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PubMed1993

Regulation of keratinocyte terminal differentiation by integrin-extracellular matrix interactions.

Suspension-induced terminal differentiation of human epidermal keratinocytes can be inhibited by fibronectin through binding to the alpha 5 beta 1 integrin. We have investigated the effect of fibronectin on expression of integrins and proteins of the actin cytoskeleton and have explored the nature of the differentiation stimulus by testing different combinations of anti-integrin monoclonal antibodies or extracellular matrix proteins in the suspension assay. Fibronectin prolonged cell surface expression of beta 1 integrins but did not overcome the inhibition of intracellular transport of integrins that occurs when keratinocytes are placed in suspension. Fibronectin did not prevent the suspension-induced decline in the level of mRNAs encoding the beta 1 integrin subunit, actin, filamin and alpha-actinin; furthermore, the inhibition of terminal differentiation did not depend on the state of assembly of microfilaments or microtubules. Terminal differentiation could be partially inhibited by an adhesion-blocking monoclonal antibody to the beta 1 integrin subunit or by a combination of adhesion blocking antibodies recognising the alpha subunits that associate with beta 1 (alpha 2, alpha 3 and alpha 5). Although laminin and type IV collagen do not inhibit terminal differentiation individually, they were inhibitory when added to cells in combination with a low concentration of fibronectin. We conclude that the proportion of keratinocyte beta 1 integrins occupied by ligand can regulate the initiation of terminal differentiation independently of the state of assembly of the actin cytoskeleton.

Actins↗

PubMed1995

Cellular growth and survival are mediated by beta 1 integrins in normal human breast epithelium but not in breast carcinoma.

We previously established a rapid three-dimensional assay for discrimination of normal and malignant human breast epithelial cells using a laminin-rich reconstituted basement membrane. In this assay, normal epithelial cells differentiate into well-organized acinar structures whereas tumor cells fail to recapitulate this process and produce large, disordered colonies. The data suggest that breast acinar morphogenesis and differentiation is regulated by cell-extra-cellular matrix (ECM) interactions and that these interactions are altered in malignancy. Here, we investigated the role of ECM receptors (integrins) in these processes and report on the expression and function of potential laminin receptors in normal and tumorigenic breast epithelial cells. Immunocytochemical analysis showed that normal and carcinoma cells in a three-dimensional substratum express profiles of integrins similar to normal and malignant breast tissues in situ. Normal cells express alpha 1, alpha 2, alpha 3, alpha 6, beta 1 and beta 4 integrin subunits, whereas breast carcinoma cells show variable losses, disordered expression, or downregulation of these subunits. Function-blocking experiments using inhibitory anti-integrin subunit antibodies showed a > 5-fold inhibition of the formation of acinar structures by normal cells in the presence of either anti-beta 1 or anti-alpha 3 antibodies, whereas anti-alpha 2 or -alpha 6 had little or no effect. In experiments where collagen type I gels were used instead of basement membrane, acinar morphogenesis was blocked by anti-beta 1 and -alpha 2 antibodies but not by anti-alpha 3. These data suggest a specificity of integrin utilization dependent on the ECM ligands encountered by the cell. The interruption of normal acinar morphogenesis by anti-integrin antibodies was associated with an inhibition of cell growth and induction of apoptosis. Function-blocking antibodies had no inhibitory effect on the rate of tumor cell growth, survival or capacity to form colonies. Thus under our culture conditions breast acinar formation is at least a two-step process involving beta 1-integrin-dependent cellular growth followed by polarization of the cells into organized structures. The regulation of this pathway appears to be impaired or lost in the tumor cells, suggesting that tumor colony formation occurs by independent mechanisms and that loss of proper integrin-mediated cell-ECM interaction may be critical to breast tumor formation.

Adenocarcinoma↗

PubMed1995

A novel role for alpha 3 beta 1 integrins in extracellular matrix assembly.

To study the biological role of alpha 3 beta 1 integrins in cell adhesion, migration, and in the deposition of extracellular matrix, we stably expressed the human alpha 3 integrin subunit in the alpha 4, alpha 5 integrin deficient CHO cell line B2. The expression of alpha 3 beta 1 integrins enhanced cell adhesion on entactin (also known as nidogen), but not on fibronectin. Using recombinant GST-fusion proteins that span the entire length of the entactin molecule, we located cell adhesive activity to the G2 domain of entactin. These results suggest that the alpha 3 beta 1 integrin functions as an adhesion receptor interacting with the G2 domain of entactin. On the other hand, the expression of alpha 3 beta 1 integrins did not confer the ability to migrate on entactin. Strikingly, the expression of alpha 3 beta 1 dramatically increased the deposition of entactin and fibronectin into the pericellular matrix. This was accompanied by increased binding activity of the 29 kDa amino-terminal domain of fibronectin. Thus, similar to alpha 5 beta 1 integrins, alpha 3 beta 1 integrins can play an important role in modulating the assembly of pericellular matrices. However, unlike fibronectin deposition supported by alpha 5 beta 1, alpha 3 beta 1 supported fibronectin deposition into pericellular matrix was not inhibited by antibodies binding to the RGD containing cell adhesion domain of fibronectin, demonstrating that the two processes are mechanistically distinct. The role of alpha 3 beta 1 in pericellular matrix assembly potentially implicates this receptor in the assembly and/or recognition of entactin-containing pericellular matrices, an observation consistent with its apparent role in the renal glomerulus of the mammalian kidney.

Amino Acid Sequence↗

PubMed2006

Alpha9beta1: a novel osteoclast integrin that regulates osteoclast formation and function.

UNLABELLED: We identified a previously unknown integrin, alpha(9)beta(1), on OCLs and their precursors. Antibody to alpha(9) inhibited OCL formation in human marrow cultures, and OCLs from alpha(9) knockout mice had a defect in actin ring reorganization and an impaired bone resorption capacity. INTRODUCTION: Integrins play important roles in osteoclast (OCL) formation and function. Mature OCLs mainly express alpha(v)beta(3) integrin, a heterodimer adhesion receptor that has been implicated in osteoclastic bone resorption. We identified ADAM8, a disintegrin and metalloproteinase, as a novel stimulator of OCL differentiation and showed that the disintegrin domain of ADAM8 mediated its effects on OCL formation. Because the disintegrin domain of ADAM8 does not bind Arg-Gly-Asp (RGD) sequences, we determined which integrin bound ADAM8 and characterized its role in OCL formation and activity. MATERIALS AND METHODS: Chinese hamster ovary cells (CHO) expressing different integrin subunits were tested for their capacity to bind the disintegrin domain of ADAM8. Mouse or human bone marrow cells and purified OCL precursors were tested for alpha(9)beta(1) integrin expression by Western blot, immunocytochemistry, and real-time RT-PCR. A monoclonal antibody to human alpha(9) was used to block alpha(9)beta(1) on OCL precursors stimulated by 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] or RANKL. Vertebrae of 7-day-old alpha(9)(-/-) mice and wildtype (WT) littermates were compared using bone histomorphometry and 3D microCT analysis. RESULTS: Alpha(9) integrin was expressed by mouse and human bone marrow-derived OCLs and their precursors. Importantly, the anti-alpha(9) antibody inhibited human OCL formation stimulated by 1alpha,25(OH)(2)D(3) or RANKL dose-dependently. Furthermore, analysis of OCLs formed in marrow cultures from alpha(9)(-/-) mice showed that the OCLs formed were more contracted and formed significantly less bone resorption pits on dentin slices. Histologic analysis of alpha(9)(-/-) vertebrae showed thickened trabecular regions and retained cartilage within vertebral bodies of alpha(9)(-/-) mice. 3D microCT analysis of alpha(9)(-/-) vertebrae also showed a significant increase in trabecular bone volume/total tissue volume and a tendency for decreased trabecular separation compared with WT mice. CONCLUSIONS: These results support a previously unknown role for alpha(9)beta(1) integrin in OCL formation and function.

ADAM Proteins↗

PubMed2004

A novel cAMP-dependent pathway activates neuronal integrin function in retinal neurons.

Retinal neurons lose the ability to attach to and extend neurites on substrata of laminin-1 (LN-1) during late embryogenesis, in a time frame that corresponds to target innervation. Although this developmental loss correlates with a modest downregulation of integrin expression, we have shown previously that these neurons use the same laminin-binding integrins for outgrowth on other laminin isoforms to which responsivity has not been lost (Ivins et al., 1998), suggesting that integrin functional states may be a critical point of regulation. Consistent with this view, expression of an activated mutant of R-ras, an activator of integrin function, restores integrin-dependent outgrowth of late embryonic retinal neurons on LN-1 (Ivins et al., 2000). Because cyclic nucleotides have been implicated in the regulation of integrin function in non-neuronal cells, as well as in the regulation of growth cone responses to various axon growth inhibitors, we asked whether raising cAMP levels in late embryonic retinal neurons could activate neuronal integrin function and restore neurite outgrowth on LN-1. We find that, similar to R-ras expression, raising cAMP levels in these neurons promotes alpha6beta1 integrin-dependent neurite outgrowth. Surprisingly, these effects of cAMP are independent of protein kinase A and the EPAC (exchange protein directly activated by cAMP)/Rap pathway and suggest the existence of a novel cAMP-dependent mechanism.

Animals↗

PubMed2006

Altered membrane dynamics of quantum dot-conjugated integrins during osteogenic differentiation of human bone marrow derived progenitor cells.

Functionalized quantum dots offer several advantages for tracking the motion of individual molecules on the cell surface, including selective binding, precise optical identification of cell surface molecules, and detailed examination of the molecular motion without photobleaching. We have used quantum dots conjugated with integrin antibodies and performed studies to quantitatively demonstrate changes in the integrin dynamics during osteogenic differentiation of human bone marrow derived progenitor cells (BMPCs). Consistent with the unusually strong BMPC adhesion previously observed, integrins on the surface of undifferentiated BMPC were found in clusters and the lateral diffusion was slow (e.g., approximately 10(-11) cm2/s). At times as early as those after a 3-day incubation in the osteogenic differentiation media, the integrin diffusion coefficients increased by an order of magnitude, and the integrin dynamics became indistinguishable from that measured on the surface of terminally differentiated human osteoblasts. Furthermore, microfilaments in BMPCs consisted of atypically thick bundles of stress fibers that were responsible for restricting the integrin lateral mobility. Studies using laser optical tweezers showed that, unlike fully differentiated osteoblasts, the BMPC cytoskeleton is weakly associated with its cell membrane. Based on these findings, it appears likely that the altered integrin dynamics is correlated with BMPC differentiation and that the integrin lateral mobility is restricted by direct links to microfilaments.

Actin Cytoskeleton↗

PubMed2005

Immunohistochemical localization of beta1 and beta4 integrins in mouse endometrium during implantation and early pregnancy.

Implantation presents the remarkable synchronisation between the development of embryo and differentiation of endometrium. Cell-cell adhesion is an important phenomenon taking place during blastocyst implantation in uterine membrane. We think that the investigation of existence and the level of integrins in women can be a guide for treatment of infertility. Our purpose in this study was to show expression beta1 and beta4 integrins on gestational days 4, 6, 12 by immunohistochemical methods and to investigate whether beta4 integrin is a useful marker for receptivity. beta1 and beta4 integrin were exhibited on surface epithelium on gestational day 4. On the other hand, strong beta4 immunoreactivity was detected on surface epithelium and glandular cells on gestational day 12 but no beta1 reactivity was present in the surface epithelium and glandular cells on day 12. In conclusion, both beta1 and beta4 integrins may have a role in implantation process because positive immunoreactivity was seen on apical membrane of surface epithelium on day 4 when implantation occurred. The localization to apical pole of surface epithelium suggest a role for beta1, beta4 integrins in initial embryo and endometrium interaction. It does not seem that beta1 integrin has a role supporting pregnancy since expression of beta4 on surface epithelium and glandular epithelium disappeared on day 12. beta4 integrin expression increasing on day 12 of pregnancy leads us to think a possible functional role supporting pregnancy.

Animals↗

PubMed1999

Alpha1beta1 integrin-mediated collagen matrix remodeling by rat mesangial cells is differentially regulated by transforming growth factor-beta and platelet-derived growth factor-BB.

Pathologic remodeling of mesangial matrix after glomerular injury is the central biologic feature of glomerular scarring (sclerosis). Transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF)-BB have been implicated in the development of glomerular scarring in rat and human glomerulonephritis. To clarify molecular and cellular mechanisms involved in abnormal mesangial remodeling, this study focused on the role of alpha1beta1 integrin, a collagen/laminin receptor, in rat mesangial cells, using collagen gel contraction as an experimental model of in vivo collagen matrix remodeling and scar formation. In addition, the influence of TGF-beta and PDGF-BB on mesangial cell (MC)-mediated collagen gel contraction in association with the alpha1beta1 integrin expression was evaluated. Integrin function blocking studies using anti-alpha1, beta1 subunit antibodies indicated that MC-alpha1beta1 integrin is essentially required not only for collagen-dependent adhesion/migration, but also for gel contraction. Protein synthesis and mRNA analysis experiments demonstrated that TGF-beta, but not PDGF-BB, increases the expression of alpha1beta1 integrin in mesangial cells cultured on plastic surface and in collagen gels. The upregulation of alpha1beta1 integrin expression by TGF-beta correlated with increases in gel contraction and collagen-dependent adhesion but not migration of mesangial cells. On the other hand, PDGF-BB enhanced MC-mediated gel contraction and migration without affecting cell adhesion to collagen I. Growth factor-induced collagen-dependent adhesion, migration, and gel contraction were significantly attenuated by incubation with anti-alpha1, beta1 subunit antibodies. Thus, these data indicate that alpha1beta1 integrin-mediated collagen matrix remodeling can be modulated by TGF-beta and PDGF-BB via different mechanisms. Alpha1 integrin-mediated mesangial matrix remodeling induced by TGF-beta or PDGF-BB may be a pathogenic mechanism leading to glomerular scarring.

Animals↗

PubMed2006

Blockade of beta1 integrin-laminin-5 interaction affects spreading and insulin secretion of rat beta-cells attached on extracellular matrix.

When attached on a matrix produced by a rat bladder carcinoma cell line (804G matrix), rat pancreatic beta-cells spread in response to glucose and secrete more insulin compared with cells attached on poly-l-lysine. The aim of this study was to determine whether laminin-5 and its corresponding cell receptor beta1 integrin are implicated in these phenomena. By using specific blocking antibodies, we demonstrated that laminin-5 is the component present in 804G matrix responsible for the effect of 804G matrix on beta-cell function and spreading. When expression of two well-known laminin-5 ligands, beta1 and beta4 integrin, was assessed by Western blot and RT-PCR, only the beta1 integrin was detected in beta-cells. Anti-beta1 integrin antibody reduced the spreading of beta-cells on 804G matrix. Blockade of the interaction between beta1 integrins and laminin-5 resulted in a reduction in glucose-stimulated insulin secretion. Blocking anti-beta1 integrin antibody also inhibited focal adhesion kinase phosphorylation induced by 804G matrix. In conclusion, anti-beta1 integrin and -laminin-5 antibodies interfere with spreading of beta-cells, resulting in decreased insulin secretion in response to glucose. Our findings indicate that outside-in signaling via engagement of beta1 integrins by laminin-5 is an important component of normal beta-cell function.

Animals↗

PubMed1993

Evaluation of integrin molecules involved in substrate adhesion.

Integrins were cross-linked to their extracellular matrix ligands using non-penetrating chemical cross-linkers. This procedure did not disturb the distribution of integrin in the adhesion structure and adhesion plaque integrin staining remained even when the cultures were extracted with ionic detergents. 80-90% of the beta 1 integrin in the cross-linked culture was extracted with RIPA buffer and the remaining 10-20% was recovered following reversal of the cross-linking. This separated two distinct integrin pools, one which can be cross-linked to substrate bound extracellular matrix and one which is not. The specificity of this procedure for cross-linking of integrins involved in substrate adhesion was demonstrated using NIH 3T3 cells which express both alpha 5 beta 1 and alpha 6 beta 1 integrins. alpha 6 was cross-linked only in cells plated on laminin whereas alpha 5 was cross-linked when fibronectin was present. Using antisera directed to the cytoplasmic domains of either alpha 5 or beta 1 integrin, it was demonstrated that these domains can be blocked in the intact cell but the blocking can be removed using ionic detergent extraction after chemical cross-linking. The extracellular matrix associated with the substrate surface but not that associated with the media exposed surface is both cross-linked and retained on the plastic dish following cross-linking.

3T3 Cells↗

PubMed1999

Reduced substratum adhesion and decreased expressions of 1 and 4 integrins in human breast cancer cells with a property of anchorage-independent growth.

The proliferation of normal human breast epithelial (HBE) cells was found to be dependent on their attachment on a solid culture substratum, whereas human breast cancer MCF-7 cells could grow in soft agar. The attachment of the dissociated single HBE cells to the substratum was complete within 4 h after replating, and most of the cells once attached were detached from the substratum by incubation with trypsin for 30 min. In contrast, the attachment of MCF-7 cells to the substratum proceeded more rapidly compared to that of HBE cells, and more than 90% of the MCF-7 cells once attached were detached within a 10-min incubation with trypsin. The attachment of HBE cells to the substratum was significantly inhibited by a function-blocking antibody to beta1 or beta4 integrin, whereas MCF-7 cells were not prevented from attaching by the anti-integrin beta1 antibody as far as examined but were by the anti-integrin beta4 antibody at the highest concentration tested. The absolute amounts of beta1 integrin in HBE and MCF-7 cells were comparable to each other, while the cell surface level of beta1 in the MCF-7 cells was only 10% of that in the HBE cells. Both the total and cell surface levels of beta4 integrin expression in the MCF-7 cells were 30-40% of those in the HBE cells. The results suggest that anchorage-dependent HBE cells attach to the culture substratum via both beta1 and beta4 integrins, thereby generating a strong adhesiveness to the substratum. In contrast, anchorage-independent MCF-7 cells had reduced adhesiveness to the substratum, and this property is suggested to be due to decreased levels of cell surface beta1 integrin and total beta4 integrin expressed compared to their levels in normal counterparts.

Antibodies↗

PubMed2005

Differences in potency of CXC chemokine ligand 8-, CC chemokine ligand 11-, and C5a-induced modulation of integrin function on human eosinophils.

The hypothesis was tested that different chemoattractants have different effects on the activity of integrins expressed by the human eosinophil. Three chemoattractants, CXCL8 (IL-8), CCL11 (eotaxin-1), and C5a were tested with respect to their ability to induce migration and the transition of eosinophils from a rolling interaction to a firm arrest on activated endothelial cells under flow conditions. CCL11 and C5a induced a firm arrest of eosinophils rolling on an endothelial surface, whereas CXCL8 induced only a transient arrest of the cells. The CXCL8- and CCL11-induced arrest was inhibited by simultaneously blocking alpha4 integrins (HP2/1) and beta2 integrins (IB4). In contrast, the C5a-induced arrest was only inhibited by 30% under these conditions. The potency differences of C5a>CCL11>CXCL8 to induce firm adhesion under flow condition was also observed in migration assays and for the activation of the small GTPase Rap-1, which is an important signaling molecule in the inside-out regulation of integrins. Interestingly, only C5a was able to induce the high activation epitope of alphaMbeta2 integrin recognized by MoAb CBRM1/5. The C5a-induced appearance of this epitope and Rap activation was controlled by phospholipase C (PLC), as was shown with the PLC inhibitor U73122. These data show that different chemoattractants are able to induce distinct activation states of integrins on eosinophils and that optimal chemotaxis is associated with the high activation epitope of the alphaMbeta2 integrin. Furthermore, PLC plays an important role in the inside-out signaling and, thus, the activation status of integrins on eosinophils.

CD18 Antigens↗

PubMed2006

Altered expression of beta1 integrins in renal carcinoma cell lines exposed to the differentiation inducer valproic acid.

Renal cell carcinoma (RCC) is the most common malignant tumor of the kidney. Adhesion receptors of the beta1 integrin family are assumed to be involved in carcinogenesis, but it is not clear how they contribute to RCC progression. In an in vitro model, we evaluated growth and adhesion capacity of Caki-I and KTC-26 kidney carcinoma cell lines compared to normal renal proximal tubular epithelial cells (PTC). alpha1-alpha6beta1 integrin subunits in malignant and non-malignant cells were evaluated by Western blotting and RT-PCR, integrin surface expression was measured by flow cytometry and confocal microscopy. Additionally, tumor cells were allowed to re-differentiate in the presence of valproic acid (VPA) and dynamic alterations of the integrin profile were analyzed. Caki-I and KTC-26 were characterized by accelerated proliferation and adhesion to an endothelial cell monolayer, compared to PTC cells. The integrin beta1 repertoire in RCC cell lines was significantly different from that detected in PTC, and included down-regulated alpha2 and alpha6, but up-regulated alpha1, alpha3 and alpha5 proteins. VPA application reduced tumor malignancy which was evidenced by reduced cell growth and adhesion capacity. The reduction in tumor malignancy was paralleled by the integrin expression profile of renal tumor cells 'matching' the pattern seen in PTC. We assume that a sensitive integrin balance exists in normal renal epithelial cells, and that dysregulation of the 'physiological' receptor equipment drives these cells towards malignancy. VPA acted on all investigated integrin subtypes and restored the receptor pattern typical for non-malignant cells. Therefore, VPA may represent a novel therapeutic option in RCC treatment.

Carcinoma, Renal Cell↗

PubMed1993

Immunolocalization of beta 1 integrins in platelets and platelet-derived microvesicles.

Human platelets contain several adhesion receptors belonging to the integrin superfamily. At least three beta 1 integrins are present on platelets and have been shown to mediate platelet adhesion to collagen, fibronectin, and laminin. To study the cellular localization of the beta 1 integrins in platelets, we produced a polyclonal antibody by immunization of goat 172 with purified beta 1 subunit from HPB-ALL cells. Antibody 172 (Ab172) specifically immunoblotted a 135-Kd protein in a lysate of whole platelets. The reactivity of Ab172 with platelet membrane proteins was further determined by immunoprecipitation of lysates of surface-radioiodinated platelets. Ab172 immunoprecipitates, resolved by nonreducing/reducing two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis consisted of three labeled proteins with migrational properties of platelet glycoprotein (GP)Ia, GPIc and GPIIa. Neither GPIIb/IIIa nor the vitronectin receptor were immunoprecipitated by Ab172, confirming a lack of cross-reactivity with the beta 3 integrins in platelets. Immunofluorescence studies using Ab172 were performed to investigate the cellular distribution of beta 1 integrins in platelets. Fluorescent labeling of intact cells demonstrated the presence of beta 1 antigen on the surface of resting cells. Permeabilization of platelets with Triton X-100 showed the presence of an intracellular pool of beta 1 antigen. Double-label experiments using Ab172 and AP-2 (anti-GPIIb/IIIa) showed identical labeling patterns, suggesting a similar subcellular distribution for these integrins. Following thrombin stimulation, permeabilized cells showed a centralized clearing of both beta 1 antigen and GPIIb/IIIa as well as an intensification of surface labeling for beta 1 antigen. These findings suggest the translocation of intracellular beta 1 antigen to the platelet surface as a result of thrombin stimulation. Because platelet-derived microvesicles have been reported to contain GPIIb/IIIa, we investigated the possible distribution of beta 1 integrins in these structures. Microvesicles, produced as a result of platelet activation, were labeled with Ab172, suggesting the distribution of beta 1 integrins in these structures as well as in intact cells.

Animals↗

PubMed1996

Changes in beta 4 integrin expression and localization in vivo in response to corneal epithelial injury.

PURPOSE: To determine whether production and localization of beta 4 integrin is altered during in vivo corneal epithelial cell migration in response to debridement wounding. METHODS: Rat corneas were wounded and animals were killed at times ranging from 3 hours to 14 days. At various time points, corneal epithelial integrins were quantitated by gel electrophoresis and immunoblotting of epithelial extracts and then were localized by immunohistochemistry. RESULTS: As early as 6 hours after wounding, an increase in the amount of the beta 4 integrin subunit expressed per microgram of total protein was observed. The level of beta 4 continued to increase until wound closure. By 14 days after wounding, beta 4 expression returned to control levels. The level of expression of beta 1 and alpha (v) integrins were found not to change significantly throughout migration. Immunohistochemical analyses using antibodies against either the beta 4 integrin subunit or HD1, a hemidesmosomal plaque component, showed that in control sections, beta 4 integrin and HD1 codistributed in a linear staining pattern above the basement membrane. As early as 4 hours after wounding, beta 4 was present in both basal and suprabasal epithelial cells, and HD1 was retained at the basal aspect of the epithelial basal cells. CONCLUSIONS: These data show that changes in expression and localization of beta 4 integrin occur in the corneal epithelium in response to debridement wounding in vivo. Previously, we had shown that quantitative changes in beta 4 integrin expression do not occur in an in vitro organ culture model used for the study of corneal epithelial cell migration. Increased beta 4 expression may not be required for migration per se, but it may be play a role in either stabilizing cell:cell or cell:substrate adhesion in vivo or in preparing cells to undergo mitosis during restratification.

Animals↗

PubMed1997

The integrin-triggered rescue of T lymphocyte apoptosis is blocked in HIV-1-infected individuals.

HIV infection is associated with a disease status-dependent impairment of Ag-specific T cell responses, resulting in anergy or unchecked apoptotic cell death. beta1 integrins play an important role in the induction of T lymphocyte responses to antigenic challenge by providing a T cell costimulatory signal, and have been shown to rescue various cell types from undergoing apoptosis. We examined the integrin-triggered cell survival signal and associated pathways in CD3+ T cells derived from 69 HIV-1-infected individuals in comparison with healthy controls. We found beta1 integrin-mediated costimulation of TCR-induced T cell proliferation and protection from aberrant cell death to be absent in the majority of patients with AIDS, but intact in asymptomatic, infected individuals. The lack of integrin-mediated rescue may be partly due to an early impairment of TCR/integrin-costimulated secretion of IFN-gamma, a type 1 lymphokine that protects against TCR-induced apoptosis of T cells from HIV-seropositive donors, but not loss of integrin expression. The mechanism of integrin hyporesponsiveness appeared to correlate with a failure of the integrin-generated signal to induce pp125FAK mRNA and protein expression. Protein kinase C activation in CD3+ T cells following integrin stimulation was also impaired in HIV-infected individuals, mostly among the symptomatic/AIDS patients. Protein kinase C inactivation in T cells was shown to have a destabilizing effect in vitro on pp125FAK mRNA that contains an AUUUA motif in the 3'-untranslated region, a consensus sequence for the AU-rich elements responsible for mRNA destabilization. These aberrant changes in pp125FAK expression may have direct significance to the overall immunopathogenesis during infection with HIV-1.

Acquired Immunodeficiency Syndrome↗

PubMed1997

Neutrophils can adhere via alpha4beta1-integrin under flow conditions.

In this study we investigated the possibility that an alternative pathway exists for neutrophil recruitment, namely an alpha4beta1-dependent pathway. A parallel plate chamber was used to investigate whether neutrophils could tether, roll, and adhere to tumor necrosis factor alpha (TNF alpha)-stimulated endothelium via alpha4beta1. alpha4beta1-integrin was induced on neutrophils using dihydrocytochalasin B and either an endogenous (endothelial-derived) chemotactic agent or an exogenous chemotactic molecule. alpha4beta1-expressing neutrophils could stably adhere under shear force (2 dyne/cm2) to TNF alpha-stimulated endothelium independent of the beta2-integrin. The firm adhesion was entirely abolished by antibodies directed against either the alpha4 or beta1-integrin subunits. However, the rolling interaction was not dependent on alpha4beta1 but was abolished by antiselectin therapy. Neutrophils expressing alpha4beta1 could also tether to the endothelium in the presence of antiselectin therapy, but at shear stresses less than 2 dyne/cm2. alpha4beta1-expressing neutrophils also tethered to and stably adhered (no rolling) to VCAM-1- but not to ICAM-1-transfected L cells. The interaction only occurred at shear stress less than 2 dyne/cm2. A cell line (Ramos) known to express high quantities of alpha4beta1-integrin interacted with VCAM-1-transfected L cells at very similar shear conditions. alpha4beta1-expressing neutrophils were also able to adhere to a second alpha4-integrin ligand, fibronectin; however, this interaction only occurred under static conditions. These data suggest that, under certain conditions, neutrophils can adhere independently of the beta2-integrin pathway and adhere via the alpha4beta1-integrin. This study refutes the concept that alpha4beta1-integrin adhesion is restricted to mononuclear leukocytes and is not functional on human neutrophils.

Animals↗

PubMed1998

Analysis of ligand-induced and ligand-attenuated epitopes on the leukocyte integrin alpha4beta1: VCAM-1, mucosal addressin cell adhesion molecule-1, and fibronectin induce distinct conformational changes.

The leukocyte integrin alpha4beta1 is a receptor for both cell surface ligands (VCAM-1 and mucosal addressin cell adhesion molecule-1 (MAdCAM-1)) and extracellular matrix components (fibronectin). Through regulated interactions with these molecules, alpha4beta1 mediates leukocyte migration from the vasculature at sites of inflammation. Regulation of integrin activity plays a key role in controlling leukocyte-adhesive events and appears to be partly determined by changes in integrin conformation. Several mAbs that recognize ligand-induced binding site epitopes on integrins have been characterized, and a subset of these mAbs are capable of stimulating integrin-ligand binding. Conversely, some mAbs recognize epitopes that are attenuated by ligand engagement and allosterically inhibit ligand binding. To gain insight into ligand-specific effects on integrin conformation, we have examined the ability of different ligands to modulate the binding of four distinct classes (A, B1, B2, and C) of anti-alpha4 Abs to alpha4beta1. VCAM-1 attenuated B (antifunctional) class epitopes via an allosteric mechanism and also allosterically inhibited the binding of the function-blocking anti-beta1 mAb 13. Additional alpha4beta1 ligands (fibronectin fragments, MAdCAM-1, and the CS1 peptide) also inhibited mAb 13-integrin binding; however, the epitopes of the class B anti-alpha4 mAbs were attenuated by the fibronectin fragments, but not by MAdCAM-1 or the CS1 peptide. Of the two anti-alpha4 class A mAbs examined, one recognized an epitope that was induced uniquely by VCAM-1. Taken together, these data suggest that overlapping but distinct binding mechanisms exist for different alpha4beta1 ligands and that distinct conformational changes are induced upon integrin engagement by different ligands.

Animals↗