beta4 was associated with the expression of cytokeratin in the glandular structures of secretory meningiomas, whereas it was associated with NCAM expression in the whorls of meningothelial meningiomas. The expression of integrin receptors by tumor cells was strongly correlated with that of their respective ligands in the extracellular matrix. In invasive meningiomas, the expression of alpha3 and alpha6 by tumor cells was significantly lower. The higher the MIB-1 proliferation index, the lower the expression of alpha3. The 6 schwannomas expressed only alpha2, alpha3, alpha6, beta1, and beta4 integrins. CONCLUSIONS: Each histologic subtype of meningioma has a specific spectrum of integrin expression. The study of alpha3 and alpha6 may have prognostic value in the assessment of meningiomas. The study of the integrin profile is valuable for the differential diagnosis of fibrous meningiomas and schwannomas.
To clarify the signal transduction in vascular endothelial cells (VEC) apoptosis induced by deprivation of FGF and serum, we investigated the function of integrin beta4 by using the monoclonal antibody (mAb) of this integrin. We added anti-beta 4 integrin mAb at the concentration of 5 microg/ml to the cells deprived of FGF and serum, apoptosis of these cells were completely inhibited 24 h after the treatment. Furthermore we plated the cells onto untreated bacterial culture plates on which the cells cannot adhere and spread in MCDB medium without FGF and serum; however, when anti-beta 4 integrin mAb was present at 5 microg/ml in the seeding medium, the cells rapidly adhered and spread. Our results first demonstrated that integrin beta4 participated in apoptotic signaling in VEC, and our findings indicate that hemidesmosome structures and keratin filament system might be important in regulation of apoptotic signaling.
Epidermolysis bullosa with pyloric atresia (EB-PA; OMIM 226730) is a clinically and genetically heterogeneous autosomal recessive blistering disorder, including lethal and nonlethal variants. Recently, expression of alpha6beta4 integrin, a transmembrane protein of the epithelial basement membranes, has been shown to be altered in these patients. In this work, we have explored the molecular pathology of the lethal form of EB-PA, and we describe novel ITGB4 mutations in five alleles of three patients. The mutation detection strategy included polymerase chain reaction amplification of each exon of ITGB4, followed by heteroduplex analysis and direct nucleotide sequencing. The novel mutations included a homozygous 2-bp deletion in exon 34 (4501delTC), compound heterozygosity for a 2-bp deletion within the paternal allele (120delTG) within exon 3 and a cysteine substitution in the maternal allele (C245G) within exon 7, and the paternal nonsense mutation within exon 4 (Q73X). Thus, three of four distinct mutations predicted truncated polypeptide chains, whereas the missense mutation in the extracellular domain of beta4 integrin may affect ligand binding or dimerization of alpha6 and beta4 integrin subunits. These mutations emphasize the critical importance of the alpha6beta4 integrin in providing stability to the association of epidermis to the underlying dermis at the cutaneous basement membrane zone.
OBJECTIVE: To compare the expression of alpha6 and beta4 integrin subunit levels throughout the menstrual cycle in the endometrium of healthy women and infertile women. DESIGN: Prospective study. SETTING: An academic teaching hospital. PATIENT(S): Endometrium was collected from 58 women, including healthy subjects (n = 28) and patients undergoing diagnostic testing for infertility (n = 30). INTERVENTION(S): Endometrial biopsies were performed throughout the menstrual cycle in healthy women and infertile women. MAIN OUTCOME MEASURE(S): Immunohistochemical staining intensity of alpha6 and beta4 using the semiquantitative immunohistochemical score, compared using regression analysis and analysis of variance with Scheffé's correction. RESULT(S): There was no correlation between menstrual cycle phase and endometrial integrin subunit alpha6 or beta4 on glandular or luminal epithelium. Even women with identified luteal phase defects had indistinguishable patterns of expression for these integrin subunits during the window of implantation. CONCLUSION(S): The integrin subunits alpha6 and beta4 are expressed uniformly throughout the menstrual cycle on glandular and luminal epithelium. These integrins may have a housekeeping role in anchoring endometrial epithelium and do not appear to be useful markers of uterine receptivity.
We identified nine single-nucleotide polymorphisms (SNPs) in the human integrin beta4 (ITGB4) gene (17q24-q25), which encodes a cell-surface receptor, by screening all exons and exon-intron boundaries. Seven of these SNPs were present in coding regions and two in intronic sequences; four of the coding SNPs involved amino-acid substitutions. As the gene is implicated in the tumorigenesis of breast cancers, the polymorphic sites will serve as useful markers not only for distinguishing alleles in loss of heterozygosity (LOH) analyses but also for studying genetic susceptibility to malignancies in humans.
Junctional epidermolysis bullosa associated with pyloric atresia (EB-PA; OMIM 226730) is a rare autosomal recessively inherited disease in which mucocutaneous fragility is associated with gastrointestinal atresia. This disease is usually fatal within the first few weeks or months of life even following surgical correction of the intestinal obstruction. Recently, mutations in the genes encoding the epithelial integrin alpha6beta4 (ITGA6 and ITGB4) have been identified in several patients with EB-PA. We report two unrelated patients with this disease who have survived into early childhood with mild cutaneous involvement, in whom we have identified pathogenetic mutations in ITGB4. The first patient was a compound heterozygote for a splice site mutation in exon 30 (3793 + 1G-to-A) and a non-sense mutation in exon 36 (W1478X), and the second was a compound heterozygote for a missense mutation in exon 3 (C38R) and a 1 bp deletion in exon 36 (4776delG). Although the non-sense and deletion mutations are predicted to result in markedly reduced beta4 integrin mRNA levels, the presence of the missense or splice site mutation on the second allele may enable the synthesis of some functional, albeit perturbed, beta4 polypeptide. Determination of the molecular mechanisms in these two cases increases our understanding of EB-PA and may enable correlation between genotype and phenotype.
A general improvement with ageing has been reported in a few cases of epidermolysis bullosa with pyloric atresia (PA-JEB), an autosomal recessive skin disease characterized by extensive disadhesion of epithelia. In a patient who improved from severe to mild PA-JEB, a search for mutations in the integrin beta4 gene (IGTB4) detected heterozygosity for a novel base substitution 3986-19T-->A in the putative branchpoint sequence of intron 31, and a point mutation 3802+1G-->A in the donor splice site of intron 30 previously associated with severe PA-JEB. Analysis of mRNA showed that the intronic mutation prevents legitimate splicing of the beta4 pre-mRNA. Functional splicing can be restored in vitro by seeding the proband's keratinocytes on feeders of irradiated fibroblasts. Study of mRNA in wild-type keratinocytes transfected with IGTB4 minigenes containing intron 31 with or without mutation 3986-19T-->A, confirmed the causative role of the intronic mutation in PA-JEB, and highlighted the influence of feeders on the maturation process of the mutated beta4 pre-mRNA. Our results show that in a context of overall reduction of the beta4 mRNA levels, activation of the legitimate splice site in the aberrant beta4 pre-mRNA underlies the transient severity of the condition. The results also point to the relevance which the interaction between epithelial and stromal cells may have in modulating expression of integrin receptors.
The integrin alpha6 beta4 is a major component of hemidesmosomes, in which it mediates firm adhesion to laminin 5. Previous studies have shown that the incorporation of alpha6 beta4 into hemidesmosomes requires a 303 amino acid stretch of the cytoplasmic domain of beta4, comprising part of the first fibronectin type III (FNIII) repeat, the second FNIII repeat and the segment that connects the second to the third FNIII repeat (connecting segment). Now, we have further defined sequences within beta4 that are critical for its localization in hemidesmosomes and we demonstrate that these sequences also induce the redistribution of HD1/plectin into junctional complexes containing the integrin alpha6 beta4 in COS-7 cells, transfected with cDNAs encoding alpha6A and beta4. Truncation of the cytoplasmic domain of beta4 after amino acids 1,382 or 1,355 in the connecting segment, by which a potential tyrosine activation motif (TAM) is removed, does not prevent the localization of alpha6 beta4 in hemidesmosomes in the rat bladder carcinoma cell line 804G and neither did it eliminate the ability of alpha6 beta4 to change the subcellular distribution of HD1/plectin in COS-7 cells. In contrast, beta4 subunits in which the entire connecting segment had been deleted or which were truncated after amino acid 1,328, which removes almost the complete segment, had lost both of these functions. Furthermore, when beta4 subunits with either a deletion of the second FNIII repeat or a small deletion in this repeat were co-expressed with alpha6, the integrins were not localized in hemidesmosomes and did not induce the redistribution of HD1/plectin in COS-7 cells. Finally, the fourth FNIII repeat of beta4 could not replace the second in either of these activities. These findings establish that a region in beta4, which encompasses the second FNIII repeat and a stretch of 27 amino acids (1,329-1,355) of the connecting segment, is critical for the localization of alpha6beta4 in hemidesmosomes and that it regulates the distribution of HD1/plectin.
Cicatricial pemphigoid is a heterogeneous group of autoimmune subepidermal blistering diseases associated most commonly with autoantibodies to bullous pemphigoid (BP)180 and less frequently with those to laminin 5 or type VII collagen. In addition, a few cases have been described with autoantibodies to the beta4 subunit of alpha6beta4 integrin. We describe a patient with extensive disease of ocular, oral, pharyngeal, laryngeal and genital mucous membranes that healed with scarring of conjunctivae. IgG autoantibodies bound to the dermal-epidermal junction on direct immunofluorescence (IF) microscopy and to the epidermal side of 1 mol L(-1) NaCl-split skin on indirect IF microscopy. Our patient's circulating IgG recognized a 205-kDa protein in extracts of 293T cells transfected with the beta4 subunit of alpha6beta4 integrin and in the cell extract of DJM-1 cells. Our patient's IgG and IgA autoantibodies also reacted with full-length BP180 derived from epidermal extracts and the ectodomain of BP180 (LAD-1) derived from culture supernatant of keratinocytes. In addition, a weak IgG reaction with BP230 was noted. The disease rapidly responded to dexamethasone-cyclophosphamide pulse therapy, and immunoblot reactivity to both beta4 integrin and BP180 decreased according to disease activity.
To study whether alpha6beta4 integrin regulates apoptosis, human A431 cells were plated on bacteria plates in the presence or absence of mAb beta4. In the absence of mAb beta4, A431 cells demonstrated morphological characteristics of apoptosis by 24 h and most cells died by 48 h. In contrast, in the presence of mAb beta4, cells remained viable, and at the end of 48 h, 70-80% of cells survived. Treatment of A431 cells with mAb beta4 resulted in tyrosine phosphorylation of the p85 subunit of PI3 kinase; PI3 kinase activity increased within 15 min and peaked at 60 min. Stimulation of beta4 in A431 cells resulted in a time-dependent phosphorylation of Akt with a concomitant and parallel phosphorylation of Bad. Inactivation of PI3 kinase with inhibitors blocked the anti-apoptotic effect induced by mAb beta4. These are the first results to suggest that ligation of alpha6beta4 integrin protects cells from apoptosis through a PI3K/Akt kinase signaling pathway.
Bullous pemphigoid antigen 180 (BP180) is a component of hemidesmosomes, i.e., cell-substrate adhesion complexes. To determine the function of specific sequences of BP180 to its incorporation in hemidesmosomes, we have transfected 804G cells with cDNA-constructs encoding wild-type and deletion mutant forms of human BP180. The results show that the cytoplasmic domain of BP180 contains sufficient information for the recruitment of the protein into hemidesmosomes because removal of the extracellular and transmembrane domains does not abolish targeting. Expression of chimeric proteins, which consist of the membrane targeting sequence of K-Ras fused to the cytoplasmic domain of BP180 with increasing internal deletions or lacking the NH2 terminus, indicates that the localization of BP180 in hemidesmosomes is mediated by a segment that spans 265 amino acids. This segment comprises two important regions located within the central part and at the NH2 terminus of the cytoplasmic domain of BP180. To investigate the effect of the alpha6beta4 integrin on the subcellular distribution of BP180, we have transfected COS-7 cells, which lack alpha6beta4 and BP180, with cDNAs for BP180 as well as for human alpha6A and beta4. We provide evidence that a mutant form of BP180 lacking the collagenous extracellular domain as well as a chimeric protein, which contains the entire cytoplasmic domain of BP180, are colocalized with alpha6beta4. In contrast, when cells were transfected with cDNAs for alpha6A and mutant forms of beta4, either lacking the cytoplasmic COOH-terminal half or carrying phenylalanine substitutions in the tyrosine activation motif of the cytoplasmic domain, the recombinant BP180 molecules were mostly not colocalized with alpha6beta4, but remained diffusely distributed at the cell surface. Moreover, in cells transfected with cDNAs for alpha6A and a beta4/beta1 chimera, in which the cytoplasmic domain of beta4 was replaced by that of the beta1 integrin subunit, BP180 was not colocalized with the alpha6beta4/beta1 chimera in focal adhesions, but remained again diffusely distributed. These results indicate that sequences within the cytoplasmic domain of beta4 determine the subcellular distribution of BP180.
Plectin is a widely expressed cytoskeletal linker. Here we report the crystal structure of the actin binding domain of plectin and show that this region is sufficient for interaction with F-actin or the cytoplasmic region of integrin alpha6beta4. The structure is formed by two calponin homology domains arranged in a closed conformation. We show that binding to F-actin induces a conformational change in plectin that is inhibited by an engineered interdomain disulfide bridge. A two-step induced fit mechanism involving binding and subsequent domain rearrangement is proposed. In contrast, interaction with integrin alpha6beta4 occurs in a closed conformation. Competitive binding of plectin to F-actin and integrin alpha6beta4 may rely on the observed alternative binding mechanisms and involve both allosteric and steric factors.
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The integrin beta4 has a long cytodomain necessary for hemidesmosome formation. A yeast two-hybrid screen using beta4 cytodomain uncovered a protein called p27(BBP) that represents a beta4 interactor. Both in yeast and in vitro, p27(BBP) binds the two NH2-terminal fibronectin type III modules of beta4, a region required for signaling and hemidesmosome formation. Sequence analysis of p27(BBP) revealed that p27(BBP) was not previously known and has no homology with any isolated mammalian protein, but 85% identical to a yeast gene product of unknown function. Expression studies by Northern analysis and in situ hybridization showed that, in vivo, p27(BBP) mRNA is highly expressed in epithelia and proliferating embryonic epithelial cells. An antibody raised against p27(BBP) COOH-terminal domain showed that all beta4-containing epithelial cell lines expressed p27(BBP). The p27(BBP) protein is insoluble and present in the intermediate filament pool. Furthermore, subcellular fractionation indicated the presence of p27(BBP) both in the cytoplasm and in the nucleus. Confocal analysis of cultured cells showed that part of p27(BBP) immunoreactivity was both nuclear and in the membrane closely apposed to beta4. These results suggest that the p27(BBP) is an in vivo interactor of beta4, possibly linking beta4 to the intermediate filament cytoskeleton.