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PubMed2006

Beta 1-integrins are required for hippocampal AMPA receptor-dependent synaptic transmission, synaptic plasticity, and working memory.

Integrins comprise a large family of cell adhesion receptors that mediate diverse biological events through cell-cell and cell-extracellular matrix interactions. Recent studies have shown that several integrins are localized to synapses with suggested roles in synaptic plasticity and memory formation. We generated a postnatal forebrain and excitatory neuron-specific knock-out of beta1-integrin in the mouse. Electrophysiological studies demonstrated that these mutants have impaired synaptic transmission through AMPA receptors and diminished NMDA receptor-dependent long-term potentiation. Despite the impairment in hippocampal synaptic transmission, the mutants displayed normal hippocampal-dependent spatial and contextual memory but were impaired in a hippocampal-dependent, nonmatching-to-place working memory task. These phenotypes parallel those observed in animals carrying knock-outs of the GluR1 (glutamate receptor subunit 1) subunit of the AMPA receptor. These observations suggest a new function of beta1-integrins as regulators of synaptic glutamate receptor function and working memory.

Animals↗
PubMed1999

The epidermotropic mycosis fungoides associated alpha1beta1 integrin (VLA-1, CD49a/CD29) is primarily a collagen IV receptor on malignant T cells.

Several of the beta1 integrin receptors [very late antigen (VLA) molecules] for extracellular matrix (ECM) proteins are expressed by malignant T cells in cutaneous T-cell lymphoma (CTCL). We evaluated the function of VLA-1, a beta1 integrin specifically expressed in epidermotropic mycosis fungoides (MF), in CD4+ leukemic T cells Jurkat line). We found that Jurkat cells adhere significantly to collagens only after their activation with phorbol 12-myristate 13-acetate (PMA). However, the adhesion to collagen IV (but not to collagen I) of Jurkat cells selected for expressing increased levels of VLA-1 (with unchanged levels of VLA-2, the second collagen integrin receptor) was significantly enhanced relative to that of "VLA-1 low" cells. Monoclonal antibody (mAb) 1B3.1, directed against the collagen binding domain of VLA-1, inhibited adhesion to collagen IV and to collagen I by 36.67%+/-5.25% and 18%+/-4.32%, respectively (p<0.05), whereas the inhibition by anti-VLA-2 mAb PIE6 was comparable on both collagens (25%+/-7.48% and 36.3%+/-0.94%, respectively; p<0.09). Immuno-histochemical studies of skin biopsies from 10 untreated MF patients showed that in all cases at least 10% of the lymphocytes residing in the epidermis are VLA-1+VLA-2-. While not directly applicable to MF, the demonstrated functions of VLA-1 in leukemic Jurkat cells, together with its expression in MF skin, suggest a role for VLA-1 integrins in epidermotropism in a small proportion of leukemic MF cells.

Antigens, CD↗
PubMed2001

Fibronectin and laminin elicit differential behaviors from SH-SY5Y growth cones contacting inhibitory chondroitin sulfate proteoglycans.

Neuronal growth cones integrate signals from outgrowth-promoting molecules, e.g., laminin (LN) or fibronectin (FN), and outgrowth-inhibiting molecules, e.g., chondroitin sulfate proteoglycans (CSPGs), to navigate through extracellular matrix (ECM). Sensory neurons on LN typically turn to avoid areas rich in inhibitory CSPGs, whereas neuron-like cells of human origin (SH-SY5Y) preferentially stop/stall. These different behaviors may reflect differences in neuron type, response to outgrowth-promoters, or the mechanisms involved in outgrowth vs. inhibition. We used image analysis to determine the effects of different outgrowth promoters on the response of SH-SY5Y cells to inhibitory CSPGs. LN increased neurite initiation and elongation compared to cells plated either on endogenous matrix or FN. On a patterned substratum consisting of alternating stripes of FN and CSPGs, 59.6 +/- 9.3% of SH-SY5Y growth cones turned upon CSPG contact, whereas only 31.9 +/- 8.2% of growth cones turned at a LN/CSPG border. Growth cones on LN spread more upon contact with CSPG than growth cones on FN, whereas growth cones on LN or FN not contacting CSPGs were morphologically similar. Because it is known that integrins are involved in outgrowth on promoters, we analyzed integrin expression in response to inhibitory CSPGs in a choice assay. CSPGs did not induce increases or redistribution of several integrin subunits in SH-SY5Y cells. Furthermore, an anti-beta1 integrin function-blocking antibody did not alter growth cone behavior at a CSPG border. These results indicate that significant mechanistic differences may exist between outgrowth on homogenous outgrowth promoters and growth cone turning at inhibitory molecules.

Antibodies↗
PubMed2000

Integrin-associated protein/CD47 regulates motile activity in human B-cell lines through CDC42.

Cell migration requires a dynamic interaction between the cell, its substrate, and the cytoskeleton-associated motile apparatus. Integrin-associated protein (IAP)/CD47 is a 50-kd cell surface protein that is physically associated with beta3 integrins and that modulates the functions of beta3 integrins in various cells. However, in B-lymphocytes that express beta1 integrins but few beta3 integrins, the roles of IAP/CD47 remain to be determined. Cross-linking of IAP/CD47 by the immobilized anti-IAP/CD47 monoclonal antibody (mAb) B6H12, but not 2D3, produced signals to promote polarization with lamellipodia, a characteristic morphology during leukocyte migration, in pre-B and mature B-cell lines (BALL, Nalm6, ONHL-1, Daudi), but not in myeloma cell lines (RPMI8226, OPM-2). In the presence of the immobilized fibronectin (FN), soluble B6H12 could increase the rate of the polarization and activate migratory activity of BALL cells to FN in a transwell filter assay. Furthermore, the dominant-negative form of CDC42 completely blocked B6H12-induced morphologic and functional changes without inhibiting phorbol 12-myristate 13-acetate-induced spreading on FN in BALL cells, whereas the dominant-negative form of Rac1 inhibited all these changes. These findings demonstrate that in B-lymphocytes, IAP/CD47 may transduce the signals to activate the migratory activity, in which CDC42 may be specifically involved, and that IAP/CD47 shows synergistic effect with alpha4beta1 on B-cell migration. These findings would provide new insight into the role of IAP/CD47 on B-cell function.

Antibodies, Monoclonal↗
PubMed2005

Hierarchy of ADAM12 binding to integrins in tumor cells.

ADAMs (a disintegrin and metalloprotease) comprise a family of cell surface proteins with protease and cell-binding activities. Using different forms and fragments of ADAM12 as substrates in cell adhesion and spreading assays, we demonstrated that alpha9beta1 integrin is the main receptor for ADAM12. However, when alpha9beta1 integrin is not expressed--as in many carcinoma cells--other members of the beta1 integrin family can replace its ligand binding activity. In attachment assays, the recombinant disintegrin domain of ADAM12 only supported alpha9 integrin-dependent tumor cell attachment, whereas full-length ADAM12 supported attachment via alpha9 integrin and other integrin receptors. Cells that attached to full-length ADAM12 in an alpha9 integrin-dependent manner also attached to ADAM12 in which the putative alpha9beta1 integrin-binding motif in the disintegrin domain had been mutated. This attachment was mediated through use of an alternate beta1 integrin. We also found that cell spreading in response to ADAM12 is dependent on the apparent level of integrin activation. Binding of cells to ADAM12 via the alpha9beta1 integrin was Mn(2+)-independent and resulted in attachment of cells with a rounded morphology; attachment of cells with a spread morphology required further activation of the alpha9beta1 integrin. We demonstrated that phosphoinositide-3-kinase appears to be central in regulating alpha9beta1 integrin cell spreading activity in response to ADAM12.

ADAM Proteins↗
PubMed2000

Colon cancer cells adhesion and spreading on autocrine laminin-10 is mediated by multiple integrin receptors and modulated by EGF receptor stimulation.

Epidermal growth factor (EGF) receptor ligands such as EGF and transforming growth factor-alpha (TGF-alpha) play an important role in controlling the proliferation, survival, morphology, and motility of colonic epithelial cells. There is also increasing evidence that growth factors and extracellular matrix (ECM) proteins cooperate to regulate these cellular processes. We have reported previously that autocrine TGF-alpha and an unidentified ECM protein in the serum-free conditioned medium of the human colon carcinoma cell line LIM1215 synergize to induce spreading of these cells in low-density cultures. We have now purified the ECM protein secreted by LIM1215 cells and show that it synergizes with EGF to induce spreading of LIM1215 cells and other human cell lines from the colon and other tissues. The purified ECM migrated as a single protein band with an apparent molecular mass of approximately 800 kDa on SDS-PAGE under nonreducing conditions and, under reducing conditions, as three protein bands of approximately 360, 210, and 200 kDa. Immunoblotting experiments and mass spectrometry analysis of tryptic digests on the purified protein identified the 360-, 210-, and 200-kDa protein bands as laminin alpha5, beta1, and gamma1 chains, respectively, indicating that LIM1215 cells secrete laminin-10 (alpha5 beta1 gamma1). In serum-free medium, LIM1215 cells adhere to laminin-10 primarily via alpha2 beta1 and alpha3 beta1 integrin receptors. EGF-induced spreading of LIM1215 cells on laminin-10 is partially inhibited by pretreatment of the cells with blocking antibodies directed against integrin alpha3 or beta1 but not alpha2, alpha6, or beta4 subunits. Spreading is almost completely inhibited by blocking alpha3 + alpha2, alpha3 + alpha6, or beta1 + beta4 integrin chains and results in cell death. Increased spreading in the presence of EGF correlates with up-regulation of alpha6 beta4 integrins in these cells after exposure to EGF. These results indicate that colon cancer cells attach and spread on laminin-10 via multiple integrin receptors and suggest a critical role for alpha3 beta1 integrins in the spreading response. Together, our results support the concept that the adhesive properties of colon cancer cells are modulated by autocrine production of TGF-alpha and laminin-10 and autocrine induction of appropriate integrins.

Amino Acid Sequence↗
PubMed1999

The compliance of collagen gels regulates transforming growth factor-beta induction of alpha-smooth muscle actin in fibroblasts.

Wound contraction is mediated by myofibroblasts, specialized fibroblasts that appear in large numbers as the wound matures and when resistance to contractile forces increases. We considered that the regulation of myofibroblast differentiation by wound-healing cytokines may be dependent on the resistance of the connective tissue matrix to deformation. We examined transforming growth factor-beta1 (TGF-beta1) induction of the putative fibroblast contractile marker, alpha-smooth muscle actin (alpha-SMA), and the regulation of this process by the compliance of collagen substrates. Cells were cultured in three different types of collagen gels with wide variations of mechanical compliance as assessed by deformation testing. The resistance to collagen gel deformation determined the levels of intracellular tension as shown by staining for actin stress fibers. For cells plated on thin films of collagen-coated plastic (ie, minimal compliance and maximal intracellular tension), TGF-beta1 (10 ng/ml; 6 days) increased alpha-SMA protein content by ninefold as detected by Western blots but did not affect beta-actin content. Western blots of cells in anchored collagen gels (moderate compliance and tension) also showed a TGF-beta1-induced increase of alpha-SMA content, but the effect was greatly reduced compared with collagen-coated plastic (<3-fold increase). In floating collagen gels (high compliance and low tension), there were only minimal differences of alpha-SMA protein. Northern analyses for alpha-SMA and beta-actin indicated that TGF-beta1 selectively increased mRNA for alpha-SMA similar to the reported protein levels. In pulse-chase experiments, [35S]methionine-labeled intracellular alpha-SMA decayed most rapidly in floating gels, less rapidly in anchored gels, and not at all in collagen plates after TGF-beta1 treatment. TGF-beta1 increased alpha2 and beta1 integrin content by 50% in cells on collagen plates, but the increase was less marked on anchored gels and was undetectable in floating gels. When intracellular tension on collagen substrates was reduced by preincubating cells with blocking antibodies to the alpha2 and beta1 integrin subunits, TGF-beta1 failed to increase alpha-SMA protein content in all three types of collagen matrices. These data indicate that TGF-beta1-induced increases of alpha-SMA content are dependent on the resistance of the substrate to deformation and that the generation of intracellular tension is a central determinant of contractile cytoskeletal gene expression.

Actins↗
PubMed1999

The spatial relationship between stem cells and their progeny in the basal layer of human epidermis: a new view based on whole-mount labelling and lineage analysis.

In order to examine the spatial organisation of stem cells and their progeny in human epidermis, we developed a method for whole-mount epidermal immunofluorescence labelling using high surface beta1 integrin expression as a stem cell marker. We confirmed that there are clusters of high beta1 integrin-expressing cells at the tips of the dermal papillae in epidermis from several body sites, whereas alpha6 integrin expression is more uniform. The majority of actively cycling cells detected by Ki67 or bromodeoxyuridine labelling were found in the beta1 integrin-dull, transit amplifying population and integrin-negative, keratin 10-positive cells left the basal layer exclusively from this compartment. When we examined p53-positive clones in sun-exposed epidermis, we found two types of clone that differed in size and position in a way that was consistent with the founder cell being a stem or transit amplifying cell. The patterning of the basal layer implies that transit amplifying cells migrate over the basement membrane away from the stem cell clusters. In support of this, isolated beta1 integrin-dull keratinocytes were more motile on type IV collagen than beta1 integrin-bright keratinocytes and EGFP-labelled stem cell clones in confluent cultured sheets were compact, whereas transit amplifying clones were dispersed. The combination of whole-mount labelling and lineage marking thus reveals features of epidermal organisation that were previously unrecognised.

Biomarkers↗
PubMed1998

Enterocytic differentiation of the human Caco-2 cell line correlates with alterations in integrin signaling.

We previously reported that the enterocytic differentiation of human colonic Caco-2 cells correlated with down-regulation of fibronectin (FN) and laminin (LN), two extracellular matrix components interacting with cell surface integrin receptors. We now investigated whether Caco-2 cell differentiation was associated with alterations in integrin signaling with special interest in the expression and activity of focal adhesion kinase (FAK) and mitogen-activated protein (MAP) kinase. The differentiation of Caco-2 cells was associated with: 1) down-regulation of beta1 integrin expression at the mRNA and protein levels; 2) increased FAK expression together with decreased FAK autophosphorylation; 3) decreased FAK's ability to associate with PI3-kinase and pp60c-src; and 4) increased MAP kinase expression along with decreased MAP activity. In addition, we show that FAK and MAP kinase belong to distinct integrin signaling pathways and that both pathways remain functional during Caco-2 cell differentiation since the coating of differentiating cells on FN and LN but not on polylysine increased the tyrosine phosphorylation of FAK and of its endogenous substrate paxillin, and stimulated MAP kinase activity. In conclusion, our results provide evidence that FAK and MAP kinase, two signaling molecules activated independently by beta1 integrins in Caco-2 cells, undergo alterations of both expression and activity during the enterocytic differentiation of this cell line.

Caco-2 Cells↗
PubMed1998

The 'ligand-induced conformational change' of alpha 5 beta 1 integrin. Relocation of alpha 5 subunit to uncover the beta 1 stalk region.

Integrin heterodimers undergo a conformational change upon the binding of ligand to their extracellular domains. An anti-beta1 integrin monoclonal antibody AG89 can detect such a conformational change since it recognizes a ligand-inducible epitope in the stalk-like region of beta1 subunits. The binding of a 125I-labeled AG89 Fab fragment to alpha5 beta1 integrins on K562 cells was assessed and analyzed by the Scatchard method. High affinity binding sites for AG89 are present on cells treated with ligand peptide. In addition, results revealed that cells treated with EDTA also express AG89 binding sites with the same affinity although the number of binding sites is 4-fold lower. AG89 immunoprecipitated alpha5 beta1 complexes from surface-labeled K562 cells treated with ligand peptide. By contrast, it immunoprecipitated only beta1 chains when the ligand peptide was absent, suggesting that high affinity binding sites on EDTA-treated cells are associated with non-functional beta1 monomer. Additional studies show that the epitope for AG89 is constitutively exposed on mutant beta1 that cannot complex with alpha5. These data suggest that the AG89 epitope is masked by the alpha5 subunit. Ligand binding and integrin activation may uncover the beta1 stalk region by triggering a conformational shift of alpha5 relative to beta1.

Animals↗
PubMed1999

Role of very late adhesion integrins in mediating repair of human airway epithelial cell monolayers after mechanical injury.

Repair of the airway epithelium after injury requires that processes such as adhesion and cell migration occur in a defined order. Both of these processes depend on interactions between extracellular matrix (ECM) proteins and appropriate integrins. To study these interactions, we examined monolayer wound repair in a cultured human airway epithelial cell line, 16HBE14o-. Wounds created in confluent monolayers grown on either collagen-IV, laminin-1, or laminin-2 matrix closed quickly in response to 15 ng/ml epidermal growth factor (EGF). Concurrent treatment of cells grown on each matrix protein with EGF and a monoclonal antibody (mAb) to beta1-integrin inhibited wound closure. Treatment with a mAb to alpha2-, alpha3-, and alpha6-integrin blocked wound repair in monolayers grown on collagen-IV but did not do so in monolayers grown either on laminin-1 or laminin-2. Inhibition was not due to cell detachment or apoptosis. These data demonstrate that integrins expressed by airway epithelial cells mediate wound closure on different constitutive ECM proteins. These data suggest that beta1-integrin subunit function is required to permit migration and spreading of epithelial cells, and that alpha-integrin subunits alone do not mediate migration of epithelial cells grown on either laminin-1 or laminin-2. These differences may become important if the matrix protein composition of airway basement membrane changes in disease states such as asthma.

Antibodies, Monoclonal↗
PubMedDate not supplied

Activation of focal adhesion kinase in human lung cancer cells involves multiple and potentially parallel signaling events.

Integrins are adhesion receptors that transmit signals bidirectionally across the plasma membrane. In our previous report we have shown that the squamous lung cancer cell line, Calu-1, binds to collagen type IV (Coll IV) through beta1-integrin and results in phosphorylation of focal adhesion kinase (FAK) (Ann Thorac Surg 2004; 78:450-457). Considering the critical role of FAK in cell migration, proliferation, and survival, here we investigated potential mechanisms of its activation and regulation in Calu-1 cells. We observed the phosphorylation of Tyr397 of FAK (the autophosphorylation site of FAK) and paxillin, the immediate downstream substrate of FAK following the adhesion of Calu-1 cells to Coll IV. FAK remains phosphorylated during proliferation either on Coll IV or on uncoated plates for 72 h, as determined by peroxivanadate treatment. Exposure of Calu-1 cells with 60 microM genistein, reduces FAK phosphorylation (7.6 fold) and cell proliferation. Extracellular signal regulated kinases (ERKs) were also phosphorylated after Coll IV attachment. Disruption of Calu-1 cell cytoskeleton integrity by 1-5 muM Cytochalasin D resulted in the inhibition of cell adhesion (50% to 75%, p<0.19 - 6.6 x 10(7)) and ERKs phosphorylation (2 fold) without any effect on FAK phosphorylation. Protein Kinase C inhibitor, Calphostin C at 100 and 250 nM concentrations did not block Coll IV induced FAK phosphorylation but activated the ERKs in a dose dependent manner. beta1-integrin is essential for Coll IV induced FAK activation, but it is not physically associated with FAK as determined by immunodetection assay. Collectively, this report defines the existence of multiple and potentially parallel Coll IV/beta1-integrin mediated signaling events in Calu-1 cells, which involve FAK, ERKs, and PKC.

Cell Adhesion↗
PubMed1997

Association of the Cas-like molecule HEF1 with CrkL following integrin and antigen receptor signaling in human B-cells: potential relevance to neoplastic lymphohematopoietic cells.

CrkL, a cellular homologue of the v-crk oncogene, belongs to the family of adaptor proteins, containing SH2 and SH3 domains, but no catalytic domain. Stimulation of normal B-cells and B-cell lines through beta1 integrin or - cell antigen receptor (BCR) promoted the association of CrkL with a set of 105-130 kD tyrosine phosphorylated substrates. The principal substrate is a recently identified molecule known as p105HEF1 (HEF1), which is highly homologous to p130Cas (Cas), the major tyrosine-phosphorylated protein detected in fibroblasts after transformation by v-crk. Immunodepletion studies indicated that all the tyrosine phosphorylated HEF1 or Cas was complexed with CrkL. Furthermore, the guanine nucleotide exchange factor C3G, which is thought to be involved in the regulation of the ras pathway and constitutively binds to the C-terminal SH3 domain of CrkL, could be detected in HEF1 immunoprecipitates. Therefore, CrkL is involved in the formation of a HEF1-CrkL-C3G ternary complex in B-cells, suggesting that it is likely to play an important role, allowing the propagation of the stimulation initiated by both BCR and beta1 integrin ligation.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Butyrate-treated colonic Caco-2 cells exhibit defective integrin-mediated signaling together with increased apoptosis and differentiation.

We previously reported that the enterocytic differentiation of human colonic Caco-2 cells correlated with alterations in integrin signaling. We now investigated whether differentiation and apoptosis of Caco-2 cells induced by the short-chain fatty acid butyrate (NaBT) was associated with alterations in the integrin-mediated signaling pathway with special interest in the expression and activity of focal adhesion kinase (FAK), of the downstream phosphatidylinositol 3'-kinase (PI 3-kinase)-Akt pathway and in the role of the nuclear factor kappaB (NF-kappaB). NaBT increased the level of sucrase. It induced apoptosis as shown by: (1) decreased Bcl-2 and Bcl-X(L) proteins and increased Bax protein; (2) activation of caspase-3; and (3) increased shedding of apoptotic cells in the medium. This effect was associated with defective integrin-mediated signaling as shown by: (1) down-regulation of beta1 integrin expression; 2) decreased FAK expression and tyrosine phosphorylation; (3) concerted alterations in cytoskeletal and structural focal adhesions proteins (talin, ezrin); and (4) decreased FAK ability to associate with PI 3-kinase. However, in Caco-2 cells, beta1-mediated signaling failed to be activated downstream of FAK and PI 3-kinase at the level of Akt. Transfection studies show that NaBT treatment of Caco-2 cells promoted a significant activation of the NF-kappaB which was probably involved in the NaBT-induced apoptosis. Our results indicate that the prodifferentiating agent NaBT induced apoptosis of Caco-2 cells probably through NF-kappaB activation together with a defective beta1 integrin-FAK-PI 3-kinase pathways signaling.

Apoptosis↗
PubMed1998

Contribution of CD11a/CD18, CD11b/CD18, ICAM-1 (CD54) and -2 (CD102) to human monocyte migration through endothelium and connective tissue fibroblast barriers.

Recently we reported that monocyte migration through a barrier of human synovial fibroblasts (HSF) is mediated by the CD11/CD18 (beta2) integrins, and the beta1 integrins VLA-4 and VLA-5 on monocytes. Here we investigated in parallel the role of beta2 integrin family members, LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) on monocytes, and the immunoglobulin supergene family members, ICAM-1 and ICAM-2 on HSF and on human umbilical vein endothelial cells (HUVEC), in monocyte migration through HSF and HUVEC monolayers. Using function blocking monoclonal antibodies (mAb), when both VLA-4 and VLA-5 on monocytes were blocked, treatment of monocytes with mAb to both LFA-1 and to Mac-1 completely inhibited monocyte migration across HSF barriers, although blocking either of these beta2 integrins alone had no effect on migration, even when VLA-4 and VLA-5 were blocked. This indicates that optimal beta2 integrin-dependent monocyte migration in synovial connective tissue may be mediated by either LFA-1 or Mac-1. Both ICAM-1 and ICAM-2 were constitutively expressed on HSF and on HUVEC, although ICAM-2 was only minimally expressed on HSF. Based on results of mAb blockade, ICAM-1 appeared to be the major ligand for LFA-1-dependent migration through the HSF. In contrast, both ICAM-1 and ICAM-2 mediated LFA-1-dependent monocyte migration through HUVEC. However, neither ICAM-1 nor ICAM-2 was required for Mac-1 -dependent monocyte migration through either cell barrier, indicating that Mac-1 can utilize ligands distinct from ICAM-1 and ICAM-2 on HSF and on HUVEC during monocyte transmigration.

Antigens, CD↗
PubMed2005

Transmigration of human ovarian adenocarcinoma cells through endothelial extracellular matrix involves alphav integrins and the participation of MMP2.

The growth of ovarian carcinoma is dependent upon their vascularistion, but the interaction of ovarian cancer cells with the endothelium and their invasion through an endothelial environment remain poorly understood at the molecular level. To investigate adhesive events underlying this process with focusing on the role of alphav integrins and MT1MMP-MMP2 proteinases, we used in vitro models of cocultures of human ovarian adenocarcinoma cell lines (IGROV1 and SKOV3) with human umbilical vein endothelial cells (HUVECs). Immunostaining of HUVECs revealed the network organisation of fibrillar fibronectin (Fn) and pericellular vitronectin (Vn). During coculture, IGROV1 and SKOV3 cells gain access to subendothelial basement membrane of HUVECs and dislocated endothelial Fn without affecting endothelial Vn. Transmigration assays revealed that tumour cells invade Vn and, with an higher efficiency, Fn. Our data also highlighted that ovarian carcinoma cells migrated through the Fn-rich HUVEC-ECM. The expression of MMP2 and MT1-MMP was revealed in tumour cells within an endothelial environment. Furthermore, we found that cell migration through the endothelial ECM was almost totally dependent on alphav integrin function, whereas beta1 integrins were not solicited. In addition, inhibitors of MMP2 activity (alone or combined with anti-alphav integrin MAb) or TSRI265 (which blocks MMP2-alphavbeta3 association) were found to impede this process. Finally, alphav integrins, MT1-MMP and MMP2 were found in ovarian carcinoma cells within the 3-dimensional architecture of intraperitoneal tumour nodes collected from nude mice xenografted with IGROV1 or SKOV3 cell lines or within human tumour tissues. alphav integrins therefore appear as essential to the migration properties of human ovarian carcinoma cells, especially in an endothelial environment, with MMP2 participating to this process.

Animals↗
PubMed1999

alpha9beta1 integrin is expressed on human neutrophils and contributes to neutrophil migration through human lung and synovial fibroblast barriers.

Accumulation of leukocytes in inflamed tissue involves their migration through vascular endothelium and then in the connective tissue. Recently we utilized a barrier of human synovial, dermal, and lung fibroblasts (HSF, HDF, and HLF) grown on polycarbonate filters as a model of human polymorphonuclear leukocyte (PMN) migration through connective tissue. The beta2 integrins (CD 11/ CD18) and alpha4, alpha5, and alpha6beta1 (VLA-4, -5, and -6) integrins each contributed to this PMN migration. Here we report that on human blood leukocytes, alpha9beta1 (VLA-9) is expressed only on PMNs and that it is up-regulated after PMN activation. Based on monoclonal antibody (mAb) blocking studies, alpha9beta1 integrin contributed to C5a-induced PMN migration through fibroblast (HLF and HSF) barriers. This role was apparent only when alternate mechanisms such as CD18, alpha4, alpha5, and alpha6beta1 integrins were blocked and then mAb to alpha9beta1 integrin inhibited the residual PMN migration (by 40-50%) through the HLF or HSF barrier, resulting in > or = 75% inhibition overall. In contrast, PMN migration across interleukin-1-activated endothelium (HUVEC) in response to a C5a gradient, which is partly (30-40%) via CD11/CD18-independent mechanisms, was not inhibited by adhesion blocking by mAbs to alpha4, alpha5, alpha6, and alpha9beta1 even in combination. These results indicate that alpha9beta1 integrin on PMN may have a special role, in conjunction with other beta1 integrins, in mediating PMN migration in the extravascular space, and may contribute to differential neutrophil function within tissues.

Animals↗
PubMed1998

Transcription of alpha2 integrin gene in osteosarcoma cells is enhanced by tumor promoters.

Integrin alpha2beta1 is a heterodimeric transmembrane receptor for collagens. In osteogenic cells the expression of alpha2beta1 integrin is induced by both Kirsten sarcoma virus and chemical transformation. The association of alpha2 integrin with transformed cell phenotype was studied further by testing the effects of two tumor promoters, 12-O-tetradecanoylphorbol 13-acetate (TPA) and okadaic acid (OA), on human MG-63 osteosarcoma cells. TPA, an activator of protein kinase C, increased the cell surface expression of alpha2 integrin and the corresponding mRNA levels. Nuclear run-on assays indicated that TPA activated the transcription of alpha2 integrin gene. TPA also slightly increased the expression of alpha3 integrin but had no effect on the transcription of alpha5, alphav, or beta1 integrin subunits. OA, an inhibitor of serine/threonine phosphatases, increased alpha2 integrin gene transcription and mRNA levels, but in contrast to TPA, OA decreased alpha3 integrin expression. The increased expression of alpha2 integrin on TPA-treated MG-63 cells led to faster cell spreading on type I collagen. Our results link the enhanced transcription of alpha2 integrin gene to tumor progression and show the independent regulation of alpha2 integrin compared to other integrin genes.

Antigens, CD↗