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Results for “Integrin alpha5”

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PubMed1997

RGDN peptide interaction with endothelial alpha5beta1 integrin causes sustained endothelin-dependent vasoconstriction of rat skeletal muscle arterioles.

The ability of an integrin-binding Arg-Gly-Asp-Asn (RGDN)- containing peptide to influence vascular tone by interacting with the alpha5beta1 integrin was studied using rat skeletal muscle arterioles. After blockade of beta3 integrin function, isolated arterioles with spontaneous tone showed concentration-dependent vasoconstrictions to topical application of GRGDNP, a peptide that shows a greater ability to interact with alpha5beta1 than with alphavbeta3. The constriction to GRGDNP (2.1 mM) was inhibited by blocking alpha5 integrin function, and was intensified by blocking beta3 integrin function. In contrast, GRGDSP, a peptide that interacts better with alphavbeta3, was unable to induce sustained constrictions. Removal of the endothelium abolished the vasoconstriction in response to GRGDNP, suggesting that the response was due to release of an endothelium-dependent factor. Indeed, blockade of ETA endothelin receptors with BQ-610 (1 microM), similar to removal of the endothelium and alpha5 integrin blockade, inhibited the vasoconstriction. These data indicate that interaction of RGD peptides, and in particular the RGDN sequence with endothelial cell alpha5beta1, causes endothelin-mediated arteriolar vasoconstriction. These results indicate that integrins are novel signaling receptors within the vascular wall that affect vasomotor tone, and may play an important role in vascular control.

Animals↗

PubMedDate not supplied

Inhibitory effect of fucoidan on the adhesion of adenocarcinoma cells to fibronectin.

Fucoidans inhibit tumour cell adhesion to various substrata, but their mechanisms of action are not fully understood. Using 3H-fucoidan, we observed that fucoidan binds to fibronectin, this binding being saturable and sensitive to ionic strength and pH. The interaction occurred on at least four different sites along the polypeptide chain, two of them being the heparin-binding sequences. Moreover, when MDA-MB-231 tumour cells were exposed to DTAF-fucoidan, internalization occurred and punctuated vesicles were observed in the perinuclear region. The treated cells also showed a different morphology with a cytoskeleton devoid of vinculin and a reorganiztion of the repartition of the integrin-alpha5 subunit on the cell surface. Based on these data, we hypothesize that fucoidan inhibits the adhesion of MDA-MB-231 cells to fibronectin i) by blocking the protein's heparin- and cell-binding domains, ii) by modulating the reorganization of the integrin alpha5 subunit and iii) by down-regulating the expression of vinculin.

Actins↗

PubMed2003

Cell adhesion-related gene expression by Helicobacter pylori in gastric epithelial AGS cells.

Helicobacter pylori (H. pylori) infection leads to gastroduodenal inflammation, peptic ulceration and gastric carcinoma. H. pylori may induce disease-specific gene expression in gastric epithelial cells. cDNA microarray for 352 cancer-related genes was used to identify the genes altered by H. pylori (cagA positive) in gastric epithelial AGS cells. Expressions of the genes identified on the microarray and other genes closely associated with these genes were determined by reverse transcriptase-polymerase chain reaction (RT-PCR). Western blot analysis and cell adhesion assay were performed to confirm the protein levels of the genes and the role of the genes on cell adhesion in H. pylori-infected AGS cells. As a result, the expression of four genes (galectin 1, aldolase A, integrin alpha5, LIM domain only 7 (LMO7)) were up-regulated by H. pylori in AGS cells, determined by cDNA microarray. RT-PCR analysis showed that the genes up-regulated by H. pylori were the genes regulating cell-cell adhesion and cell-extracellular matrix interaction, such as galectin-1 and galectin-3, integrin alpha5, and LIM domain only 7 (LMO7), and cancer-related glycolytic enzyme aldolase A and C. Cell adhesion to extracellular matrix proteins such as poly-L-lysine and fibronectin was mediated by H. pylori-induced expression of integrin alpha5. RT-PCR and Western blot analysis showed that E-cadherin, regulating cell adhesion and contact cell inhibition, was decreased by H. pylori in AGS cells. In conclusion, the increased expression of cell adhesion molecules and decrease in E-cadherin expression by H. pylori might contribute to cell adhesion, invasion and possibly cell proliferation in gastric epithelial cells.

Adenocarcinoma↗

PubMed1999

New human hepatocellular carcinoma (HCC) cell line with highly metastatic potential (MHCC97) and its expressions of the factors associated with metastasis.

A new human hepatocellular carcinoma (HCC) cell line with a highly metastatic potential was established from subcutaneous xenograft of a metastatic model of human HCC in nude mice (LCI-D20) by means of alternating cell culture in vitro and growth in nude mice. The line, designated MHCC97, has been cultivated for 18 months and subcultured for more than 90 passages. The line was showed to be of human origin by karyotype analysis. The cells were either grown as compact colonies (in clusters) or as a monolayered sheet with about 31 h of population-doubling time, exhibited typical malignant epithelial in morphology and were positive for alpha-fetoprotein (AFP). Flow cytometric analysis of the cell DNA content showed an aneuploid pattern, and its index was 1.5 as compared to that of normal human peripheral blood lymphocytes. Karyotypic analyses of G- and C-banding techniques revealed that all cells presented chromosome abnormalities in number and structure. The number of cell line MHCC97 chromosome ranged from 59 to 65 with a modal number of 60 and 61. At least two common chromosome markers, i(1q) and der(4)t(4;?)(4pter-->q35::?), were present in all cells, and deletion of Y chromosome also occurred in all cells. The subcutaneous and intrahepatic xenografts were formed and metastatic lesions in lungs were found after the cells were inoculated into nude mice. The rate of metastasis to lungs was 100% using orthotopic inoculation. Reverse transcription polymerase chain reaction products revealed positive expressions of integrin alpha5 and beta1, urokinase type plasminogen activator receptor (uPAR), vascular endothelial growth factor and nm23-H1 mRNAs of cell line MHCC97. Immunostaining of c-Met, uPAR showed strongly positive in both subcutaneous xenografts and lung metastatic lesions; while positive in xenografts and negative in metastatic lesions for integrin alpha5, beta1. E-cadherin and P53 was not expressed either in xenograft or in the metastatic lesions. PCR products of HBsAg and HBxAg were both positive. The cell line MHCC97 still retained some characteristic features of original tumour. Establishment of cell line MHCC97 should be beneficial to the studies of HCC metastatic mechanisms.

Adult↗

PubMedDate not supplied

Differential regulation of focal adhesion kinase and paxillin phosphorylation by the small GTP-binding protein Rho in human corneal epithelial cells.

PURPOSE: Lysophosphatidic acid (LPA), which activates the small guanosine triphosphate (GTP)-binding protein Rho, was previously shown to promote migration of the rabbit corneal epithelium in culture. The signaling pathway responsible for this effect of LPA was examined in this study with a human corneal epithelial (HCE) cell line. METHODS: The activation of Rho was detected with a pull-down assay. Tyrosine phosphorylation of paxillin and of focal adhesion kinase (FAK) were examined both by immunofluorescence staining and by immunoprecipitation and immunoblot analyses. Expression of integrins alpha5 and beta1 was evaluated by immunoblot analysis. RESULTS: Incubation of cells with LPA (10 micro M) for 2 min resulted in marked activation of Rho, and this effect was blocked by pretreatment with the Rho inhibitor exoenzyme C3 (2 micro g/ml) for 24 h. Tyrosine phosphorylation of both paxillin and FAK was detected in HCE cells under basal conditions by immunofluorescence staining, immunoprecipitation, and immunoblot analyses. LPA induced a concentration- and time-dependent increase in the tyrosine phosphorylation of paxillin, which was maximal at a concentration of 10 micro M and a time of 2 min. Exoenzyme C3 inhibited LPA-induced paxillin phosphorylation. Neither LPA nor exoenzyme C3 affected tyrosine phosphorylation of FAK or expression of integrins alpha5 and beta1. CONCLUSIONS: LPA induces Rho activation and the consequent tyrosine phosphorylation of paxillin in HCE cells, and these effects likely contribute to the promotion of corneal epithelial migration by this agent.

Cell Line↗

PubMed1998

Physical and biochemical regulation of integrin release during rear detachment of migrating cells.

Cell migration can be considered as a repeated cycle of membrane protrusion and attachment, cytoskeletal contraction and rear detachment. At intermediate and high levels of cell-substratum adhesiveness, cell speed appears to be rate-limited by rear detachment, specifically by the disruption of cytoskeleton-adhesion receptor-extracellular matrix (ECM) linkages. Often, cytoskeletal linkages fracture to release integrin adhesion receptors from the cell. Cell-extracellular matrix bonds may also dissociate, allowing the integrins to remain with the cell. To investigate molecular mechanisms involved in fracturing these linkages and regulating cell speed, we have developed an experimental system to track integrins during the process of rear retraction in Chinese hamster ovary (CHO) cells. Integrin expression level was varied by transfecting CHO B2 cells, which express very little endogenous alpha5 integrin, with a plasmid containing human alpha5 integrin cDNA and sorting the cells into three populations with different alpha5 expression levels. Receptor/ligand affinity was varied using CHO cells transfected with either alphaIIbbeta3 or alphaIIbbeta3(beta1-2), a high affinity variant. alphaIIbbeta3(beta1-2) is activated to a higher affinity state with an anti-LIBS2 antibody. Fluorescent probes were conjugated to non-adhesion perturbing anti-integrin antibodies, which label integrins in CHO cells migrating on a matrix-coated glass coverslip. The rear retraction area was determined using phase contrast microscopy and integrins initially in this area were tracked by fluorescence microscopy and a cooled CCD camera. We find that rear retraction rate appears to limit cell speed at intermediate and high adhesiveness, but not at low adhesiveness. Upon rear retraction, the amount of integrin released from the cell increases as extracellular matrix concentration, receptor level and receptor-ligand affinity increase. In fact, integrin release is a constant function of cell-substratum adhesiveness and the number of cell-substratum bonds. In the adhesive regime where rear detachment limits the rate of cell migration, cell speed has an inverse relationship to the amount of integrin released at the rear of the cell. At high cell-substratum adhesiveness, calpain, a Ca2+-dependent protease, is also involved in release of cytoskeletal linkages during rear retraction. Inhibition of calpain results in decreased integrin release from the cell membrane, and consequently a decrease in cell speed, during migration. These observations suggest a model for rear retraction in which applied tension and calpain-mediated cytoskeletal linkage cleavage are required at high adhesiveness, but only applied tension is required at low adhesiveness.

Animals↗

PubMed2003

Regulation of the integrin subunit alpha5 gene promoter by the transcription factors Sp1/Sp3 is influenced by the cell density in rabbit corneal epithelial cells.

PURPOSE: Expression of the alpha5beta1 fibronectin (Fn) integrin is well recognized in the corneal epithelium and has been postulated to increase during wound healing. In the present study, the regulatory influence of the positive transcription factors Sp1/Sp3 on the activity directed by the promoter of the alpha5 gene was examined in rabbit corneal epithelial cells (RCECs) primary cultured at various cell densities. METHODS: Expression of the alpha5 subunit was assessed at the transcriptional level by semiquantitative RT-PCR analyses. The regulatory elements necessary to direct expression of the alpha5 gene were identified by transfecting RCECs with recombinant plasmids bearing various lengths from the alpha5 gene promoter fused to the CAT reporter gene. Binding of Sp1/Sp3 to the alpha5 promoter was assessed by both electrophoretic mobility shift assays (EMSAs) and DNaseI footprinting. Endogenous levels of Sp1/Sp3 were determined by Western blot and supershift analyses. The regulatory influence exerted by Sp1/Sp3 on the alpha5 promoter was evaluated both by site-directed mutagenesis and cotransfection in Sp1-deficient Drosophila SL-2 Schneider cells. RESULTS: Subconfluent RCECs expressed nearly five times more alpha5 transcript than 48-hour postconfluent RCECs. The activity directed by the alpha5 promoter was found to be affected by cell density. Strong promoter activity was observed in subconfluent RCECs, whereas a dramatic repression was measured in postconfluent cells. EMSA and DNaseI footprinting provided evidence for the binding of Sp1 to both a proximal site located within the previously reported alpha5 fibronectin responsive element (FRE), and a distal site located between positions -117 and -101. Cotransfection experiments in Schneider cells, as well as transfection of RCECs with recombinant constructs bearing mutations into the distal Sp1 site, confirmed the positive regulatory influence of Sp1 on both the -42/-92 and -92/-132 alpha5 promoter segments. Most of all, EMSA and Western blot analyses demonstrated the expression of substantial amounts of Sp1/Sp3 in subconfluent but not postconfluent RCECs. CONCLUSIONS: These results provide support to the hypothesis that the strong reduction in the activity of the alpha5 promoter when RCECs reach a high cell density is the consequence of a reduced expression of Sp1/Sp3 under such cell culture conditions.

Animals↗

PubMed2006

RGD-peptide presents anti-adhesive effect, but not direct pro-apoptotic effect on endothelial progenitor cells.

Circulating endothelial progenitor cells (EPCs) contribute to neovascularization in tumor or ischemic tissues by multi-step events, including adhesion, migration, chemoattraction, and differentiation to endothelial cells. Anti-angiogenic RGD-peptides have been shown to directly induce apoptosis in human umbilical vein endothelial cells (HUVECs) and T cells. Here, we examined the effects of RGD-peptides on EPCs in terms of adhesive differentiation and apoptosis. When mononuclear cells (MNCs) isolated from human cord blood were cultured on fibronectin-coated plates for 7 days, RGD-peptide treatment decreased dose-dependently the number of adherent cells double positive for DiI-ac-LDL uptake and UEA-1 binding. The cells treated with RGD peptide were also stained less strongly by vWF or KDR antibody by immunofluorescence staining. Immobilization of the RGD-peptide promoted cell adhesion, but resulted in a deficiency in the development of ability of ac-LDL uptake and UEA-1 binding, showing an antagonistic effect. Accordingly, ex vivo-cultivated EPCs expressed integrin alpha5, alphav, beta1, beta3, and beta5, and antibodies to integrins alpha5, alphav, and beta1 decreased the number of adherent cells. However, viability of total MNCs containing early EPCs was not affected by RGD-peptide. In addition, neither an increase in apoptotic cell death nor a direct activation of caspase-3 by RGD-peptide was detected in ex vivo-cultivated EPCs, unlike in HUVECs. Interestingly, RGD-peptide rather enhanced Bcl-2 expression in ex vivo-cultivated EPCs and the EPCs themselves with a high Bcl-2/Bax ratio are comparatively resistant to apoptosis. Therefore, these results suggest that RGD-peptides may inhibit EPC differentiation by anti-adhesive effect, but not by a direct pro-apoptotic effect.

Apoptosis↗

PubMed2006

The dependence of fibrillar adhesions in human fibroblasts on substratum chemistry.

Little is known about the influence of substratum properties and composition on the ability of cells to translocate alpha5beta1 integrins and to form fibrillar adhesion. We have examined the impact of self-assembled monolayers (SAMs) bearing different functional end groups (amines (NH2) or carboxylic acids (COOH)) on the presence of fibrillar adhesions in human fibroblasts attached to fibronectin-coated SAMs. Most of the fibroblasts incubated in serum-free medium for 2 h on COOH showed segregation of focal contact components (alphav integrin subunits, phosphotyrosine proteins) and fibrillar adhesions (alpha5 integrin subunits, tensin) while the majority of cells plated on NH(2) did not. Analysis of fibronectin fibril formation confirmed also that human fibroblasts plated on COOH formed matrix fibrils significantly better. The surface-associated alpha5 to alphav integrin ratio was smaller in cells on COOH than on NH2 due to a decreased alpha5 integrin binding. In addition, human fibroblasts migrated more readily on COOH in comparison to NH2 which points to a different binding strength of integrins to the substratum. Overall, the results indicate that the molecular composition of substrata has a strong influence on FN matrix formation by promoting or inhibiting segregation of focal and fibrillar adhesions.

Cell Adhesion↗

PubMed2002

Analysis of IL-2, IFN-gamma and TNF-alpha production, alpha5 beta1 integrins and actin filaments distribution in peritoneal mouse macrophages treated with homeopathic medicament.

The newer forms of immune modulatory therapy are aimed at specific cells or cytokines that contribute to the immune response. These forms of immunotherapy have been referred to as 'biological response modifiers'. Our lab was interested in investigating if a homeopathic medicament 'Metodo Canova' (MC), sold in homeopathic drugstores, does enhance immunological system responses acting through macrophages pathway. Mice peritoneal macrophages were cultivated with or without homeopathic medicament for 24 h for alpha5, beta1 and actin filaments distribution analyses through immunolabelling for confocal microscopy. To detect the IL-2, IFN-gamma and TNF-alpha production these cells were cultivated for 48 h with or without medicament, followed by analyses of these cytokines in supernatant culture with ELISA kits. It was observed differences in morphology and molecular distribution (alpha5 and beta1 integrins, actin filaments and Fc receptors) between the groups control and treated with MC. In control group macrophages had the morphology of resident cells and in MC treated group macrophages were more spread, had many cellular projections and a substantial increase in cytoplasmic volume. In addition, macrophages culture with two doses of MC showed that TNF-alpha production decreased when compared with control group.

Actin Cytoskeleton↗

PubMed2006

The effect of gamma-tocopherol on proliferation, integrin expression, adhesion, and migration of human glioma cells.

The effect of vitamin E on proliferation, integrin expression, adhesion, and migration in human glioma cells has been studied. gamma-tocopherol at 50 microM concentration exerted more inhibitory effect than alpha-tocopherol at the same concentration on glioma cell proliferation. Integrin alpha5 and beta1 protein levels were increased upon both alpha- and gamma-tocopherol treatments. In parallel, an increase in the alpha5beta1 heterodimer cell surface expression was observed. The tocopherols inhibited glioma cell-binding to fibronectin where gamma-tocopherol treatment induced glioma cell migration. Taken together, the data reported here are consistent with the notion that the inhibition of glioma cell proliferation induced by tocopherols may be mediated, at least in part, by an increase in integrin alpha5 and beta1 expression. Cell adhesion is also negatively affected by tocopherols, despite a small increase in the surface appearance of the alpha5beta1 heterodimer. Cell migration is stimulated by gamma-tocopherol. It is concluded that alpha5 and beta1 integrin expression and surface appearance are not sufficient to explain all the observations and that other integrins or in general other factors may be associated with these events.

Cell Adhesion↗

PubMed2002

Adhesion of synchronized human hematopoietic progenitor cells to fibronectin and vascular cell adhesion molecule-1 fluctuates reversibly during cell cycle transit in ex vivo culture.

Ex vivo expansion of hematopoietic stem/progenitor cells may result in defective engraftment. Human cord blood CD34(+) progenitor cells were synchronized and assayed for adhesion and migration onto fibronectin (Fn) and vascular cell adhesion molecule-1 (VCAM-1) at different stages of a first cell cycle executed ex vivo. During S phase transit, adhesion to Fn was transiently increased while binding to VCAM-1 was reversibly decreased, after which adhesion to both ligands returned to baseline levels with cell cycle completion. Transmigration across Fn and VCAM-1 decreased irreversibly during S phase progression. The function of alpha4 and alpha5 integrins was assessed with specific neutralizing antibodies. In uncultured CD34(+) cells and long-term culture-initiating cells (LTC-ICs), both adhesion and migration on Fn were inhibited by anti-alpha4 but not by anti-alpha5 antibodies. In mitotically activated CD34(+) cells and LTC-ICs, adhesion and migration on Fn were mainly dependent on alpha5 integrin and to a lesser extent on alpha4 integrin. Changes in integrin function were not dependent on parallel modulation of integrin expression. In conclusion, Fn and VCAM-1 binding of progenitor cells fluctuates reversibly during cell cycle transit ex vivo. In addition, our data show that mitogenic activation induces a shift from a dominant alpha4 to a preferential alpha5 integrin-dependent interaction with Fn.

Antigens, CD↗

PubMed2003

The homeobox transcription factor Hox D3 promotes integrin alpha5beta1 expression and function during angiogenesis.

Neovascularization promotes wound healing, tumor growth, and arthritis. Endothelial cell migration and survival during neovascularization are regulated by adhesion proteins, including integrin alpha5beta1. Integrin alpha5beta1 is poorly expressed on normal quiescent blood vessels, but its expression is induced on tumor blood vessels and in response to angiogenic factors such as basic fibroblast growth factor, interleukin-8, tumor necrosis factor-alpha, and the angiomatrix protein Del-1. We show here that alpha5beta1 expression, and hence function, during angiogenesis is regulated by the transcription factor Hox D3, a homeobox gene that also controls the expression of endothelial cell integrin alphavbeta3 and urokinase-type plasminogen activator. Hox D3 expression in endothelial cells enhances integrin alpha5 protein and message expression, whereas Hox D3 antisense inhibits its expression. Hox D3 promotes alpha5 expression during angiogenesis in vivo, whereas inhibition of alpha5 expression by Hox D3 antisense suppresses angiogenesis. Hox D3 binds directly to the promoters of the integrin alpha5 and beta3 subunits, inducing subunit expression. As Hox D3, integrin alphavbeta3, and integrin alpha5beta1 are expressed on tumor blood vessels but not on normal quiescent vessels, these studies suggest that Hox D3 coordinately regulates the expression of integrin alpha5beta1 and integrin alphavbeta3 during angiogenesis in vivo. These studies also suggest that Hox D3 inhibition could be a useful approach to inhibit tumor angiogenesis.

Animals↗

PubMed1998

Enhancement of metastatic activity of colon cancer as influenced by expression of cell surface antigens.

BACKGROUND: Cell surface antigens are contributory factors toward metastatic activity. There have been no detailed studies on changes in cell surface antigens of colon cancer cell lines. To control life-threatening metastasis, it is necessary to evaluate what types of changes in cell surface antigens exert an influence on metastatic activity. MATERIALS AND METHODS: In vivo selection was performed using the human colon cancer-derived cell line KM12SM to obtain variants of metastatic activity. A murine spleen injection-liver metastasis procedure reflecting the latter half of the metastatic process was adopted and repeated four times. Flow cytometric analyses were carried out to detect expression of antigens: Lewis a (Lea), Lewis x (Lex), sialyl Lewis a (sLea), sialyl Lewis x (sLex), E-cadherin, CD44v6, integrin alpha2 (CD49b), integrin alpha3 (CD49c), integrin alpha4 (CD49d), integrin alpha5 (CD49e), and integrin beta1 (CD29). RESULTS: In vivo selection produced variants with higher metastatic activity. In the original line KM12SM, sLea, E-cadherin, CD49b, CD49c, or CD29 were positive in more than 40% of the cells. After selection, the percentage of cells positive for Lea, sLea, and all examined integrins significantly increased. Lex, sLex, and CD44v6 increased slightly, while E-cadherin decreased slightly. CONCLUSIONS: In vivo selection and flow cytometric analysis revealed that Lea, sLea, CD49b, CD49c, and CD29 appear to be involved in the increase of metastatic activity. The changes of integrin expression in this study suggest that integrins collaborate in the promotion of adhesion to an extracellular matrix.

Animals↗

PubMed1999

Tumor necrosis factor-alpha stimulates attachment of small cell lung carcinoma to endothelial cells.

Tumor cell attachment to endothelial cells (ECs) is an important step in the metastasis of small cell lung carcinoma (SCLC). Tumor necrosis factor-alpha (TNF-alpha) stimulation of ECs increases the attachment of some malignant cell types to ECs by affecting the expression of cell adhesion molecules (CAMs). Similarly, the inhibition of EC protein kinase C (PKC) and tyrosine kinase (TK) pathways modulates TNF-alpha-mediated effects on CAM expression. We hypothesized that TNF-alpha would increase SCLC attachment to ECs by affecting CAM expression through activation of PKC and TK pathways. To test this hypothesis, human umbilical vein endothelial cells (HUVECs) were stimulated with TNF-alpha (0 to 500 U/mL) for variable time periods (1 to 24 hours), and the attachment of H82 cells (an SCLC cell line) to the HUVECs was quantified. TNF-alpha stimulation of the HUVECs increased H82 attachment from 28.1% +/- 1.6% to 48.8% +/- 1.7% (P < .05). Preincubation of HUVECs with the PKC inhibitors bis-indolylmaleimide (BIN) or calphostin C or the TK inhibitors genistein or herbimycin A (HMA) blocked the TNF-alpha-induced increase in H82 cell attachment. The addition of antibodies to vitronectin (Vn) or beta1-integrin to TNF-alpha-activated HUVECs before the addition of the H82 cells also significantly decreased H82 attachment, whereas the addition of antibodies to E-selectin, P-selectin, vascular cell adhesion molecule (VCAM), intercellular adhesion molecule (ICAM), neural cell adhesion molecule (NCAM), sialyl-Lewis(x), fibronectin (Fn), alpha(v)-integrin, alpha3-integrin, alpha4-integrin, or alpha5-integrin had no effect on SCLC attachment. In summary, the TNF-alpha-mediated increase in SCLC attachment to ECs appears to be mediated by the activation of EC PKC and TK pathways as well as through effects on the function or expression of EC Vn and beta1 integrin.

Carcinoma, Small Cell↗

PubMed2003

Alpha-smooth muscle actin is crucial for focal adhesion maturation in myofibroblasts.

Cultured myofibroblasts are characterized by stress fibers, containing alpha-smooth muscle actin (alpha-SMA) and by supermature focal adhesions (FAs), which are larger than FAs of alpha-SMA-negative fibroblasts. We have investigated the role of alpha-SMA for myofibroblast adhesion and FA maturation. Inverted centrifugation reveals two phases of initial myofibroblast attachment: during the first 2 h of plating microfilament bundles contain essentially cytoplasmic actin and myofibroblast adhesion is similar to that of alpha-SMA-negative fibroblasts. Then, myofibroblasts incorporate alpha-SMA in stress fibers, develop mature FAs and their adhesion capacity is significantly increased. When alpha-SMA expression is induced in 5 d culture by TGFbeta or low serum levels, fibroblast adhesion is further increased correlating with a "supermaturation" of FAs. Treatment of myofibroblasts with alpha-SMA fusion peptide (SMA-FP), which inhibits alpha-SMA-mediated contractile activity, reduces their adhesion to the level of alpha-SMA negative fibroblasts. With the use of flexible micropatterned substrates and EGFP-constructs we show that SMA-FP application leads to a decrease of myofibroblast contraction, shortly followed by disassembly of paxillin- and beta3 integrin-containing FAs; alpha5 integrin distribution is not affected. FRAP of beta3 integrin-EGFP demonstrates an increase of FA protein turnover following SMA-FP treatment. We conclude that the formation and stability of supermature FAs depends on a high alpha-SMA-mediated contractile activity of myofibroblast stress fibers.

Actins↗

PubMed2003

Alpha5beta1 integrin mediates strong tissue cohesion.

Integrins and cadherins are considered to have distinct and opposing functions. Integrins are traditionally cited for their role in cell-substratum interactions, whereas cadherins are thought to mediate strong intercellular cohesion. Together, these adhesion systems play crucial roles in a wide variety of cellular and developmental processes including cell migration, morphology, differentiation and proliferation. In this manuscript we present evidence that integrins possess the ability to mediate strong intercellular cohesion when cells are grown as 3D aggregates. Much of the data elucidating the role of integrins as mediators of cell-extracellular matrix (ECM) interactions have been generated using conventional cell culture techniques in which cells are plated onto ECM-coated 2D surfaces. In vivo, cells are embedded in a 3D meshwork of ECM proteins. We hypothesized that, within this meshwork, integrin-ECM interactions may impart cohesivity to an aggregate of cells by linking adjacent cells together. To test this hypothesis, we transfected Chinese hamster ovary (CHO-B2) cells to express alpha5beta1 integrin and found that these cells formed compact, spherical aggregates. We measured aggregate cohesivity using tissue surface tensiometry, a novel technique that quantifies cell-cell cohesivity of spheroids under physiological conditions. We determined that alpha5beta1 integrin is capable of conferring strong cohesivity (sigma=8.22+/-0.68 dynes/cm) to aggregates of alpha5-integrin-transfected cells. This cohesion was found to be independent of cadherin expression and was significantly greater than the cohesivity conferred onto CHO-B2 cells transfected with N-cadherin (sigma=3.14+/-0.20 dynes/cm, P</=0.0001), a more traditional cell-cell cohesion system. Fibronectin-null CHO cells that express alpha5beta1 integrin but do not secrete endogenous fibronectin do not form aggregates in fibronectin-depleted medium. Addition of increasing amounts of exogenous dimeric fibronectin to these cells resulted in a dose-dependent compaction. However, compaction failed to occur in the presence of fibronectin monomers. These data indicate that fibronectin is required for alpha5beta1-mediated compaction and that the dimeric structure of fibronectin is essential for this process. Additionally, aggregate formation of the alpha5 integrin transfectants was inhibited by an RGD peptide thus confirming alpha5beta1 integrin specificity. Collectively, these data confirm our hypothesis that alpha5beta1 integrin acts through fibronectin to link adjacent cells together, thus promoting strong intercellular cohesion in 3D cellular aggregates.

Animals↗

PubMed2002

Regulation of fibronectin fibrillogenesis by protein kinases in cultured rat osteoblasts.

Fibronectin (Fn) plays an important role in the regulation of adhesion, migration, and maturation of osteoblasts. Fn fibrillogenesis is involved in the process of bone mineralization. To elucidate the regulatory role of protein kinases in the formation of fibrillar Fn matrix, Fn synthesis and assembly were examined in cultured osteoblasts. Osteoblasts assembled the endogenously released soluble Fn into immobilized form on the substratum in a time-dependent manner. Both 12-O-tetradecanoylphorbol-13 acetate (TPA) and forskolin increased the synthesis of Fn. However, the extracellular assembly of Fn fibril from both endogenously released and exogenously applied soluble Fn was increased by TPA but decreased by forskolin. Protein kinase C (PKC) inhibitors, such as H7, Ro 318220, and Gö 6976, inhibited Fn fibrillogenesis. These results suggest that the dynamic of Fn fibrillogenesis is differentially regulated by the activation of PKC and protein kinase A (PKA). Both classic and novel isoforms of PKC are involved in the action of TPA in osteoblasts. It has been reported that alpha5beta1 integrin is related to Fn fibrillogenesis. Immunocytochemistry and flow cytometry showed that TPA and forskolin increased and inhibited, respectively, the clustering and surface expression of alpha5 integrins. TPA and forskolin did not affect protein levels of alpha5 integrins. The Western blot and reverse transcriptase-polymerase chain reaction showed that protein and mRNA levels of beta1 integrins also were not affected by TPA and forskolin. These results suggest that TPA and forskolin may affect the surface expression of alpha5beta1 integrins. cAMP response element-binding protein phosphorylation is involved in the action of forskolin but not that of TPA. Our results suggest that PKC activation enhanced Fn fibrillogenesis, whereas PKA activation inhibited extracellular Fn fibrillogenesis in primary cultured osteoblasts. Cytosolic Fn synthesis and extracellular Fn assembly may be differentially regulated by the activation of PKA.

Animals↗