The role of integrins alpha 2 beta 1 and alpha 3 beta 1 in cell-cell and cell-substrate adhesion of human epidermal cells

We have examined cultures of neonatal human foreskin keratinocytes (HFKs) to determine the ligands and functions of integrins alpha 2 beta 1, and alpha 3 beta 1 in normal epidermal stratification and adhesion to the basement membrane zone (BMZ) in skin. We used three assay systems, HFK adhesion to purified extracellular matrix (ECM) ligands and endogenous secreted ECM, localization of integrins in focal adhesions (FAs), and inhibition of HFK adhesion with mAbs to conclude: (a) A new anti-alpha 3 beta 1 mAb, P1F2, localized alpha 3 beta 1 in FAs on purified laminin greater than fibronectin/collagen, indicating that laminin was the best exogeneous ligand for alpha 3 beta 1. However, in long term culture, alpha 3 beta 1 preferentially codistributed in and around FAs with secreted laminin-containing ECM, in preference to exogenous laminin. Anti-alpha 3 beta 1, mAb P1B5, detached prolonged cultures of HFKs from culture plates or from partially purified HFK ECM indicating that interaction of alpha 3 beta 1 with the secreted laminin-containing ECM was primarily responsible for HFK adhesion in long term culture. (b) In FA assays, alpha 2 beta 1 localized in FAs conincident with initial HFK adhesion to exogenous collagen, but not laminin or fibronectin. However, in inhibition assays, anti-alpha 2 beta 1 inhibited initial HFK adhesion to both laminin and collagen. Thus, alpha 2 beta 1 contributes to initial HFK adhesion to laminin but alpha 3 beta 1 is primarily responsible for long-term HFK adhesion to secreted laminin-containing ECM. (c) Serum or Ca2(+)-induced aggregation of HFKs resulted in relocation of alpha 2 beta 1 and alpha 3 beta 1 from FAs to cell-cell contacts. Further, cell-cell adhesion was inhibited by anti-alpha 3 beta 1 (P1B5) and a new anti-beta 1 mAb (P4C10). Thus, interaction of alpha 3 beta 1 with either ECM or membrane coreceptors at cell-cell contacts may facilitate Ca2(+)-induced HFK aggregation. (d) It is suggested that interaction of alpha 3 beta 1 with a secreted, laminin-containing ECM in cultured HFKs, duplicates the role of alpha 3 beta 1 in basal cell adhesion to the BMZ in skin. Further, relocation of alpha 2 beta 1 and alpha 3 beta 1 to cell-cell contacts may result in detachment of cells from the BMZ and increased cell-cell adhesion in the suprabasal cells contributing to stratification of the skin.     

Laminin alpha 5 is required for lobar septation and visceral pleural basement membrane formation in the developing mouse lung

Laminin alpha/beta/gamma heterotrimers are the major noncollagenous components of all basement membranes. To date, five alpha, three beta, and three gamma chains have been identified. Laminin alpha 5 is expressed early in lung development and colocalizes with laminin alpha1. While laminin alpha1 expression in the lung is restricted to the embryonic period, laminin alpha 5 expression persists throughout embryogenesis and adulthood. Targeted mutation of the mouse laminin alpha 5 gene Lama5 causes embryonic lethality at E14-E17 associated with exencephaly, syndactyly, placentopathy, and kidney defects, all attributable to abnormal basement membranes. In this investigation, lung development in Lama5(-/-) mice up to E16.5 was examined. We observed normal lung branching morphogenesis and vasculogenesis, but incomplete lobar septation and absence of the visceral pleura basement membrane. Preservation of branching morphogenesis was associated with ectopic deposition of laminin alpha 4 in the airway basement membrane. Perturbation of pleural basement membrane formation and right lung septation correlated with absence of laminin alpha 5, which was found to be the only laminin alpha chain present in the normal visceral pleura basement membrane. Our finding of normal lung branching morphogenesis with abnormal lobar septation demonstrates that these processes are not obligatorily linked.     

Distinct functions for integrins alpha 3 beta 1 in focal adhesions and alpha 6 beta 4/bullous pemphigoid antigen in a new stable anchoring contact (SAC) of keratinocytes: relation to hemidesmosomes

Basal cells of stratified epidermis are anchored to the basement membrane zone (BMZ) of skin via hemidesmosomes. We previously identified integrin alpha 3 beta 1, in focal adhesions (FAs), of cultured human keratinocytes (HFKs) as a mediator of HFK adhesion to secreted BMZ-like extracellular matrix (ECM; Carter, W.G., E.A. Wayner, T.S. Bouchard, and P. Kaur. 1990. J. Cell Biol. 110: 1387-1404). Here, we have examined the relation of integrins alpha 6 beta 4 and alpha 3 beta 1, to bullous pemphigoid antigen (BPA), a component of hemidesmosomes. We conclude that alpha 6 beta 4 in HFKs localizes in a new stable anchoring contact (SAC) that cooperates with alpha 3 beta 1- FAs to mediate adhesion to ECM, based on the following. (a) Comparison of secreted ECM, with exogenous laminin, fibronectin and collagen identified ECM as the preferred ligand for HFK adhesion and spreading and for formation of both alpha 6 beta 4-SACs and alpha 3 beta 1-FAs. (b) Inhibition of HFK adhesion with combined anti-alpha 3 beta 1 (P1B5) and anti-alpha 6 beta 4 (GoH3) antibodies indicated that both receptors were functional in adhesion to ECM while alpha 3 beta 1 played a dominant role in spreading. (c) alpha 6 beta 4 colocalized with BPA in SACs that were proximal to but excluded from FAs. Both alpha 6 beta 4- SACs and alpha 3 beta 1-FAs were in contact with the adhesion surface as indicated by antibody exclusion and interference reflection microscopy. (d) In contrast to alpha 3 beta 1-FAs, alpha 6 beta 4-SACs were present only in nonmotile cells, not associated with stress fibers, and were relatively stable to detergents and urea, suggesting a nonmotile, or anchoring function for SACs and motility functions for alpha 3 beta 1-FAs. (e) alpha 6 beta 4 formed a detergent-insoluble complex with exogenous ECM in an affinity isolation procedure, confirming the ability of an unidentified ECM ligand to interact with alpha 6 beta 4. (f) We suggest that alpha 6 beta 4/BPA-SACs in culture restrict migration of HFKs on ECM while alpha 3 beta 1-FAs form dynamic adhesions in spreading and migrating cells. alpha 6 beta 4/BPA-SACs in culture bear functional and compositional similarities to hemidesmosomes in skin.     

Induction of extracellular matrix gene expression in normal human keratinocytes by transforming growth factor beta is altered by cellular differentiation

Changes in epithelial substrate have been related to the cellular capacity for proliferation and to changes in cellular behavior. The effect of TGF beta 1 on the expression of the basement membrane genes, fibronectin, laminin B1, and collagen alpha 1 (IV), was examined. Northern analysis revealed that treatment of normal human epidermal keratinocytes with 100 pM TGF beta 1 increased the expression of each extracellular matrix (ECM) gene within 4 h of treatment. Maximal induction was reached within 24 h after treatment. The induction of ECM mRNA expression was dose dependent and was observed at doses as low as 1-3 pM TGF beta 1. Incremental doses of TGF beta 1 also increased cellular levels of fibronectin protein in undifferentiated keratinocytes and resulted in increased secretion of fibronectin. Squamous-differentiated cultures of keratinocytes expressed lower levels of the extracellular matrix RNAs than did undifferentiated cells. Treatment of these differentiated cells with TGF beta 1 induced the expression of fibronectin mRNA to levels seen in TGF beta-treated, undifferentiated keratinocytes but only marginally increased the expression of collagen alpha 1 (IV) and laminin B1 mRNA. The increased fibronectin mRNA expression in the differentiated keratinocytes was also reflected by increased accumulation of cellular and secreted fibronectin protein. The inclusion of cycloheximide in the protocol indicated that TGF beta induction of collagen alpha 1 (IV) mRNA was signaled by proteins already present in the cells but that TGF beta required the synthesis of a protein(s) to fully induce expression of fibronectin and laminin B1 mRNA. The differential regulation of these genes in differentiated cells may be important to TGF beta action in regulating reepithelialization.     

Unaltered distribution of laminins, fibronectin, and tenascin in celiac intestinal mucosa.

Extensive remodeling of the extracellular matrix (ECM) occurs in inflammatory tissues. The celiac lesion in the small intestine is characterized by inflammation accompanied by profound morphological alterations. We used immunohistochemistry to determine the distribution of laminin, fibronectin, and tenascin isoforms in small intestinal biopsies of untreated patients with celiac disease. In normal mucosa, the distribution of laminin isoforms defines three epithelial basement membrane (BM) zones. We found that the organization of these zones was maintained in the celiac mucosa. Thus, components of laminin-5 (alpha3 and beta3) were found in the surface epithelial BM, laminin alpha2 chain was found selectively at crypt bottoms, and laminin alpha5 chain was the sole alpha-type chain in middle crypt BMs. Likewise, the distribution of fibronectin and tenascin resembled that of the normal gut. The organization of pericryptal fibroblasts and lamina propria smooth muscle strands, as defined by immunostaining for alpha-smooth muscle actin, also remained unchanged in the celiac mucosa. Unexpectedly, major ECM changes were not detected in the celiac lesion.     

Expression of integrin beta 6 enhances invasive behavior in oral squamous cell carcinoma.

Oral squamous cell carcinoma (SCC) is characterized by invasive growth and the propensity for distant metastasis. The expression of specific adhesion receptors promotes defined interactions with the specific components found within the extracellular matrix (ECM). We previously showed that the alpha v beta 6 fibronectin receptor is highly expressed in oral SCC. Here we forced expression of the beta 6 subunit into poorly invasive SCC9 cells to establish the SCC9 beta 6 cell line and compared these two cell lines in several independent assays. Whereas adhesion to fibronectin was unaffected by the expression of beta 6, migration on fibronectin and invasion through a reconstituted basement membrane (RBM) were both increased. Function-blocking antibodies to alpha v beta 6 (10D5) reduced both migration on fibronectin and invasion through an RBM, whereas anti-alpha 5 antibodies were effective only in suppressing migration on fibronectin, not invasion. Expression of beta 6 also promoted tumor growth and invasion in vivo and modulated fibronectin matrix deposition. When grown as a co-culture with SCC9 cells, peritumor fibroblasts (PTF) organized a dense fibronectin matrix. However, fibronectin matrix assembly was decreased in co-cultures of SCC9 beta 6 cells and PTF and this decrease was reversed by the addition of function-blocking anti-alpha v beta 6 antibodies. The expression of beta 6 also resulted in increased levels of matrix metalloproteinase 3. Addition of the general MMP inhibitor GM6001 to SCC9 beta 6/PTF co-cultures dramatically increased fibronectin matrix assembly in a similar fashion as incubation with anti-alpha v beta 6 antibodies. These results demonstrate that expression of beta 6 (1) increases oral SCC cell motility and growth in vitro and in vivo; (2) negatively affects fibronectin matrix assembly; and (3) stimulates the expression and activation of MMP3. We suggest that the integrin alpha v beta 6 is a key component of oral SCC invasion and metastasis through modulation of MMP-3 activity.     

Alterations in integrin receptor expression on chemically transformed human cells: specific enhancement of laminin and collagen receptor complexes

The abilities of malignant tumor cells to bind and migrate through basement membranes are important steps in invasion and metastasis. Malignant tumor cells would therefore be expected to express receptors on their surfaces for basement membrane and stromal components, such as collagens, laminin, and fibronectin, although the pattern of expression of these receptors on the malignant cells may be different from that on their normal progenitors. We report here that chemically transformed tumorigenic human cells express an altered pattern of integrin receptors on their cell surfaces as compared with their untransformed nontumorigenic counterparts. Specifically, N-methyl-N'-nitro-N-nitrosoguanidine transformation of HOS cells into highly tumorigenic cells results in a significant specific increase in the expression of (in descending order of level of cell surface expression) the integrins alpha 6/beta 1, alpha 2/beta 1, and alpha 1/beta 1, which are receptors for laminin, collagens, and collagen type IV and laminin, respectively. The level of expression of two fibronectin receptor integrins, alpha 5/beta 1 and alpha 3/beta 1, are, however, unaltered, whereas the level of expression of vitronectin receptor integrin, alpha v/beta 3, is drastically reduced on the transformed cells. Consistent with the increased expression of laminin and collagen receptors and the decreased expression of vitronectin receptors on the transformed cells, these cells attached three- to fivefold more strongly to laminin and collagen but attached very poorly to vitronectin. The MNNG-HOS cells were also found to have a greater potential for invasion through reconstituted basement membrane, matrigel, the major components of which are laminin and type IV collagen. The invasion of both the HOS and MNNG-HOS cells was inhibited 45-50% by a polyclonal anti-fibronectin receptor antibody. However, although the invasion of HOS cells could be inhibited up to 75% by an anti-alpha 6 monoclonal antibody, a similar concentration of this antibody had no effect on the alpha 6-overproducing MNNG-HOS cells. A fivefold higher concentration of this antibody did result in partial inhibition of MNNG-HOS invasion. These data indicate a critical role for the alpha 6/beta 1 laminin receptor in the invasion of these cells through basement membranes and demonstrate that chemical transformation of nontumorigenic human cells to highly tumorigenic cells is associated with an altered pattern of integrin expression which may play a direct role in the increased capacity of these cells to bind and invade through basement membranes.     

Integrin alpha 3 beta 1 participates in the phagocytosis of extracellular matrix molecules by human breast cancer cells.

The mechanisms and receptors involved in phagocytosis by nonhematopoietic cells are not well understood. The involvement of the alpha 3 beta 1 integrin in phagocytosis of the extracellular matrix by human breast cancer cells was studied. The possible role of this integrin was suggested since alpha 3 and beta 1 but not alpha 2 subunits are concentrated at membrane sites where local degradation of fluorescently labeled gelatin occurs. Strikingly, anti-alpha 3 integrin monoclonal antibodies (mAbs) stimulate the phagocytosis of fluorescently labeled gelatin films, gelatin beads, and Matrigel films in a quantitative phagocytosis assay. Stimulation of the gelatin uptake by the anti-alpha 3 mAb is dose responsive, saturable, and time dependent. Antibodies against other integrin subunits have a lower stimulatory effect (anti-beta 1) or no significant effect (anti-alpha 2, -alpha 5, -alpha 6, and -alpha v) on gelatin phagocytosis. The synthetic HGD-6 human laminin peptide that binds specifically the alpha 3 beta 1 integrin, but not the scrambled HSGD-6 control peptide, also markedly stimulates gelatin uptake in a dose-responsive way. Furthermore, the stimulatory effects of the HGD-6 peptide and the anti-alpha 3 mAb are additive, suggesting that they might promote phagocytosis in different ways. Other laminin (YIGSR, IKVAV) and fibronectin (GRGDS) peptides have no effect on gelatin phagocytosis. Immunofluorescence shows that the alpha 3 and the beta 1, but not the alpha 2 integrin subunit, concentrate into patches on the cell surface after treatment with their respective mAbs. And, both gelatin and the alpha 3 beta 1 but not the alpha 2 beta 1 integrin are cointernalized and routed to acidic vesicles such as lysosomes. In conclusion, we demonstrate that human breast cancer cells locally degrade and phagocytose the extracellular matrix and show for the first time that the alpha 3 beta 1 integrin participates in this phagocytosis. We hypothesize that the anti-alpha 3 antibodies and the laminin peptide HGD-6 activate the alpha 3 beta 1 integrin, which results in a downstream signaling cascade stimulating phagocytosis.     

Rapid expression and activation of MMP-2 and MMP-9 upon exposure of human breast cancer cells (MCF-7) to fibronectin in serum free medium

Interactions between tumour cells and the extracellular matrix (ECM) strongly influence tumour development, affecting cell survival, proliferation and migration. Many of these interactions are mediated through a family of cell surface receptors named integrins. Fibronectin and its integrin receptors play important roles in tumour development. The alpha5beta 1 integrin interacts with the central cell adhesive region of fibronectin and requires both the RGD and synergy sites for maximal binding. Matrix metalloproteinases (MMPs) are a family of zinc dependent endopeptidases. They are capable of digesting the different components of the ECM and basement membrane. The ECM gives structural support to cells and plays a central role in cell adhesion, differentiation, proliferation and migration. Binding of ECM to integrins modulates expression and activity of the different MMPs. Our experimental findings demonstrate that cultivation of human breast cancer cells, MCF-7, in serum free medium in the presence of fibronectin upregulates the activity of MMP-2 and MMP-9. Blocking of alpha5beta 1 integrin with anti-alpha5 monoclonal antibody inhibits the fibronectin-induced MMP activation response appreciably. This strongly indicates alpha5beta 1 mediated signalling events in activation of MMP-2 and MMP-9. Phosphorylation of FAK and PI-3 kinase and the nuclear translocation of ERK and NF-kappaB upon fibronectin binding demonstrate possible participation of the FAK/PI-3K/ERK signalling pathways in the regulation of MMP-2 activity.     

Integrin alphavbeta3 binding to human alpha5-laminins facilitates FGF-2- and VEGF-induced proliferation of human ECV304 carcinoma cells

Human ECV304 cells respond reproducibly by tube formation to complex basement membrane matrices. Laminins are major glycoproteins of basement membranes. We therefore studied the ability of ECV304 cells to attach to defined laminin isoforms and to fibronectin, and identified the involved laminin receptors. The cells bound poorly to fibronectin, to some extent to laminin-1, whereas laminin-2/4 and -10/11 were strong adhesive substrates. Antibody perturbation assays showed that adhesion to laminin-1 was mediated by integrin alpha6beta1, and adhesion to laminin-2/4 by cooperative activity of integrins alpha3beta1 and alpha6beta1. Adhesion of ECV 304 cells to laminin-10/11 was mainly mediated by integrins alpha3beta1, with minor involvement of alpha6beta1/4 and alphavbeta3. Solid-phase binding assays confirmed that integrin alphavbeta3 binds human laminin-10/11 and -10, in an RGD-dependent fashion. Although integrin alphavbeta3 played a very minor role in cell adhesion to laminin-10/11, this interaction facilitated growth factor-induced proliferation of ECV304 cells. In response to FGF-2 or VEGF, the cells proliferated better when attached on laminin-10/11 than on laminin-1, -2/4, or gelatin. The proliferation induced by the joint application of laminin-10/11 and either one of the growth factors could be blocked by antibodies against integrin alphavbeta3. Fragments of several other basement membrane components are known to interact with alphavbeta3. The current data show that that integrin alphavbeta3 can bind intact alpha5-containing laminin trimers. Since the laminin alpha5 chain is broadly expressed in adult basement membranes, this interaction could be physiologically important. Our data suggest that this interaction is involved in the regulation of cellular responses to growth factors known to be involved in epithelial and endothelial development.     

Role of hypoxia and extracellular matrix-integrin binding in the modulation of angiogenic growth factors secretion by retinal pigmented epithelial cells.

The retinal pigmented epithelium (RPE) is a monolayer of polarized cells located between retinal photoreceptors and blood vessels of the choroid. The basal surface of RPE cells rests on Bruch's membrane, a complex extracellular matrix structure which becomes abnormal in several disease processes, including age-related macular degeneration (AMD). Ruptures or abnormalities in Bruch's membrane are frequently accompanied by choroidal neovascularization. Disturbed interaction of RPE cells with their extracellular matrix (ECM) could play a role in this process. The present study was undertaken to examine the complex interactions between hypoxia, integrin, and ECM in the regulation of RPE functions. Antibody blocking experiments demonstrated that RPE cell adhesion to vitronectin is mediated primarily through alphavbeta5 and adhesion to fibronectin occurs through alpha5beta1. RPE adhesion to immobilized laminin demonstrated highest level of non-RGD-mediated adhesion as compared to that with collagen IV or the RGD matrices such as vitronectin (alphavalpha5) , fibronectin (alpha5beta1), or thrombospondin (alpha5beta1 + alphavbeta5). Addition of soluble vitronectin, or fibrinogen to RPE cell cultures resulted in a small to moderate increase in VEGF and FGF2 in the media, while each of these growth factors was dramatically increased after addition of thrombospondin 1 (TSP1). In contrast, soluble fibronectin resulted in differential upregulation of VEGF but not FGF2. Similarly, immobilized TSP1 resulted in differential greater upregulation in VEGF but not FGF2 release from RPE as compared to other ECMs under either normoxic or hypoxic conditions. Additionally, hypoxia resulted in a time-dependent increase in VEGF, but not FGF2 release in the media. RPE cells grown on TSP1-coated plates showed increased VEGF and FGF2 in their media compared to cells grown on plates coated with type IV collagen, laminin, vitronectin, or fibronectin. The TSP1-induced increase in secretion of growth factors was partially blocked by anti-alpha5beta1, anti-alphavbeta3, and anti-alphavbeta5 antibodies indicating that it may be mediated in part by TSP1 binding to those integrins. These data suggest that alterations in oxygen levels (hypoxia/ischemia) and ECM of RPE cells, a prominent feature of AMD, can cause increased secretion of angiogenic growth factors that might contribute to the development of choroidal neovascularization. These data also suggest the potential modulatory role of VEGF release from RPE by ECM and alphavbeta5 and alpha5beta1 integrins.     

Basic fibroblast growth factor modulates integrin expression in microvascular endothelial cells.

During angiogenesis capillary endothelial cells undergo a coordinated set of modifications in their interactions with extracellular matrix components. In this study we have investigated the effect of the prototypical angiogenic factor basic fibroblast growth factor (bFGF) on the expression and function of several integrins in microvascular endothelial cells. Immunoprecipitation experiments with antibodies to individual subunits indicated that microvascular cells express at their surface several integrins. These include the alpha 1 beta 1, alpha 2 beta 1, and alpha 3 beta 1 laminin/collagen receptors; the alpha 6 beta 1 laminin receptor; the alpha 5 beta 1 and alpha v beta 1 fibronectin receptors; the alpha 6 beta 4 basement membrane receptor; and the alpha v beta 3 and alpha v beta 5 vitronectin receptors. Treatment with bFGF caused a significant increase in the surface expression of the alpha 2 beta 1, alpha 3 beta 1, alpha 5 beta 1, alpha 6 beta 1, alpha 6 beta 4, and alpha v beta 5 integrins. In contrast, the level of expression of the alpha 1 beta 1 and alpha v beta 3 integrins was decreased in bFGF-treated cells. Immunoprecipitation of metabolically labeled cells indicated that bFGF increases the biosynthesis of the alpha 3, alpha 5, alpha 6, beta 4, and beta 5 subunits and decreases the production of the alpha v and beta 3 subunits. These results suggest that bFGF modulates integrin expression by altering the biosynthesis of individual alpha or beta subunits. In accordance with the upregulation of several integrins observed in bFGF-treated cells, these cells adhered better to fibronectin, laminin, vitronectin, and type I collagen than did untreated cells. The largest differences in beta 1 integrin expression occurred approximately 72 h after exposure to bFGF, at a time when the expression of the endothelial cell-to-cell adhesion molecule endoCAM was also significantly upregulated. In contrast, a shorter exposure to bFGF (24-48 h) was required for the maximal induction of plasminogen activator production in the same cells. Taken together, these results show that bFGF causes significant changes in the level of expression and function of several integrins in microvascular endothelial cells.     

The receptor for the basement membrane glycoprotein entactin is the integrin alpha 3/beta 1.

Entactin is a glycoprotein found in basement membranes in complex with laminin, and purified entactin can promote the attachment and spreading of cells. We report here the isolation and identification of the plasma membrane receptor for entactin from PC-3 human prostate carcinoma cells which attach and spread on entactin. The receptor was isolated by affinity chromatography on mouse recombinant entactin-Sepharose of 125I surface-labeled octyl glucoside cell extracts. The receptor, which consisted of two polypeptides of relative molecular masses of 150 and 116 kDa, bound to the entactin-Sepharose matrix in the presence of CaCl2, MgCl2, and MnCl2, and was eluted with EDTA, but not with Arg-Gly-Asp-containing peptides. Utilizing anti-integrin antibodies, the heterodimeric receptor was identified as the integrin alpha 3 beta 1. Purified alpha 3 beta 1 bound to entactin Sepharose in a divalent cation-dependent manner and liposomes prepared with fractions eluted from the entactin-Sepharose matrix, as well as purified alpha 3 beta 1 also bound to entactin. Liposomes prepared with other integrins such as alpha 2 beta 1 did not bind to entactin. Antibody inhibition assays demonstrated that an anti-alpha 3 antibody (P1B5) inhibited the attachment of PC-3 cells to entactin whereas this antibody did not inhibit the attachment of these cells to laminin. Attachment to laminin was, however, blocked by anti-alpha 6 antibody (G0H3). These data demonstrate that the cell surface receptor for entactin on these prostate carcinoma cells is the integrin alpha 3 beta 1 and that these cells utilize alpha 6 beta 1 as the receptor for laminin.     

Laminin alpha5-chain is expressed early and widely during the development of the anterior segment of rat eye

The distribution of a novel laminin alpha5-chain in the basement membranes of the anterior segment of rat eye was studied. Frozen sections of embryonic day (E)16--17, post-natal day (P)2, 5, 10, 15 and 30 and adult rat eyes were immunostained for laminin chains alpha2, alpha5, beta1, beta2 and gamma1 and for laminin-5, as well as for EHS-laminin, to visualize all basement membranes. Laminin alpha5-, beta1- and gamma1-chain immunoreactivities were found in the basement membranes of the inner and outer layers of optic cup, lens epithelium, further corneal epithelium and skin of the eyelids in E16--17 rat eyes. In P2 and older rat eyes, laminin alpha5-, beta1- and gamma1-chains were all seen in the basement membranes of the corneal and conjunctival epithelium, Descemet's membrane, lens epithelium, ciliary processes, blood vessels and skin of the eyelids. There was a change in the expression pattern of laminin alpha5, beta1- and gamma1-chains in Descemet's membrane from the endothelial side of the membrane (P2--P15 eyes) to both sides of the membrane after P30. Immunoreactivity for laminin-5 was weak in the basement membrane of E16--17 epidermis, but strong in the basement membrane of corneal, conjunctival and eyelid epithelium in P2 and older rat eyes. Laminin alpha2- and beta2-chains were seen in conjunctival and uveal blood vessels in P15 and older rat eyes. The laminin beta2-chain emerged into the basement membrane of conjunctival epithelium in P30 and older rat eyes, suggesting a role for the laminin beta2-chain in the maturation of conjunctiva. The results suggest that laminin alpha5-chain, possibly in laminin-10 (alpha5beta1gamma1), is early and widely expressed in the basement membranes of developing and adult rat eye and, further, that laminin alpha5-chain is a major laminin alpha-chain, partly in coexpression with the alpha3-chain of laminin-5 in the basement membranes of the anterior segment of the eye in developing and adult rats. © 1998 Chapman & Hall     

Changes in keratinocyte adhesion during terminal differentiation: reduction in fibronectin binding precedes alpha 5 beta 1 integrin loss from the cell surface

During terminal differentiation keratinocytes move out of the basal layer of the epidermis and thereby lose contact with the basement membrane. We show that terminal differentiation in culture involves loss of adhesiveness to fibronectin, laminin, and collagen types I and IV. The adhesive changes precede, by several hours, loss of the alpha 2 beta 1, alpha 3 beta 1, and alpha 5 beta 1 integrins from the cell surface. Keratinocyte adhesion to fibronectin is mediated by the alpha 5 beta 1 integrin, and the decrease in adhesion of intact cells to fibronectin is correlated with a decrease in the ability of alpha 5 beta 1 receptors to bind fibronectin. Thus modulation of integrin function early in terminal differentiation may be an early event determining cell migration out of the basal layer.     

Fibronectin promotes brain capillary endothelial cell survival and proliferation through alpha5beta1 and alphavbeta3 integrins via MAP kinase signalling

We showed previously that blood vessel maturation in the CNS is associated with a developmental switch in brain capillary endothelial cells (BCEC), from fibronectin signalling during angiogenesis to laminin signalling in the adult. To investigate the functional significance of this switch, we have examined the response of BCEC to different extracellular matrix (ECM) proteins. This showed that BCEC proliferation was significantly promoted by fibronectin (28.2 +/- 4.0%) and by vitronectin (14.8 +/- 2.1%) compared with uncoated glass (7.2 +/- 0.7%), while BCEC survival was significantly promoted by fibronectin (1130 +/- 131 cells), vitronectin (830 +/- 63 cells), collagen IV (703 +/- 77 cells) and laminin (680 +/- 34 cells) compared with the uncoated glass (367 +/- 48 cells). Biochemical studies showed that BCEC express a limited repertoire of integrins, including the beta1 integrins, alpha3beta1, alpha5beta1 and alpha6beta1, and the alphavbeta3 integrin. Function-blocking studies showed that the response to fibronectin was mediated equally by the alpha5beta1 and alphavbeta3 integrins. Analysis of signalling pathways revealed that fibronectin stimulated activation of the p44/p42 MAP kinase signalling pathway and pharmacological inhibitors of this pathway blocked BCEC proliferation on fibronectin. Taken together, these findings show that fibronectin exerts a strong angiogenic influence on endothelial cells (EC) in the CNS, and that this is mediated through the alpha5beta1 and alphavbeta3 integrins via MAP kinase signalling. In addition to a fundamental role in development, these findings may also have implications in pathological conditions of the CNS where fibronectin is re-expressed.     

Human bronchial epithelial cells secrete laminin 5, express hemidesmosomal proteins, and assemble hemidesmosomes.

Epithelial cells attach to the basement membrane through adhesive contacts between the basal cells of the epithelium and the proteins of the extracellular matrix (ECM). The hemidesmosome (HD) is a specialized cell-ECM contact, that mediates the attachment of the epithelial cell basal surface to the ECM. In bronchial epithelial cells, the protein components that constitute the HD have not been demonstrated. Using immunohistochemical techniques, we determined that normal human bronchial epithelial (NHBE) cells express the HD cell surface integrin alpha6beta4 and produce laminin 5, the ECM protein associated with HDs. Furthermore, expression of the HD-associated structural proteins, bullous pemphigoid antigens 1 (BPAG 1) and 2 (BPAG 2), was demonstrated in NHBE cells by immunofluorescence microscopy and immunoblot analyses. In addition, we confirmed the presence of laminin 5 in the basement membrane (BM) of bronchial epithelial biopsy specimens and of BP230, BP180, and the alpha6beta4 integrin heterodimer at the site of bronchial epithelial cell-ECM interaction in vivo. Finally, using electron microscopy, we were able to demonstrate intact HDs in a glutaraldehyde-fixed NHBE cell monolayer. These findings suggest that bronchial epithelium forms HDs and that the laminin 5-alpha6beta4 integrin interaction may be important in stabilizing epithelial cell adhesion to the BM in the lung.     

Human microvascular endothelial cells use beta 1 and beta 3 integrin receptor complexes to attach to laminin

Microvascular endothelial cells (MEC) use a set of surface receptors to adhere not only to the vascular basement membrane but, during angiogenic stimulation, to the interstitium. We examined how cultured human MEC interact with laminin-rich basement membranes. By using a panel of monoclonal antibodies, we found that MEC cells express a number of integrin-related receptor complexes, including alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, alpha 5 beta 1, alpha 6 beta 1, alpha V beta 3. Attachment to laminin, a major adhesive protein in basement membranes, was studied in detail. Blocking monoclonal antibodies specific to different integrin receptor complexes showed that the alpha 6 beta 1 complex was important for MEC adhesion to laminin. In addition, blocking antibody also implicated the vitronectin receptor (alpha V beta 3) in laminin adhesion. We used ligand affinity chromatography of detergent-solubilized receptor complexes to further define receptor specificity. On laminin-Sepharose columns, we identified several integrin receptor complexes whose affinity for the ligand was dependent on the type of divalent cation present. Several beta 1 complexes, including alpha 1 beta 1, alpha 2 beta 1, and alpha 6 beta 1 bound strongly to laminin. In agreement with the antibody blocking experiments, alpha V beta 3 was found to bind well to laminin. However, unlike binding to its other ligands (e.g., vitronectin, fibrinogen, von Willebrand factor), alpha V beta 3 interaction with laminin did not appear to be Arg-Gly-Asp (RGD) sensitive. Finally, immunofluorescent staining demonstrated both beta 1 and beta 3 complexes in vinculin-positive focal adhesion plaques on the basal surface of MEC adhering to laminin-coated substrates. The results indicate that both these subfamilies of integrin heterodimers are involved in promoting MEC adhesion to laminin and the vascular basement membrane.     

Asn-linked oligosaccharide-dependent interaction between laminin and gp120/140. An alpha 6/beta 1 integrin

Receptor-mediated recognition and adhesion to laminin, a specific glycoprotein from basement membranes, exert an important role in many biological phenomena. Studying cell surface proteins of B16-F10, a metastatic murine melanoma cell line, we identified a 120-140 kDa glycoprotein (gp120/140) that binds laminin. This glycoprotein was recognized by a polyclonal antibody raised against the human fibronectin receptor beta 1-integrin chain, as well as immunoprecipitated by an anti-alpha 6 chain (monoclonal antibody GoH3), characterizing it as an alpha 6/beta 1-integrin. Its binding to laminin was specific and displayed moderate affinity, as its apparent dissociation constant was 18 nM. To characterize the influence of carbohydrate moieties on the laminin-gp120/140 interaction, metaperiodate oxidation, metabolic inhibition of glycosylation, and enzymatic deglycosylation studies were performed. Our results indicate that gp120/140 Asn-linked oligosaccharides play a part in this interaction. Reciprocally, both metaperiodate and N-glycanase treatment of native laminin reduced its binding to gp120/140, characterizing the latter as a lectin-like molecule. These results point to glycosylation processes as a possible mechanism for variable binding specificity profiles among integrins.