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PubMed1998

Integrin alpha5beta1: a potent inhibitor of experimental lung metastasis.

The integrin alpha5beta1 seems to be the most relevant receptor of tumor cells for binding to fibronectin. Although numerous studies suggest a role of tumor cell fibronectin interaction in tumor metastasis, differential integrin expression on tumor cells has, however, not been correlated with metastatic capabilities. We addressed this question by transfection of the integrin alpha5beta1 cDNA into HT-29 human colon carcinoma cells which led to de novo expression of functional integrin alpha5beta1. Similar to other reports, expression of the integrin alpha5beta1 in HT-29 tumor cells exerted an inhibitory action on cell proliferation as indicated in our study by formation of fewer colonies in soft agar. The tumor growth inhibitory property of the integrin alpha5beta1 was also shown by reduction of subcutaneous xenograft growth in nude mice to approximately 50% of that of control transfectants. For the first time, we found that several clones of integrin alpha5 subunit transfectants displayed dramatically reduced formation of lung colonies and cutaneous metastasis after intravenous injection into nude mice. While most animals inoculated with control transfectant cells formed macroscopically visible lung colonies ranging from 12.6 +/- 2.6 to 22.0 +/- 6.6 (mean colony number +/- SEM), mice inoculated with HT-29 cell clones expressing the integrin alpha5beta1 were almost completely free of lung colonies (ranging from 0.0 +/- 0 to 0.2 +/- 0.1). Our results imply that integrin alpha5beta1 expression inhibits circulating tumor cells in pursuing late steps of the metastatic process as represented by the artificial metastasis (lung colonisation) model.

Animals↗

PubMed1999

Quantitative changes in integrin and focal adhesion signaling regulate myoblast cell cycle withdrawal.

We previously demonstrated contrasting roles for integrin alpha subunits and their cytoplasmic domains in controlling cell cycle withdrawal and the onset of terminal differentiation (Sastry, S., M. Lakonishok, D. Thomas, J. Muschler, and A.F. Horwitz. 1996. J. Cell Biol. 133:169-184). Ectopic expression of the integrin alpha5 or alpha6A subunit in primary quail myoblasts either decreases or enhances the probability of cell cycle withdrawal, respectively. In this study, we addressed the mechanisms by which changes in integrin alpha subunit ratios regulate this decision. Ectopic expression of truncated alpha5 or alpha6A indicate that the alpha5 cytoplasmic domain is permissive for the proliferative pathway whereas the COOH-terminal 11 amino acids of alpha6A cytoplasmic domain inhibit proliferation and promote differentiation. The alpha5 and alpha6A cytoplasmic domains do not appear to initiate these signals directly, but instead regulate beta1 signaling. Ectopically expressed IL2R-alpha5 or IL2R-alpha6A have no detectable effect on the myoblast phenotype. However, ectopic expression of the beta1A integrin subunit or IL2R-beta1A, autonomously inhibits differentiation and maintains a proliferative state. Perturbing alpha5 or alpha6A ratios also significantly affects activation of beta1 integrin signaling pathways. Ectopic alpha5 expression enhances expression and activation of paxillin as well as mitogen-activated protein (MAP) kinase with little effect on focal adhesion kinase (FAK). In contrast, ectopic alpha6A expression suppresses FAK and MAP kinase activation with a lesser effect on paxillin. Ectopic expression of wild-type and mutant forms of FAK, paxillin, and MAP/erk kinase (MEK) confirm these correlations. These data demonstrate that (a) proliferative signaling (i.e., inhibition of cell cycle withdrawal and the onset of terminal differentiation) occurs through the beta1A subunit and is modulated by the alpha subunit cytoplasmic domains; (b) perturbing alpha subunit ratios alters paxillin expression and phosphorylation and FAK and MAP kinase activation; (c) quantitative changes in the level of adhesive signaling through integrins and focal adhesion components regulate the decision of myoblasts to withdraw from the cell cycle, in part via MAP kinase.

Animals↗

PubMedDate not supplied

In vitro analysis of adhesion molecule expression and gel contraction of human granulation fibroblasts.

The objective of this study was to determine whether human fibroblasts obtained at different times from normally healing wounds were phenotypically distinct with respect to cell-matrix interactions. We cultured human granulation fibroblasts obtained from repeated biopsies from the same punch area. On days 3, 6, 9, and 14 the expression of key adhesion molecules and extracellular matrix components at the protein level by flow cytometry analysis were compared with quiescent fibroblasts. Intercellular adhesion molecule-1 levels were significantly elevated only around day 3, which implies a phenotypic change during the early phase of wound healing. Homing cell adhesion molecule, as well as the alpha4 integrin subunit (CD49d), were essentially unaltered. The alpha5 integrin subunit (CD49e), which imparts the specificity of the fibronectin receptor and alphav (CD51), a vitronectin receptor component, are upregulated around days 3 and 6; the alpha2 and 3 integrin subunits (CD49b/c) were only increased on day 6. In granulation fibroblasts, at days 3 and 14, the level of the beta1 integrin subunit was enhanced. In addition, collagen gel contraction with granulation fibroblasts increases constantly from day 3 to day 14. These results show that human granulation fibroblasts differentially express cell surface adhesion molecules depending on the age of the healing wound. Further, changes in the ability of these cells to contract collagen gels indicate a synchronicity between wound bed contraction and different stages of wound healing.

Journal Article↗

PubMed1999

Selective increase of alpha2-integrin sub-unit expression on human carcinoma cells upon EGF-receptor activation.

The effects of chronic EGF exposure on expression of the alpha2beta1 collagen and alpha5beta1 fibronectin receptor in a pair of human carcinoma cell lines (A431 and A549) with differential responses to EGF in a short-term ECM-cell adhesion assay were investigated. Treatment with EGF at 10 ng/ml for 24 hr increased on both cell lines the expression of the alpha2- but not the beta1- or alpha5-integrin sub-units, and concomitantly cellular adhesion was increased on collagen IV but not on fibronectin. Increased collagen adhesion of A549 cells could be blocked by alpha2- and beta1-integrin-sub-unit antibodies down to control levels, while it was blocked by alpha2-integrin-sub-unit antibody only by 60% and completely by the beta1-integrin-sub-unit antibody on A431 cells. EGF induced disparate shifts in cell morphologies (dome-like structures, A431, vs. spindle-like fibroblastoid, A549) with concomitant opposite changes in the expression/localization of E-cadherin in cell-cell contacts. This could be taken as an indication for cell-type-specific differential changes in the ratio of cell-ECM vs. cell-cell contacts. The EGF-induced up-regulation of the alpha2beta1 integrin was instrumental in increasing collagen adhesion of A549 but only partly in the case of A431 cells, in which cells the alpha2beta1 integrin may have additional functions besides serving as cell-ECM receptor.

Adenocarcinoma↗

PubMed2001

Integrin receptors are required for cell survival and proliferation during development of the peripheral glial lineage.

Proliferation and survival of Schwann cells are important for nerve development and for disease processes in peripheral nerves. We have analyzed embryos lacking alpha4- or alpha5-integrins and show here that these integrins contribute to the control of glial cell numbers. To overcome early embryonic lethality an explant and grafting system that allows the study of isolated glial progenitor cells both in vitro and in vivo was used. Schwann cells differentiate in the absence of alpha5 but their numbers and the proliferation rate of early progenitor cells are reduced, suggesting that alpha5 is essential for normal proliferation. Survival, rather than proliferation, is compromised in alpha4-deficient explants. Conditional immortalization allowed further characterization and revealed that alpha4 contributes to survival in a cell-density-dependent fashion. In addition, transplants into chicken embryos were used to analyze in vivo cell migration and showed that cell death occurs mainly in highly motile, individually migrating cells. The cell death patterns in vitro and in vivo argue that alpha4-integrins play a role in survival during cell migration. Neural crest migration has been suggested to require these integrins; however, no defects in migration were observed in the absence of alpha4 or alpha5. We conclude that integrins can complement growth factors in the control of glial cell numbers.

Animals↗

PubMed1998

Expression of integrins in experimental autoimmune neuritis and Guillain-Barré syndrome.

Integrins are a subclass of adhesion molecules that mediate cell-cell and cell-extracellular matrix interactions. Integrins influence transendothelial migration of lymphocytes and monocytes and are suitable targets for experimental immunotherapy. They are critically involved in the pathogenesis of autoimmune neuritis and abnormally expressed in human neuropathies. Also, the role of integrins in myelination, neurite outgrowth, and nerve regeneration suggests that they could be involved in the recovery phase of immune-mediated neuropathies. We investigated by immunohistochemistry the expression of a number of integrin subunits during the course of experimental autoimmune neuritis (EAN). Results were compared with the human immune neuropathy Guillain-Barre syndrome (GBS) and extended in vitro. Inflammation and demyelination in both EAN and GBS induced the down-regulation of beta4 integrin in Schwann cells (SCs), whereas loss of alpha2 was noted only in EAN. When axonal loss was present, SCs displayed alpha5 integrin, in both EAN and GBS. In vitro, basal lamina and inflammatory cytokines modulated the expression of beta4 in SCs, but they did not influence alpha2 and alpha5 expression. Finally, integrins were differentially expressed in blood vessels during EAN. In conclusion, the spatiotemporal changes in integrin expression may be used to characterize, stage, and better understand the pathogenesis and evolution of inflammation during GBS and EAN. This may help to establish useful, novel therapy for immune-mediated neuropathies.

Animals↗

PubMed2004

Differential susceptibility of osteosarcoma cells and primary osteoblasts to cell detachment caused by snake venom metalloproteinase protein.

The interaction of extracellular matrix with cells plays a key role in the regulation of cell adhesion, migration, proliferation as well as differentiation. Transformed cells express a different profile of adhesion molecules, which may mediate metastasis under specific matrix microenvironment. We here found that ROS 17/2.8 osteosarcoma cells and osteoblasts have different expression of alpha5 integrin, executing different fibronectin fibrillogenesis. As compared with ROS 17/2.8 cells, osteoblasts have higher expression of fibronectin, collagen, alpha5, beta1, alpha2 integrins and focal adhesion kinase as examined by immunostaining and flow cytometry. Crovidisin, a PIII snake venom metalloproteinase (SVMP) purified from venom of Crotalus viridis, exhibits collagen-binding activity and matrix metalloproteinase activity. Crovidisin selectively caused the detachment of ROS 17/2.8 osteosarcoma cells but not of primary cultured osteoblasts. On the other hand, triflavin, an RGD-dependent disintegrin purified from venom of Trimeresurus flavoviridis, did not cause the detachment of both osteoblasts and ROS 17/2.8 cells. Although ROS 17/2.8 cells detached from substratum after crovidisin treatment for 24 h, the loss of mitochondrial membrane potential was not observed unless a prolonged treatment for longer than 36 h. These results suggest that cultured primary rat osteoblasts and ROS 17/2.8 osteosarcoma cells possess different expression of integrins and matrix environment, and ROS 17/2.8 is much more susceptible to be detached by crovidisin. The matrix degradation by crovidisin may be responsible for the preferential detachment of ROS 17/2.8 osteosarcoma cells.

Animals↗

PubMed1998

Neural precursor cell chain migration and division are regulated through different beta1 integrins.

Proliferation and tangential migration of neural precursor cells are essential determinants of CNS development. We have established cell culture models of both these processes using neural precursor cells grown as neurospheres. The pattern of migration that we observe in these cells is homotypic and occurs in the absence of a glial or neuronal scaffold, and is therefore equivalent to that previously described as chain migration. To determine the role of integrins in proliferation and migration, we have analysed the expression pattern of integrins on neurosphere cells and then performed blocking peptide and antibody experiments. Neurosphere cells express five major integrins, alpha5 beta1, alpha 6Abeta1, alphav beta1, alphav beta5 and alpha vbeta8 and, in addition, express low levels of alpha 6Bbeta1. Chain migration is inhibited by blocking the alpha 6beta1 integrin. Proliferation, by contrast, is inhibited by blocking the other beta1 integrins, alphav beta1 and alpha5 beta1. These results show that integrins are important regulators of neural precursor cell behaviour, with distinct beta1 integrins regulating proliferation and migration. They also demonstrate a novel role for the alpha6 beta1 integrin in the cell-cell interactions underlying homotypic chain migration.

Animals↗

PubMed2002

Fibronectin combined with stem cell factor plays an important role in melanocyte proliferation, differentiation and migration in cultured mouse neural crest cells.

Stem cell factor (SCF) is essential to the migration and differentiation of melanocytes during embryogenesis because mutations in either the SCF gene, or its ligand, KIT, result in defects in coat pigmentation in mice. Using a neural crest cell (NCC) primary culture system from wild-type mice, we previously demonstrated that KIT-positive and/or L-3, 4-dihydroxyphenylalanine (DOPA)-positive melanocyte precursors proliferate following the addition of SCF to the culture medium. Extracellular matrix (ECM) proteins are considered to play a role in the migration and differentiation of various cells including melanocytes. We cultured mouse NCCs in the presence of SCF in individual wells coated with ECM; fibronectin (FN), collagen I (CLI), chondroitin sulphate, or dermatan sulphate. More KIT-positive cells and DOPA-positive cells were detected in the presence of SCF on ECM-coated wells than on non-coated wells. A statistically significant increase in DOPA-positive cells was evident in FN and CLI wells. In contrast, in the absence of SCF, few DOPA-positive cells and KIT-positive cells were detected on either the ECM-coated or non-coated wells. We concluded that ECM affect melanocyte proliferation and development in the presence of SCF. To determine the key site of FN function, RGDS peptides in the FN sequence, which supports spreading of NCCs, were added to the NCC culture. The number of DOPA-positive cells decreased with RGDS concentration in a dose-dependent fashion. Immunohistochemical staining revealed the presence of integrin alpha5, a receptor of RGDS, in NCCs. These results suggest the RGDS domain of FN plays a contributory role as an active site in the induction of FN function in NCCs. In addition, we examined the effect of FN with SCF on the NCC migration by measuring cluster size, and found an increase in size following treatment with FN.

Animals↗

PubMed2001

Functional immaturity of cord blood monocytes as detected by impaired response to hepatocyte growth factor.

BACKGROUND: Monocytes as antigen-presenting cells play an important role in host defense and transplantation. However, there are little reports on cord blood monocytes, and the role of monocytes in cord blood transplantation is largely unknown. METHODS AND RESULTS: There are several cytokines affecting monocyte function. These include interferon-gamma, interleukin-4, interleukin-10, granulocyte macrophage-colony stimulating factor and hepatocyte growth factor (HGF). We investigated the effect of these cytokines on antigen-presenting capacity (APC) of cord and adult blood monocytes. Using either mononuclear cells or purified CD4+ T cells as responder cells, HGF enhanced APC of adult monocytes most effectively among these cytokines. In contrast, cord blood monocytes failed to respond to HGF. As HLA, costimulatory and adhesion molecules may affect APC function, we examined these antigens of monocytes following HGF stimulation. The HGF upregulated integrin alpha5 subunit (CD49e) and intercellular adhesion molecule-1 (CD54) was expressed in adult blood monocytes, but not in cord blood. In kinetic studies, HGF downregulated c-met protein/HGF receptor expression of adult monocytes in lower concentrations and at shorter incubation time as compared with that of cord blood. CONCLUSIONS: The results suggest that impaired response of cord blood monocytes to HGF may be responsible, in large part, for their functional immaturity.

Adult↗

PubMed2005

Global epithelial cell transcriptional responses reveal Streptococcus pyogenes Fas regulator activity association with bacterial aggressiveness.

The bacterial human pathogen Streptococcus pyogenes (group A streptococci, GAS) is able to adhere to, internalize into and cross-talk on multiple levels with its host cells. To gain insight into the Fas function in pathogenesis we used Affymetrix human genome DNA-arrays to measure temporal and global transcriptional responses of HEp-2 cells infected with M49 S. pyogenes wild-type bacteria and DeltafasX, an isogenic S. pyogenes two-component-signal-transduction system mutant. A modified stringent statistical analysis method identified a total of 86 HEp-2 cell genes as differentially transcribed upon infection over the investigated time course. Increased expression of genes encoding proteins involved in GAS host cell adherence and internalization (fibronectin, integrin-alpha5) was found as a common response. In contrast to earlier reports investigating other GAS serotype strains, Ras superfamily and RhoA pathways are exploited by M49 GAS, suggesting serotype specific interactions with the host cell cytoskeleton. Despite transcriptional induction, secreted IL-8 levels of deltafasX mutant infected cells were below those of non-infected cells, indicating an absence of Fas expression could be important for GAS tissue colonization and long-term intracellular persistence. Oppositely, activity of the S. pyogenes Fas-system apparently promotes high adherence and internalization rates, massive cytokine gene transcription and cytokine release, host cell apoptosis via a caspase-2 activation pathway, and cytotoxicity. Thus, the S. pyogenes Fas two-component signal transduction system could be involved in local tissue destruction and general bacterial aggressiveness towards host cells.

Apoptosis↗

PubMed2003

Mesothelial differentiation as reflected by differential gene expression.

Human mesothelial cells obtained from benign effusions retain their proliferative capacity and grow uniformly either with a fibroblastic or epithelioid morphology in vitro. These cultures therefore provide a model for the process of mesothelial differentiation in vivo. To study this differentiation, we isolated differentially expressed genes obtained by suppression subtractive hybridization. Of the nine genes found to be overexpressed in fibroblastic mesothelial cells, three are matrix-associated (integrin alpha5, collagen binding protein 2, human cartilage glycoprotein 39), whereas the others are associated with a proliferative cell type (14-3-3 epsilon, plexin B2, N33, and three genes encoding ribosomal elements). Seven of the eight genes upregulated in the epithelioid phenotype are related rather to specialized functions, such as metabolism (aldose reductase, lecithin:cholesterol acyltransferase, ATPase 6), cytoskeletal composition (cytokeratins 7 and 8), and regulation of differentiation (granulin, annexin II). Immunohistochemistry with available antibodies to six of the differentially expressed gene products confirmed the differences also in pleural tissues, where submesothelial cells displayed the fibroblastic markers, whereas surface cells displayed the epithelioid markers. In summary, this approach revealed a pattern of genes coordinately regulated during mesothelial differentiation and suggests that mesothelium may regenerate also by recruiting cells from the submesothelial layer. Some of the gene products may also be useful markers for differentiation and activation in serosal tissues.

14-3-3 Proteins↗

PubMed2002

Differential gene expression in human abdominal aorta: aneurysmal versus occlusive disease.

OBJECTIVE: Inflammation and atherosclerosis are present in both abdominal aortic aneurysm (AAA) and arterial occlusive disease (AOD). Changes in gene expression that underlie the development of AAA versus AOD are poorly defined. This study evaluated differences in gene expression in AAA, AOD, and control aortic tissue with human gene array technology. METHODS: RNA was isolated from human aortic specimens (seven AAA, five AOD, and five control), and complementary DNA (cDNA) probes were generated. The cDNA probes were hybridized to a human cell interaction array of 265 genes and quantitated with phosphorimaging. The data were corrected for background and were standardized to housekeeping genes. Statistical differences in individual gene expression were determined with the Kruskal-Wallis test. RESULTS: Of 265 genes studied, 11 showed statistically different expression in diseased aorta as compared with control. The following three genes were downregulated in AAA: collagen VI alpha1 (P <.037), glycoprotein IIIA (P <.006), and alpha2-macroglobulin (P <.020). The following two genes were upregulated in AOD: laminin alpha4 (P <.034) and insulin-like growth factor 2 receptor (P <.049). The following three genes were upregulated in both AAA and AOD: matrix metalloproteinase-9 (MMP-9; P <.005), intercellular adhesion molecule-1 (P <.012), and tumor necrosis factor--beta receptor (P <.022). The following three genes were downregulated in both AAA and AOD: integrin alpha5 (P <.012), ephrin A5 (P <.037), and rho/rac guanine nucleotide exchange factor (P <.028). Of 16 MMPs evaluated, only MMP-9 was significantly (P <.005) upregulated in both AAA and AOD. Evaluation results of four tissue inhibitors of metalloproteinases showed no significant difference in expression for all tissue types, although tissue inhibitor of metalloproteinase-1 trended toward upregulation in AAA (P =.053). Eight of the fifteen most highly expressed genes in all the groups were extracellular matrix or secreted proteins. Of these, only collagen VI alpha1 (P <.037) showed a significant change, although biglycan trended toward downregulation in AAA (P =.076). CONCLUSION: This study used cDNA array technology in the comparison of human control and pathologic aortic tissue. Six genes had similar differential expression in both AAA and AOD as compared with control. Even more interesting were differences between AAA and AOD in the expression of five genes. These data suggest a similarity in genetic expression for both AAA and AOD, with altered expression of several genes playing a role in disease differentiation.

Adult↗

PubMedDate not supplied

Differential integrin expression by cardiac fibroblasts from hypertensive and exercise-trained rat hearts.

The cardiac fibroblast is the principal cell type responsible for extracellular matrix (ECM) synthesis in the heart during growth and pathophysiological conditions. A dynamic interaction exists between the cardiac ECM and fibroblasts that is sensitive to the local mechanical and chemical tissue environment. We propose here that cardiac fibroblasts structurally and functionally adapt to changing local environments by altering their expression of receptor integrins. Changes in the extracellular environment are communicated in part by integrins, which link the ECM to the cell and regulate phenotype and function. In this report, we analyze integrin protein expression, migration and gel contraction by cardiac fibroblasts from rats subjected to 10 weeks of treadmill exercise (XTR), experimental hypertension (HYP) or controls (CONT). Immunoprecipitation shows that beta1 protein increases in XTR and HYP. Also, alpha1 and alpha2 integrins are lower in XTR and HYP, and alpha5 integrin is higher in XTR and lower in HYP. Functional assays show that XTR and HYP migrate slower on collagen, while XTR migrate faster and HYP slower on fibronectin. Cell isolation procedure, population expansion number or a general adaptation to culture conditions does not explain the differences observed. No significant differences in collagen gel contraction are detected. These results indicate that cardiac fibroblasts retain their in vivo patterns in vitro for a limited number of population expansions. This tissue-specific phenotype is exhibited in early passage (< or =6). However, by late passage (>8), cells begin to show adaptation to the in vitro conditions. These results show that cardiac fibroblasts respond to changing environments in pathophysiological conditions by modulating integrin expression, which is associated with changes in cell migration. They also suggest a pragmatic use for primary cardiac fibroblasts as a model to study the cardiac matrix remodeled by physiological (exercise) and pathological (hypertension) stressors.

Animals↗

PubMed2003

De novo expression of the integrin alpha5beta1 regulates alphavbeta3-mediated adhesion and migration on fibrinogen.

Recent evidence demonstrates that interactions between different integrins that are present on the cell surface can strongly influence the adhesive function of individual receptors. In this report, we show that Chinese hamster ovary cells that express the integrin alphavbeta3 in the absence of alpha5beta1 demonstrate increased adhesion and migration on fibrinogen. Furthermore, alphavbeta3-mediated adhesion to fibrinogen is not augmented by the soluble agonist, MnCl2, suggesting that alphavbeta3 exists in a higher affinity state in these cells. De novo expression of wild-type alpha5beta1 negatively regulates alphavbeta3-mediated adhesion and migration. This effect is not seen with expression of a chimeric alpha5beta1 integrin in which the cytoplasmic portion of the alpha5 integrin subunit is replaced by the cytoplasmic portion of the alpha4 integrin. In addition, it does not require ligation of alpha5beta1 by fibronectin. Cells that express a constitutively active beta3 integrin that contains a point mutation in the conserved membrane proximal region of the cytoplasmic tail, D723R, are resistant to the effect of alpha5beta1 expression. These data provide additional evidence of "cross-talk" between the integrins alpha5beta1 and alphavbeta3, and support the idea that alpha5beta1 regulates alphavbeta3-mediated ligand binding. This provides a relevant biological mechanism whereby variations in alpha5beta1 expression in vivo may modulate activation of alphavbeta3 to influence its adhesive function.

Animals↗

PubMed1998

Tetraspanin CD9 is associated with very late-acting integrins in human vascular smooth muscle cells and modulates collagen matrix reorganization.

CD9, a member of the tetraspanin family, and very late-acting (VLA) integrins are known to associate and form functional units on the surface of several cell types. We studied the changes in expression of CD9 and beta1-integrins (CD29, VLA) in human vascular smooth muscle cells (VSMCs) under in vitro culture conditions mimicking proliferative vascular diseases. We also investigated possible interactions between CD9 and VLA integrins in VSMCs. We found that CD9 is highly expressed in VSMCs and is subject to modulation, depending on the proliferative/contractile state of the cells. In the contractile phenotype, the levels of CD9, CD81, another tetraspanin, and CD29 are approximately 50% of those found in the proliferative phenotype. Coimmunoprecipitation experiments showed physical association between CD9 and CD29. CD9 was mainly associated with alpha2 and alpha3-integrins (CD49b and c) and also with alpha5-integrin to a weaker extent. Functionally, the addition of anti-CD9 monoclonal antibodies (MoAbs) doubled the extent of collagen gel contraction mediated by VSMCs, a model for the reorganization of the extracellular collagen matrix occurring in the vessel wall. Anti-CD29 MoAbs inhibited gel contraction, but anti-CD9 MoAbs counteracted this inhibitory effect of anti-CD29 MoAbs. Transfection of human CD9 into Chinese hamster ovary cells more than doubled the extent of Chinese hamster ovary cell-mediated collagen gel contraction (130% stimulation), confirming a role for CD9 in extracellular matrix reorganization. Thus, CD9 seems to be involved in the modulation of VLA integrin-mediated collagen matrix reorganization by VSMCs. These findings suggest that high CD9 expression is associated with a proliferative state of VSMCs. The role of CD9 could be to modulate the function of VLA integrins on the surface of VSMCs.

Animals↗

PubMed1998

De novo expression of the alpha5beta1-fibronectin receptor in HT29 colon-cancer cells reduces activity of C-SRC. Increase of C-SRC activity by attachment on fibronectin.

Changes in integrin expression during malignant transformation have been observed in many tumors. Colon-carcinoma cells show reduced expression or even loss of the alpha5beta1 integrin compared to normal or adenoma cells. To determine the significance of absent alpha5beta1 integrin signaling, we transfected the cDNA coding for the alpha5 integrin sub-unit into the human colon-carcinoma cell line HT29, which constitutively lacks this subunit but does express the beta1 subunit. We show here that the newly expressed fibronectin receptor alpha5beta1 generates multiple signals, causing marked changes in cytoskeletal arrangements within a few minutes of adhesion to fibronectin. Cells expressing the alpha5beta1 integrin exhibit the formation of actin stress fibers and focal adhesions, as well as the induction of tyrosine phosphorylation of several proteins, within 10 min. We identified the focal adhesion kinase pp125FAK and the cytoskeletal protein paxillin as major phosphorylation substrates in these cells. These proteins remained hypophosphorylated when alpha5-negative control cells were plated on fibronectin. The tyrosine kinase pp60c-src, regarded as central in the regulation of cellular proliferation and constitutively over-expressed in HT29 and in colon-carcinoma cells, showed reduced intrinsic kinase activity in unstimulated HT29alpha5 cells. In contrast, fibronectin-induced signaling through alpha5beta1 increased pp60c-src activity. Moreover, immunoprecipitation of pp60c-src from extracts of HT29alpha5 cells cultivated on fibronectin for 20 min revealed complex formation of pp60c-src and tyrosine-phosphorylated pp125FAK. Our data suggest that de novo expression of the alpha5beta1 integrin in HT29 colon-cancer cells restores signaling via pp125FAK and pp60c-src. Thus, loss of this receptor during malignant transformation may contribute to tumor-cell autonomy, while reduced activity of pp60c-src in HT29alpha5-cells may participate directly in growth control.

Cell Adhesion↗

PubMed1998

Distribution of alpha3, alpha5 and alpha(v) integrin subunits in mature and immature human oocytes.

The distribution of three integrin subunits, alpha3, alpha5 and alpha(v), in immature and mature human oocytes has been examined using immunofluorescence and confocal microscopy. The results demonstrate that both alpha5 and alpha(v) are present at the germinal vesicle stage, while alpha3 was only detected in oocytes after germinal vesicle breakdown, in metaphase I and II stage oocytes. The cortical concentration of integrin subunits alpha3 and alpha5 is consistent with their localization in the oolemma. In contrast, the homogeneous distribution of alpha(v) throughout the oocyte suggests the existence of cytoplasmic reservoirs of this protein in the oocyte.

Antigens, CD↗