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PubMed2000

Thrombospondin-1 promotes alpha3beta1 integrin-mediated adhesion and neurite-like outgrowth and inhibits proliferation of small cell lung carcinoma cells.

Although human small cell lung carcinoma (SCLC) cell lines are typically anchorage-independent and do not attach on most extracellular matrix proteins, OH-1, and several other SCLC cell lines attached on substrates coated with thrombospondin-1 (TSP1). SCLC cells grew long-term as adherent cells on a TSP1-coated substrate. Adhesion of SCLC cells on TSP1 was inhibited by heparin, function-blocking antibodies recognizing alpha3 or beta1 integrin subunits, and by soluble alpha3beta1 integrin ligands. SCLC cells extended neurite-like processes on a TSP1 substrate, which was also mediated by alpha3beta1 integrin. Process formation on a TSP1 substrate was specifically stimulated by epidermal growth factor and somatostatin. Adhesion on TSP1 weakly inhibited SCLC cell proliferation, but this inhibition was strongly enhanced in the presence of epidermal growth factor. TSP1 and an alpha3beta1 integrin-binding peptide from TSP1 also inhibited proliferation when added in solution. High-affinity binding of 125I-labeled TSP1 to OH-1 cells was heparin-dependent and may be mediated by sulfated glycolipids, which are the major sulfated glycoconjugates synthesized by these cells. Synthesis or secretion of TSP1 by SCLC cells could not be detected. On the basis of these results, the alpha3beta1 integrin and sulfated glycolipids cooperate to mediate adhesion of SCLC cells on TSP1. Interaction with TSP1 through this integrin inhibits growth and induces neurotypic differentiation, which suggests that this response to TSP1 may be exploited to inhibit the progression of SCLC.

Carcinoma, Small Cell↗
PubMed2006

A critical role for tetraspanin CD151 in alpha3beta1 and alpha6beta4 integrin-dependent tumor cell functions on laminin-5.

The basement membrane protein laminin-5 supports tumor cell adhesion and motility and is implicated at multiple steps of the metastatic cascade. Tetraspanin CD151 engages in lateral, cell surface complexes with both of the major laminin-5 receptors, integrins alpha3beta1 and alpha6beta4. To determine the role of CD151 in tumor cell responses to laminin-5, we used retroviral RNA interference to efficiently silence CD151 expression in epidermal carcinoma cells. Near total loss of CD151 had no effect on steady state cell surface expression of alpha3beta1, alpha6beta4, or other integrins with which CD151 associates. However, CD151-silenced carcinoma cells displayed markedly impaired motility on laminin-5, accompanied by unusually persistent lateral and trailing edge adhesive contacts. CD151 silencing disrupted alpha3beta1 integrin association with tetraspanin-enriched microdomains, reduced the bulk detergent extractability of alpha3beta1, and impaired alpha3beta1 internalization in cells migrating on laminin-5. Both alpha3beta1- and alpha6beta4-dependent cell adhesion to laminin-5 were also impaired in CD151-silenced cells. Reexpressing CD151 in CD151-silenced cells reversed the adhesion and motility defects. Finally, loss of CD151 also impaired migration but not adhesion on substrates other than laminin-5. These data show that CD151 plays a critical role in tumor cell responses to laminin-5 and reveal promotion of integrin recycling as a novel potential mechanism whereby CD151 regulates tumor cell migration.

Antigens, CD↗
PubMed1997

Generation of monoclonal antibodies to integrin-associated proteins. Evidence that alpha3beta1 complexes with EMMPRIN/basigin/OX47/M6.

The alpha3beta1 integrin forms complexes with other cell-surface proteins, including transmembrane-4 superfamily (TM4SF) proteins (e. g. CD9, CD53, CD63, CD81, and CD82). To identify additional cell-surface proteins associated with alpha3beta1 integrin, a monoclonal antibody selection protocol was developed. Mice were immunized with integrin alpha3beta1-containing complexes isolated from HT1080 fibrosarcoma cells, and then 712 hybridoma clones were produced, and 95 secreted antibodies that recognized the HT1080 cell surface. Among these, 12 antibodies directly recognizing integrin alpha3 or beta1 subunits were eliminated. Of the remaining 83, 16 co-immunoprecipitated proteins that resembled integrins under non-stringent detergent conditions. These 16 included 15 monoclonal antibodies recognizing EMMPRIN/basigin/OX-47/M6, a 45-55-kDa transmembrane protein with two immunoglobulin domains. The EMMPRIN protein associated with alpha3beta1 and alpha6beta1, but not alpha2beta1 or alpha5beta1, as shown by reciprocal immunoprecipitation experiments. Also, association with alpha3beta1 was confirmed by cell-surface cross-linking and immunofluorescence co-localization experiments. Importantly, EMMPRIN-alpha3beta1 complexes appear not to contain TM4SF proteins, suggesting that they are distinct from TM4SF protein-alpha3beta1 complexes.

Animals↗
PubMed1998

Cell adhesion to laminin 1 or 5 induces isoform-specific clustering of integrins and other focal adhesion components.

Laminin 1 (alpha1beta1gamma1) and laminin 5 (alpha3beta3gamma2) induce cell adhesion with different involvement of integrins: both are ligands for the alpha6beta1 integrin, while alpha3beta1 integrin has affinity for laminin 5 only. These two laminin isoforms therefore provide good models to investigate whether alpha3beta1 and alpha6beta1 integrins play different roles in signal transduction and in focal adhesion formation. Laminin 1 or 5 induced adhesion of normal human skin fibroblasts to a similar extent but promoted different overall cell shapes. On laminin 1 the fibroblasts formed mainly filopodia-like structures, while on laminin 5 they developed lamellipodias. Staining of fibrillar actin with fluorescein-phalloidin revealed a similar organisation of the actin cytoskeleton on both substrates. However, integrin subunits and several cytoskeletal linker proteins, including vinculin, talin, and paxillin, showed an isoform-specific arrangement into focal adhesions. On laminin 1 they were recruited into thick and short aggregates localized at the termini of actin stress fibers, while on laminin 5 they appeared as dots or streaks clustered on a long portion of actin microfilaments. To test whether the differing affinity of laminin 1 or 5 for alpha3beta1 integrin would explain the formation of morphologically different focal adhesions, cells were seeded on laminin 1 under conditions in which alpha3beta1 integrins were occupied by a function-blocking antibody. This resulted in the formation of focal adhesions similar to that observed on laminin 5, where the integrin is occupied by its natural ligand. These results provide the first evidence for a cross-talk between alpha3beta1 and alpha6beta1 integrins and indicate that occupancy of alpha3beta1 integrins results in a trans-dominant regulation of alpha6beta1 integrin clustering and of focal adhesions. It suggests that recruitment of integrins and cytoskeletal linker proteins are laminin isoform-specific and that tissue specific expression of laminin isoforms might modulate cell behavior by the activation of distinct sets of integrins and by the induction of distinct molecular assemblies within the cell adhesion signaling complexes.

Breast↗
PubMed2003

EWI-2 regulates alpha3beta1 integrin-dependent cell functions on laminin-5.

EWI-2, a cell surface immunoglobulin SF protein of unknown function, associates with tetraspanins CD9 and CD81 with high stoichiometry. Overexpression of EWI-2 in A431 epidermoid carcinoma cells did not alter cell adhesion or spreading on laminin-5, and had no effect on reaggregation of cells plated on collagen I (alpha2beta1 integrin ligand). However, on laminin-5 (alpha3beta1 integrin ligand), A431 cell reaggregation and motility functions were markedly impaired. Immunodepletion and reexpression experiments revealed that tetraspanins CD9 and CD81 physically link EWI-2 to alpha3beta1 integrin, but not to other integrins. CD81 also controlled EWI-2 maturation and cell surface localization. EWI-2 overexpression not only suppressed cell migration, but also redirected CD81 to cell filopodia and enhanced alpha3beta1-CD81 complex formation. In contrast, an EWI-2 chimeric mutant failed to suppress cell migration, redirect CD81 to filopodia, or enhance alpha3beta1-CD81 complex formation. These results show how laterally associated EWI-2 might regulate alpha3beta1 function in disease and development, and demonstrate how tetraspanin proteins can assemble multiple nontetraspanin proteins into functional complexes.

Animals↗
PubMed1997

An antibody to the tetraspan membrane protein CD9 promotes neurite formation in a partially alpha3beta1 integrin-dependent manner.

The tetraspan cell surface glycoprotein, CD9, has been implicated in cellular signaling during growth and differentiation in the hematopoietic and nervous systems. Because CD9 expression is induced early in development in sensory and sympathetic neuroblasts, we investigated the role of CD9 in neurite outgrowth. We plated dissociated cells from neonatal sympathetic ganglia on immobilized anti-CD9 antibodies or antibodies against other cell surface molecules. We show here that B2C11, an anti-CD9 antibody that has been shown previously to activate Schwann cells in vitro, promotes robust neurite outgrowth from sympathetic neurons that is greater than that on other antibody surfaces and is comparable to neurite outgrowth on a collagen substratum. In addition, B2C11 causes dramatic morphological changes in neurons and glia from dissociated ganglia, including a flattening of these cells. Because CD9 interacts with integrins in many cell types including Schwann cells, and specifically with the alpha3beta1 integrin in some cells, we tested whether the effect of B2C11 on neurite outgrowth is mediated by this integrin. An anti-alpha3beta1 antibody, Ralph 3-1, attenuates the extent of neurite outgrowth on B2C11 and collagen, but not on laminin. Because the alpha3beta1 integrin has been shown to mediate neurite outgrowth on different substrates, these results provide a functional significance for the CD9-alpha3beta1 interaction; downstream signaling may be activated by this cis interaction on the cell surface in response to external cues that promote neurite outgrowth.

Animals↗
PubMed1998

Integrin alpha3beta1-mediated interaction with laminin-5 stimulates adhesion, migration and invasion of malignant glioma cells.

Gliomas, characterized by their progressively invasive phenotype, express integrin alpha3beta1 as a major receptor for the extracellular matrix both in vivo and in vitro. Since the integrin alpha3beta1 has been shown to be a specific receptor for laminin-5 (alpha3beta3gamma2), we examined the effects of purified human laminin-5 on adhesion, migration and invasion of human glioma cells. Among different types of laminin variants and other matrix proteins including fibronectin and vitronectin, laminin-5 was most potent in promoting adhesion and migration of different kinds of glioma cells. Laminin-5-mediated adhesion and migration were specifically inhibited by monoclonal antibodies against integrin alpha3 and beta1 chains, confirming the role of integrin alpha3beta1 as the major laminin-5 receptor. Invasion of the reconstituted basement membrane (i.e., Matrigel) by glioma cells was also selectively stimulated by laminin-5. Out results show that laminin-5 is the major extracellular stimulant for glioma cell adhesion, migration and invasion. The immunohistochemical distribution of laminin gamma2 chain, a laminin subunit unique to laminin-5, showed that it was expressed in the tumor parenchyma of human glioma tissues. Expression of laminin alpha3, beta3 and gamma2 chains in glioma tissues and in glioma cell lines was also demonstrated at the messenger RNA level by reverse transcription polymerase chain reaction. Our results, taken together, show that laminin-5 may be involved in the invasive phenotype of malignant gliomas both in vitro and in vivo.

Antigens, CD↗
PubMed2000

Active zones on motor nerve terminals contain alpha 3beta 1 integrin.

Active zones are the sites along nerve terminals where synaptic vesicles dock and undergo calcium-dependent exocytosis during synaptic transmission. Here we show, by immunofluorescent staining with antibodies generated against Xenopus laevis integrins, that alpha3beta1 integrin is concentrated at the active zones of Xenopus motor nerve terminals. Because integrins can link extracellular matrix molecules to cytoskeletal elements and participate in the formation of signaling complexes, the localization of integrin at active zones suggests that it may play a role in the adhesion of the nerve terminals to the synaptic basal lamina, in the formation and maintenance of active zones, and in some of the events associated with calcium-dependent exocytosis of neurotransmitter. Our findings also indicate that the integrin composition of the terminal Schwann cells differs from that of the motor nerve terminals, and this may account at least in part for differences in their adhesiveness to the synaptic basal lamina.

Animals↗
PubMed2006

Integrin alpha3beta1-dependent activation of FAK/Src regulates Rac1-mediated keratinocyte polarization on laminin-5.

Upon epidermal wounding, keratinocytes at the wound edge become activated, deposit newly synthesized laminin-5 into the extracellular matrix, and migrate into the wound bed. The interaction between integrin alpha3beta1 and laminin-5 is essential for establishment of a stable, leading lamellipodium and persistent keratinocyte migration. We previously showed that integrin alpha3beta1 activates the Rho family GTPase Rac1 and regulates Rac1-dependent formation of polarized, leading lamellipodia in migrating keratinocytes. In the present study, we explored the role of focal adhesion kinase (FAK) and src signaling in this process. We show that overexpression of the FAK inhibitor FAK-related non-kinase or of the FAK(Y397F) auto-phosphorylation mutant, induced abnormal, non-polarized spreading of keratinocytes on laminin-5. Integrin alpha3beta1 was required for full FAK auto-phosphorylation at Y397, and subsequent src kinase-dependent phosphorylation of FAK at residues Y861 and Y925, sites responsible for promoting signal transduction downstream of FAK, indicating that alpha3beta1 regulates the coordination of FAK/src signal transduction. Inhibiting either src kinase activity or FAK signaling interfered with alpha3beta1-mediated Rac1 activation and polarized cell spreading. These findings reveal a novel pathway in migratory keratinocytes wherein alpha3beta1-laminin-5 interactions regulate src kinase signaling through FAK, promoting Rac1 activation and polarized lamellipodium extension.

Animals↗
PubMed1998

Effect of ligand conformation on melanoma cell alpha3beta1 integrin-mediated signal transduction events: implications for a collagen structural modulation mechanism of tumor cell invasion.

The importance of three-dimensional interactions between receptors with their respective ligands has been extensively explored during the binding process, but considerably less so for postbinding events such as induction of signaling pathways. Tumor cell receptor association with basement membrane proteins is believed to facilitate the metastatic process. Melanoma and ovarian carcinoma cells have been shown to utilize the alpha3beta1 integrin to bind to models of the alpha1(IV)531-543 sequence from basement membrane (type IV) collagen [Miles, A. J., et al. (1994) J. Biol. Chem. 269, 30939-30945; Miles, A. J., et al. (1995) J. Biol. Chem. 270, 29047-29050]. In the present study, the effects of ligand three-dimensional structure on possible signal transduction pathways induced by alpha3beta1 integrin binding have been evaluated. Human melanoma cell binding to type IV collagen resulted in Tyr phosphorylation of p125(FAK), consistent with prior studies correlating beta1 integrin subunit binding to collagen and p125(FAK) Tyr phosphorylation. Cross-linking of an anti-alpha3 integrin subunit monoclonal antibody also induced p125(FAK) Tyr phosphorylation. Incubation of melanoma cells with single-stranded or triple-helical peptide models of alpha1(IV)531-543 induced Tyr phosphorylation of intracellular proteins. Immunoprecipitation analysis identified one of these proteins as pp125(FAK). Induction of p125(FAK) Tyr phosphorylation was enhanced and the time of induction was shortened when the ligand was used in triple-helical conformation. Subsequent clustering of either the single-stranded or the triple-helical ligand also increased the level of p125(FAK) phosphorylation compared to unclustered ligand. The clustered triple-helical peptide ligand induced more rapid paxillin Tyr phosphorylation than the single-stranded ligand. In addition, the induction of activated proteases was found to be more rapid due to ligand triple helicity. Overall, these studies have shown that (i) a model of an isolated sequence from type IV collagen, alpha1(IV)531-543, can induce alpha3beta1 integrin-mediated signal transduction in melanoma cells and (ii) ligand conformation (secondary, tertiary, and/or quaternary structure) can directly influence several alpha3beta1 integrin-mediated signal transduction events. The effects of ligand conformation suggest that a "collagen structural modulation" mechanism may exist for tumor cell invasion, whereby triple-helical collagen promotes cell binding and induction of signal transduction, subsequently leading to collagen dissolution by proteases, decreased signal transduction, and enhanced tumor cell motility.

Cell Adhesion↗
PubMed2003

alpha3beta1 integrin-CD151, a component of the cadherin-catenin complex, regulates PTPmu expression and cell-cell adhesion.

The beta1 family of integrins has been primarily studied as a set of receptors for the extracellular matrix. In this paper, we define a novel role for alpha3beta1 integrin in association with the tetraspanin CD151 as a component of a cell-cell adhesion complex in epithelial cells that directly stimulates cadherin-mediated adhesion. The integrin-tetraspanin complex affects epithelial cell-cell adhesion at the level of gene expression both by regulating expression of PTPmu and by organizing a multimolecular complex containing PKCbetaII, RACK1, PTPmu, beta-catenin, and E-cadherin. These findings demonstrate how integrin-based signaling can regulate complex biological responses at multiple levels to determine cell morphology and behavior.

Animals↗
PubMed2006

Contribution of alpha2beta1, alpha3beta1, alpha6beta4 integrins and 67 kDa laminin receptor to the interaction of epidermoid carcinoma A-431 cells with laminin-2/4.

Laminin-2/4 is the major laminin isoform of normal muscle and nerve tissues and plays an important role in tumor invasion and metastasis. Despite the fact that laminin-2/4 has been found in the skin basement membrane, insufficient evidence is available on the effect of laminin-2/4 on the behavior of both normal and transformed skin cells. A comparison of the contribution of alpha2beta1, alpha3beta1, alpha6beta4 integrins and 67 kDa laminin receptor on the surface of the human epidermoid carcinoma cell, A-431, to interaction with laminin-2/4 was carried out. The cell interaction with extracellular matrix component is a multistage process. We employed new methods for studying different stages of the interaction of A-431 cells with laminin-2/4. We demonstrated that integrins alpha2beta1, alpha3beta1, alpha6beta4 and 67 kDa laminin receptor are involved in the interaction of A-431 cells with laminin-2/4. We found that contribution of the same receptors to different stages of the interaction with laminin can be different. alpha2beta1 integrins are involved in EGF-induced A-431 cells' migration on laminin-2/4. We demonstrated the cooperation between alpha2beta1 and alpha3beta1 integrins during adhesion and spreading of A-431 cells on laminin-2/4-coated substrate. These results provide information about laminin-2/4 receptors and their contribution to different stages of the interaction with cells.

Animals↗
PubMed1997

alpha2,6-Sialyltransferase gene transfection into a human glioma cell line (U373 MG) results in decreased invasivity.

Glycosyltransferase gene transfection into cell lines has been an approach used successfully to elucidate the functional role of cell surface glycoconjugates. We have transfected the rat CMP-NeuAc:Galbeta1,4GlcNAc alpha2,6-sialyltransferase (EC 2.4.99.1) gene into a human, tumorigenic, glioma cell line, U373 MG. This transfection led to a marked inhibition of invasivity, alterations in adhesivity to fibronectin and collagen matrices, and inappropriately sialylated alpha3beta1 integrin. Adhesion-mediated protein tyrosine phosphorylation was reduced in the transfectants despite increased expression of focal adhesion kinase, p125fak. Furthermore, the transfectants showed a distinct cell morphology, an increased number of focal adhesion sites, and different sensitivity to cytochalasin D treatment than control U373 MG cells. These results suggest that inappropriate sialylation of cell surface glycoconjugates, such as integrins, can change focal adhesion as well as adhesion-mediated signal transduction and block glioma cell invasivity in vitro.

Animals↗
PubMed2002

C5b-9 and adhesion molecules in human idiopathic membranous nephropathy.

BACKGROUND: Cellular immune responses and C5b-9 seem to play an important role in the pathogenesis and progression of idiopathic membranous nephropathy (IMN). The aim of the study was to investigate the role of C5b-9 and adhesion molecules in the pathogenesis of the disease. METHODS: The clinical and pathological data of 35 patients with biopsy-proven IMN were correlated with immunohistochemical findings using monoclonal antibodies against T lymphocytes, monocytes/macrophages (MM), HLA-DR antigens, C5b-9, and adhesion molecules such as alpha3beta1, LFA-1beta, and ICAM-1. RESULTS: In the glomeruli, C5b-9 deposits showed a significant correlation with the intensity of IgG and C3 deposition. The stage of the disease had a significant negative relationship with the glomerular alpha3beta1 expression. In the tubulointerstitium (TIN), the number of HLA-DR(+) cells was highly correlated with the numbers of total T lymphocytes, MM, and LFA-1beta(+) cells, as well as with the percentage of tubules with C5b-9 deposits. The extent of ICAM-1 expression in the TIN was significantly correlated with the numbers of interstitial MM, HLA-DR(+), and LFA-1beta(+) cells, as well as with the extent of tubular C5b-9 deposition. The severity of tubular atrophy and interstitial fibrosis had a relationship with the numbers of total T lymphocytes, MM, HLA-DR(+), and LFA-1beta(+) cells and with the extent of tubular C5b-9 deposition and ICAM-1 expression in the TIN. Serum creatinine (Scr) was highly correlated with the numbers of interstitial total T lymphocytes, MM, HLA-DR(+), and LFA-1beta(+) cells. Moreover, Scr had a significant relationship with the severity of tubular atrophy and interstitial fibrosis, as well as with the extent of tubular C5b-9 deposition and ICAM-1 expression in the TIN. Proteinuria was significantly correlated with the extent of tubular alpha3beta1 expression. CONCLUSIONS: In IMN, C5b-9 formation may be secondary to IgG and C3 deposition. Proteinuria may contribute to the TIN damage by altering the expression of alpha3beta1 integrins in tubular cells. De novo ICAM-1 and C5b-9 expression within the TIN as well as the activated interstitial cells may be important factors leading to renal damage and renal function impairment.

Adult↗
PubMed1998

At 40 years of the 'Golden Chain'. Which are the stem cells in ectocervical epithelium?

In normal vaginal and ectocervical epithelium there is no consistent drop in density of the cytoplasmic ultrastructure at the transition between basal cells and parabasal cells. A peculiar golden-brownish argyrophilia, limited to the basal cells and vanishing in dysplasias, had been imputed to this density [2]. The reproducibility of the pertinent silver stain was improved. The phenomenon coincides with the known distribution of cytokeratin 19 and alpha3beta1 integrin. Ultrastructural signs of a specialized anchoring function of the basal cells, published autoradiographic and immunohistological evidence, clues as to the interference with differentiation by persistent papilloma virus DNA, and striking differences in wound healing between keratinizing and nonkeratinizing squamous epithelia prompted the thesis that the true stem cells in this epithelium are not the basal cells but the next higher parabasal cells.

Cervix Uteri↗
PubMed2006

N-acetylglucosaminyltransferase III antagonizes the effect of N-acetylglucosaminyltransferase V on alpha3beta1 integrin-mediated cell migration.

N-acetylglucosaminyltransferase V (GnT-V) catalyzes the addition of beta1,6-GlcNAc branching of N-glycans, which contributes to metastasis. N-acetylglucosaminyltransferase III (GnT-III) catalyzes the formation of a bisecting GlcNAc structure in N-glycans, resulting in the suppression of metastasis. It has long been hypothesized that the suppression of GnT-V product formation by the action of GnT-III would also exist in vivo, which will consequently lead to the inhibition of biological functions of GnT-V. To test this, we draw a comparison among MKN45 cells, which were transfected with GnT-III, GnT-V, or both, respectively. We found that alpha3beta1 integrin-mediated cell migration on laminin 5 was greatly enhanced in the case of GnT-V transfectant. This enhanced cell migration was significantly blocked after the introduction of GnT-III. Consistently, an increase in bisected GlcNAc but a decrease in beta1,6-GlcNAc-branched N-glycans on integrin alpha3 subunit was observed in the double transfectants of GnT-III and GnT-V. Conversely, GnT-III knockdown resulted in increased migration on laminin 5, concomitant with an increase in beta1,6-GlcNAc-branched N-glycans on the alpha3 subunit in CHP134 cells, a human neuroblastoma cell line. Therefore, in this study, the priority of GnT-III for the modification of the alpha3 subunit may be an explanation for why GnT-III inhibits GnT-V-induced cell migration. Taken together, our results demonstrate for the first time that GnT-III and GnT-V can competitively modify the same target glycoprotein and furthermore positively or negatively regulate its biological functions.

Cell Line, Tumor↗
PubMed2001

Conformational regulation of the fibronectin binding and alpha 3beta 1 integrin-mediated adhesive activities of thrombospondin-1.

The recognition of extracellular matrix components can be regulated by conformational changes that alter the activity of cell surface integrins. We now demonstrate that conformational regulation of the matrix glycoprotein thrombospondin-1 (TSP1) can also modulate its binding to an integrin receptor. F18 1G8 is a conformation-sensitive TSP1 antibody that binds weakly to soluble TSP1 in the presence of divalent cations. However, binding of the antibody to melanoma cells was strongly stimulated by adding exogenous TSP1 in the presence of calcium, suggesting that TSP1 undergoes a conformational change following its binding to the cell surface. This conformation was not induced by known cell surface TSP1 receptors, whereas binding of F18 was stimulated when TSP1 bound to fibronectin but not to heparin or fibrinogen. Conversely, binding of F18 to TSP1 enhanced TSP1 binding to fibronectin. Exogenous fibronectin also stimulated TSP1-dependent binding of F18 to melanoma cells. Binding of the fibronectin-TSP1 complex to melanoma cells was mediated by alpha4beta1 and alpha5beta1 integrins. Furthermore, binding to F18 or fibronectin strongly enhanced the adhesive activity of immobilized TSP1 for some cell types. This enhancement of adhesion was mediated by alpha3beta1 integrin and required that the alpha3beta1 integrin be in an active state. Fibronectin also enhanced TSP1 binding to purified alpha3beta1 integrin. Therefore, both fibronectin and the F18 antibody induce conformational changes in TSP1 that enhance the ability of TSP1 to be recognized by alpha3beta1 integrin. The conformational and functional regulation of TSP1 activity by fibronectin represents a novel mechanism for extracellular signal transduction.

Animals↗
PubMed2004

Laminin-2 stimulates the proliferation of epithelial cells in a conjunctival epithelial cell line.

Laminin-2 (LN-2, alpha2beta1gamma1) is a basement membrane-associated laminin isoform usually considered in the context of muscle and nerve tissues. To test the hypothesis that LN-2 can additionally modulate epithelial cell biology, an analysis of the role of LN-2 in cell adhesion, activation of signalling intermediates and proliferation was undertaken. A virally transformed human conjunctival epithelial cell line (HC0597) was utilized in this study. Adhesion assays using function-inhibiting antibodies demonstrated that alpha3beta1 integrin is essential for the rapid attachment of conjunctival epithelial cells to LN-2. Bromodeoxyuridine (BrdU) incorporation analyses revealed that, compared with LN-1 or LN-10, LN-2 significantly promotes epithelial proliferation. Phosphorylation of the signalling intermediates Erk1/2 and Akt-1 was observed within 15 min of cell adhesion to LN-2. Inhibiting alpha3beta1 integrin function decreased total cellular phosphotyrosine levels, specifically inhibited phosphorylation of Erk1/2 and Akt-1, and dampened the proliferation response of epithelial cells adherent to LN-2. Inhibition of Erk or Akt activation inhibited cell proliferation in a dose-dependent manner. However, the inhibition of Erk resulted in a stronger suppression of proliferation compared with Akt inhibition. From these results, it is concluded that human conjunctival epithelial cells adhere to immobilized LN-2 using alpha3beta1 integrin. alpha3beta1 integrin/LN-2 signalling, transduced primarily through an Erk pathway, enhances epithelial cell proliferation. These results demonstrate that LN-2 can impact on epithelial cell biology in addition to nerve and muscle, and provide information regarding the role of this isoform in ocular surface epithelial cells.

Cell Adhesion↗