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PubMed2002

PPARalpha inhibits TGF-beta-induced beta5 integrin transcription in vascular smooth muscle cells by interacting with Smad4.

Integrins play an important role in vascular smooth muscle cell (VSMC) migration, a crucial event in the development of restenosis and atherosclerosis. Transforming growth factor-beta (TGF-beta) is highly expressed in restenotic and atherosclerotic lesions, and known to induce integrin expression. Peroxisome proliferator-activated receptor alpha (PPARalpha), a member of the nuclear receptor superfamily, regulates gene expression in a variety of vascular cells. We investigated the effects of PPARalpha ligands on TGF-beta-induced beta3 and beta5 integrin expression and potential interaction between PPARalpha and TGF-beta signaling. PPARalpha ligands WY-14643 (100 micromol/L) and 5,8,11,14-eicosatetranoic acid (ETYA, 50 micromol/L) inhibited TGF-beta-induced beta5 integrin protein expression by 72+/-6.8% and 73+/-7.1%, respectively (both P<0.05). TGF-beta-stimulated beta3 integrin expression was not affected by PPARalpha ligands. Both PPARalpha ligands also suppressed TGF-beta-induced beta5 integrin mRNA levels. PPARalpha ligands inhibited TGF-beta-inducible transcription of beta5 integrin by an interaction with a TGF-beta response element between nucleotides -63 and -44, which contains a Sp1/Sp3 transcription factor binding site. Nuclear complexes binding to the TGF-beta response region contained Sp1/Sp3 and TGF-beta-regulated Smad 2, 3, and 4 transcription factors. TGF-beta-stimulated Sp1/Smad4 nuclear complex formation was inhibited by WY-14643 and ETYA with a parallel induction of PPARalpha/Smad4 interactions. However, in vitro pull-down experiments failed to demonstrate direct binding between PPARalpha/Smad4. Both PPARalpha ligands blocked PDGF-directed migration of TGF-beta-pretreated VSMCs, a process mediated, in part, by beta5 integrins. The present study demonstrates that PPARalpha activators inhibit TGF-beta-induced beta5 integrin transcription in VSMCs through a novel indirect interaction between ligand-activated PPARalpha and the TGF-beta-regulated Smad4 transcription factors. The full text of this article is available at http://www.circresaha.org.

5,8,11,14-Eicosatetraynoic Acid↗

PubMed1994

The anti-inflammatory agent alpha-trinositol exerts its edema-preventing effects through modulation of beta 1 integrin function.

Edema formation in acute inflammation can be induced through lowering of interstitial fluid pressure (Pif) and seems to involve dynamic beta 1 integrin-mediated interactions between dermal cells and extracellular matrix fibers. The present experiments investigate the role of beta 1 integrins in the control of Pif. The anti-inflammatory drug alpha-trinositol (1,2,6-D-myo-inositol trisphosphate) stabilizes Pif in acute inflammation. Pretreatment with 5 mg IV alpha-trinositol in pentobarbital-anesthetized rats inhibited the lowering in Pif and the edema formation induced by subdermal injection of anti-beta 1 integrin IgG. This stabilization of the beta 1 integrin function in vivo was paralleled by effects of alpha-trinositol on contraction of fibroblast-populated three-dimensional collagen lattices in vitro. alpha-Trinositol was additive to the known stimulatory effect of platelet-derived growth factor-BB on the final gel size in the collagen gel contraction assay. Furthermore, alpha-trinositol counteracted the inhibitory effect of anti-beta 1 integrin Fab fragments on collagen gel contraction. Finally, subdermal injection of dibutyryl-cAMP (db-cAMP) induced increased negativity of Pif to the same extent as did anti-beta 1 integrin antibodies, and in vitro db-cAMP reduced the ability of fibroblasts to contract collagen gels. The latter effect was opposed by alpha-trinositol. The data demonstrate that alpha-trinositol modulates beta 1 integrin function and may do so via intracellular pathways in turn affecting the function and/or cell surface expression of beta 1 integrins and suggest that alpha-trinositol can serve as a tool to study integrin function. Furthermore, the data indicate that the collagen contraction assays may provide important information of the control of Pif in vivo.

Acute Disease↗

PubMed1996

Differential regulation of airway epithelial integrins by growth factors.

The pattern of integrin expression on human airway epithelium changes significantly in injury or inflammation. In particular, two integrins, the fibronectin receptor, alpha 5 beta 1 and the fibronectin/tenascin receptor alpha v beta 6, are expressed at low or undetectable levels in normal airways in vivo but are induced in response to airway epithelial injury. We investigated the effects of various growth factors known to be present in the airways on the expression of constitutively expressed and inducible airway epithelial integrins using flow cytometry. In primary cultures of human airway epithelial cells, transforming growth factor-beta 1 (TGF beta 1) dramatically increased expression of alpha v beta 6 and essentially did not affect the expression of any other integrin, including alpha 5 beta 1. In contrast, epidermal growth factor (EGF) upregulated surface levels of both alpha v beta 6 and alpha 5 beta 1. Together, TGF beta 1 and EGF had an additive effect on alpha v beta 6 and alpha 5 beta 1 expression while increasing levels of alpha 2 beta 1 and decreasing expression of alpha 3 beta 1- and alpha 6-containing integrins. In contrast, the transformed airway epithelial cell line, BEAS-2B, expressed a markedly different repertoire of integrins. Integrin expression on BEAS-2B cells was not affected by any of the growth factors tested in this study. These results demonstrate that, in primary cultures of human airway epithelial cells, the pattern of integrin expression can be dramatically altered by growth factors. The inducible integrins, alpha v beta 6, and alpha 5 beta 1 are most subject to regulation by growth factors and expression of each of these can be differentially regulated. The differential regulation of the two principal fibronectin receptors on airway epithelial cells suggests that they may mediate different cellular responses to fibronectin.

Antibodies, Monoclonal↗

PubMed2006

Beta1 and beta3 integrins cooperate to induce syndecan-4-containing cross-linked actin networks in human trabecular meshwork cells.

PURPOSE: To characterize the molecular composition of cross-linked actin networks (CLANs) and the regulation of their formation by integrins in normal human trabecular meshwork (TM) cells. CLANs have been observed in steroid-treated and glaucomatous TM cells and have been suggested to contribute to decreased outflow facility by altering the contractility of the TM. METHODS: Immunofluorescence microscopy was used to identify molecular components of CLANs and quantitate CLAN formation in HTM cells plated on coverslips coated with various extracellular matrix (ECM) proteins (fibronectin, types I and IV collagen, and vitronectin), vascular cell adhesion molecule (VCAM)-1, or activating antibodies against beta1, beta3, or alpha2beta1 integrins. These integrin antibodies were also used as soluble ligands. RESULTS: CLAN vertices contained the actin-binding proteins alpha-actinin and filamin and the signaling molecules syndecan-4 and PIP2. CLANs lacked Arp3 and cortactin. CLAN formation was dependent on the ECM substrate and was significantly higher on fibronectin and VCAM-1 compared with vitronectin, types I or IV collagen. Adsorbed beta1 integrin antibodies also induced CLANs, whereas adsorbed beta3 or alpha2beta1 integrin antibodies did not. Soluble beta3 integrin antibodies, however, induced CLANs and actually enhanced CLAN formation in cells spread on fibronectin, VCAM-1, type I or type IV collagen, or beta1 integrin antibodies. CONCLUSIONS: CLANs are unique actin-branched networks whose formation can be regulated by beta1 and beta3 integrin signaling pathways. Thus, integrin-mediated signaling events can modulate the organization of the actin cytoskeleton in TM cells and hence could participate in regulating cytoskeletal events previously demonstrated to be involved in controlling outflow facility.

Actin Cytoskeleton↗

PubMed1996

Distinct spatial and temporal functions for PS integrins during Drosophila wing morphogenesis.

At the onset of pupariation in the Drosophila wing, the PS1 and PS2 integrins are expressed preferentially on the dorsal and ventral wing epithelia, respectively. Clonal analysis experiments have indicated that integrins are required to maintain the tight association of the wing surfaces. Surprisingly, we find that even in clones of cells lacking integrins the wing layers become apposed early in metamorphosis. However, following the normal period of wing separation, large integrin mutant clones do not become re-apposed in the pupa, and integrins are not organized in basal plaques in cells opposite a mutant clone. Paradoxically, our experiments indicate that at least one integrin function requires different integrins on the dorsal and ventral wing surfaces, however in some cases both alphaPS subunits can function to some degree on each wing surface. Finally, overexpression of an alphaPS subunit throughout the wing leads to a dominant wing blister phenotype, and the critical period for this phenotype is the beginning of pupariation. These data indicate that integrin requirements in wing morphogenesis can be separated into early (prepupal) and late (pupal) functions. The late function seems to reflect the traditional view of integrins as cell-matrix adhesion proteins. The early requirement, which probably requires dorsoventral segregation of PS1 and PS2, suggests functions for PS1 and PS2 in signaling events that regulate morphogenesis.

Animals↗

PubMed2004

Spatial mapping of integrin interactions and dynamics during cell migration by image correlation microscopy.

Image correlation microscopy methodology was extended and used to determine retrospectively the density, dynamics and interactions of alpha5-integrin in migrating cells. Alpha5-integrin is present in submicroscopic clusters containing 3-4 integrins before it is discernibly organized. The integrin in nascent adhesions, as identified by the presence of paxillin, is approximately 1.4 times more concentrated, approximately 4.5 times more clustered and much less mobile than in surrounding regions. Thus, while integrins are clustered throughout the cell, they differ in nascent adhesions and appear to initiate adhesion formation, despite their lack of visible organization. In more mature adhesions where the integrin is visibly organized there are approximately 900 integrins microm(-2) (about fivefold higher than surrounding regions). Interestingly, alpha5-integrin and alpha-actinin, but not paxillin, reside in a complex throughout the cell, where they diffuse and flow together, even in regions where they are not organized. During adhesion disassembly some integrins diffuse away slowly, alpha-actinin undergoes a directed movement at speeds similar to actin retrograde flow (0.29 microm min(-1)), while all of the paxillin diffuses away rapidly.

Actinin↗

PubMed2002

The involvement of lipid rafts in the regulation of integrin function.

Integrin activity on cells such as T lymphocytes is tightly controlled. Here we demonstrate a key role for lipid rafts in regulating integrin function. Without stimulation integrin LFA-1 is excluded from lipid rafts, but following activation LFA-1 is mobilised to the lipid raft compartment. An LFA-1 construct from which the I domain has been deleted mimics activated integrin and is constitutively found in lipid rafts. This correlation between integrin activation and raft localisation extends to a second integrin, alpha4beta1, and the clustering of alpha4beta1 is also raft dependent. Both LFA-1 and alpha4beta1-mediated adhesion is dependent upon intact lipid rafts providing proof of the functional relevance of the lipid raft localisation. Finally we find that non-raft integrins are excluded from the rafts by cytoskeletal constraints. The presence of integrin in lipid rafts under stimulating conditions that activate these receptors strongly indicates that the rafts have a key role in positively regulating integrin activity.

Bridged Bicyclo Compounds, Heterocyclic↗

PubMed1999

B1 integrin activation inhibits in vitro tube formation: effects on cell migration, vacuole coalescence and lumen formation.

Human endothelial cells (EC), when plated onto gels of extracellular matrix proteins such as Matrigel or collagen form capillary tubes in a process thought to mimic angiogenesis. We have shown previously that the extent of tube formation and the phenotype of the lumen are regulated by integrins (Gamble et al 1993) and lumen formation occurs through a process of vacuolization, coalescence and ultimate directional fusion of these vacuoles with the plasma membrane (Meyer 1997 et al). We now show here that activation of beta1 integrins on endothelial cells inhibits tube formation. On collagen gels, endothelial cells treated with 31 activating antibody 8A2 failed to migrate into the gel and tube formation was inhibited. Although several integrins mediate EC attachment to collagen alpha2beta1 is the chief determinant of EC behaviour since a blocking antibody to (alpha2beta1 reversed the effect of 8A2. On Matrigel tube formation was also inhibited by 8A2 treatment although cell alignment and sprout formation was still evident. Electron microscopy revealed the organisation of normal numbers of cells into solid sprouts and the formation of small intracellular vacuoles suggesting that initial stages of tube formation including cell migration were unaffected. However, beta1 integrin activation inhibited the coalescence of these small vacuoles into larger vacuoles, the recruitment of more cells into the sprout and the subsequent formation of mature lumen. The inhibition of capillary tube formation by beta1 activation was time dependent and long lasting. The critical time for activation of the beta1 integrin was the initial 1-2h after plating in order to inhibit tube formation although once activated, the beta1 mediated inhibition on Matrigel was still evident 4 days later. Our results suggest that beta1 integrins are critical in capillary tube formation in at least two phases. beta1 integrins are essential for migration of EC through collagen gels. Independently, beta1 integrins, although not involved in initial vacuole formation, are involved in the process of vacuole coalescence and subsequent lumen formation since beta1 integrin activation inhibits these processes.

Antibodies, Monoclonal↗

PubMed1995

Calcium-induced changes in distribution and solubility of cadherins, integrins and their associated cytoplasmic proteins in human keratinocytes.

Studies with cultured human epidermal keratinocytes have shown that stratification, the movement of differentiating cells out of the basal layer, involves changes in cell-extracellular matrix and cell-cell adhesiveness mediated by receptors of the integrin and cadherin families, respectively. Keratinocytes normally lose their integrins when they initiate terminal differentiation. However, when stratification is inhibited by a low concentration of calcium ions in the medium (0.1 mM) or by addition of antibodies to P- and E-cadherin in standard medium (1.8 mM calcium ions), differentiating, involucrin-positive, cells continue to express functional integrins. In order to investigate the mechanism by which cadherins may regulate integrin expression, we have examined the distribution and detergent solubility of the receptors and associated cytoplasmic proteins in keratinocytes grown as a monolayer in low calcium medium or transferred to standard medium to induce stratification. Within 1 hour of raising the concentration of calcium ions, integrins, cadherins, alpha-catenin, beta-catenin, plakoglobin, vinculin and alpha-actinin appeared to accumulate at cell-cell borders, whereas the focal contact proteins, paxillin and talin, did not. The change in distribution was correlated with decreased solubility in 0.5% Triton X-100 of some of the proteins examined, but the integrins, alpha-actinin, paxillin and talin remained completely soluble. Addition of cytochalasin D inhibited both the redistribution of proteins and subsequent stratification of involucrin-positive cells. Cycloheximide treatment allowed protein redistribution and stratification, but involucrin-positive cells continued to express integrins. These results suggest that stratification requires the interactions of cadherins and integrins with the actin cytoskeleton and that the selective loss of integrins from differentiating cells requires de novo protein synthesis.

Antibodies, Monoclonal↗

PubMed2001

Resting murine neutrophils express functional alpha 4 integrins that signal through Src family kinases.

There is mounting evidence that alpha(4) (CD49d) integrins are involved in neutrophil recruitment and function during inflammatory responses. We report that all resting murine neutrophils derived from bone marrow or peripheral blood express easily detectable levels of alpha(4) integrins on their surface. These alpha(4) integrins were functional, as demonstrated by stimulation of respiratory burst when neutrophils adhered to surfaces coated with the murine vascular cell adhesion molecule-1 (mVCAM-1). Adhesion occurred via alpha(4) integrins, as preincubation of neutrophils with an anti-alpha(4)-specific Ab inhibited attachment to mVCAM-1. Direct cross-linking of the alpha(4) integrin subunit by surface-bound mAbs also elicited superoxide release and release of the secondary granule marker, lactoferrin. The functional responses that occurred downstream of alpha(4) integrin cross-linking required signaling by Src family kinases. Neutrophils derived from hck(-/-)fgr(-/-)lyn(-/-) triple-knockout or hck(-/-)fgr(-/-) double-knockout mice failed to undergo respiratory burst when plated on mVCAM-1. Triple mutant neutrophils were also defective in release of both superoxide and lactoferrin when plated on surfaces coated with mAbs directed against alpha(4). Correlated with impaired alpha(4)-induced functional responses, triple-mutant neutrophils also failed to spread and tightly adhere to anti-alpha(4) mAb-coated surfaces. This is the first direct evidence that functional alpha(4) integrins are expressed by murine PMNs, and that these surface molecules can mediate cellular responses such as tight adhesion, spreading, sustained respiratory burst, and specific granule release in vitro. Moreover the alpha(4) integrins, like all other integrins tested, use the Src family kinases to transduce intracellular signals.

Animals↗

PubMed2003

Threonine phosphorylation sites in the beta 2 and beta 7 leukocyte integrin polypeptides.

The cytoplasmic domains of integrins play a key role in a variety of integrin-mediated events including adhesion, migration, and signaling. The molecular mechanisms that enhance integrin function are still incompletely understood. Because protein kinases are known to be involved in the signaling and the activation of integrins, the role of phosphorylation has been studied by several groups. The beta(2) leukocyte integrin subunit has previously been shown to become phosphorylated in leukocytes on cytoplasmic serine and functionally important threonine residues. We have now mapped the phosphorylated threonine residues in activated T cells. After phorbol ester stimulation, all three threonine residues (758-760) of the threonine triplet became phosphorylated but only two at a time. CD3 stimulation leads to a strong threonine phosphorylation of the beta(2) integrin, but differed from phorbol ester activation in that phosphorylation occurred only on threonine 758. The other leukocyte-specific integrin, beta(7), has also been shown to need the cytoplasmic domain and leukocyte-specific signal transduction elements for integrin activation. Cell activation with phorbol ester, and interestingly, through the TCR-CD3 complex, caused beta(7) integrin binding to VCAM-1. Additionally, cell activation led to increased phosphorylation of the beta(7) subunit, and phosphoamino acid analysis revealed that threonine residues became phosphorylated after cell activation. Sequence analysis by manual radiosequencing by Edman degradation established that threonine phosphorylation occurred in the same threonine triplet as in beta(2) phosphorylation.

Alanine↗

PubMed1999

Integrin alpha3beta1 expressed by human colon cancer cells is a major carrier of oncodevelopmental carbohydrate epitopes.

Oncodevelopmental carbohydrate epitopes are a common feature of human colorectal carcinoma, yet their biological significance remains unclear. We have shown previously that monoclonal antibody (MAb) 3A7, which recognizes a determinant on type 2 chain blood group A and B oligosaccharides, detects oncodevelopmental changes in azoxymethane-induced rat colon tumors and some human colon cancer cell lines. (Laferté S et al. [19951 J. Cell. Biochem. 57:101-119). In this study, we set out to purify gp140, the major glycoprotein carrier of the 3A7 epitope expressed by human colon cancer cells, as a first step towards elucidating the contribution of the 3A7 epitope and its major glycoprotein carrier to colon cancer progression. To this end, gp140 was purified from HT29 cells and used for the preparation of polypeptide-specific monoclonal antibodies. Five monoclonal antibodies (7A8, 7B11, 8C7, 8H7, and 11D4) immunoprecipitated a 3A7-immunoreactive glycoprotein complex of 140 kDa which was subsequently identified by partial protein sequencing as alpha3beta1 integrin. Flow cytometric analysis of Chinese hamster ovary (CHO) cells expressing different human integrin chains revealed that MAbs 7A8 and 7B11 detect the alpha3 integrin subunit whereas MAbs 8C7 and 8H7 detect the beta1 integrin subunit. MAb 11D4, which did not bind to any of the CHO transfectants, detected type 2 chain blood group A determinant. Flow cytometric analysis of a panel of human colon carcinoma cell lines obtained from blood group A, AB, or B individuals revealed a direct correlation between cell-surface expression of the 3A7 epitope and alpha3 integrin subunit, suggesting that alpha3beta1 integrin is a preferred target of the 3A7 epitope in colon cancer cells. Using lectins and glycosidases to examine further the carbohydrate structure of alpha3beta1 integrin, we demonstrated that it is a sialoglycoprotein containing both N- and O-linked oligosaccharides. In addition, both alpha3 and beta1 integrin subunits express beta1-6 branched Asn-linked oligosaccharides and short poly-N-acetyllactosamine units (Galbeta1-4GlcNAc-R; n < or = 3), glycans previously implicated in cancer metastasis.Thus, alpha3beta1 integrin expressed by human colon carcinoma cells is a major carrier of oncodevelopmental carbohydrate epitopes whose presence may modulate tumor cell adhesion, migration, and/or invasion.

Animals↗

PubMed2006

[Regulatory effect of integrin alpha5 and beta1 on proliferation inhibition of K562 cells induced by interferon alpha-2b].

BACKGROUND & OBJECTIVE: Integrin beta1 can inhibit the proliferation of chronic myelocytic leukemia (CML) ph+ cells. The dysfunction of integrin beta1 might accelerate the growth of CML ph+ cells. This study was to explore the effects of integrin alpha5 and beta1 on the proliferation inhibition of K562 cells induced by IFNalpha-2b. METHODS: The expression indexes of integrin alpha5 and beta1 on K562 cells, the binding capability of K562 cells to fibronectin (FN), and K562 cell-FN binding blocking induced by integrin alpha5 and beta1 antibodies were evaluated by flow cytometry (FCM). The viability of K562 cells, treated with IFNalpha-2b (10,000 u/ml), was observed by MTT assay. The mRNA level of focal adhesion kinase (FAK) in K562 cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) 48 h after treatment of interferon alpha-2b (IFNalpha-2b). RESULTS: The positive rates of integrin alpha5 and beta1 were significantly higher on K562 cells than on bone marrow mononuclear cells from healthy donors [(97.59+/-1.04)% vs. (64.05+/-2.38)%, (99.24+/-0.52)% vs. (72.40+/-3.56)%, P<0.05). IFNalpha-2b could not change the expression of integrin alpha5 and beta1 on K562 cells, but improved the binding capability of K562 cells to FN, which could be blocked by anti-alpha5 and/or anti-beta1 antibodies. IFNalpha-2b enhanced the expression of FAK gene, and inhibited the proliferation of K562 cells. The anti-alpha5 and anti-beta1 antibodies improved the inhibitory effect of IFNalpha-2b on the proliferation of K562 cells, and blocked IFNalpha-2b-induced increase of FAK gene expression. CONCLUSION: IFNalpha-2b could inhibit the proliferation of K562 cells through restoring the function of integrin alpha5 and beta1, enhancing binding capability of integrin alpha5 and beta1 to FN, and up-regulating FAK gene expression.

Antibodies↗

PubMed2006

Right on the spot. Chemokine triggering of integrin-mediated arrest of rolling leukocytes.

The arrest of rolling leukocytes on various target vascular beds is crucial for their recruitment at inflammatory sites and secondary lymphoid tissues. Leukocyte arrest is predominantly mediated by integrins interacting with either constitutive or inducible endothelial ligands. Integrins are cytoskeletally regulated heterodimers maintained in largely low affinity conformational states on circulating leukocytes. For arrest to occur, the affinity of integrin heterodimers must be enhanced in situ upon leukocyte encounter with proper endothelial-displayed chemokines or chemoattractants which bind and signal through specific G-protein coupled receptors (GPCRs) on the leukocyte surface. Recent studies suggest that this integrin activation involves rapid conformational alterations locally induced at confined leukocyte-endothelial contact sites. Following binding, integrin microclustering reinforced by associations with the cortical actin cytoskeleton further enhances integrin-mediated adhesiveness under shear stress. These events are controlled by complex signaling events, involving a series of small GTPases, as well as protein and lipid kinases which are triggered by chemokine bound GPCRs. To rapidly mediate this specialized function, subsets of signaling proteins and their specific targets are thought to preexist in pre-assembled multi-molecular complexes or signalosomes. Recent in vitro dissection of chemokine-triggered integrin activation on lymphocytes and neutrophils suggests that these signalosomes may vary both in composition and mode of activity between different immune cell types and distinct integrins. We review in this article recent findings on key elements implicated in chemokine triggering of integrin activation on rolling leukocytes, and discuss the possible existence of preformed proadhesive signaling networks in different subsets of leukocytes.

Animals↗

PubMed1994

Regulation of epithelial cell surface polarity reversal by beta 1 integrins.

The role of extracellular matrix in the regulation of epithelial cell surface polarity development was studied using MDCK cells. Previous work has demonstrated that MDCK cells cultured in suspension form epithelial cysts having polarized cell surface distributions of several membrane proteins. When MDCK suspension cysts are incubated within collagen gel, a dynamic epithelial membrane remodeling occurs that is accompanied by the reversal of cell surface polarity (Wang et al., 1990b, J. Cell Sci. 95, 153-165), suggesting that extracellular matrix is important in the modulation of epithelial polarity development. To determine if members of the integrin receptor family were involved, MDCK cyst binding studies were done utilizing antifunctional monoclonal antibodies (AIIB2 and AJ2) against the beta 1 integrin subunit. These antibodies inhibited cyst binding to type I collagen, type IV collagen and laminin, providing evidence that functional beta 1 integrin heterodimers were present on the cyst outer membrane. Integrin localization on suspension cysts demonstrated that the alpha 2, alpha 3 and alpha 6 integrin subunits had a non-polarized cell surface distribution and were localized to both the apical and basolateral membranes. Interestingly, immunofluorescence microscopy determined that the beta 1 subunit had a polarized, basolateral membrane distribution although cyst binding studies using inhibitory monoclonal antibodies suggested that functional beta 1 subunits were present on the cyst outer membrane. After incubation of suspension cysts in collagen gel for 8 hours, the beta 1 integrin subunit was detected on the outer membrane, suggesting that the formation of additional integrin alpha/beta heterodimers could be involved in epithelial remodeling. To establish the role of beta 1 integrins in polarity reversal, experiments were done on cysts incubated in collagen gel. After 6 hours in collagen gel, considerable membrane remodeling had occurred as determined by a reduction in outer membrane microvilli. However, the presence of monoclonal antibody AIIB2 inhibited membrane remodeling by preventing both microvillar loss and the endocytosis of the apical membrane glycoprotein gp135. These results provide strong evidence that members of the beta 1 integrin family are involved in the regulation of epithelial polarity reversal, and demonstrate that MDCK cysts constitute an excellent model system for studying the role of cell-extracellular matrix interactions in the regulation of epithelial plasticity and cell surface polarity development.

Animals↗

PubMed1994

Transforming growth factor beta 1 suppresses transformation in hepatocytes by regulating alpha 1 beta 1 integrin expression.

We previously reported that (a) treatment of the ras-transformed hepatocyte cell line NR4 with transforming growth factor (TGF) beta 1 suppresses many characteristics associated with the transformed phenotype including altered morphology, actin cytoskeleton reorganization, and anchorage-independent growth such that the cells more closely resemble the immortalized CWSV1 parent cell line; (b) transformed NR4 cells expressed significantly less alpha 1 integrin RNA than the immortalized CWSV1 cells; and (c) TGF-beta 1 treatment of NR4 cells stimulated the expression of alpha 1 and beta 1 integrin RNAs. In this report, the role of the alpha 1 beta 1 integrin in TGF-beta 1-mediated suppression of the ras-transformed phenotype was investigated. We determined that (a) the cell surface integrin that increased in response to TGF-beta 1 treatment of NR4 cells was alpha 1 integrin; (b) TGF-beta 1 altered the ability of NR4 cells to attach to collagen and laminin, the extracellular matrix components that interact with the alpha 1 beta 1 integrin receptor; (c) TGF-beta 1 treatment resulted in relocalization of the alpha 1 integrin on the NR4 cell surface; and (d) TGF-beta 1-mediated inhibition of anchorage-independent growth was blocked by the presence of alpha 1 integrin antibody. A cell line that overexpresses alpha 1 integrin was derived from NR4 cells; characterization of these cells indicated that they continued to express H-ras RNA but were less transformed than the parent NR4 cells. Specifically, they had an altered morphology, an organized actin cytoskeleton, and reduced ability to demonstrate anchorage-independent growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1996

Integrin-mediated interactions between primary/T-sv40 immortalized human glomerular epithelial cells and type IV collagen.

The use of human glomerular epithelial cells (HGEC) in research has been severely restricted by several obstacles, which have been circumvented by the generation of T-SV40 immortalized human visceral glomerular epithelial cells (Delarue et al, 1991). In this work, we compared the primary and immortalized HGEC for expression of integrin and some nonintegrin surface receptors. We also studied the adhesion of both types of HGEC to glomerular basement membrane (GBM), type IV collagen (tIV), and its major noncollagenous NC1 domain. The integrins mediating adhesion of HGEC to tIV were also examined. Expression of integrin and some nonintegrin cell surface receptors was analyzed by flow cytometry. Adhesion to GBM, tIV, and its major noncollagenous NC1 domain was studied by direct solid phase cell adhesion assays. Identification of integrins mediating adhesion of HGEC to tIV was achieved by inhibition of cell adhesion using monoclonal antibodies to integrin subunits. The primary and immortalized HGEC share phenotypic characteristics, and alpha3beta1 appeared to be the major integrin present on both HGEC types. The kinetics of binding to GBM, tIV, and its noncollagenous NCI domain were similar in both the primary and immortalized HGEC, although the latter displayed a somewhat weaker binding. Both the primary and immortalized HGEC displayed significantly better adhesion to NC1-alpha3 compared with NC1-alpha1, alpha3beta1 appears to be the major integrin mediating the adhesion of HGEC to tIV. Our studies suggest that alpha3beta1 is the major integrin present on HGEC. This has been confirmed by flow cytometric analysis. In addition, we demonstrated a functional role for this integrin in mediating attachment of HGEC to tIV. Our data also demonstrate a preference in binding of HGEC to alpha3 chains of NC1 compared with alpha1 chains of NC1. These findings were seen in both the primary and immortalized HGEC. The T-SV40 immortalized HGEC can therefore serve as a very useful tool to study glomerular visceral cell biology.

Animals↗

PubMed1998

Alterations in the expression of alpha6beta4 integrin and p21/WAF1/Cip1 in N-nitrosomethylbenzylamine-induced rat esophageal tumorigenesis.

Integrin alpha6beta4 is altered in many neoplastic cells, but no data exist to show this happens in esophageal neoplasms. To examine the expression of this integrin in rat esophageal tumorigenesis induced by N-nitrosomethylbenzylamine (NMBA), (alpha6 and beta4 expression was evaluated in normal esophageal epithelium, in NMBA-induced preneoplastic lesions, and in papillomas by quantitative reverse transcription (RT)-polymerase chain reaction (PCR) and immunohistochemical analysis. Because the 34 subunit of this integrin has been found to cause cell-cycle arrest by the induction of p21/WAF1/Cip1, the expression of p21/WAF1/Cip1 was also analyzed by RT-PCR. Compared with the levels in normal epithelium, the alpha6A, alpha6B, and beta4 integrin levels in esophageal papillomas were 1.9-, 2.2-, and 2.1-fold lower, respectively. RT-PCR analysis showed no significant differences in integrin levels between preneoplastic and normal samples, and northern blot analysis of the beta4 integrin produced results in agreement with the RT-PCR results. The p21/WAF1/Cip1 level was decreased 1.6-fold in preneoplastic tissues and 3.1-fold in papilloma samples when compared with the mRNA levels in normal epithelium. Immunostaining showed that alpha6beta4 integrin was localized at the basolateral surface of the basal cells in normal esophageal epithelium. In preneoplastic lesions, however, the expression of this integrin was not polarized and was expressed in basal cells as well as in suprabasal cells. Beta4 expression was significantly reduced and alpha6A expression was decreased and delocalized in papillomas. These findings suggest that alteration in alpha6beta4 integrin and p21/WAF1/Cip1 expression may be an important biomarker for tumor progression in NMBA-induced rat esophageal tumorigenesis.

Animals↗