Alpha9 and beta8 integrin expression correlates with the merger of the developing mouse eyelids.
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As previously reported, alpha9 integrin is expressed between the merged or fused eyelids of mice at birth, and changes in alpha9 localization occur during lid opening. To determine whether alpha9 and/or additional integrin subunits mediate the emergence and temporary fusion of the eyelids, immunofluorescence and confocal microscopy were used to evaluate the localization of various integrin subunits in the developing ocular surface of the mouse. No detectable beta5, beta6, or beta7 integrins were observed on the epithelia of the ocular surface. alpha2, alpha3, alphav, and beta1 integrins were most abundant in the basal cells beginning at 13.5 days post conception and remained primarily localized to the basal cell layers throughout development. beta4 was localized at the basal surface of the epidermal basal cells beginning at 13.5 days post conception but was not found on the corneal epithelial basal cells until after birth. alpha9 and beta8 integrins were present on suprabasal cells of the epidermis at the leading edge of the eyelid before merger and on the epithelial bridge that forms immediately after these tissues merge, suggesting that they play a role in the initial fusion of the epithelial tissues of the eyelid and in stabilizing the epithelial junction. After birth and into adulthood, beta8 was retained within the suprabasal cell layers of the epidermis, whereas alpha9 became localized to the basal cells of the epidermis, the conjunctiva, and the limbus. The lack of co-localization of beta4 with either alpha9 or beta8 in double-labeling studies suggests that alpha9 and beta8 are restricted to the lateral and apical aspects of those cells in which they are expressed. The presence of tenascin-C and laminin-5 at the epithelial junction site suggests that alpha9: tenascin-C and beta4: laminin-5 interactions may play a role in stabilizing the fusion between lids early on but do not appear to be involved in the movement of the lids across the cornea. The data presented identify specific integrins and matrix proteins that are likely to mediate eyelid fusion. (+info)
The role of beta7 integrins in CD8 T cell trafficking during an antiviral immune response.
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The requirement of beta7 integrins for lymphocyte migration was examined during an ongoing immune response in vivo. Transgenic mice (OT-I) expressing an ovalbumin-specific major histocompatibility complex class I-restricted T cell receptor for antigen were rendered deficient in expression of all beta7 integrins or only the alphaEbeta7 integrin. To quantitate the relative use of beta7 integrins in migration in vivo, equal numbers of OT-I and OT-I-beta7(-/-) or OT-I-alphaE-/- lymph node (LN) cells were adoptively transferred to normal mice. Although OT-I-beta7(-/-) LN cells migrated to mesenteric LN and peripheral LN as well as wild-type cells, beta7 integrins were required for naive CD8 T cell and B cell migration to Peyer's patch. After infection with a recombinant virus (vesicular stomatitis virus) encoding ovalbumin, beta7 integrins became critical for migration of activated CD8 T cells to the mesenteric LN and Peyer's patch. Naive CD8 T cells did not enter the lamina propria or the intestinal epithelium, and the majority of migration of activated CD8 T cells to the small and large intestinal mucosa, including the epithelium, was beta7 integrin-mediated. The alphaEbeta7 integrin appeared to play no role in migration during a primary CD8 T cell immune response in vivo. Furthermore, despite dramatic upregulation of alphaEbeta7 by CD8 T cells after entry into the epithelium, long-term retention of intestinal intraepithelial lymphocytes was also alphaEbeta7 independent. (+info)
Mechanotransduction in response to shear stress. Roles of receptor tyrosine kinases, integrins, and Shc.
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Shear stress, the tangential component of hemodynamic forces, activates many signal transduction pathways in vascular endothelial cells. The conversion of mechanical stimulation into chemical signals is still unclear. We report here that shear stress (12 dynes/cm2) induced a rapid and transient tyrosine phosphorylation of Flk-1 and its concomitant association with the adaptor protein Shc; these are accompanied by a concurrent clustering of Flk-1, as demonstrated by confocal microscopy. Our results also show that shear stress induced an association of alphavbeta3 and beta1 integrins with Shc, and an attendant association of Shc with Grb2. These associations are sustained, in contrast to the transient Flk-1. Shc association in response to shear stress and the transient association between alphavbeta3 integrin and Shc caused by cell attachment to substratum. Shc-SH2, an expression plasmid encoding the SH2 domain of Shc, attenuated shear stress activation of extracellular signal-regulated kinases and c-Jun N-terminal kinases, and the gene transcription mediated by the activator protein-1/12-O-tetradecanoylphorbol-13-acetate-responsive element complex. Our results indicate that receptor tyrosine kinases and integrins can serve as mechanosensors to transduce mechanical stimuli into chemical signals via their association with Shc. (+info)
Human T-cell leukaemia/lymphoma virus type 1 syncytium formation is regulated in a cell-specific manner by ICAM-1, ICAM-3 and VCAM-1 and can be inhibited by antibodies to integrin beta2 or beta7.
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Human T-cell leukaemia/lymphoma virus type 1 (HTLV-1) is a pathogenic retrovirus responsible for a number of inflammatory pathologies and adult T-cell leukaemia. Although T-cell tropic in vivo, HTLV-1 can infect a wide variety of cell types in vitro. Cell-to-cell spread of HTLV-1 may require specific binding of envelope to its cellular receptor, with other cell-surface molecules facilitating fusion. Here it is shown that intercellular adhesion molecule-1 or -3 (ICAM-1, ICAM-3) or vascular cell adhesion molecule-1 (VCAM-1) are required for syncytium formation of K562 with HTLV-1-infected MT2 cells but not C91-PL cells. The effect of ICAMs and VCAM-1 on MT2-induced fusion can be blocked by antibodies that bind beta-integrins. These fusion co-factor molecules are effective only when present in combination with HTLV-1 receptor-bearing cells and are not sufficient to mediate syncytium formation alone. The results suggest that engagement of HTLV-1-infected cells with susceptible target cells requires the simultaneous binding of viral envelope glycoprotein to the cellular receptor and co-factor molecules to beta-integrins. The tissue-specific expression of adhesion molecules might therefore influence HTLV-1 virus tropism and pathogenic changes associated with syncytium formation. (+info)
Inflammatory cytokines synergize with the HIV-1 Tat protein to promote angiogenesis and Kaposi's sarcoma via induction of basic fibroblast growth factor and the alpha v beta 3 integrin.
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The Tat protein of HIV-1, a transactivator of viral gene expression, is released by acutely infected T cells and, in this form, exerts angiogenic activities. These have linked the protein to the pathogenesis of Kaposi's sarcoma (KS), a vascular tumor frequent and aggressive in HIV-1-infected individuals (AIDS-KS). In this study, we show that a combination of the same inflammatory cytokines increased in KS lesions, namely IL-1 beta, TNF-alpha, and IFN-gamma, synergizes with Tat to promote in nude mice the development of angioproliferative KS-like lesions that are not observed with each factor alone. Inflammatory cytokines induce the tissue expression of both basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), two angiogenic molecules highly produced in primary KS lesions. However, bFGF, but not VEGF, synergizes with Tat in vivo and induces endothelial cells to migrate, to adhere, and to grow in response to Tat in vitro. Tat angiogenic effects correlate with the expression of the alpha v beta 3 integrin that is induced by bFGF and binds the arginine-glycine-aspartic acid (RGD) region of Tat. In contrast, no correlation is observed with the expression of alpha v beta 5, which is promoted by VEGF and binds Tat basic region. Finally, KS lesion formation induced by bFGF and Tat in nude mice is blocked by antagonists of RGD-binding integrins. Because alpha v beta 3 is an RGD-binding integrin that is highly expressed in primary KS lesions, where it colocalizes with extracellular Tat on vessels and spindle cells, these results suggest that alpha v beta 3 competitors may represent a new strategy for the treatment of AIDS-KS. (+info)
Calpain cleavage of integrin beta cytoplasmic domains.
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We showed previously that the calcium-dependent protease, calpain, cleaves the cytoplasmic domain of the integrin beta3 subunit. To investigate whether susceptibility to calpain is a common feature of all integrin beta subunits, and to map calpain cleavage sites in different integrin beta tails, we treated recombinant cytoplasmic domains of integrin beta1A, beta1D, beta2, beta3 and beta7 subunits with purified calpain in vitro. We found that the cytoplasmic domains of all these integrin chains were cleaved by calpain. HPLC followed by mass spectrometry was used to identify calpain cleavage sites. These sites were clustered in the C-terminal half of the integrin beta cytoplasmic domains in regions flanking the two NXXY motifs, suggesting the possibility that the structural framework provided by these motifs is recognized by calpain. We used the knowledge of these cleavage sites to develop cleavage site-specific antibodies and to demonstrate cleavage of the beta1A cytoplasmic domain in intact platelets stimulated with calcium ionophore or thrombin. Thus susceptibility to calpain cleavage is common to integrin beta subunits, can be induced in intact cells, and appears to favor regions surrounding two conserved NXXY motifs. (+info)
Enhancement of endothelial cell migration and in vitro tube formation by TAP20, a novel beta 5 integrin-modulating, PKC theta-dependent protein.
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Migration, proliferation, and tube formation of endothelial cells are regulated by a protein kinase C isoenzyme PKCtheta. A full-length cDNA encoding a novel 20-kD protein, whose expression was PKCtheta-dependent, was identified in endothelial cells, cloned, characterized, and designated as theta-associated protein (TAP) 20. Overexpression of TAP20 decreased cell adhesion and enhanced migration on vitronectin and tube formation in three-dimensional culture. An antiintegrin alphavbeta5 antibody prevented these TAP20 effects. Overexpression of TAP20 also decreased focal adhesion formation in alphavbeta3-deficient cells. The interaction between TAP20 and beta5 integrin cytoplasmic domain was demonstrated by protein coprecipitation and immunoblotting. Thus, the discovery of TAP20, which interacts with integrin beta5 and modulates cell adhesion, migration, and tube formation, further defines a possible pathway to angiogenesis dependent on PKCtheta. (+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)