The role of antibody to human beta4 integrin in conjunctival basement membrane separation: possible in vitro model for ocular cicatricial pemphigoid.
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PURPOSE: To demonstrate the specific binding of autoantibodies present in the sera of patients with ocular cicatricial pemphigoid (OCP) to human beta4 integrin present in the normal human conjunctiva (NHC) and to study the role of OCP autoantibodies and antibody to human beta4 integrin in the pathogenesis of subepithelial lesion formation in OCP. METHODS: Indirect immunofluorescence assay and in vitro organ culture method using NHC were used. Sera and IgG fractions from 10 patients with OCP; immunoaffinity-purified OCP autoantibody; antibodies to human beta4, beta1, alpha6, and alpha5 integrins; and sera from patients with pemphigus vulgaris, bullous pemphigoid (BP), and chronic atopic and chronic ocular rosacea cicatrizing conjunctivitis; and normal human serum (NHS) were used. RESULTS: Nine of 10 OCP sera or IgG fractions, immunoaffinity-purified OCP autoantibody, antibodies to human beta4 and alpha6 integrins, and sera from patients with BP showed homogenous, smooth linear binding along the basement membrane zone (BMZ) of the NHC. NHS, antibodies to other integrins, and sera from patients with chronic cicatrizing conjunctivitis from other causes showed no such binding. When NHC was first absorbed with OCP sera and then reacted with anti-beta4 antibodies or vice versa, the intensity of the BMZ binding was dramatically reduced or completely eliminated, indicating that there were autoantibodies in OCP sera specific for the beta4 integrin. BMZ separation developed 48 to 72 hours after addition of total OCP sera, IgG fractions from OCP sera, immunoaffinity-purified autoantibodies from sera of patients with OCP, or anti-beta4 antibodies to the NHC cultures, but not after addition of normal control sera, sera from patients with chronic cicatrizing conjunctivitis from causes other than OCP, or sera from patients with OCP in clinical remission. CONCLUSION: Circulating anti-beta4 integrin antibody may have an important role in the pathogenesis of OCP. (+info)
Splicing modulation of integrin beta4 pre-mRNA carrying a branch point mutation underlies epidermolysis bullosa with pyloric atresia undergoing spontaneous amelioration with ageing.
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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. (+info)
Expression of functionally distinct variants of the beta(4)A integrin subunit in relation to the differentiation state in human intestinal cells.
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Integrins are important mediators of cell-laminin interactions. In the small intestinal epithelium, which consists of spatially separated proliferative and differentiated cell populations located, respectively, in the crypt and on the villus, laminins and laminin-binding integrins are differentially expressed along the crypt-villus axis. One exception to this is the integrin alpha(6)beta(4), which is thought to be ubiquitously expressed by intestinal cells. However, in this study, a re-evaluation of the beta(4) subunit expression with different antibodies revealed that two forms of beta(4) exist in the human intestinal epithelium. Furthermore, we show that differentiated enterocytes express a full-length 205-kDa beta(4)A subunit, whereas undifferentiated crypt cells express a novel beta(4)A subunit that does not contain the COOH-terminal segment of the cytoplasmic domain (beta(4)A(ctd-)). This new form was not found to arise from alternative beta(4) mRNA splicing. Moreover, we found that these two beta(4)A forms can associate into alpha(6)beta(4)A complexes; however, the beta(4)A(ctd-) integrin expressed by the undifferentiated crypt cells is not functional for adhesion to laminin-5. Hence, these studies identify a novel alpha(6)beta(4)A(ctd-) integrin expressed in undifferentiated intestinal crypt cells that is functionally distinct. (+info)
Partial rescue of epithelial phenotype in integrin beta4 null mice by a keratin-5 promoter driven human integrin beta4 transgene.
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Integrin beta4 null mice exhibit extensive epidermal detachment, reminiscent of the human skin blistering disease junctional epidermolysis bullosa associated with pyloric atresia. Hemidesmosomes, the stable adhesion structures of squamous epithelia, are not formed in the absence of alpha6beta4. Null mutant mice die shortly after birth, but apart from their striking epithelial phenotype, no obvious developmental defects have been observed. To elucidate the cause of death in these mice, we generated transgenic mice with a heterologous construct consisting of the squamous epithelial-specific keratin-5 promoter and a human integrin beta4 subunit cDNA. The transgene was not expressed in the presence of endogenous beta4, probably as a result of competition for a limited pool of alpha6 subunits. In a beta4 null background, however, the transgene was expressed, and its expression pattern followed that of squamous epithelial-specific keratins. These rescued pups appeared healthy and ultrastructural analysis revealed that the interspecies heterodimer alpha6(mouse)/beta4(human) was sufficient to trigger the assembly of hemidesmosomes. After a variable period of up to 48 hours after birth these animals began to exhibit haemorrhages at the plantar and palmar areas. We observed the formation of small blisters and found that the transgene was not detectably expressed in this region, which is devoid of hair follicles. The rescued neonates became increasingly cyanotic and died soon after the onset of this phenomenon. We performed a developmental study of the expression of beta4 in the complete respiratory tract, but we found no correlation between the spatiotemporal distribution of beta4 and the onset of the respiratory insufficiency. It became clear, however, that there was a gradual detachment of squamous epithelia in the oral and nasal cavities which led to obstruction of the respiratory tract, suggesting that in beta4 null and rescued mice, neonatal death was a direct consequence of decreased adhesion properties of hairless squamous epithelia, rather than a developmental defect of the lungs. (+info)
Analysis of gene expression in human bullous keratopathy corneas containing limiting amounts of RNA.
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PURPOSE: To validate the use of polymerase chain reaction (PCR)-amplified full-length cDNA as a substitute for mRNA in nucleic acid array and gene expression analysis. METHODS: Total RNA was isolated from age-matched normal autopsy corneas and pseudophakic bullous keratopathy (PBK) corneas. Full-length cDNA was generated and PCR amplified using the Smart cDNA synthesis technology. Southern blot analysis of this cDNA was compared with Northern blot analysis of the RNA. Amplified cDNA was used to probe a commercial gene array. By immunohistochemistry, the expression pattern of several adhesion molecules represented on the array was assessed. RESULTS: The cDNA produced by the Smart cDNA system gave results very similar to those of northern blot analysis when examined for beta2-microglobulin, Rab geranylgeranyl transferase, and tenascin-C. This cDNA obtained from normal or PBK corneas was labeled and used to probe a 588 gene array (Clontech). Among other differences, beta6 integrin was detected only with the PBK probe, beta-catenin was markedly elevated in PBK, and beta4 integrin appeared to be reduced in PBK. Immunohistochemical patterns of these proteins were consistent with the hybridization signals on the gene array. CONCLUSIONS: Smart cDNA synthesis and nucleic acid arrays were combined and validated for the first time to identify differential gene expression in normal and diseased corneas. These techniques require very little RNA such as that equivalent to a half of a single cornea, which is useful when the amount of tissue is limiting. Altered expression of adhesive proteins beta6 integrin and beta-catenin may be related to the formation of epithelial bullae and microcystic changes in PBK patients. (+info)
Expression of a highly conserved protein, p27BBP, during the progression of human colorectal cancer.
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The highly conserved protein p27BBP is a cytoplasmic interactor of integrin beta4 expressed in epithelia. p27BBP is found in two pools: one nuclear pool enriched in the perinucleolar region, and one cytoplasmic pool. Deletion of p27BBP in yeast is lethal as a result of loss of the ribosomal 60S subunit. The aim of this study was to investigate the distribution of p27BBP in gut epithelium and its behavior during progression of human colorectal carcinomas. Results indicated that p27BBP is high in rapidly cycling cells and decreased in villous cells committed to apoptotic cell death. In dysplastic adenomas and carcinomas, p27BBP displayed a large increase of its nucleolar component that was superimposable to argyrophylic nucleolar organizing region-associated proteins and was associated with the nuclear matrix. Western blotting confirmed increased p27BBP in dysplastic adenomas and in carcinomas. In particular, p27BBP increased progressively from adenomas to carcinomas and, in the latter, was related to the tumor stage. The overexpression of p27BBP corresponded to mRNA up-regulation in carcinomas, supporting the idea of transcriptional or post-transcriptional regulation of its expression. Results suggested that p27BBP alterations are an early event in the transition from benign to malignant colorectal phenotypes and provide a novel tool in surgical pathology. (+info)
Formation of hemidesmosome-like structures in the absence of ligand binding by the (alpha)6(beta)4 integrin requires binding of HD1/plectin to the cytoplasmic domain of the (beta)4 integrin subunit.
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Hemidesmosomes are adhesion structures that mediate anchorage of epithelial cells to the underlying basement membrane. We have previously shown that the (alpha)6(beta)4 integrin can induce the assembly of these multi-protein structures independent of binding to its ligand laminin-5 (ligand-independent formation of hemidesmosomes). Our results suggested a role for HD1/plectin, which binds to the cytoplasmic domain of the (beta)4 integrin subunit, in controlling the clustering of hemidesmosomal components at the basal side of the cell. Using keratinocytes derived from patients lacking HD1/plectin, we now show that ligand-independent formation of hemidesmosomal clusters indeed requires HD1/plectin, in contrast to the ligand-dependent assembly of hemidesmosomes. No clustering of the (alpha)6(beta)4 integrin, or of the bullous pemphigoid antigens BP180 and BP230, was seen when HD1/plectin-deficient keratinocytes were plated on fibronectin or type IV collagen. In (&bgr;)4-deficient keratinocytes, expression of an interleukin 2 receptor (IL2R) transmembrane chimera containing the (beta)4 cytoplasmic tail with the mutation R1281W, which abrogates HD1/plectin binding, resulted in a diffuse distribution of the chimeric receptor. In contrast, a (beta)4(R1281W) mutant that can associate with (alpha)6 and bind ligand, was found to be directed to the basal surface of the cells, at sites where laminin-5 was deposited. In addition, this mutant induced clustering of BP180 and BP230 at these sites. Together, these results show that the formation of hemidesmosomes requires binding of either ligand or HD1/plectin to the (beta)4 integrin subunit. Intriguingly, we found that IL2R/(beta)4 chimeras become localized in pre-existing hemidesmosomes of HD1/plectin-deficient keratinocytes, and that this localization requires a domain in the (beta)4 cytoplasmic tail that is also required for HD1/plectin binding (residues 1115-1356). Because this part of (beta)4 lacks the BP180 binding site, and since we show in this study that it is unable to interact with the same part on another (beta)4 molecule, we suggest that the chimera becomes incorporated into hemidesmosomes of HD1/plectin-deficient keratinocytes by interacting with an as yet unidentified hemidesmosomal component. (+info)
Expression of alpha(v)beta6 integrin in oral leukoplakia.
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The distribution of alpha(v)beta6 integrin was examined in oral leukoplakia, lichen planus and squamous cell carcinomas using immunohistochemistry. Controls included oral mucosal wounds, chronically inflamed and normal oral mucosa. Integrins beta1, beta3, beta4, beta5, fibronectin and tenascin were also studied. The integrin alpha(v)beta6 was highly expressed throughout the whole lesion of 90% of the squamous cell carcinomas but was not present in any of the normal specimens. alpha(v)beta6 integrin was also expressed in 41% of the leukoplakia specimens, and 85% of the lichen planus samples, but in none of the tissues with inflammatory hyperplasia or chronic inflammation. The expression of beta1 integrins was localized in the basal layer, and that of the beta4 at the cell surface facing the basement membrane of all specimens. The integrins beta3 and beta5 were absent from all normal and leukoplakia specimens. Fibronectin and tenascin were present in the connective tissue underneath the epithelium of all the sections, and their expression was similar in both alpha(v)beta6-positive and alpha(v)beta6-negative tissues. A group of 28 leukoplakia patients were followed 1-4 years after first diagnosis. In this group, initially alpha(v)beta6 integrin-positive leukoplakia specimens had high tendency for disease progression while alpha(v)beta6-negative specimens did not progress. These results suggest that the expression of alpha(v)beta6 integrin could be associated in the malignant transformation of oral leukoplakias. (+info)