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Squamous cell carcinoma of the oral cavity is characterized by persistent, disorganized expression of integrin alpha3beta1 and enhanced production of urinary-type plasminogen activator (uPA) and its receptor (uPAR) relative to normal oral mucosa. Because multivalent aggregation of alpha3beta1 integrin up-regulates uPA and induces a dramatic co-clustering of uPAR, we explored the hypothesis that lateral ligation of alpha3beta1 integrin by uPAR contributes to uPA regulation in oral mucosal cells. To investigate mechanisms by which uPAR/alpha3beta1 binding enhances uPA expression, integrin-dependent signal activation was assessed. Both Src and ERK1/2 were phosphorylated in response to integrin aggregation, and blocking Src kinase activity completely abrogated ERK1/2 activation and uPA induction, whereas inhibition of epidermal growth factor receptor tyrosine kinase activity did not alter uPA expression. Proteinase up-regulation occurred at the transcriptional level and mutation of the AP1 (-1967) site in the uPA promoter blocked the uPAR/integrin-mediated transcriptional activation. Because uPAR is redistributed to clustered alpha3beta1 integrins, the requirement for uPAR/alpha3beta1 interaction in uPA regulation was assessed. Clustering of alpha3beta1 in the presence of a peptide (alpha325) that disrupts uPAR/alpha3beta1 integrin binding prevented uPA induction. Depletion of cell surface uPAR using small interfering RNA also blocked uPA induction following integrin alpha3beta1 clustering. These results were confirmed using a genetic strategy in which alpha3 null epithelial cells reconstituted with wild type alpha3 integrin, but not a mutant alpha3 unable to bind uPAR, induced uPA expression upon integrin clustering, confirming the critical role of uPAR in integrin-regulated proteinase expression. Disruption of uPAR/alpha3beta1 binding using peptide alpha325 or small interfering RNA blocked filopodia formation and matrix invasion, indicating that this interaction stimulates invasive behavior. Together these data support a model wherein matrix-induced clustering ofalpha3beta1 integrin promotes uPAR/alpha3beta1 interaction, thereby potentiating cellular signal transduction pathways culminating in activation of uPA expression and enhanced uPA-dependent invasive behavior.

Neuroblastoma, a common tumor of nervous system origin in young children, is usually detected only after the primary tumor has metastasized and the chances of its complete removal are low. Metastatic neuroblastoma cells commonly suppress expression of the gene encoding caspase-8. In a neuroblastoma murine model, expression of caspase-8 and integrin alpha3beta1 was dramatically reduced during tumor development. Analysis of clinical biopsies supported the observation that expression of both genes is low in human patients with metastatic disease. These data suggest that loss of expression of both caspase-8 and unligated integrins contribute to the survival of tumor cells migrating from the primary tumor. Integrin receptors that are unable to find appropriate ligands can form a large molecular complex containing caspase-8, explaining why cells that have diminished expression of either of these two genes have a significant survival advantage in foreign microenvironments. Thus, upregulating expression of caspase-8 and integrins, or alternatively, antagonizing integrins within the primary tumor may be important therapeutically in halting neuroblastoma metastasis.

At the GABAergic synapses between inhibitory interneurons and a Purkinje neuron (PN) in the cerebellum, the postsynaptic depolarization induces the long-term potentiation (called rebound potentiation; RP) of GABAA receptor responsiveness. Here, we show that integrins, a type of cell-adhesion molecules, are involved in the regulation of RP. Integrin activation by Mn2+ impaired the RP induction of GABA responsiveness and mIPSCs in PNs, which was abolished by the function blocking antibody against either integrin alpha3 or beta1 subunit, but not by that against alpha5 or alphaV subunit. Furthermore, overexpression of integrin alpha3 subunit in a PN by itself impaired the RP induction. We also show that Src-family of protein tyrosine kinases mediated the suppressive effect of integrin activity on the RP induction. Thus, the integrin/Src pathway negatively regulates the induction of long-term plasticity at inhibitory synapses on a cerebellar PN.

Integrins attach cells to extracellular matrix (ECM) and mediate signals from ECM to cells or from cells to ECM. They regulate cell functions, including adhesion, migration, cell cycle regulation, and differentiation. Podocytes may detach from the glomerular basement membrane (GBM) and be excreted in the urine, and proteinuria is found in patients with primary focal segmental glomerulosclerosis (FSGS); both may be associated with loss of alpha3beta1integrins. In this study, we have examined the podocyte number in patients with primary FSGS and normal controls, and the alpha3- and beta1-integrin subunits expression of podocytes in patients with primary FSGS and chronic puromycin aminonucleoside (PAN)-treated rats by the morphometric, immunoperoxidase histochemical, and immunoelectron microscopic examination. We also measured their expression serially in rats that received repeated PAN injection. The results showed that the podocyte number was significantly decreased in patients with primary FSGS than in normal control (P0.05). The immunostaining score showed that both alpha3- and beta1-integrin subunits on podocytes in patients with primary FSGS were significantly lower than in normal controls (both P0.01). The number of immuno-gold particles of alpha3- and beta1-integrins at the effaced foot process area of patients with primary FSGS were also significantly decreased than that of normal controls (both P0.05). The immunostaining score of both alpha3- and beta1-integrin subunits was negatively correlated with the degree of glomerular sclerosing score and the amount of daily protein loss, and they were positively correlated with the number of podocytes. Chronic 12-week PAN-treated rats showed similar findings with decreased immunostaining expression and immuno-gold particles of alpha3-integrin on podocytes than in normal control (both P0.05). The chronic PAN-treated rats also showed a trend toward gradually decreased immunostaining expression of alpha3-integrin subunit on podocyte during the progress from normal to FSGS state. These studies indicate that podocyte expression of alpha3- and beta1-integrin subunits is significantly reduced in humans with primary FSGS and chronic PAN-treated rats, before the morphological changes of FSGS are observed. The decreased podocyte expression of alpha3beta1 integrins is closely related with podocyte depletion, glomerular sclerosis, and daily protein loss in patients with primary FSGS.

Human herpesvirus 8 (HHV-8) causes Kaposi’s sarcoma and pleural effusion lymphoma. In this study, we show that dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN; CD209) is a receptor for HHV-8 infection of myeloid DCs and macrophages. DC-SIGN was required for virus attachment to these cells and DC-SIGN-expressing cell lines. HHV-8 binding and infection were blocked by anti-DC-SIGN mAb and soluble DC-SIGN, and mannan, a natural ligand for DC-SIGN. Infection of DCs and macrophages with HHV-8 led to production of viral proteins, with little production of viral DNA, similar to HHV-8 infection of vascular endothelial cells. Infection of DCs resulted in down-regulation of DC-SIGN, a decrease in endocytic activity, and an inhibition of Ag stimulation of CD8+ T cells. We propose that DC-SIGN serves as a portal for immune dysfunction and oncogenesis caused by HHV-8 infection.

Binding of integrins to proteins of the extracellular matrix (ECM) provides structural and signaling information for biological processes such as cell proliferation, migration, neurite outgrowth, and differentiation. Integrins represent a family of heterodimeric transmembrane cell surface receptors. Besides connecting the ECM with the cytoskeleton, integrins also induce various signaling pathways in response to ligand binding. Integrin ligation leads to cytoplasmic protein-protein interactions requiring both integrin cytoplasmic tails. These sequences are initiation points for focal adhesion formation and subsequent signal transduction cascades. In this study, we addressed the question of whether the short cytoplasmic tail of the alpha(3) integrin subunit of alpha(3)beta(1) integrin is required for alpha(3)beta(1) integrin-dependent processes. For this purpose, cDNA representing the extracellular and transmembrane domain of the interleukin 2 receptor (IL2R) alpha subunit and the cytoplasmic sequence of the alpha(3) integrin subunit was transfected into PC12 cells. Autonomous expression of the cytoplasmic alpha(3) tail does not affect attachment but leads to inhibition of neuronal differentiation on laminin 5. This indicates that the cytoplasmic alpha(3) sequence is not required for cell attachment but is necessary for long-term adhesion and for the reorganization of the cytoskeleton that precedes neuronal differentiation. Inhibition of neurite outgrowth by chimeric IL2R-alpha(3) can be rescued by treatment of transfected cells with the pharmacological inhibitor Y27632, which inhibits the RhoA downstream effector Rho kinase alpha.

The Rho-like guanosine triphosphatase Rac1 regulates various signaling pathways, including integrin-mediated adhesion and migration of cells. However, the mechanisms by which integrins signal toward Rac are poorly understood. We show that the Rac-specific guanine nucleotide exchange factor Tiam1 (T-lymphoma invasion and metastasis 1) is required for the integrin-mediated laminin (LN)-5 deposition, spreading, and migration of keratinocytes. In contrast to wild-type keratinocytes, Tiam1-deficient (Tiam1−/−) keratinocytes are unable to adhere to and spread on a glass substrate because they are unable to deposit their own LN5 substrate. Both Tiam1 and V12Rac1 can rescue the defects of Tiam1−/− keratinocytes, indicating that these deficiencies are caused by impaired Tiam1-mediated Rac activation. Tiam1−/− cells are unable to activate Rac upon α3β1-mediated adhesion to an exogenous LN5 substrate. Moreover, Tiam1 deficiency impairs keratinocyte migration in vitro and reepithelialization of excision wounds in mouse skin. Our studies indicate that Tiam1 is a key molecule in α3β1-mediated activation of Rac, which is essential for proper production and secretion of LN5, a requirement for the spreading and migration of keratinocytes.

Metastasis of primary tumors leads to a very poor prognosis for patients suffering from cancer. Although it is well established that not every tumor will eventually metastasize, it is less clear whether primary tumors acquire genetic alterations in a stochastic process at a late stage, which make them invasive, or whether genetic alterations acquired early in the process of tumor development drive primary tumor growth and determine whether this tumor is going to be metastatic. To address this issue, we tested genes identified in a large-scale comparative genomic hybridization analysis of primary tumor for their ability to confer metastatic properties on a cancer cell. We identified amplification of the ACK1 gene in primary tumors, which correlates with poor prognosis. We further show that overexpression of Ack1 in cancer cell lines can increase the invasive phenotype of these cells both in vitro and in vivo and leads to increased mortality in a mouse model of metastasis. Biochemical studies show that Ack1 is involved in extracellular matrix-induced integrin signaling, ultimately activating signaling processes like the activation of the small GTPase Rac. Taken together, this study supports a theory from Bernards and Weinberg [Bernards, R. & Weinberg, R. A. (2002) Nature 418, 823], which postulates that the tendency to metastasize is largely predetermined.

Cell motility is highly dependent on the organization and function of microdomains composed of integrin, proteolipid/tetraspanin CD9, and ganglioside (Ono, M., Handa, K., Sonnino, S., Withers, D. A., Nagai, H., and Hakomori, S. (2001) Biochemistry 40, 6414-6421; Kawakami, Y., Kawakami, K., Steelant, W. F. A., Ono, M., Baek, R. C., Handa, K., Withers, D. A., and Hakomori, S. (2002) J. Biol. Chem. 277, 34349-34358), later termed "glycosynapse 3" (Hakomori, S., and Handa, K. (2002) FEBS Lett. 531, 88-92, 2002). Human bladder cancer cell lines KK47 (noninvasive and nonmetastatic) and YTS1 (highly invasive and metastatic), both derived from transitional bladder epithelia, are very similar in terms of integrin composition and levels of tetraspanin CD9. Tetraspanin CD82 is absent in both. The major difference is in the level of ganglioside GM3, which is several times higher in KK47 than in YTS1. We now report that the GM3 level reflects glycosynapse function as follows: (i) a stronger interaction of integrin alpha3 with CD9 in KK47 than in YTS1; (ii) conversion of benign, low motility KK47 to invasive, high motility cells by depletion of GM3 by P4 (D-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol) treatment or by knockdown of CD9 by the RNA interference method; (iii) reversion of high motility YTS1 to low motility phenotype like that of KK47 by exogenous GM3 addition, whereby the alpha3-to-CD9 interaction was enhanced; (iv) low GM3 level activated c-Src in YTS1 or in P4-treated KK47, and high GM3 level by exogenous addition caused Csk translocation into glycosynapse, with subsequent inhibition of c-Src activation; (v) inhibition of c-Src by "PP2" in YTS1 greatly reduced cell motility. Thus, GM3 in glycosynapse 3 plays a dual role in defining glycosynapse 3 function. One is by modulating the interaction of alpha3 with CD9; the other is by activating or inhibiting the c-Src activity, possibly through Csk translocation. High GM3 level decreases tumor cell motility/invasiveness, whereas low GM3 level enhances tumor cell motility/invasiveness. Oncogenic transformation and its reversion can be explained through the difference in glycosynapse organization.

We studied the adhesion characteristics of immortalized human corneal epithelial (HCE) cells on purified human laminin (Ln)-10 (alpha5beta1gamma1). Immunofluorescence studies with monoclonal antibodies (MAb) revealed the presence of laminin alpha5 chain receptor Lutheran (Lu) in vivo on the basal cells of cornea and as punctate cell surface-confined reactivity on adhering and spread HCE cells, while alpha- and beta-dystroglycans could not be found. Northern blot analysis showed the presence of 4.0 and 2.8 kb Lu transcripts in HCE cells. The results showed that Ln-10 induced adhesion and spreading of HCE cells without formation of focal adhesions. Quantitative adhesion assay showed that Lu together with integrin (Int) alpha3beta1 independently mediated the adhesion of HCE cells to Ln-10.

Beta ig-h3 is an extracellular matrix protein whose expression is highly induced by transforming growth factor (TGF)-beta1. Whereas beta ig-h3 is known to mediate keratinocyte adhesion and migration, its effects on keratinocyte differentiation remain unclear. In the present study, it was demonstrated that expression of both beta ig-h3 and TGF-beta1 was enhanced during keratinocyte differentiation and that expression of the former was strongly induced by that of the latter. This study also asked whether changes in beta-h3 expression would affect keratinocyte differentiation. Indeed, down-regulation of beta ig-h3 by transfection with antisense beta ig-h3 cDNA constructs effectively inhibited keratinocyte differentiation by decreasing the promoter activities and thus expression of involucrin and transglutaminase. The result was an approximately 2-fold increase in mitotic capacity of the cells. Conversely, overexpression of beta ig-h3, either by transfection with beta ig-h3 expression plasmids or by exposure to recombinant beta ig-h3, enhanced keratinocyte differentiation by inhibiting cell proliferation and concomitantly increasing involucrin and transglutaminase expression. Recombinant beta ig-h3 also promoted keratinocyte adhesion through interaction with integrin alpha3beta1. Changes in beta ig-h3 expression did not affect intracellular calcium levels. Subsequent analysis revealed not only induction of Akt phosphorylation by recombinant beta ig-h3 but also blockage of Akt phosphorylation by LY294002, an inhibitor of phosphatidylinositol 3-kinase. Taken together, these findings indicate that enhanced beta ig-h3, induced by enhanced TGF-beta during keratinocyte differentiation, provoked cell differentiation by enhancing involucrin and transglutaminase expression through the integrin alpha3beta1 and phosphatidylinositol 3-kinase/Akt signaling pathway. Lastly, it was observed that beta ig-h3-mediated keratinocyte differentiation was caused by promotion of cell adhesion and not by calcium regulation.

The high affinity interaction between the urokinase-type plasminogen activator (uPA) and its glycolipid-anchored cellular receptor (uPAR) promotes plasminogen activation and the efficient generation of pericellular proteolytic activity. We demonstrate here that expression of the tetraspanin CD82/KAI1 (a tumor metastasis suppressor) leads to a profound effect on uPAR function. Pericellular plasminogen activation was reduced by approximately 50-fold in the presence of CD82, although levels of components of the plasminogen activation system were unchanged. uPAR was present on the cell surface and molecularly intact, but radioligand binding analysis with uPA and anti-uPAR antibodies revealed that it was in a previously undetected cryptic form unable to bind uPA. This was not due to direct interactions between uPAR and CD82, as they neither co-localized on the cell surface nor could be co-immunoprecipitated. However, expression of CD82 led to a redistribution of uPAR to focal adhesions, where it was shown by double immunofluorescence labeling to co-localize with the integrin alpha(5)beta(1), which was also redistributed in the presence of CD82. Co-immunoprecipitation experiments showed that, in the presence of CD82, uPAR preferentially formed stable associations with alpha(5)beta(1), but not with a variety of other integrins, including alpha(3)beta(1). These data suggest that CD82 inhibits the proteolytic function of uPAR indirectly, directing uPAR and alpha(5)beta(1) to focal adhesions and promoting their association with a resultant loss of uPA binding. This represents a novel mechanism whereby tetraspanins, integrins, and uPAR, systems involved in cell adhesion and migration, cooperate to regulate pericellular proteolytic activity and may suggest a mechanism for the tumor-suppressive effects of CD82/KAI1.

Laminin-5 regulates various cellular functions, including cell adhesion, spreading, and motility. Here, we expressed the five human laminin alpha3 chain globular (LG) domains as monomeric, soluble fusion proteins, and examined their biological functions and signaling. Recombinant LG3 (rLG3) protein, unlike rLG1, rLG2, rLG4, and rLG5, played roles in cell adhesion, spreading, and integrin alpha3beta1 binding. More significantly, we identified a novel motif (PPFLMLLKGSTR) in the LG3 domain that is crucial for these responses. Studies with the synthetic peptides delineated the PPFLMLLKGSTR peptide within LG3 domain as a major site for both integrin alpha3beta1 binding and cell adhesion. Substitution mutation experiments suggest that the Arg residue is important for these activities. rLG3 protein- and PPFLMLLKGSTR peptide-induced keratinocyte adhesion triggered cell signaling through FAK phosphorylation at tyrosine-397 and -577. To our knowledge, this is the first report demonstrating that the PPFLMLLKGSTR peptide within the LG3 domain is a novel motif that is capable of supporting integrin alpha3beta1-dependent cell adhesion and spreading.

CD151, one of the tetraspanins, forms a stable complex with integrin alpha3beta1, the major laminin receptor on the cell surface. We found that 8C3, an anti-CD151 mAb obtained by screening for reactivity with integrin alpha3beta1-CD151 complexes, was capable of dissociating CD151 from integrin alpha3beta1, thereby allowing us to deplete CD151 from purified integrin alpha3beta1-CD151 complexes. The CD151-free integrin alpha3beta1 thus obtained showed a significant reduction in its ability to bind to laminin-10/11, a high-affinity ligand for integrin alpha3beta1, with a concomitant reduction in its reactivity with mAb AG89, which recognizes activated beta1-containing integrins. These results raised the possibility that the association of integrin alpha3beta1 with CD151 potentiates the ligand-binding activity of the integrin through sustaining its activated conformation. In support of this possibility, the ligand-binding activity was restored when CD151-free integrin alpha3beta1 was reassociated with purified CD151. 8C3-induced dissociation of CD151 from integrin alpha3beta1 was also demonstrated on the surface of living cells by fluorescent resonance energy transfer imaging, accompanied by a concomitant reduction in the cell adhesion to laminin-10/11-coated substrates. CD151 knock-down by RNA interference also resulted in a reduction in the adhesive activity of the cells. Taken together, these results indicate that CD151 association modulates the ligand-binding activity of integrin alpha3beta1 through stabilizing its activated conformation not only with purified proteins but also in a physiological context.

We show that alpha3 integrin mutation disrupts distinct aspects of neuronal migration and placement in the cerebral cortex. The preplate develops normally in alpha3 integrin mutant mice. However, time lapse imaging of migrating neurons in embryonic cortical slices indicates retarded radial and tangential migration of neurons, but not ventricular zone-directed migration. Examination of the actin cytoskeleton of alpha3 integrin mutant cortical cells reveals aberrant actin cytoskeletal dynamics at the leading edges. Deficits are also evident in the ability of developing neurons to probe their cellular environment with filopodial and lamellipodial activity. Calbindin or calretinin positive upper layer neurons as well as the deep layer neurons of alpha3 integrin mutant mice expressing EGFP were misplaced. These results suggest that alpha3beta1 integrin deficiency impairs distinct patterns of neuronal migration and placement through dysregulated actin dynamics and defective ability to search and respond to migration modulating cues in the developing cortex.

Laminin-5 is an important constituent of the basal lamina. The receptors for laminin-5, the integrins alpha3beta1 and alpha6beta4, have been associated with epithelial wound migration and carcinoma invasion. The signal transduction mechanisms that regulate these integrins are not well understood. We report here that the small GTPase Rap1 regulates the adhesion of a number of cell lines to various extracellular matrix proteins including laminin-5. cAMP also mediates cell adhesion and spreading on laminin-5, a process that is independent of protein kinase A but rather dependent on Epac1, a cAMP-dependent exchange factor for Rap. Interestingly, although both alpha3beta1 and alpha6beta4 mediate adhesion to laminin-5, only alpha3beta1-dependent adhesion is dependent on Rap1. These results provide evidence for a function of the cAMP-Epac-Rap1 pathway in cell adhesion and spreading on different extracellular matrix proteins. They also define different roles for the laminin-binding integrins in regulated cell adhesion and subsequent cell spreading.

In addition to the three known beta(1) integrin recognition sites in the N-module of thrombospondin-1 (TSP1), we found that beta(1) integrins mediate cell adhesion to the type 1 and type 2 repeats. The type 1 repeats of TSP1 differ from typical integrin ligands in that recognition is pan-beta(1)-specific. Adhesion of cells that express one dominant beta(1) integrin on immobilized type 1 repeats is specifically inhibited by antagonists of that integrin, whereas adhesion of cells that express several beta(1) integrins is partially inhibited by each alpha-subunit-specific antagonist and completely inhibited by combining the antagonists. beta(1) integrins recognize both the second and third type 1 repeats, and each type 1 repeat shows pan-beta(1) specificity and divalent cation dependence for promoting cell adhesion. Adhesion to the type 2 repeats is less sensitive to alpha-subunit antagonists, but a beta(1) blocking antibody and two disintegrins inhibit adhesion to immobilized type 2 repeats. beta(1) integrin expression is necessary for cell adhesion to the type 1 or type 2 repeats, and beta(1) integrins bind in a divalent cation-dependent manner to a type 1 repeat affinity column. The widely used TSP1 function blocking antibody A4.1 binds to a site in the third type 2 repeat. A4.1 proximally inhibits beta(1) integrin-dependent adhesion to the type 2 repeats and indirectly inhibits integrin-dependent adhesion mediated by the TSP1 type 1 repeats. Although antibody A4.1 is also an antagonist of CD36 binding to TSP1, these data suggest that some biological activities of A4.1 result from antagonism of these novel beta(1) integrin binding sites.

Regulation of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF-A) expression in podocytes. Background Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF-A) is expressed constitutively in the adult glomerular podocytes at high levels; however, the regulation of its production is unclear. Recent data from podocyte-specific knockout mice suggest that VPF/VEGF-A is critical for the proper maintenance of glomerular filtration barrier and the glomerular endothelial fenestrae. We hypothesized that the glomerular basement membrane (GBM) matrix–podocyte interaction may play a role in the constitutive expression of VPF/VEGF-A in the adult glomerulus. Methods VPF/VEGF-A mRNA levels in a human podocyte cell line grown in the presence of various extracellular matrices were quantitated by real-time polymerase chain reaction (PCR) experiments. VPF/VEGF-A protein levels in the culture supernatant from the same conditions were measured by enzyme-linked immunosorbent assay (ELISA). Promoter activity of VPF/VEGF-A gene in these cells was performed by transfecting full length (2.6 kb) VPF/VEGF-A promoter, which is fused with luciferase reporter gene. Immunoprecipitation and Western blot experiments were carried out in order to detect the association of hypoxia-inducible factor-α (HIF-α) and p300 in podocyte cells. Results In this study, we provide preliminary evidence that signaling through the extracellular matrix proteins and, in particular, laminin and its receptor α3β1 integrin may regulate VPF/VEGF-A production in cultured podocytes in vitro. We also present data that increased activity of the transcription factor HIF-αs in podocyte is not related to hypoxia and may lead to up-regulation of VPF/VEGF-A transcription. The classical type protein kinase C (PKC) may be a potential intermediate signaling molecule in this event. Conclusion These data suggest a novel nonhypoxic regulation of VPF/VEGF-A production in the glomerulus of the kidney during physiologic states. These observations may form the basis of more elaborate studies that will finally provide the detailed signaling pathway for VPF/VEGF-A synthesis in podocytes and will help our understanding of the pathogenesis of various VPF/VEGF-A–related diseases in the glomerulus of the kidney.

Fibronectin induces ureteric bud cells branching and cellular cord and tubule formation. Background The extracellular matrix (ECM) protein fibronectin is involved in several stages of embryogenesis. Fibronectin exerts its effect through interaction with cellular integrin and nonintegrin receptors. Methods We investigated the effect of fibronectin on branching and tubulogenesis of ureteric bud cells in a three-dimensional gel culture system. Primary ureteric bud cells from mouse embryos at gestation 11 days (E11) were isolated and established in culture. Fibronectin and integrin subunits were localized using immunoperoxidase staining. Results In three-dimensional collagen type I gel culture of ureteric bud cell, fibronectin dose dependently induces cord and tubule formation. Both ureteric bud cells and ureteric bud branches in embryonic kidney express the same multiple integrin subunits that include β 1 , β 3 , α 3 , α 4 and α v . Embryonic kidneys examined at E12, E14, and E16 days of gestation express fibronectin in the undifferentiated mesenchyme especially next to ureteric bud branches and in the interstitium around glomerulotubular structures and blood vessels. Fibronectin expression was similar at the tips and stalks of branching ureteric bud. Fibronectin expression is maximum at E12 and decreases with advanced gestation. Cultured ureteric bud cells also express fibronectin. RGD peptides inhibit cord and tubular formation in the three-dimensional gel. Anti-α 3 β 1 antibody partially inhibits fibronectin-induced cord and tubule formation. Hepatocyte growth factor (HGF), fibroblast growth factor (FGF), and glial cell line-derived neurotrophic factor (GDNF) induce ureteric bud cell cord formation in three-dimensional gel. The effects of growth factors are delayed and quantitatively less compared to the effect of fibronectin. Conclusion Fibronectin induces ureteric bud cells branching and tubulogenesis through interaction with multiple integrin receptors. Cultured ureteric bud cells express febronectin and the origin of fibronectin at mesenchyme-ureteric bud interface is likely both the metanephric mesenchyme and ureteric bud epithelium. Addition of individual neutralizing antibodies to β 1 , β 3 , α 3 , α 4, α 6 and α v integrin subunits does not block the effect of fibronectin. Only an antibody to α 3 β 1 integrin substantially blocks the effect of fibronectin. Other mechanisms, including unidentified integrins, are likely involved in fibronectin-induced cord and tubule formation.

In cancer and angiogenesis, coagulation-independent roles of tissue factor (TF) in cell migration are incompletely understood. Immobilized anti-TF extracellular domain antibodies induce cell spreading, but this phenomenon is epitope specific and is not induced by anti-TF 5G9. Spreading on anti-TF is beta1 integrin-dependent, indicating functional interactions of the TF extracellular domain 5G9 epitope (a presumed integrin-binding site) and integrins. Recombinant TF extracellular domain supports adhesion of cells expressing alphavbeta3 or certain beta1 integrin heterodimers (alpha3beta1, alpha4beta1, alpha5beta1, alpha6beta1, alpha9beta1) and adhesion is blocked by specific anti-integrin antibodies or mutations in the integrin ligand-binding site. Although several studies have linked TF to cell migration, we here demonstrate that TF specifically regulates alpha3beta1-dependent migration on laminin 5. Expression of TF suppresses alpha3beta1-dependent migration, but only when the TF cytoplasmic domain is not phosphorylated. Suppression of migration can be reversed by 5G9, presumably by disrupting integrin interaction, or by the protease ligand VIIa, known to induce PAR-2-dependent phosphorylation of TF. In both cases, release of alpha3beta1 inhibition is prevented by mutation of critical phosphorylation sites in the TF cytoplasmic domain. Thus, TF influences integrin-mediated migration through cooperative intra- and extracellular interactions and phosphorylation regulates TF's function in cell motility.

Inadequate or inappropriate adhesion of epithelial cells to extracellular matrix leads to a form of apoptosis known as anoikis. During various tissue remodelling events, such as wound healing or carcinoma invasion, changes in the physical properties, and/or composition of the extracellular matrix, can lead to anoikis of epithelial cells that lack appropriate receptor-matrix interactions. Laminin-5 is the major ligand for keratinocyte adhesion in the epidermis, and it also promotes keratinocyte survival in vivo and in vitro. Integrins alpha 3 beta 1 and alpha 6 beta 4 are the major receptors for laminin-5; however, specific roles for these integrins in keratinocyte survival have not been determined. In the current study, we exploited keratinocyte cell lines derived from wild-type or alpha 3 integrin knockout mice to reveal a critical role for alpha 3 beta 1 in protecting keratinocytes from apoptosis upon serum withdrawal. We show that alpha 3 beta 1-mediated adhesion to laminin-5 extracellular matrix inhibits proteolytic activation of caspase-3 and TUNEL-staining, both hallmarks of apoptosis. We also show that alpha 3 beta1-mediated adhesion activates focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), and that inhibition of either FAK or ERK signaling leads to apoptosis of keratinocytes attached to laminin-5. alpha 6 beta 4-mediated adhesion to laminin-5 only partially protects cells from apoptosis in the absence of alpha 3 beta 1, and alpha 6 beta 4 is not necessary for cell survival in the presence of alpha 3 beta 1. These results suggest that alpha 3 beta 1 is necessary and sufficient for maximal keratinocyte survival on laminin-5. We propose a model to address the potential importance of alpha 3 beta 1-mediated survival for migrating keratinocytes at the leading edge of a cutaneous wound.

Epithelial cell migration is a crucial event in wound healing, yet little is known about mechanisms whereby integrins regulate epithelial cell polarization and migration. In the present work, we demonstrate the importance of adhesion through the alpha3beta1 integrin in promoting the stabilization of leading lamellipodia in migrating keratinocytes. We demonstrate that this integrin is found at the leading edge of migrating keratinocytes and that inhibition of alpha3beta1 binding to laminin-5 prevents the formation of stable leading lamellipodia. Consistent with this observation, keratinocytes derived from alpha3beta1-deficient mice fail to form stable leading lamellipodia but retain the ability to form actin-containing protrusions that rapidly extend and retract from the cell membrane. Formation of a leading lamellipodium also requires alpha3beta1-dependent activation of Rac1, because alpha3beta1-deficient keratinocytes show decreased activation of Rac1 compared with alpha3beta1-expressing cells, and formation of stable leading lamellipodia can be inhibited in the latter cells by expression of the dominant negative Rac1 mutant Rac1N17. Furthermore, alpha3beta1-deficient keratinocytes expressing constitutively active Rac1L61 failed to form stable lamellipodia when plated onto laminin-5, demonstrating that alpha3beta1 is required for Rac1-mediated formation of a stable lamellipodium. These observations identify a crucial role for integrin-mediated adhesion and signaling in the formation of large, polarized, stable lamellipodia by migrating epithelial cells. To our knowledge, this study is the first to demonstrate that signal transduction through a specific integrin is required to direct the development of a lamellipodium from an initial protrusion and promote persistent epithelial cell migration.

Alpha3beta1 integrin has been considered to be a mysterious adhesion molecule due to the pleiotropy in its ligand-binding specificity. However, recent studies have identified laminin isoforms as high-affinity ligands for this integrin, and demonstrated that alpha3beta1 integrin plays a number of essential roles in development and differentiation, mainly by mediating the establishment and maintenance of epithelial tissues. Furthermore, alpha3beta1 integrin is also implicated in many other biological phenomena, including cell growth and apoptosis, angiogenesis and neural functions. This integrin receptor forms complexes with various other membrane proteins, such as the transmembrane-4 superfamily proteins (tetraspanins), cytoskeletal proteins and signaling molecules. Recently, lines of evidence have been reported showing that complex formation regulates integrin functions in cell adhesion and migration, signal transduction across cell membranes, and cytoskeletal organization. In addition to these roles in physiological processes, alpha3beta1 integrin performs crucial functions in various pathological processes, especially in wound healing, tumor invasion and metastasis, and infection by pathogenic microorganisms.

One of the basement membrane (BM) proteins, laminin-5 (LN5), is known to support efficient cell adhesion and migration through interaction with integrins on the basal plasma membrane. Here, we show that a soluble form of LN5 induced migration of human epithelial cells and carcinoma cells by interacting with integrins on the apical cell surface. Although both LN5 and laminin-10/11 (LN10/11) promoted cell migration when coated onto a plastic surface as insoluble substrata, only LN5 stimulated cell migration in its soluble form on other substrata such as fibronectin (FN), vitronectin (VN) and collagen. Soluble LN5 interacted with integrins alpha3beta1 and alpha6beta1 on the apical cell surface and stimulated cell migration, while the cell morphology was largely dependent on the underlying substratum. Thus, integrin signals from the apical surface and the basal surface synergistically regulated cytoskeletal organization and cell motility. Soluble and insoluble LN5 induced cell motility by activating signal pathways via protein kinase C (PKC), phosphoinositide 3-OH kinase (PI3-K) and MAP kinase. The PKC dependency was more prominent for soluble LN5 than insoluble LN5, and was absent in the stimulation by insoluble LN10/11. In vitro scratch assays with keratinocytes, self-produced soluble LN5 bound to the apical cell surface of migrating cells at the scratched edges, suggesting that soluble LN5 may contribute to cell migration in pathological conditions such as wound healing and tumor invasion.

Selective antitumor chemotherapy can be achieved by using antibody-drug conjugates that recognize surface proteins upregulated in cancer cells. One such receptor is integrin α 3 β 1 , which is overexpressed on malignant melanoma, prostate carcinoma, and glioma cells. We previously identified a human single-chain Fv antibody (scFv), denoted Pan10, specific for integrin α 3 β 1 that is internalized by human pancreatic cancer cells. Herein, we describe the chemical introduction of reactive thiol groups onto Pan10, the specific conjugation of the modified scFv to maleimide-derivatized analogs of the potent cytotoxic agent duocarmycin SA, and the properties of the resultant conjugates. Our findings provide evidence that Pan10-drug conjugates maintain the internalizing capacity of the parent scFv and are cytotoxic at nanomolar concentrations. Our Pan10-drug conjugates may be promising candidates for targeted chemotherapy of malignant diseases associated with overexpression of integrin α 3 β 1 .

Human herpesvirus 8 (HHV-8; Kaposi's sarcoma-associated herpesvirus) envelope glycoprotein gB possesses an RGD motif, interacts with alpha 3 beta 1 integrin, and uses it as one of the entry receptors. HHV-8 induces the integrin-dependent focal adhesion kinase (FAK), a critical step in the outside-in signaling pathways necessary for the subsequent phosphorylation of other cellular kinases, cytoskeletal rearrangements, and other functions. As an initial step toward deciphering the role of HHV-8 gB-integrin interaction in infection, signal pathways induced by gB were examined. A truncated form of gB without the transmembrane and carboxyl domains (gB Delta TM), a gB Delta TM mutant form (gB Delta TM-RGA) with an RGD-to-RGA mutation, and inhibitors of cellular kinases were used. HHV-8 gB Delta TM, but not gB Delta TM-RGA, induced FAK phosphorylation in target cells, which was in part dependent on the presence of alpha 3 beta 1 integrin. FAK was critical for the subsequent phosphorylation of Src by gB Delta TM, and Src induction was essential for the phosphorylation of phosphatidylinositol 3-kinase (PI-3K). HHV-8 gB Delta TM-induced PI-3K was essential for the induction of RhoA and Cdc42 Rho GTPases that was accompanied by the cytoskeletal rearrangements. These gB-induced morphological changes were inhibited by the PI-3K inhibitors. Ezrin, one of the essential elements required to cross-link the actin cytoskeleton with the plasma membrane and to induce the morphological changes, was induced by the Rho GTPases. Inhibition of cellular tyrosine kinases by the brief treatment of cells with 4',5,7-trihydroxyisoflavone (genistein) blocked the entry of HHV-8 into target cells. These findings suggest that, independently of other viral glycoproteins and via its RGD motif, HHV-8 gB induces integrin-dependent pre-existing FAK-Src-PI-3K-Rho GTPase kinases. Since these signal pathways play vital roles in host cell endocytosis and movement of particulate materials in the cytoplasm, the early stages of HHV-8 gB interaction with host cells may provide a very conducive environment for the successful infection of target cells.

Repair of wounded epidermis requires both keratinocyte migration and deposition of laminin 5 over exposed dermal collagen. To understand the coupling between leading cell migration and laminin 5 deposition, we developed a novel migration assay using time-lapse microscopy. We demonstrate that in migrating, human keratinocytes the deposition of laminin 5 promoted 'processive migration', characterized by stable cell polarization that was tightly coupled to persistent, linear migration in the absence of a chemotactic gradient. Processive migration required deposition of laminin 5, which was restricted to the rear of the polar cell. Integrin alpha 3 beta 1 interacted with these laminin 5 deposits at contact sites that did not require actin-dependent cross-linking. Further, we show that the migrating cells switched adhesion by integrin alpha 2 beta 1 on collagen at the front of the cell to integrin alpha 3 beta 1 on exogenous laminin 5 at the rear of the cell. Along with this switch of integrin usage was the removal of collagen from sites under the cell that precisely correlated with deposition of laminin 5. Processive migration was blocked with suppressors of microtubule dynamics (nocodazole and taxol) or rottlerin, a PKC-delta inhibitor. These drugs were also shown to block deposition of laminin 5 but, surprisingly, constitutive secretion was unimpaired, suggesting deposition was a regulated event. Thus, at the front of the cell, the leading lamellipodium was stabilized through integrin interactions in focal complexes with the exogenous substratum. However, at the rear of the cell, stable cell polarization and linear migration was promoted by laminin 5 deposits and integrin alpha 3 beta 1.

This research was originally published in the Journal of Biological Chemistry. Hiroyuki Ido, Kenji Harada, Sugiko Futaki, Yoshitaka Hayashi, Ryoko Nishiuchi, Yuko Natsuka, Shaoliang Li, Yoshinao Wada, Ariana C. Combs, James M. Ervasti and Kiyotoshi Sekiguchi. Molecular Dissection of the α-Dystroglycan- and Integrin-binding Sites within the Globular Domain of Human Laminin-10. J. Biol. Chem. 2004; 279: 10946-10954 © the American Society for Biochemistry and Molecular Biology, The adhesive interactions of cells with laminins are mediated by integrins and non-integrin-type receptors such as α-dystroglycan and syndecans. Laminins bind to these receptors at the C-terminal globular domain of their α chains, but the regions recognized by these receptors have not been mapped precisely. In this study, we sought to locate the binding sites of laminin-10 (α5β1γ1) for α3β1 and α6β1 integrins and α-dystroglycan through the production of a series of recombinant laminin-10 proteins with deletions of the LG (laminin G-like) modules within the globular domain. We found that deletion of the LG4–5 modules did not compromise the binding of laminin-10 to α3β1 and α6β1 integrins but completely abrogated its binding to α-dystroglycan. Further deletion up to the LG3 module resulted in loss of its binding to the integrins, underlining the importance of LG3 for integrin binding by laminin-10. When expressed individually as fusion proteins with glutathione S-transferase or the N-terminal 70-kDa region of fibronectin, only LG4 was capable of binding to α-dystroglycan, whereas neither LG3 nor any of the other LG modules retained the ability to bind to the integrins. Site-directed mutagenesis of the LG3 and LG4 modules indicated that Asp-3198 in the LG3 module is involved in the integrin binding by laminin-10, whereas multiple basic amino acid residues in the putative loop regions are involved synergistically in the α-dystroglycan binding by the LG4 module.

To clarify the roles of integrin and extracellular matrix (ECM) in the process of non-small cell lung cancer (NSCLC) brain metastasis, we established an in vivo model of brain metastasis of human NSCLC cell line EBC-1/original in athymic mice, and established highly brain metastatic subclone EBC-1/brain and highly bone metastatic subclone EBC-1/bone. Integrin expression of these subclones was evaluated by flow cytometry. In vitro cell attachment, migration and proliferation assays with ECMs were performed using these subclones. Expression of integrin α3 subunit was higher in EBC-1/brain than in both EBC-1/original and EBC-1/ bone. In vitro cell attachment, migration, and proliferation assays revealed that EBC-1/brain had higher affinity and higher reactivity to laminin than EBC-1/original and EBC-1/bone. Blocking of integrin α3β β β β1 significantly (P

Many adenovirus serotypes enter cells by high-affinity binding to the coxsackievirus-adenovirus receptor (CAR) and integrin-mediated internalization. In the present study, we analyzed the possible receptor function of α3β1 for adenovirus serotype 5 (Ad5). We found that penton base and integrin α3β1 could interact in vitro. In vivo, both Ad5-cell binding and virus-mediated transduction were inhibited in the presence of anti-α3 and anti-β1 function-blocking antibodies, and this occurred in both CAR-positive and CAR-negative cell lines. Peptide library screenings and data from binding experiments with wild-type and mutant penton base proteins suggest that the Arg-Gly-Asp (RGD) in the penton base protein, the best known integrin binding motif, is only part of the binding interface with α3β1, which involved multiple additional contact sites.

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 (α2β1 integrin ligand). However, on laminin-5 (α3β1 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 α3β1 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 α3β1–CD81 complex formation. In contrast, an EWI-2 chimeric mutant failed to suppress cell migration, redirect CD81 to filopodia, or enhance α3β1–CD81 complex formation. These results show how laterally associated EWI-2 might regulate α3β1 function in disease and development, and demonstrate how tetraspanin proteins can assemble multiple nontetraspanin proteins into functional complexes.

A mouse monoclonal antibody (MAb-9) produced by immunization with a human esophageal carcinoma cell line, TE-2 (derived from undifferentiated squamous cell carcinoma) reacted specifically with about 30% of esophageal carcinoma cell lines and tissue sections from clinical samples. MAb-9 showed minimal reactivity with normal esophageal tissue. (125)I, fluorescent or gold particle labeled MAb-9 bound to TE-2 cell surfaces. (125)I-radiolabeled MAb-9 was used to detect reactive material from cell extracts in Western blot. Treatment of TE-2 membrane proteins with neuraminidase, N-glycanase or O-glycanase reduced antigen detection. Treatment of cells with periodic acid destroyed antibody binding in ELISA. Lipid extracts from cell membranes, containing glycolipids, also reacted with MAb-9. MAb-9 was used to purify target antigen from detergent solubilized membrane proteins and the prominent bands from subsequent gel electrophoresis were trypsin digested and analyzed by mass spectrometry. Peptides from alpha(3) and beta(1) integrin chains were identified. These data indicate that alpha3beta1integrin is prominently expressed on certain esophageal carcinomas and that a specific carbohydrate unit is selectively displayed on the alpha(3) integrin subunit as well as on glycolipid on the cell surface. The alpha3beta1 integrin expressed on A-431 carcinoma cells does not display this carbohydrate epitope and is not detected by MAb-9. Thus, expression of the carbohydrate epitope is the basis for the tumor selective reaction of MAb-9 with a subset of esophageal carcinomas.

Integrins alpha3beta1 and alpha6beta1 are two major laminin receptors expressed on the surface of mammalian cells. Interactions of cells with laminins through these integrins play important roles in cell adhesion, differentiation, motility, and matrix assembly. To determine the binding specificity and affinity of these integrins toward various types of laminins at the level of direct protein-protein interactions, we purified integrins alpha3beta1 and alpha6beta1 from human placenta, and examined their binding to a panel of laminin isoforms, each containing distinct alpha chains (i.e., laminin-1, laminin-2/4, laminin-5, laminin-8, and laminin-10/11). Integrin alpha3beta1 showed clear specificity for laminin-5 and laminin-10/11, with no significant binding to laminin-1, laminin-2/4, and laminin-8. In contrast, integrin alpha6beta1 showed a broad spectrum of specificity, with apparent binding affinity in the following order: laminin-10/11 > laminin-5 > laminin-1 > laminin-2/4 congruent with laminin-8. Integrin titration assays demonstrated that laminin-10/11 was the most preferred ligand among the five distinct laminin isoforms for both alpha3beta1 and alpha6beta1 integrins. Given that laminin-10/11 is the major basement membrane component of many adult tissues, the interaction of laminin-10/11 with these integrins should play a central role in the adhesive interactions of epithelial cells with underlying basement membranes.

alpha3beta1-integrin is abundantly expressed in the epidermis, and in mice, ablation of the alpha3 gene results in embryonic defects and perinatal lethality. To determine the role of alpha3-integrin in adult skin development, we grafted skin from newborn alpha3-integrin-deficient mice on to ICRF nu/nu recipients. We report that adult alpha3-integrin-deficient skin has severe abnormalities restricted to hair follicle morphology, which include stunted hair follicle growth, increased hair follicle fragility, aberrant pigment accumulation and formation of hair follicle clusters. These abnormalities are caused by a combination of defects in: (1) keratinocyte cytoskeletal organisation, (2) outer root sheath architecture and (3) integrity of the lamina densa. Our results indicate that alpha3beta1 is not essential for adult interfollicular epidermal differentiation, but it is required to direct several processes important in hair follicle maintenance and morphogenesis.

Purpose To report the expression pattern of key molecules by the reconstructed corneal epithelium after a keratolimbal allograft (KLAL) and amniotic membrane transplantation (AMT) for total limbal stem cell deficiency. Design Interventional case report. Method A 50-year-old woman with severe chemical burns in both eyes received an AMT as a temporary patch at the acute stage, and a KLAL with AMT as a graft at the chronic stage for total limbal stem cell deficiency. The corneal button removed during subsequent corneal transplantation was submitted for immunofluorescence staining with monoclonal antibodies against keratin K3, MUC5AC, connexin 43, integrins α3β1 and α6β4, and laminin 5 for comparison with a normal cornea. Results Histologically, a normal stratified corneal epithelium has five to six cell layers that lay on the thick amniotic membrane basement membrane. The phenotype was of a corneal origin, based on expression of positive keratin K3, negative MUC5AC, and positive connexin 43. Furthermore, intact basement membrane complexes were present, evidenced by positive staining to integrins α3β1 and α6β4 and to laminin 5. Conclusions A normal corneal epithelial phenotype with normal basement membrane complexes was restored after a KLAL and AMT in a case with total limbal stem cell deficiency.

Analyses of mice with targeted deletions in the genes for alpha3 and beta1 integrin suggest that the alpha3beta1 integrin heterodimer likely determines the organization of the extracellular matrix within the basement membrane of skin. Here we tested this hypothesis using keratinocytes derived from alpha3 integrin-null mice. We have compared the organizational state of laminin-5, a ligand of alpha3beta1 integrin, in the matrix of wild-type keratinocytes with that of laminin-5 in the matrix of alpha3 integrin-null cells. Laminin-5 distributes diffusely in arc structures in the matrix of wild-type mouse keratinocytes, whereas laminin-5 is organized into linear, spike-like arrays by the alpha3 integrin-null cells. The fact that alpha3 integrin-null cells are deficient in their ability to assemble a proper laminin-5 matrix is also shown by their failure to remodel laminin-5 when plated onto surfaces coated with purified laminin-5 protein. In sharp contrast, wild-type keratinocytes organize exogenously added laminin-5 into discrete ring-like organizations. These findings led us next to assess whether differences in laminin-5 organization in the matrix of the wild-type and alpha3 integrin-null cells impact cell behavior. Our results indicate that alpha3 integrin-null cells are more motile than their wild-type counterparts and leave extensive trails of laminin-5 over the surface on which they move. Moreover, HEK 293 cells migrate significantly more on the laminin-5-rich matrix derived from the alpha3 integrin-null cells than on the wild-type keratinocyte laminin-5 matrix. In addition, alpha3 integrin-null cells show low strength of adhesion to surfaces coated with purified laminin-5 compared to wild-type cells although both the wild type and the alpha3 integrin-null keratinocytes adhere equally strongly to laminin-5 that has been organized into arrays by other epithelial cells. These data suggest: (1) that alpha3beta1 integrin plays an important role in determining the incorporation of laminin-5 into its proper higher-order structure within the extracellular matrix of keratinocytes and (2) that the organizational state of laminin-5 has an influence on laminin-5 matrix function.

The integrin alpha3beta1 mediates cellular adhesion to the matrix ligand laminin-5. A second integrin ligand, the urokinase receptor (uPAR), associates with alpha3beta1 via a surface loop within the alpha3 beta-propeller (residues 242-246) but outside the laminin binding region, suggesting that uPAR-integrin interactions could signal differently from matrix engagement. To explore this, alpha3-/- epithelial cells were reconstituted with wild-type (wt) alpha3 or alpha3 with Ala mutations within the uPAR-interacting loop (H245A or R244A). Wt or mutant-bearing cells showed comparable expression and adhesion to laminin-5. Cells expressing wt alpha3 and uPAR dissociated in culture, with increased Src activity, up-regulation of SLUG, and down-regulation of E-cadherin and gamma-catenin. Src kinase inhibition or expression of Src 1-251 restored the epithelial phenotype. The H245A and R244A mutants were unaffected by coexpression of uPAR. We conclude that alpha3beta1 regulates both cell-cell contact and matrix adhesion, but through distinct protein interaction sites within its beta-propeller. These studies reveal an integrin- and Src-dependent pathway for SLUG expression and mesenchymal transition.

BACKGROUND Interferon-alpha and -gamma are glycoproteins with antiviral and immunoregulatory properties. In vitro studies have shown a role for these cytokines in the regulation of epidermal keratinocyte growth and differentiation. In the same way, integrins are adhesion molecules which regulate keratinocyte proliferation and differentiation. AIM To determine whether the regulatory activity of interferons on keratinocyte proliferation and differentiation is related to a modulation of keratinocyte integrins. METHODS Two different methods were used: monolayers and reconstituted skin, incubated either with 1,200 U/mL interferon-alpha or 500 U/mL interferon-gamma or control medium for 48 h. The integrin expression was assessed by flow cytometry and immunohistochemistry. RESULTS In monolayers, only the alpha3 subunit was significantly inhibited by interferon-gamma. In reconstituted skin, where keratinocytes are differentiated, both interferons had an inductive effect on beta1 expression and interferon-alpha had an inhibitory effect on alpha6 expression. CONCLUSION Interferon-alpha and -gamma induce a modulatory effect on alpha3, alpha6 and beta1 which appears to be related to the state of differentiation. Moreover, the decreased expression of alpha6 and alpha3 could be one of the mechanisms involved in the formation of bullous lesions during long-term interferon therapy.

The cell surface glycoprotein CD9 is a member of a group of proteins known as the "tetraspan" or "transmembrane 4 superfamily." Previous work with non-neural cells has shown that CD9 associates in cis with integrins and small GTP-binding proteins on the cell surface. To extend our recent findings showing that perturbation of CD9 alters Schwann cell adhesion, proliferation, and migration, as well as neurite outgrowth in sympathetic neurons, we have searched for CD9-associated proteins in S-16 Schwann cells. We demonstrate here that CD9 is specifically coprecipitated from S-16 cell extracts by antibodies against integrins alpha 3, alpha 6, and beta 1. In addition, double immunofluorescence labeling and co-capping experiments indicate that CD9 is specifically co-localized with these integrins on the cell membrane of S-16 Schwann cells.

Cultured human foreskin keratinocytes (HFKs) adhere to and grow on nonfibrous collagen via integrin alpha2beta1. During incubation, the receptors used for adhesion are changed to integrins alpha3beta1 and alpha6beta4 and those receptors bind to laminin 5 which is deposited by keratinocytes themselves. In this report, we examined the behaviors of HFKs and transformed keratinocytes on collagen fibril gels. These cells adhered to and spread on collagen gels using integrin alpha2beta1. After several hours on collagen gels, however, cells became round and apoptosis occurred. The behavior of keratinocytes contrasted to that of fibroblasts that grew well even on collagen gel. At the point of apoptosis, integrins alpha2beta1 and alpha3beta1 were not found in the contact region of HFKs. Also, deposition of laminin 5 on collagen gel was not found despite the synthesis of mRNA for laminin 5 and laminin 10/11, while soluble laminin 5 protein is readily detectable. Phosporylation of Akt, which is known as a survival signal, was detected in HFKs cultured on coated collagen; however, the protein level and signals of Akt were dramatically decreased on collagen gel after 1 day of culture. These results indicate that collagen gel has different effects than nonfibrous collagen on HFKs and transformed keratinocytes and the interactions of integrin alpha3beta1 and laminin 5/10/11 are indispensable for maintenance of keratinocyte adhesion and survival.

The alpha 3 beta 1 integrin shows strong, stoichiometric, direct lateral association with the tetraspanin CD151. As shown here, an extracellular CD151 site (QRD(194-196)) is required for strong (i.e., Triton X-100-resistant) alpha 3 beta 1 association and for maintenance of a key CD151 epitope (defined by monoclonal antibody TS151r) that is blocked upon alpha 3 integrin association. Strong CD151 association with integrin alpha 6 beta 1 also required the QRD(194-196) site and masked the TS151r epitope. For both alpha 3 and alpha 6 integrins, strong QRD/TS151r-dependent CD151 association occurred early in biosynthesis and involved alpha subunit precursor forms. In contrast, weaker associations of CD151 with itself, integrins, or other tetraspanins (Triton X-100-sensitive but Brij 96-resistant) were independent of the QRD/TS151r site, occurred late in biosynthesis, and involved mature integrin subunits. Presence of the CD151-QRD(194-196)--INF mutant disrupted alpha 3 and alpha 6 integrin-dependent formation of a network of cellular cables by Cos7 or NIH3T3 cells on basement membrane Matrigel and markedly altered cell spreading. These results provide definitive evidence that strong lateral CD151-integrin association is functionally important, identify CD151 as a key player during alpha 3 and alpha 6 integrin-dependent matrix remodeling and cell spreading, and support a model of CD151 as a transmembrane linker between extracellular integrin domains and intracellular cytoskeleton/signaling molecules.

betaig-h3 is an extracellular matrix protein and its expression is highly induced by TGF-beta and it has also been suggested to play important roles in skin wound healing. In this paper, we demonstrate that betaig-h3 is present in the papillary layer of dermis and synthesized in the basal keratinocytes in vivo and its expression is induced by TGF-beta in normal human keratinocytes (NHEK) and HaCaT cells. betaig-h3 mediates not only adhesion and spreading of keratinocytes but also supports migration and proliferation. These activities are mediated through interacting with alpha3beta1 integrin. Previously identified two alpha3beta1 integrin-interacting motifs of betaig-h3, EPDIM, and NKDIL, are responsible for these activities. The results suggest that betaig-h3 may regulate keratinocyte functions in normal skin and potentially during wound-healing process.

Here we demonstrate that multiple tetraspanin (transmembrane 4 superfamily) proteins are palmitoylated, in either the Golgi or a post-Golgi compartment. Using CD151 as a model tetraspanin, we identified and mutated intracellular N-terminal and C-terminal cysteine palmitoylation sites. Simultaneous mutations of C11, C15, C242, and C243 (each to serine) eliminated >90% of CD151 palmitoylation. Notably, palmitoylation had minimal influence on the density of tetraspanin protein complexes, did not promote tetraspanin localization into detergent-resistant microdomains, and was not required for CD151-α3β1 integrin association. However, the CD151 tetra mutant showed markedly diminished associations with other cell surface proteins, including other transmembrane 4 superfamily proteins (CD9, CD63). Thus, palmitoylation may be critical for assembly of the large network of cell surface tetraspanin-protein interactions, sometimes called the “tetraspanin web.” Also, compared with wild-type CD151, the tetra mutant was much more diffusely distributed and showed markedly diminished stability during biosynthesis. Finally, expression of the tetra-CD151 mutant profoundly altered α3 integrin-deficient kidney epithelial cells, such that they converted from a dispersed, elongated morphology to an epithelium-like cobblestone clustering. These results point to novel biochemical and biological functions for tetraspanin palmitoylation.

Human herpesvirus-8 (HHV-8) is implicated in the pathogenesis of Kaposi's sarcoma. HHV-8 envelope glycoprotein B possesses the RGD motif known to interact with integrin molecules, and HHV-8 infectivity was inhibited by RGD peptides, antibodies against RGD-dependent alpha3 and beta1 integrins, and by soluble alpha3beta1 integrin. Expression of human alpha3 integrin increased the infectivity of virus for Chinese hamster ovary cells. Anti-gB antibodies immunoprecipitated the virus-alpha3 and -beta1 complexes, and virus binding studies suggest a role for alpha3beta1 in HHV-8 entry. Further, HHV-8 infection induced the integrin-mediated activation of focal adhesion kinase (FAK). These findings implicate a role for alpha3beta1 integrin and the associated signaling pathways in HHV-8 entry into the target cells.

Integrin receptors are well-known mediators of cell adhesion that also have a fundamental role in controlling the migration of cells through tissues. Among the numerous members of a still growing family, two particular molecular complexes have turned out to be of key importance in tumor cell invasion of basement membranes, the α3β1 and α6β4 integrins. In this Review, we will focus on the role of these two receptors and the mechanisms by which they influence the invasion process.

We evaluated the role of soluble factors produced from epidermal cells in melanoma cell motility by using the Boyden chamber chemoinvasion system. The migration of two melanoma cell lines, A375 and Mewo, was potentiated by conditioned media of A431 epidermoid cells in a concentration-dependent manner. The enhancement of A375 melanoma cell motility induced by the conditioned medium was blocked by antibodies against either alpha3 or beta1 integrin subunit. The motility-stimulating activity was recovered in the same fraction as the alpha3 integrin-dependent adhesion-promoting activity in a high-molecular-weight (>200 kDa) fraction on Superose 12 gel chromatography, and adsorbed with an anti-laminin-5 antibody. Purified laminin-5 was capable of potentiating melanoma cell migration as measured in either the chemotaxis assay with a soluble form of laminin-5 or the haptotaxis assay with membranes coated with a mixture of laminin-5 and Matrigel. Furthermore, immobilized laminin-5 induced A375 melanoma cells to secrete matrix metalloproteinase-9 (type IV collagenase) into the culture medium. These results strongly suggest that the interaction of laminin-5 produced in the epidermis with alpha3beta1 integrin on melanoma cells is involved in cell migration, invasion, and degradation of extracellular matrix proteins.

Integrin-mediated migration of neutrophils to infected tissue sites is vital for pathogen clearance and therefore host survival. Although β2 integrins have been shown to mediate neutrophil transendothelial migration during systemic and local inflammation, relatively little information is available regarding neutrophil migration in sepsis beyond the endothelial cell layer. In this study, we report that integrin α3β1 (VLA-3; CD49c/CD29) is dramatically upregulated on neutrophils isolated from both human septic patients and in mouse models of sepsis. Compared with the α3β1 (low) granulocytes, α3β1 (high) cells from septic animals displayed hyperinflammatory phenotypes. Administration of a α3β1 blocking peptide and conditional deletion of α3 in granulocytes significantly reduced the number of extravasating neutrophils and improved survival in septic mice. In addition, expression of α3β1 on neutrophils was associated with Toll-like receptor-induced inflammatory responses and cytokine productions. Thus, our results show that α3β1 is a novel marker of tissue homing and hyperresponsive neutrophil subtypes in sepsis, and blocking of α3β1 may represent a new therapeutic approach in sepsis treatment.

Dermatopontin (DPT), a tyrosine-rich, acidic matricellular protein, has been implicated in several human cancers. However, its biological functions and molecular mechanisms in cancer progression, particular hepatocellular carcinoma (HCC), remain unknown. We demonstrated that DPT was significantly down-regulated in 202 HCC clinical samples and that its expression level was closely correlated with cancer metastasis and patient prognosis. The overexpression of DPT dramatically suppressed HCC cell migration in vitro and intrahepatic metastasis in vivo. We further revealed that the down-regulation of DPT in HCC was due to epigenetic silencing by promoter DNA methylation. And the inhibitory effects of DPT on HCC cell motility were associated with dysregulated focal adhesion assembly, decreased RhoA activity and reduced focal adhesion kinase (FAK) and c-Src tyrosine kinase (Src) phosphorylation, and all of these alterations required the involvement of integrin signaling. Furthermore, we determined that the inhibitory effects of DPT on HCC cell motility were primarily mediated through α3β1 integrin. Our study provides new evidence for epigenetic control of tumor microenvironment, and suggests matricellular protein DPT may serve as a novel prognostic marker and act as a HCC metastasis suppressor.

The constitutive activation of β-catenin signaling in the mammary basal epithelial cell layer in transgenic K5ΔNβcat mice leads to basal-type tumor development. Integrins of the β1 family and integrin-mediated signaling events have an important role in breast tumor growth and progression. We show here that the deletion of α3β1 integrin, a major laminin receptor, from the basal layer of the mammary epithelium of K5ΔNβcat mice completely prevented the tumorigenesis induced by β-catenin signaling. Moreover, the depletion of α3β1 integrin from a spontaneously transformed mouse mammary basal epithelial cell line (MEC) prevented the cells from forming colonies in soft agar and greatly reduced tumor development in orthotopic grafts. Inhibition of the integrin signaling intermediates Rac1 or PAK1 (P21-activated Kinase 1) in MEC affected tumor cell growth in soft agar, whereas the expression of activated forms of these effectors in α3-depleted cells rescued the capacity of these cells to grow in non-adherent conditions. Similarly, the tumorigenic potential of α3-depleted cells was restored by the expression of activated PAK1, as assessed by orthotopic transplantation assay. In three-dimensional Matrigel culture, MEC survival and proliferation were affected by the depletion of α3β1 integrin, which also significantly decreased the activation of focal adhesion kinase (FAK), mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK). Our data suggest that the activation of signaling cascades downstream from α3β1 and involving the Rac1/PAK1 pathway, MAPK and JNK, promotes prosurvival and proproliferative signals required for the malignant growth of basal mammary epithelial cells, providing further insight into the molecular mechanisms underlying breast cancer initiation and progression.

Although type IV collagen is heavily glycosylated, the influence of this post-translational modification on integrin binding has not been investigated. In the present study, galactosylated and nongalactosylated triple-helical peptides have been constructed containing the α1(IV)382-393 and α1(IV)531-543 sequences, which are binding sites for the α2β1 and α3β1 integrins, respectively. All peptides had triple-helical stabilities of 37 °C or greater. The galactosylation of Hyl(393) in α1(IV)382-393 and Hyl(540) and Hyl(543) in α1(IV)531-543 had a dose-dependent influence on melanoma cell adhesion that was much more pronounced in the case of α3β1 integrin binding. Molecular modeling indicated that galactosylation occurred on the periphery of α2β1 integrin interaction with α1(IV)382-393 but right in the middle of α3β1 integrin interaction with α1(IV)531-543. The possibility of extracellular deglycosylation of type IV collagen was investigated, but no β-galactosidase-like activity capable of collagen modification was found. Thus, glycosylation of collagen can modulate integrin binding, and levels of glycosylation could be altered by reduction in expression of glycosylation enzymes but most likely not by extracellular deglycosylation activity.