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Sample records for beta1 integrins differentially

  1. Beta1 integrin cytoplasmic variants differentially regulate expression of the antiangiogenic extracellular matrix protein thrombospondin 1.

    PubMed

    Goel, Hira Lal; Moro, Loredana; Murphy-Ullrich, Joanne E; Hsieh, Chung-Cheng; Wu, Chin-Lee; Jiang, Zhong; Languino, Lucia R

    2009-07-01

    Beta(1) integrins play an important role in regulating cell proliferation and survival. Using small interfering RNA or an inhibitory antibody to beta(1), we show here that, in vivo, beta(1) integrins are essential for prostate cancer growth. Among the five known beta(1) integrin cytoplasmic variants, two have been shown to differentially affect prostate cell functions. The beta(1A) variant promotes normal and cancer cell proliferation, whereas the beta(1C) variant, which is down-regulated in prostate cancer, inhibits tumor growth and appears to have a dominant effect on beta(1A). To investigate the mechanism by which beta(1C) inhibits the tumorigenic potential of beta(1A), we analyzed changes in gene expression in cells transfected with either beta(1C) or beta(1A). The results show that beta(1C) expression increases the levels of an extracellular matrix protein, thrombospondin 1 (TSP1), an angiogenesis inhibitor. TSP1 protein levels are increased upon beta(1C) expression in prostate cancer cells as well as in beta(1)-null GD25 cells. We show that TSP1 does not affect proliferation, apoptosis, or anchorage-independent growth of prostate cancer cells. In contrast, the newly synthesized TSP1, secreted by prostate cancer cells expressing beta(1C), prevents proliferation of endothelial cells. In conclusion, our novel findings indicate that expression of the beta(1C) integrin variant in prostate glands prevents cancer progression by up-regulation of TSP1 levels and inhibition of angiogenesis.

  2. Ablation of beta1 integrin in mammary epithelium reveals a key role for integrin in glandular morphogenesis and differentiation.

    PubMed

    Naylor, Matthew J; Li, Na; Cheung, Julia; Lowe, Emma T; Lambert, Elise; Marlow, Rebecca; Wang, Pengbo; Schatzmann, Franziska; Wintermantel, Timothy; Schüetz, Günther; Clarke, Alan R; Mueller, Ulrich; Hynes, Nancy E; Streuli, Charles H

    2005-11-21

    Integrin-mediated adhesion regulates the development and function of a range of tissues; however, little is known about its role in glandular epithelium. To assess the contribution of beta1 integrin, we conditionally deleted its gene in luminal epithelia during different stages of mouse mammary gland development and in cultured primary mammary epithelia. Loss of beta1 integrin in vivo resulted in impaired alveologenesis and lactation. Cultured beta1 integrin-null cells displayed abnormal focal adhesion function and signal transduction and could not form or maintain polarized acini. In vivo, epithelial cells became detached from the extracellular matrix but remained associated with each other and did not undergo overt apoptosis. beta1 integrin-null mammary epithelial cells did not differentiate in response to prolactin stimulation because of defective Stat5 activation. In mice where beta1 integrin was deleted after the initiation of differentiation, fewer defects in alveolar morphology occurred, yet major deficiencies were also observed in milk protein and milk fat production and Stat5 activation, indicating a permissive role for beta1 integrins in prolactin signaling. This study demonstrates that beta1 integrin is critical for the alveolar morphogenesis of a glandular epithelium and for maintenance of its differentiated function. Moreover, it provides genetic evidence for the cooperation between integrin and cytokine signaling pathways.

  3. Coordination of chondrocyte differentiation and joint formation by alpha5beta1 integrin in the developing appendicular skeleton.

    PubMed

    Garciadiego-Cázares, David; Rosales, Carlos; Katoh, Masaru; Chimal-Monroy, Jesús

    2004-10-01

    The control point by which chondrocytes take the decision between the cartilage differentiation program or the joint formation program is unknown. Here, we have investigated the effect of alpha5beta1 integrin inhibitors and bone morphogenetic protein (BMP) on joint formation. Blocking of alpha5beta1 integrin by specific antibodies or RGD peptide (arginine-glycine-aspartic acid) induced inhibition of pre-hypertrophic chondrocyte differentiation and ectopic joint formation between proliferating chondrocytes and hypertrophic chondrocytes. Ectopic joint expressed Wnt14, Gdf5, chordin, autotaxin, type I collagen and CD44, while expression of Indian hedgehog and type II collagen was downregulated in cartilage. Expression of these interzone markers confirmed that the new structure is a new joint being formed. In the presence of BMP7, inhibition of alpha5beta1 integrin function still induced the formation of the ectopic joint between proliferating chondrocytes and hypertrophic chondrocytes. By contrast, misexpression of alpha5beta1 integrin resulted in fusion of joints and formation of pre-hypertrophic chondrocytes. These facts indicate that the decision of which cell fate to make pre-joint or pre-hypertrophic is made on the basis of the presence or absence of alpha5beta1 integrin on chondrocytes.

  4. Differential expression of beta 1 integrins in nonneoplastic smooth and striated muscle cells and in tumors derived from these cells.

    PubMed Central

    Mechtersheimer, G.; Barth, T.; Quentmeier, A.; Möller, P.

    1994-01-01

    Integrins are a superfamily of transmembrane alpha beta heterodimers that play an important role in cell-matrix and cell-cell interactions by acting as receptors for extracellular matrix proteins and for cell adhesion molecules. Using monoclonal antibodies against beta 1, alpha 1 to alpha 6, and alpha v subunits, the in situ distribution pattern of beta 1 integrins was examined immunohistochemically in nonneoplastic smooth and striated muscle cells and in their tumors. Nonneoplastic smooth muscle cells were beta 1+, alpha 1+, alpha 3+, alpha v+ and, in diverse localizations, also alpha 5+ or even alpha 6+. The expression of the beta 1 chain was conserved in all leiomyomas and leiomyosarcomas. The distribution pattern of the alpha subunits by contrast underwent several changes during malignant transformation of smooth muscle cells. These alterations consisted in a neoexpression of alpha 2, alpha 4, and alpha 6 as well as in an abnormal abrogation of alpha 1 and alpha 3 in some leiomyosarcomas. Except for the absence of alpha 5 in the majority of epithelioid leiomyosarcomas, expression of the alpha 5 and alpha v subunits was mainly conserved. In addition, tumors with epithelioid differentiation differed from typical cases by the absence of alpha 1 and the simultaneous presence of alpha 4. Adult striated muscle cells were beta 1+ but alpha 1- to alpha 6- and alpha v-, whereas fetal striated muscle cells were not only beta 1+ but also alpha 3+/-, alpha 4+/-, alpha 5+ and alpha 6+. In all rhabdomyosarcomas the expression of beta 1 was retained. Furthermore, the majority of cases showed the expression of one or more alpha subunits most of which, ie, alpha 4, alpha 5, and alpha 6, were also found in fetal striated muscle cells. In conclusion, beta 1 integrins exhibited a differential expression pattern along the two lines of myogenic differentiation. This integrin profile underwent characteristic changes during malignant transformation. Nevertheless, the compiled

  5. Beta-1 integrin is important for the structural maintenance and homeostasis of differentiating fiber cells

    PubMed Central

    Scheiblin, David A.; Gao, Junyuan; Caplan, Jeffrey L.; Simirskii, Vladimir N.; Czymmek, Kirk J.; Mathias, Richard T.; Duncan, Melinda K.

    2014-01-01

    β1-integrin is a heterodimeric transmembrane protein that has roles in both cell-extracellular matrix and cell-cell interactions. Conditional deletion of β1-integrin from all lens cells during embryonic development results in profound lens defects, however, it is less clear whether this reflects functions in the lens epithelium alone or whether this protein plays a role in lens fibers. Thus, a conditional approach was used to delete β1-integrin solely from the lens fiber cells. This deletion resulted in two distinct phenotypes with some lenses exhibiting cataracts while others were clear, albeit with refractive defects. Analysis of “clear” conditional knockout lenses revealed that they had profound defects in fiber cell morphology associated with the loss of the F-actin network. Physiological measurements found that the lens fiber cells had a two-fold increase in gap junctional coupling, perhaps due to differential localization of connexins 46 and 50, as well as increased water permeability. This would presumably facilitate transport of ions and nutrients through the lens, and may partially explain how lenses with profound structural abnormalities can maintain transparency. In summary, β1-integrin plays a role in maintaining the cellular morphology and homeostasis of the lens fiber cells. PMID:24607497

  6. Distinct ErbB2 receptor populations differentially interact with beta1 integrin in breast cancer cell models

    PubMed Central

    Toscani, Andrés Martín; Sampayo, Rocío G.; Barabas, Federico Martín; Fuentes, Federico; Simian, Marina

    2017-01-01

    ErbB2 is a member of the ErbB family of tyrosine kinase receptors that plays a major role in breast cancer progression. Located at the plasma membrane, ErbB2 forms large clusters in spite of the presence of growth factors. Beta1 integrin, membrane receptor of extracellular matrix proteins, regulates adhesion, migration and invasiveness of breast cancer cells. Physical interaction between beta1 integrin and ErbB2 has been suggested although published data are contradictory. The aim of the present work was to study the interaction between ErbB2 and beta1 integrin in different scenarios of expression and activation. We determined that beta1 integrin and ErbB2 colocalization is dependent on the expression level of both receptors exclusively in adherent cells. In suspension cells, lack of focal adhesions leave integrins free to diffuse on the plasma membrane and interact with ErbB2 even at low expression levels of both receptors. In adherent cells, high expression of beta1 integrin leaves unbound receptors outside focal complexes that diffuse within the plasma membrane and interact with ErbB2 membrane domains. Superresolution imaging showed the existence of two distinct populations of ErbB2: a major population located in large clusters and a minor population outside these structures. Upon ErbB2 overexpression, receptors outside large clusters can freely diffuse at the membrane and interact with integrins. These results reveal how expression levels of beta1 integrin and ErbB2 determine their frequency of colocalization and show that extracellular matrix proteins shape membrane clusters distribution, regulating ErbB2 and beta1 integrin activity in breast cancer cells. PMID:28306722

  7. Distinct ErbB2 receptor populations differentially interact with beta1 integrin in breast cancer cell models.

    PubMed

    Toscani, Andrés Martín; Sampayo, Rocío G; Barabas, Federico Martín; Fuentes, Federico; Simian, Marina; Coluccio Leskow, Federico

    2017-01-01

    ErbB2 is a member of the ErbB family of tyrosine kinase receptors that plays a major role in breast cancer progression. Located at the plasma membrane, ErbB2 forms large clusters in spite of the presence of growth factors. Beta1 integrin, membrane receptor of extracellular matrix proteins, regulates adhesion, migration and invasiveness of breast cancer cells. Physical interaction between beta1 integrin and ErbB2 has been suggested although published data are contradictory. The aim of the present work was to study the interaction between ErbB2 and beta1 integrin in different scenarios of expression and activation. We determined that beta1 integrin and ErbB2 colocalization is dependent on the expression level of both receptors exclusively in adherent cells. In suspension cells, lack of focal adhesions leave integrins free to diffuse on the plasma membrane and interact with ErbB2 even at low expression levels of both receptors. In adherent cells, high expression of beta1 integrin leaves unbound receptors outside focal complexes that diffuse within the plasma membrane and interact with ErbB2 membrane domains. Superresolution imaging showed the existence of two distinct populations of ErbB2: a major population located in large clusters and a minor population outside these structures. Upon ErbB2 overexpression, receptors outside large clusters can freely diffuse at the membrane and interact with integrins. These results reveal how expression levels of beta1 integrin and ErbB2 determine their frequency of colocalization and show that extracellular matrix proteins shape membrane clusters distribution, regulating ErbB2 and beta1 integrin activity in breast cancer cells.

  8. Cytoplasmic tails of beta 1, beta 2, and beta 7 integrins differentially regulate LFA-1 function in K562 cells.

    PubMed Central

    Lub, M; van Vliet, S J; Oomen, S P; Pieters, R A; Robinson, M; Figdor, C G; van Kooyk, Y

    1997-01-01

    The beta 2 integrin lymphocyte function-associated antigen 1 (LFA-1) mediates activation-dependent adhesion of lymphocytes. To investigate whether lymphocyte-specific elements are essential for LFA-1 function, we expressed LFA-1 in the erythroleukemic cell line K562, which expresses only the integrin very late antigen 5. We observed that LFA-1-expressing K562 cannot bind to intercellular adhesion molecule 1-coated surfaces when stimulated by phorbol 12-myristate 13-acetate (PMA), whereas the LFA-1-activating antibody KIM185 markedly enhanced adhesion. Because the endogenously expressed beta 1 integrin very late antigen 5 is readily activated by PMA, we investigated the role of the cytoplasmic domain of distinct beta subunits in regulating LFA-1 function. Transfection of chimeric LFA-1 receptors in K562 cells reveals that replacement of the beta 2 cytoplasmic tail with the beta 1 but not the beta 7 cytoplasmic tail completely restores PMA responsiveness of LFA-1, whereas a beta 2 cytoplasmic deletion mutant of LFA-1 is constitutively active. Both deletion of the beta 2 cytoplasmic tail or replacement by the beta 1 cytoplasmic tail alters the localization of LFA-1 into clusters, thereby regulating LFA-1 activation and LFA-1-mediated adhesion to intercellular adhesion molecule 1. These data demonstrate that distinct signaling routes activate beta 1 and beta 2 integrins through the beta-chain and hint at the involvement of lymphocyte-specific signal transduction elements in beta 2 and beta 7 integrin activation that are absent in the nonlymphocytic cell line K562. Images PMID:9247650

  9. Muscle beta1D integrin reinforces the cytoskeleton-matrix link: modulation of integrin adhesive function by alternative splicing.

    PubMed

    Belkin, A M; Retta, S F; Pletjushkina, O Y; Balzac, F; Silengo, L; Fassler, R; Koteliansky, V E; Burridge, K; Tarone, G

    1997-12-15

    Expression of muscle-specific beta1D integrin with an alternatively spliced cytoplasmic domain in CHO and GD25, beta1 integrin-minus cells leads to their phenotypic conversion. beta1D-transfected nonmuscle cells display rounded morphology, lack of pseudopodial activity, retarded spreading, reduced migration, and significantly enhanced contractility compared with their beta1A-expressing counterparts. The transfected beta1D is targeted to focal adhesions and efficiently displaces the endogenous beta1A and alphavbeta3 integrins from the sites of cell-matrix contact. This displacement is observed on several types of extracellular matrix substrata and leads to elevated stability of focal adhesions in beta1D transfectants. Whereas a significant part of cellular beta1A integrin is extractable in digitonin, the majority of the transfected beta1D is digitonin-insoluble and is strongly associated with the detergent-insoluble cytoskeleton. Increased interaction of beta1D integrin with the actin cytoskeleton is consistent with and might be mediated by its enhanced binding to talin. In contrast, beta1A interacts more strongly with alpha-actinin, than beta1D. Inside-out driven activation of the beta1D ectodomain increases ligand binding and fibronectin matrix assembly by beta1D transfectants. Phenotypic effects of beta1D integrin expression in nonmuscle cells are due to its enhanced interactions with both cytoskeletal and extracellular ligands. They parallel the transitions that muscle cells undergo during differentiation. Modulation of beta1 integrin adhesive function by alternative splicing serves as a physiological mechanism reinforcing the cytoskeleton- matrix link in muscle cells. This reflects the major role for beta1D integrin in muscle, where extremely stable association is required for contraction.

  10. beta1-integrin cytoplasmic subdomains involved in dominant negative function.

    PubMed

    Retta, S F; Balzac, F; Ferraris, P; Belkin, A M; Fässler, R; Humphries, M J; De Leo, G; Silengo, L; Tarone, G

    1998-04-01

    The beta1-integrin cytoplasmic domain consists of a membrane proximal subdomain common to the four known isoforms ("common" region) and a distal subdomain specific for each isoform ("variable" region). To investigate in detail the role of these subdomains in integrin-dependent cellular functions, we used beta1A and beta1B isoforms as well as four mutants lacking the entire cytoplasmic domain (beta1TR), the variable region (beta1COM), or the common region (beta1 deltaCOM-B and beta1 deltaCOM-A). By expressing these constructs in Chinese hamster ovary and beta1 integrin-deficient GD25 cells (Wennerberg et al., J Cell Biol 132, 227-238, 1996), we show that beta1B, beta1COM, beta1 deltaCOM-B, and beta1 deltaCOM-A molecules are unable to support efficient cell adhesion to matrix proteins. On exposure to Mn++ ions, however, beta1B, but none of the mutants, can mediate cell adhesion, indicating specific functional properties of this isoform. Analysis of adhesive functions of transfected cells shows that beta1B interferes in a dominant negative manner with beta1A and beta3/beta5 integrins in cell spreading, focal adhesion formation, focal adhesion kinase tyrosine phosphorylation, and fibronectin matrix assembly. None of the beta1 mutants tested shows this property, indicating that the dominant negative effect depends on the specific combination of common and B subdomains, rather than from the absence of the A subdomain in the beta1B isoform.

  11. The involvement of Gab1 and PI 3-kinase in {beta}1 integrin signaling in keratinocytes

    SciTech Connect

    Kuwano, Yoshihiro; Fujimoto, Manabu . E-mail: fujimoto-m@umin.ac.jp; Watanabe, Rei; Ishiura, Nobuko; Nakashima, Hiroko; Komine, Mayumi; Hamazaki, Tatsuo S.; Tamaki, Kunihiko; Okochi, Hitoshi

    2007-09-14

    The control of the stem cell compartment in epidermis is closely linked to the regulation of keratinocyte proliferation and differentiation. {beta}1 integrins are expressed 2-fold higher by stem cells than transit-amplifying cells. Signaling from these {beta}1 integrins is critical for the regulation of the epidermal stem cell compartment. To clarify the functional relevance of this differential expression of {beta}1 integrins, we established HaCaT cells with high {beta}1integrin expression by repeated flow cytometric sorting of this population from the parental cell line. In these obtained cells expressing {beta}1 integrins by 5-fold, MAPK activation was markedly increased. Regarding the upstream of MAPK, Gab1 phosphorylation was also higher with high {beta}1 integrin expression, while Shc phosphorylation was not altered. In addition, enhanced phosphatidylinositol 3-kinase activation was also observed. These observations suggest that Gab1 and phosphatidylinositol 3-kinase play pivotal roles in the {beta}1 integrin-mediated regulation of the epidermal stem cell compartment.

  12. The muscle-specific laminin receptor alpha7 beta1 integrin negatively regulates alpha5 beta1 fibronectin receptor function.

    PubMed

    Tomatis, D; Echtermayer, F; Schöber, S; Balzac, F; Retta, S F; Silengo, L; Tarone, G

    1999-02-01

    alpha7 beta1 is the major integrin complex expressed in differentiated muscle cells where it functions as a laminin receptor. In this work we have expressed the alpha7 integrin subunit in CHO cells to investigate the functional properties of this receptor. After transfection with alpha7 CHO cells acquired the ability to adhere and spread on laminin 1 consistent with the laminin receptor activity of the alpha7 beta1. alpha7 transfectants, however, showed a 70% reduction in the ability to adhere to fibronectin and were unable to assemble a fibronectin matrix. The degree of reduction was inversely related to the level of alpha7 expression. To define the mechanisms underlying this adhesive defect we analyzed surface expression and functional properties of the alpha5 beta1 fibronectin receptor. Although cell surface expression of alpha5 beta1 was reduced by a factor of 20-25% in alpha7 transfectants compared to control untransfected cells, this slight reduction was not sufficient to explain the dramatic reduction in cell adhesion (70%) and matrix assembly (close to 100%). Binding studies showed that the affinity of 125I-fibronectin for its surface receptor was decreased by 50% in alpha7 transfectants, indicating that the alpha5 beta1 integrin is partially inactivated in these cells. Inactivation can be reversed by Mn2+, a cation known to increase integrin affinity for their ligands. In fact, incubation of cells with Mn2+ restored fibronectin binding affinity, adhesion to fibronectin, and assembly of fibronectin matrix in alpha7 transfectants. These data indicate that alpha7 expression leads to the functional down regulation of alpha5beta1 integrin by decreasing ligand binding affinity and surface expression. In conclusion, the data reported establish the existence of a negative cooperativity between alpha7 and alpha5 integrins that may be important in determining functional regulation of integrins during myogenic differentiation.

  13. Integrin alpha v beta 3 differentially regulates adhesive and phagocytic functions of the fibronectin receptor alpha 5 beta 1

    PubMed Central

    1994-01-01

    The plasma protein fibronectin is an important opsonin in wound repair and host defense. To better understand the process of fibronectin- mediated phagocytosis, we have transfected K562 cells, which endogenously express alpha 5 beta 1, with alpha v beta 3. In these transfectants, antibodies to alpha v beta 3 block phagocytosis of fibronectin-opsonized beads completely, even though half the ingestion occurs through endogenous alpha 5 beta 1 receptors. alpha 5 beta 1- mediated adhesion to fibronectin-coated surfaces is unaffected by alpha v beta 3 ligation. Neither alpha v beta 5 nor alpha M beta 2 ligation affects alpha 5 beta 1 phagocytic function in transfectants expressing these receptors. Pharmacologic data suggest that alpha v beta 3 ligation suppresses the phagocytic competence of high affinity alpha 5 beta 1 receptors through a signal transduction pathway, perhaps involving protein kinase C. In addition to its significance for phagocytosis, alpha v beta 3 regulation of alpha 5 beta 1 function may be significant for its roles in cell migration, metastasis, and angiogenesis. PMID:7525603

  14. Conditional deletion of beta1-integrin from the developing lens leads to loss of the lens epithelial phenotype.

    PubMed

    Simirskii, Vladimir N; Wang, Yan; Duncan, Melinda K

    2007-06-15

    Beta1-integrins are cell surface receptors that participate in sensing the cell's external environment. We used the Cre-lox system to delete beta1-integrin in all lens cells as the lens vesicle transitions into the lens. Adult mice lacking beta1-integrin in the lens are microphthalmic due to apoptosis of the lens epithelium and neonatal disintegration of the lens fibers. The first morphological alterations in beta1-integrin null lenses are seen at 16.5 dpc when the epithelium becomes disorganized and begins to upregulate the fiber cell markers beta- and gamma-crystallins, the transcription factors cMaf and Prox1 and downregulate Pax6 levels demonstrating that beta1-integrin is essential to maintain the lens epithelial phenotype. Furthermore, beta1-integrin null lens epithelial cells upregulate the expression of alpha-smooth muscle actin and nuclear Smad4 and downregulate Smad6 suggesting that beta1-integrin may brake TGFbeta family signaling leading to epithelial-mesenchymal transitions in the lens. In contrast, beta1-integrin null lens epithelial cells show increased E-cadherin immunoreactivity which supports the proposed role of beta1-integrins in mediating complete EMT in response to TGFbeta family members. Thus, beta1-integrin is required to maintain the lens epithelial phenotype and block inappropriate activation of some aspects of the lens fiber cell differentiation program.

  15. Physical association and functional interaction between beta1 integrin and CD98 on human T lymphocytes

    NASA Technical Reports Server (NTRS)

    Miyamoto, Yuko J.; Mitchell, Jason S.; McIntyre, Bradley W.

    2003-01-01

    CD98 is a cell surface protein previously characterized as a cell activation marker, an amino acid transporter, and has recently been implicated in integrin-related functions. Integrins are cell surface proteins, important for homotypic cell aggregation, cell adhesion, and coactivation of T lymphocytes. We have previously shown that the anti-CD98 mAb 80A10, when coimmobilized with anti-CD3 mAb OKT3, is able to mediate human T cell coactivation that is inhibited by anti-beta1 integrin specific mAb 18D3. These results indicated a functional association of CD98 and beta1 integrin signaling but left open the question of a physical association. We now show the induction of homotypic aggregation through CD98 among human T cells and this aggregation was inhibited by anti-beta1 integrin mAb. Therefore, CD98-dependent lymphocyte proliferation and adhesion may involve integrins. Competitive binding assays and fluorescence colocalization analysis suggested that CD98 and beta1 integrin were physically associated. Differential extraction techniques and immunoprecipitations provided the first evidence that the alpha4beta1 integrin and CD98 are specifically associated on human T lymphocytes.

  16. Physical association and functional interaction between beta1 integrin and CD98 on human T lymphocytes

    NASA Technical Reports Server (NTRS)

    Miyamoto, Yuko J.; Mitchell, Jason S.; McIntyre, Bradley W.

    2003-01-01

    CD98 is a cell surface protein previously characterized as a cell activation marker, an amino acid transporter, and has recently been implicated in integrin-related functions. Integrins are cell surface proteins, important for homotypic cell aggregation, cell adhesion, and coactivation of T lymphocytes. We have previously shown that the anti-CD98 mAb 80A10, when coimmobilized with anti-CD3 mAb OKT3, is able to mediate human T cell coactivation that is inhibited by anti-beta1 integrin specific mAb 18D3. These results indicated a functional association of CD98 and beta1 integrin signaling but left open the question of a physical association. We now show the induction of homotypic aggregation through CD98 among human T cells and this aggregation was inhibited by anti-beta1 integrin mAb. Therefore, CD98-dependent lymphocyte proliferation and adhesion may involve integrins. Competitive binding assays and fluorescence colocalization analysis suggested that CD98 and beta1 integrin were physically associated. Differential extraction techniques and immunoprecipitations provided the first evidence that the alpha4beta1 integrin and CD98 are specifically associated on human T lymphocytes.

  17. Simultaneous changes in the function and expression of beta 1 integrins during the growth arrest of poorly differentiated colorectal cells (LISP-1).

    PubMed

    Roela, R A; Brentani, M M; Katayama, M L H; Reis, M; Federico, M H H

    2003-08-01

    Cells usually lose adhesion and increase proliferation and migration during malignant transformation. Here, we studied how proliferation can affect the other two characteristics, which ultimately lead to invasion and metastasis. We determined the expression of beta 1 integrins, as well as adhesion and migration towards laminin-1, fibronectin, collagens type I and type IV presented by LISP-1 colorectal cancer cells exposed to 2.5% dimethyl sulfoxide (DMSO), an agent capable of decreasing proliferation in this poorly differentiated colorectal cell line. Untreated cells (control), as shown by flow cytometry and monoclonal antibodies, expressed alpha 2 (63.8 11.3% positive cells), alpha 3 (93.3 7.0%), alpha 5 (50.4 12.0%) and alpha 6 (34.1 4.9%) integrins but not alpha1, alpha 4, alpha v or 4. Cells adhered well to laminin-1 (73.4 6.0%) and fibronectin (40.0 2.0%) substrates but very little to collagens. By using blocking monoclonal antibodies, we showed that alpha 2, alpha 3 and alpha 6 mediated laminin-1 adhesion, but neither alpha 3 nor alpha 5 contributed to fibronectin adherence. DMSO arrested cells at G0/G1 (control: 55.0 2.4% vs DMSO: 70.7 2.5%) while simultaneously reducing alpha 5 (24.2 19%) and alpha 6 (14.3 10.8%) expression as well as c-myc mRNA (7-fold), the latter shown by Northern blotting. Although the adhesion rate did not change after exposure to DMSO, alpha 3 and alpha 5 played a major role in laminin-1 and fibronectin adhesion, respectively. Migration towards laminin-1, which was clearly increased upon exposure to DMSO (control: 6 2 cells vs DMSO: 64 6 cells), was blocked by an antibody against alpha 6. We conclude that the effects of DMSO on LISP-1 proliferation were accompanied by concurrent changes in the expression and function of integrins, consequently modulating adhesion/migration, and revealing a complex interplay between function/expression and the proliferative state of cells.

  18. Beta 1D integrin displaces the beta 1A isoform in striated muscles: localization at junctional structures and signaling potential in nonmuscle cells.

    PubMed

    Belkin, A M; Zhidkova, N I; Balzac, F; Altruda, F; Tomatis, D; Maier, A; Tarone, G; Koteliansky, V E; Burridge, K

    1996-01-01

    The cytoplasmic domains of integrins provide attachment of these extracellular matrix receptors to the cytoskeleton and play a critical role in integrin-mediated signal transduction. In this report we describe the identification, expression, localization, and initial functional characterization of a novel form of beta 1 integrin, termed beta 1D. This isoform contains a unique alternatively spliced cytoplasmic domain of 50 amino acids, with the last 24 amino acids encoded by an additional exon. Of these 24 amino acids, 11 are conserved when compared to the beta 1A isoform, but 13 are unique (Zhidkova, N. I., A. M. Belkin, and R. Mayne. 1995. Biochem. Biophys. Res. Commun. 214:279-285; van der Flier, A., I. Kuikman, C. Baudoin, R, van der Neuf, and A. Sonnenberg. 1995. FEBS Lett. 369:340-344). Using an anti-peptide antibody against the beta 1D integrin subunit, we demonstrated that the beta 1D isoform is synthesized only in skeletal and cardiac muscles, while very low amounts of beta 1A were detected by immunoblot in striated muscles. Whereas beta 1A could not be detected in adult skeletal muscle fibers and cardiomyocytes by immunofluorescence, beta 1D was localized to the sarcolemma of both cell types. In skeletal muscle, beta 1D was concentrated in costameres, myotendinous, and neuromuscular junctions. In cardiac muscle this beta 1 isoform was found in costamers and intercalated discs. beta 1D was associated with alpha 7A and alpha 7B in adult skeletal muscle. In cardiomyocytes of adult heart, alpha 7B was the major partner for the beta 1D isoform. beta 1D could not be detected in proliferating C2C12 myoblasts, but it appeared immediately after myoblast fusion and its amount continued to rise during myotube growth and maturation. In contrast, expression of the beta 1A isoform was downregulated during myodifferentiation in culture and it was completely displaced by beta 1D in mature differentiated myotubes. We also analyzed some functional properties of the beta 1D

  19. Regulation of cellular interactions with laminin by integrin cytoplasmic domains: the A and B structural variants of the alpha 6 beta 1 integrin differentially modulate the adhesive strength, morphology, and migration of macrophages.

    PubMed Central

    Shaw, L M; Mercurio, A M

    1994-01-01

    Several integrin alpha subunits have structural variants that are identical in their extracellular and transmembrane domains but that differ in their cytoplasmic domains. The functional significance of these variants, however, is unknown. In the present study, we examined the possibility that the A and B variants of the alpha 6 beta 1 integrin laminin receptor differ in function. For this purpose, we expressed the alpha 6A and alpha 6B cDNAs, as well as a truncated alpha 6 cDNA (alpha 6-delta CYT) in which the cytoplasmic domain sequence was deleted after the GFFKR pentapeptide, in P388D1 cells, an alpha 6 deficient macrophage cell line. Populations of stable alpha 6A, alpha 6B, and alpha 6-delta CYT transfectants that expressed equivalent levels of cell surface alpha 6 were obtained by fluorescence-activated cell sorter and shown to form heterodimers with endogenous beta 1 subunits. Upon attachment to laminin, the alpha 6A transfectants extended numerous pseudopodia. In contrast, the alpha 6B transfectants remained rounded and extended few processes. The transfectants were also examined for their ability to migrate toward a laminin substratum using Transwell chambers. The alpha 6A transfectants were three- to fourfold more migratory than the alpha 6B transfectants. The alpha 6-delta CYT transfectants did not attach to laminin in normal culture medium, but they did attach in the presence of Mn2+. The alpha 6-delta CYT transfectants migrated to a lesser extent than either the alpha 6A or alpha 6B transfectants in the presence of Mn2+. The alpha 6 transfectants differed significantly in the concentration of substratum bound laminin required for half-maximal adhesion in the presence of Mn2+:alpha 6A (2.1 micrograms/ml), alpha 6B (6.3 micrograms/ml), and alpha 6-delta CYT (8.8 micrograms/ml). Divalent cation titration studies revealed that these transfectants also differed significantly in both the [Ca2+] and [Mn2+] required to obtain half-maximal adhesion to laminin

  20. Beta1-integrins are critical for cerebellar granule cell precursor proliferation.

    PubMed

    Blaess, Sandra; Graus-Porta, Diana; Belvindrah, Richard; Radakovits, Randor; Pons, Sebastian; Littlewood-Evans, Amanda; Senften, Mathias; Guo, Huailian; Li, Yuqing; Miner, Jeffrey H; Reichardt, Louis F; Müller, Ulrich

    2004-03-31

    We have previously shown that mice with a CNS restricted knock-out of the integrin beta1 subunit gene (Itgb1-CNSko mice) have defects in the formation of lamina and folia in the cerebral and cerebellar cortices that are caused by disruption of the cortical marginal zones. Cortical structures in postnatal and adult Itgb1-CNSko animals are also reduced in size, but the mechanism that causes the size defect has remained unclear. We now demonstrate that proliferation of granule cell precursors (GCPs) is severely affected in the developing cerebellum of Itgb1-CNSko mice. In the absence of beta1 expression, GCPs lose contact with laminin in the meningeal basement membrane, cease proliferating, and differentiate prematurely. In vitro studies provide evidence that beta1 integrins act at least in part cell autonomously in GCPs to regulate their proliferation. Previous studies have shown that sonic hedgehog (Shh)-induced GCP proliferation is potentiated by the integrin ligand laminin. We show that Shh directly binds to laminin and that laminin-Shh induced cell proliferation is dependent on beta1 integrin expression in GCPs. Taken together, these data are consistent with a model in which beta1 integrin expression in GCPs is required to recruit a laminin-Shh complex to the surface of GCPs and to subsequently modulate the activity of signaling pathways that regulate proliferation.

  1. Integrin alpha3beta1 inhibits directional migration and wound re-epithelialization in the skin.

    PubMed

    Margadant, Coert; Raymond, Karine; Kreft, Maaike; Sachs, Norman; Janssen, Hans; Sonnenberg, Arnoud

    2009-01-15

    Re-epithelialization after skin wounding requires both migration and hyperproliferation of keratinocytes. Laminin-332 is deposited during migration over the provisional matrix. To investigate the function of the laminin-332 binding integrin alpha3beta1 in wound re-epithelialization, we generated Itga3flox/flox; K14-Cre mice lacking the alpha3 subunit specifically in the basal layer of the epidermis. These mice are viable but display several skin defects, including local inflammation, hair loss, basement membrane duplication and microblistering at the dermal-epidermal junction, whereas hemidesmosome assembly and keratinocyte differentiation are not impaired. Wound healing is slightly faster in the absence of integrin alpha3beta1, whereas proliferation, the distribution of other integrins and the deposition of basement membrane proteins in the wound bed are unaltered. In vitro, cell spreading is rescued by increased surface expression of alpha6beta1 integrin in the absence of integrin alpha3. The alpha3-deficient keratinocytes migrate with an increased velocity and persistence, whereas proliferation, growth factor signaling, hemidesmosome assembly, and laminin-332 deposition appeared to be normal. We suggest that integrin alpha3beta1 delays keratinocyte migration during wound re-epithelialization, by binding to the laminin-332 that is newly deposited on the wound bed.

  2. Role for beta1 integrins in cortical osteocytes during acute musculoskeletal disuse.

    PubMed

    Phillips, Jonathan A; Almeida, Eduardo A C; Hill, Esther L; Aguirre, J Ignacio; Rivera, Mercedes F; Nachbandi, Inaam; Wronski, Thomas J; van der Meulen, Marjolein C H; Globus, Ruth K

    2008-09-01

    The mammalian skeleton adjusts bone structure and strength in response to changes in mechanical loading, however the molecular and cellular mechanisms governing this process in vivo are unknown. Terminally differentiated osteoblasts, the osteocytes, are presumptive mechanosensory cells for bone, and cell culture studies demonstrate that beta1 integrins participate in mechanical signaling. To determine the role of beta1 integrins in osteoblasts in vivo, we used the Cre-lox system to delete beta1 integrin from cells committed to the osteoblast lineage. While pCol2.3 Cre-mediated recombination was widespread in bones from Colalpha1(I)-Cre+/beta1fl/fl conditional knockout mice (cKO), beta1 integrin protein was depleted from cortical osteocytes, but not from cancellous osteocytes or cells lining bone surfaces in adults. Bones from cKO mice that were normally loaded were similar in structure to WT littermates. However, hindlimb unloading of adult cKO mice for one week intended to cause bone loss (disuse osteopenia), resulted in unexpected, rapid changes in the geometry of cortical bone; hindlimb unloading increased the cross-sectional area, marrow area, and moments of inertia in cKO, but not WT mice. Furthermore, these hindlimb unloading-induced geometric changes in cortical bone of cKO mice resulted in increased whole bone bending stiffness and strength of the femur. Together, these results confirmed the concept that osteocytes are mechanosensory cells and showed beta1 integrins in cortical osteocytes limited changes in cortical geometry in response to disuse, thus providing the first in vivo evidence that beta1 integrins on osteocytes mediate specific aspects of mechanotransduction.

  3. Platelet collagen receptor integrin alpha2beta1 activation involves differential participation of ADP-receptor subtypes P2Y1 and P2Y12 but not intracellular calcium change.

    PubMed

    Jung, S M; Moroi, M

    2001-06-01

    In agonist-induced platelet activation, the collagen platelet receptor integrin alpha2beta1 is activated to high-affinity states through ADP involvement [Jung, S.M. & Moroi, M. (2000) J. Biol. Chem. 275, 8016-8026]. Here we determined the ADP-receptor subtypes involved and their relative contributions to alpha2beta1 activation (assessed by soluble-collagen binding) using the P2Y12 antagonist AR-C69931MX and P2Y1 antagonists adenosine 3',5'-diphosphate (Ado(3,5)PP) and adenosine 3'-phosphate 5'-phosphosulfate (AdoPPS). All three inhibited alpha2beta1 activation induced by low or high ADP, low thrombin, or low collagen-related peptide (CRP) concentrations; however, AR-C69931MX was markedly more inhibitory than the P2Y1 antagonists, suggesting the greater contribution of P2Y12. Inhibition patterns by various combinations of AR-C69931MX, AdoPPS, and wortmannin suggested that P2Y1 and P2Y12 mediate alpha2beta1 activation through different pathways, with possible involvement of phosphoinositide 3-kinase in both. Low concentrations of the acetoxy-methyl derivative of 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetra-acetic acid (calcium chelator) markedly decreased alpha2beta1 activation by low thrombin or CRP, but did not affect that by low or high ADP. Measurements of intracellular Ca2+ level (fluorimetric method) and alpha2beta1 activation (soluble-collagen binding) in the same platelet preparation indicated that alpha2beta1 activation via ADP receptors was independent of intracellular Ca2+ release. Our data indicate that integrin alpha2beta1 activation by ADP occurs through an inside-out signaling mechanism involving differential contributions by P2Y1 and P2Y12 wherein each contributes to some portion of the activation, with the stronger contribution of P2Y12. Furthermore, intracellular Ca2+ increase is not directly related to integrin alpha2beta1 activation, meaning that it is separate from the calcium mobilization pathways that these two ADP receptors are involved in.

  4. The NPIY motif in the integrin beta1 tail dictates the requirement for talin-1 in outside-in signaling.

    PubMed

    Nieves, Bethsaida; Jones, Christopher W; Ward, Rachel; Ohta, Yasutaka; Reverte, Carlos G; LaFlamme, Susan E

    2010-04-15

    GAP(DeltaGAP), which lacks GAP activity restores spreading in cells adhered by constitutively activated integrins containing the beta1A tail, but not by integrins containing the beta1D tail, which is known to bind poorly to FLNa. Together, these results suggest that the binding of talin-1 to the NPIY motif is required downstream of integrin activation to promote cell spreading by preventing the inappropriate recruitment of FLNa and FilGAP to the beta1A tail. Our studies emphasize the importance of understanding the mechanisms that regulate the differential binding FLNa and talin-1 to the beta1 tail downstream of integrin activation in promoting integrin function.

  5. Pivotal role for beta-1 integrin in neurovascular remodelling after ischemic stroke.

    PubMed

    Lathia, Justin D; Chigurupati, Srinivasulu; Thundyil, John; Selvaraj, Pradeep K; Mughal, Mohamed R; Woodruff, Trent M; Chan, Sic L; Karamyan, Vardan T; Mattson, Mark P; Arumugam, Thiruma V

    2010-01-01

    beta1 integrin is a cell surface molecule that is critical for endothelial cell adhesion, migration and survival during angiogenesis. In the present study we employed in vivo and in vitro models to elucidate the role of beta1 integrin in vascular remodelling and stroke outcomes. At 24 h after cerebral ischemia and reperfusion (I/R), the ischemic cortex (ipsilateral area) exhibited modest beta1 integrin immunoreactivity and a robust increase was observed at 72 h. Double-label immunohistochemical analysis for beta1 integrin with neuronal (NeuN), microglial (Iba-1), astrocyte (GFAP), progenitor cell (Ng2) and blood vessel (collagen 4) markers showed that beta1 integrin expression only localized to blood vessels. In vitro studies using cultured endothelial cells and a beta1 integrin blocking antibody confirmed that beta1 integrin is required for endothelial cell migration, proliferation and blood vessel formation. In vivo studies in the cerebral I/R model using the beta1 integrin blocking antibody further confirmed that beta1 integrin signaling is involved in vascular formation and recovery following ischemic stroke. Finally, we found that beta1 integrin is critically involved in functional deficits and survival after a stroke. These results suggest that beta1 integrin plays important roles in neurovascular remodelling and functional outcomes following stroke, and that targeting the beta1 integrin signalling may provide a novel strategy for modulating angiogenesis in ischemic stroke and other pathological conditions.

  6. Role of Integrin-Beta 1 in Polycystic Kidney Disease

    DTIC Science & Technology

    2011-04-01

    Role of Integrin-Beta 1 in Polycystic Kidney Disease PRINCIPAL INVESTIGATOR: Gabriele Luca Gusella, Ph.D...13. SUPPLEMENTARY NOTES 14. ABSTRACT Autosomal Dominant Polycystic Kidney Disease (ADPKD) is caused by the dysregulation of the PKD1 or PKD2...characterized a novel cell line from human loop of Henle epithelium that can serve as a unique model to study medullary cystic kidney disease -2 (MCKD2) and

  7. Tissue inhibitor of metalloproteinase-2 (TIMP-2) regulates myogenesis and {beta}1 integrin expression in vitro

    SciTech Connect

    Lluri, Gentian; Langlois, Garret D.; Soloway, Paul D.; Jaworski, Diane M.

    2008-01-01

    Myogenesis in vitro involves myoblast cell cycle arrest, migration, and fusion to form multinucleated myotubes. Extracellular matrix (ECM) integrity during these processes is maintained by the opposing actions of matrix metalloproteinase (MMP) proteases and their inhibitors, the tissue inhibitor of metalloproteinases (TIMPs). Here, we report that TIMP-2, MMP-2, and MT1-MMP are differentially expressed during mouse myoblast differentiation in vitro. A specific role for TIMP-2 in myogenesis is demonstrated by altered TIMP-2{sup -/-} myotube formation. When differentiated in horse serum-containing medium, TIMP-2{sup -/-} myotubes are larger than wild-type myotubes. In contrast, when serum-free medium is used, TIMP-2{sup -/-} myotubes are smaller than wild-type myotubes. Regardless of culture condition, myotube size is directly correlated with MMP activity and inversely correlated with {beta}1 integrin expression. Treatment with recombinant TIMP-2 rescues reduced TIMP-2{sup -/-} myotube size and induces increased MMP-9 activation and decreased {beta}1 integrin expression. Treatment with either MMP-2 or MMP-9 similarly rescues reduced myotube size, but has no effect on {beta}1 integrin expression. These data suggest a specific regulatory relationship between TIMP-2 and {beta}1 integrin during myogenesis. Elucidating the role of TIMP-2 in myogenesis in vitro may lead to new therapeutic options for the use of TIMP-2 in myopathies and muscular dystrophies in vivo.

  8. Beta 1-integrin ligation and TLR ligation enhance GM-CSF–induced ALDH1A2 expression in dendritic cells, but differentially regulate their anti-inflammatory properties

    PubMed Central

    Yokota-Nakatsuma, Aya; Ohoka, Yoshiharu; Takeuchi, Hajime; Song, Si-Young; Iwata, Makoto

    2016-01-01

    Retinoic acid (RA)–producing CD103+ mature dendritic cells (DCs) in mesenteric lymph nodes (MLNs) play crucial roles in gut immunity. GM-CSF and RA contribute to the expression of the RA-producing enzyme ALDH1A2. However, additional signals appeared to be required for inducing ALDH1A2high mature DCs from immature DCs. We found here that TLR ligands (Ls) and immobilized E-cadherin could provide such signals in FLT3-L–generated bone marrow (BM)–derived DCs after treatment with GM-CSF and the RA receptor agonist Am80. The TLR-L-treated DCs produced proinflammatory cytokines unlike normal ALDH1A2high MLN-DCs, whereas the E-cadherin-treated DCs did not. Immobilized VCAM-1 and semaphorin 7 A exerted effects similar to those of E-cadherin. Soluble anti-integrin β1 antibodies or inhibitors of integrin signaling molecules suppressed the effects of these immobilized proteins, whereas immobilized anti-integrin β1 antibodies enhanced the GM-CSF/Am80-induced ALDH1A2 expression without inducing proinflammatory cytokines. Sequential stimulation of splenic pre-DCs with GM-CSF/Am80 and immobilized E-cadherin or anti-integrin β1 antibody also induced differentiation to mature DCs with high ALDH activity. The E-cadherin-treated BM-DCs induced gut-tropic Foxp3+ T cells and alleviated DSS–induced colitis, whereas the TLR-L-treated DCs aggravated DSS–induced colitis. The results suggest that integrin β1-mediated signals contribute to the differentiation and maturation of RA-producing anti-inflammatory DCs. PMID:27897208

  9. Alpha4beta1 integrin and erythropoietin mediate temporally distinct steps in erythropoiesis: integrins in red cell development.

    PubMed

    Eshghi, Shawdee; Vogelezang, Mariette G; Hynes, Richard O; Griffith, Linda G; Lodish, Harvey F

    2007-06-04

    Erythropoietin (Epo) is essential for the terminal proliferation and differentiation of erythroid progenitor cells. Fibronectin is an important part of the erythroid niche, but its precise role in erythropoiesis is unknown. By culturing fetal liver erythroid progenitors, we show that fibronectin and Epo regulate erythroid proliferation in temporally distinct steps: an early Epo-dependent phase is followed by a fibronectin-dependent phase. In each phase, Epo and fibronectin promote expansion by preventing apoptosis partly through bcl-xL. We show that alpha(4), alpha(5), and beta(1) are the principal integrins expressed on erythroid progenitors; their down-regulation during erythropoiesis parallels the loss of cell adhesion to fibronectin. Culturing erythroid progenitors on recombinant fibronectin fragments revealed that only substrates that engage alpha(4)beta(1)-integrin support normal proliferation. Collectively, these data suggest a two-phase model for growth factor and extracellular matrix regulation of erythropoiesis, with an early Epo-dependent, integrin-independent phase followed by an Epo-independent, alpha(4)beta(1)-integrin-dependent phase.

  10. Beta-1 Integrin Signaling and Function in MLO-Y4 Osteocyte-Like Cells

    NASA Technical Reports Server (NTRS)

    Searby, N. D.; vanderMeulen, M. C. H.; Dovi, J.; Roden, C.; Banerjee, I.; Kim, J.-B.; Damsky, C. D.; Almeida, E. A. C.; Globus, R. K.

    2004-01-01

    In osteocyte-like cells, disruption of beta-1 integrin signaling by the Beta-1 tail construct: 1) Altered cell morphology; 2) Reduced cell motility; 3) Increased proliferation and final cell density; 4) Reduced cell's ability to maintain shape when subjected to uniaxial strain (1%, 30 min). Thus, beta-1 integrin is important in the response of osteocytic cells to mechanical loading.

  11. Beta-1 Integrin Signaling and Function in MLO-Y4 Osteocyte-Like Cells

    NASA Technical Reports Server (NTRS)

    Searby, N. D.; vanderMeulen, M. C. H.; Dovi, J.; Roden, C.; Banerjee, I.; Kim, J.-B.; Damsky, C. D.; Almeida, E. A. C.; Globus, R. K.

    2004-01-01

    In osteocyte-like cells, disruption of beta-1 integrin signaling by the Beta-1 tail construct: 1) Altered cell morphology; 2) Reduced cell motility; 3) Increased proliferation and final cell density; 4) Reduced cell's ability to maintain shape when subjected to uniaxial strain (1%, 30 min). Thus, beta-1 integrin is important in the response of osteocytic cells to mechanical loading.

  12. alpha2beta1 integrin controls association of Rac with the membrane and triggers quiescence of endothelial cells.

    PubMed

    Cailleteau, Laurence; Estrach, Soline; Thyss, Raphael; Boyer, Laurent; Doye, Anne; Domange, Barbara; Johnsson, Nils; Rubinstein, Eric; Boucheix, Claude; Ebrahimian, Teni; Silvestre, Jean-Sebastien; Lemichez, Emmanuel; Meneguzzi, Guerrino; Mettouchi, Amel

    2010-07-15

    Integrin receptors and their extracellular matrix ligands provide cues to cell proliferation, survival, differentiation and migration. Here, we show that alpha2beta1 integrin, when ligated to the basement membrane component laminin-1, triggers a proliferation arrest in primary endothelial cells. Indeed, in the presence of strong growth signals supplied by growth factors and fibronectin, alpha2beta1 engagement alters assembly of mature focal adhesions by alpha5beta1 and leads to impairment of downstream signaling and cell-cycle arrest in the G1 phase. Although the capacity of alpha5beta1 to signal for GTP loading of Rac is preserved, the joint engagement of alpha2beta1 interferes with membrane anchorage of Rac. Adapting the 'split-ubiquitin' sensor to screen for membrane-proximal alpha2 integrin partners, we identified the CD9 tetraspanin and further establish its requirement for destabilization of focal adhesions, control of Rac subcellular localization and growth arrest induced by alpha2beta1 integrin. Altogether, our data establish that alpha2beta1 integrin controls endothelial cell commitment towards quiescence by triggering a CD9-dependent dominant signaling.

  13. Constitutive activation of integrin alpha 4 beta 1 defines a unique stage of human thymocyte development

    PubMed Central

    1994-01-01

    Our understanding of thymocyte development and of the positive and negative selection events involved in shaping the repertoire of mature T lymphocytes has been greatly facilitated by the use of transgenic and gene knockout animals. Much less is known about the factors that control the homing and population of the thymus by T cell precursors and the subsequent migration of developing thymocytes through the thymic architecture. As the integrins represent a candidate group of cell surface receptors that may regulate thymocyte development, we have analyzed the expression and function of alpha 4 beta 1 and alpha 5 beta 1 on human thymocytes. A major portion of double positive (CD4+ CD8+) human thymocytes express alpha 4 beta 1 in a constitutively active form and adhere to fibronectin and vascular cell adhesion molecule 1. alpha 4 beta 1 expression is similar on adherent and nonadherent populations, thus, activity reflects the receptor state and not simple expression. The adherent cells are immature, expressing high levels of CD4/CD8 and low levels of CD3 and CD69. In contrast, nonadherent cells possess the phenotype of thymocytes after positive selection, expressing intermediate levels of CD4 and/or CD8 and high levels of CD3 and CD69. The adherent population fails to respond to activation with anti-CD3 and fibronectin, whereas nonadherents exhibit an alpha 5 beta 1- dependent proliferation. Differential regulation of alpha 4 beta 1 and alpha 5 beta 1 receptors may provide a mechanism controlling cellular traffic, differentiation, and positive selection of thymocytes. PMID:8163937

  14. Alpha6beta1 integrin expressed by sperm is determinant in mouse fertilization

    PubMed Central

    Barraud-Lange, Virginie; Naud-Barriant, Nathalie; Saffar, Line; Gattegno, Liliane; Ducot, Beatrice; Drillet, Anne-Sophie; Bomsel, Morgane; Wolf, Jean-Philippe; Ziyyat, Ahmed

    2007-01-01

    Background Based on inhibition tests, the alpha6beta1 integrin was suggested to be a sperm receptor, but further experiments using gene deletion techniques have shown that neither oocyte alpha6, nor beta1 integrin subunits were essential for mouse fertilization. Results Using Western blot analysis and immunofluorescence, we showed that the mouse sperm expresses the alpha6beta1 integrin. As for oocyte, binding of GoH3 anti-alpha6 antibody to sperm induces a specific inhibition of sperm fertilizing ability. Comparing zona-intact and zona-free eggs in fusion tests, we showed that the removal of the zona pellucida by acid treatment bypasses fertilizing oocyte alpha6beta1 integrin's function in the adhesion/fusion process. Conclusion These findings show that alpha6beta1 integrin is expressed by both gametes and is functional in their membranes interaction. These results and previous reports, about fertilization of alpha6 or beta1 integrin subunits deleted oocytes by wild type sperm, suggest that the presence of alpha6beta1 integrin on one of the two gamete membranes can rescue the fertilization process. This hypothesis is further supported by the exchange of membrane fragments occurring between gametes prior to fusion that we recently reported. PMID:17850654

  15. SHARPIN is an endogenous inhibitor of beta1-integrin activation

    PubMed Central

    Rantala, Juha K.; Pouwels, Jeroen; Pellinen, Teijo; Veltel, Stefan; Laasola, Petra; Potter, Christopher S.; Duffy, Ted; Sundberg, John P.; Kallioniemi, Olli; Askari, Janet A.; Humphries, Martin; Parsons, Maddy; Salmi, Marko; Ivaska, Johanna

    2012-01-01

    Regulated activation of integrins is critical for cell adhesion, motility and tissue homeostasis. Talin and Kindlins activate β1-integrins, but the counteracting inhibiting mechanisms are poorly defined. Here we identified SHARPIN as an important inactivator of β1-integrins in an RNAi-screen. SHARPIN inhibited β1-integrin functions in human cancer cells and primary leukocytes. Fibroblasts, leukocytes and keratinocytes from SHARPIN-deficient mice exhibited increased β1-integrin activity which was fully rescued by re-expression of SHARPIN. SHARPIN directly bound to a conserved cytoplasmic region of integrin α-subunits and inhibited recruitment of Talin and Kindlin to the integrin. Therefore, SHARPIN inhibits the critical switching of β1-integrins from inactive to active conformations. PMID:21947080

  16. Altered localization and cytoplasmic domain-binding properties of tyrosine-phosphorylated beta 1 integrin

    PubMed Central

    1994-01-01

    We describe a novel approach to study tyrosine-phosphorylated (PY) integrins in cells transformed by virally encoded tyrosine kinases. We have synthesized a peptide (PY beta 1 peptide) that represents a portion of the cytoplasmic domain of the beta 1 integrin subunit and is phosphorylated on the tyrosine residue known to be the target of oncogenic tyrosine kinases. Antibodies prepared against the PY beta 1 peptide, after removal of cross-reacting antibodies by absorption and affinity purification, recognized the PY beta 1 peptide and the tyrosine-phosphorylated form of the intact beta 1 subunit, but did not bind the nonphosphorylated beta 1 peptide, the nonphosphorylated beta 1 subunit or other unrelated tyrosine-phosphorylated proteins. The anti- PY beta 1 antibodies labeled the podosomes of Rous sarcoma virus- transformed fibroblasts, but did not detectably stain nontransformed fibroblasts. The localization of the tyrosine phosphorylated beta 1 subunits appeared distinct from that of the beta 1 subunit. Adhesion plaques were stained by the anti-beta 1 subunit antibodies in Rous sarcoma virus-transformed fibroblasts plated on fibronectin, whereas neither podosomes nor adhesion plaques were labeled on vitronectin or on uncoated plates. Anti-phosphotyrosine antibodies labeled podosomes, adhesion plaques and cell-cell boundaries regardless of the substratum. One of the SH2 domains of the p85 subunit of phosphatidylinositol-3- kinase bound to the PY beta 1 peptide, but not to the non- phosphorylated beta 1 cytoplasmic peptide. Other SH2 domains did not bind to the PY beta 1 peptide. These results show that the phosphorylated form of the beta 1 integrin subunit is detected in a different subcellular localization than the nonphosphorylated form and suggest that the phosphorylation on tyrosine of the beta 1 subunit cytoplasmic domain may affect cellular signaling pathways. PMID:7520449

  17. Uniform overexpression and rapid accessibility of alpha5beta1 integrin on blood vessels in tumors.

    PubMed

    Parsons-Wingerter, Patricia; Kasman, Ian M; Norberg, Scott; Magnussen, Anette; Zanivan, Sara; Rissone, Alberto; Baluk, Peter; Favre, Cecile J; Jeffry, Ursula; Murray, Richard; McDonald, Donald M

    2005-07-01

    Integrin alpha5beta1 is among the proteins overexpressed on tumor vessels and is a potential target for diagnostics and therapeutics. Here, we mapped the distribution of alpha5beta1 integrin in three murine tumor models and identified sites of expression that are rapidly accessible to intravascular antibodies. When examined by conventional immunohistochemistry, alpha5beta1 integrin expression was strong on most blood vessels in RIP-Tag2 transgenic mouse tumors, adenomatous polyposis coli (apc) mouse adenomas, and implanted MCa-IV mammary carcinomas. Expression increased during malignant progression in RIP-Tag2 mice. However, immunoreactivity was also strong in normal pancreatic ducts, intestinal smooth muscle, and several other sites. To determine which sites of expression were rapidly accessible from the bloodstream, we intravenously injected anti-alpha5beta1 integrin antibody and 10 minutes to 24 hours later examined the amount and distribution of labeling. The injected antibody strongly labeled tumor vessels at all time points but did not label most normal blood vessels or gain access to pancreatic ducts or intestinal smooth muscle. Intense vascular labeling by anti-alpha5beta1 integrin antibody co-localized with the uniform CD31 immunoreactivity of tumor vessels and contrasted sharply with the patchy accumulation of nonspecific IgG at sites of leakage. This strategy of injecting antibodies revealed the uniform overexpression and rapid accessibility of alpha5beta1 integrin on tumor vessels and may prove useful in assessing other potential therapeutic targets in cancer.

  18. Sialylation of Integrin beta1 is Involved in Radiation-Induced Adhesion and Migration in Human Colon Cancer Cells

    SciTech Connect

    Lee, Minyoung; Lee, Hae-June; Seo, Woo Duck; Park, Ki Hun; Lee, Yun-Sil

    2010-04-15

    Purpose: Previously, we reported that radiation-induced ST6 Gal I gene expression was responsible for an increase of integrin beta1 sialylation. In this study, we have further investigated the function of radiation-mediated integrin beta1 sialylation in colon cancer cells. Methods and Materials: We performed Western blotting and lectin affinity assay to analyze the expression and level of sialylated integrin beta1. After exposure to ionizing radiation (IR), adhesion and migration of cells were measured by in vitro adhesion and migration assay. Results: IR increased sialylation of integrin beta1 responsible for its increased protein stability and adhesion and migration of colon cancer cells. However, for cells with an N-glycosylation site mutant of integrin beta1 located on the I-like domain (Mu3), these effects were dramatically inhibited. In addition, integrin beta1-mediated radioresistance was not observed in cells containing this mutant. When sialylation of integrin beta1 was targeted with a sulfonamide chalcone compound, inhibition of radiation-induced sialylation of integrin beta1 and inhibition of radiation-induced adhesion and migration occurred. Conclusion: The increase of integrin beta1 sialylation by ST6 Gal I is critically involved in radiation-mediated adhesion and migration of colon cancer cells. From these findings, integrin beta1 sialylation may be a novel target for overcoming radiation-induced survival, especially radiation-induced adhesion and migration.

  19. Role of alpha 5 beta 1 integrin in determining malignant properties of colon carcinoma cells.

    PubMed

    Gong, J; Wang, D; Sun, L; Zborowska, E; Willson, J K; Brattain, M G

    1997-01-01

    We characterized the expression of alpha 5 beta 1 integrin in two distinct phenotypes of colon carcinoma cell lines. Highly invasive colon cell lines (designated Group I cell lines) expressed higher levels of integrin alpha 5 beta 1 mRNA and protein than did poorly invasive colon cell lines (designated Group III cell lines). The relatively high expression of integrin alpha 5 beta 1 in Group I cell lines resulted in strong enhancement of cell adhesion to fibronectin (FN) tissue culture plates, whereas Group III cell lines showed little or no enhancement of cell adhesion by coating. There was no significant difference between Group I and Group III cell lines with respect to cell adhesion to laminin and collagen IV. Cell adhesion to FN in Group I cells was mainly mediated by integrin alpha 5 beta 1 because a monoclonal anti-alpha 5 subunit antibody could block cell adhesion to FN, whereas anti-alpha 2 and anti-alpha 3 antibodies had no effect on cell adhesion to FN. The divergence of alpha 5 beta 1 expression in these two distinct colon carcinoma phenotypes suggested that high expression of alpha 5 beta 1 might contribute to malignant progression in this model system. To test this hypothesis, GEO cells, a Group III cell line that did not express alpha 5 integrin, were transfected with the alpha 5 subunit. Stable transfection of alpha 5 sense cDNA into a typical GEO-limiting dilution clone led to the expression of alpha 5 subunit mRNA and cell surface alpha 5 beta 1 protein. The alpha 5 sense transfectants showed enhanced attachment to FN-coated plates and were more tumorigenic when the cells were injected into athymic nude mice. These results indicate that inappropriately high alpha 5 beta 1 integrin expression contributes to malignant progression in colon carcinoma.

  20. The disintegrin domain of ADAM9: a ligand for multiple beta1 renal integrins.

    PubMed

    Mahimkar, Rajeev M; Visaya, Orvin; Pollock, Allan S; Lovett, David H

    2005-01-15

    Renal tubular epithelial cells in all nephron segments express a distinct member of the metalloprotease-disintegrin family, ADAM9 (a disintegrin and metalloprotease 9), in a punctate basolateral distribution co-localized to the beta1 integrin chain [Mahimkar, Baricos, Visaya, Pollock and Lovett (2000) J. Am. Soc. Nephrol. 11, 595-603]. Discrete segments of the nephron express several defined beta1 integrins, suggesting that ADAM9 interacts with multiple renal integrins and thereby regulates epithelial cell-matrix interactions. Intact ADAM9 and a series of deletion constructs sequentially lacking the metalloprotease domain and the disintegrin domain were assembled as chimaeras with a C-terminal GFP (green fluorescent protein) tag. Stable expression of the ADAM9/GFP protein on the surface of HEK-293 cells (human embryonic kidney 293 cells) significantly decreased adhesion to types I and IV collagen, vitronectin and laminin, but had little effect on adhesion to fibronectin. Expression of the disintegrin/cysteine-rich/GFP construct yielded a similar, but more marked pattern of decreased adhesion. Expression of the cysteine-rich/GFP construct had no effect on adhesion, indicating that the disintegrin domain was responsible for the competitive inhibition of cell-matrix binding. To define the specific renal tubular beta1 integrins interacting with the ADAM9 disintegrin domain, a recombinant GST (glutathione S-transferase)-disintegrin protein was used as a substrate in adhesion assays in the presence or absence of specific integrin-blocking antibodies. Inclusion of antibodies to alpha1, alpha3, alpha6, alphav and beta1 blocked adhesion of HEK-293 cells to GST-disintegrin protein. Immobilized GST-disintegrin domain perfused with renal cortical lysates specifically recovered the alpha3, alpha6, alphav and beta1 integrin chains by Western analysis. It is concluded that ADAM9 is a polyvalent ligand, through its disintegrin domain, for multiple renal integrins of the beta1

  1. PAK proteins and YAP-1 signalling downstream of integrin beta-1 in myofibroblasts promote liver fibrosis

    PubMed Central

    Martin, Katherine; Pritchett, James; Llewellyn, Jessica; Mullan, Aoibheann F.; Athwal, Varinder S.; Dobie, Ross; Harvey, Emma; Zeef, Leo; Farrow, Stuart; Streuli, Charles; Henderson, Neil C.; Friedman, Scott L.; Hanley, Neil A.; Piper Hanley, Karen

    2016-01-01

    Fibrosis due to extracellular matrix (ECM) secretion from myofibroblasts complicates many chronic liver diseases causing scarring and organ failure. Integrin-dependent interaction with scar ECM promotes pro-fibrotic features. However, the pathological intracellular mechanism in liver myofibroblasts is not completely understood, and further insight could enable therapeutic efforts to reverse fibrosis. Here, we show that integrin beta-1, capable of binding integrin alpha-11, regulates the pro-fibrotic phenotype of myofibroblasts. Integrin beta-1 expression is upregulated in pro-fibrotic myofibroblasts in vivo and is required in vitro for production of fibrotic ECM components, myofibroblast proliferation, migration and contraction. Serine/threonine-protein kinase proteins, also known as P21-activated kinase (PAK), and the mechanosensitive factor, Yes-associated protein 1 (YAP-1) are core mediators of pro-fibrotic integrin beta-1 signalling, with YAP-1 capable of perpetuating integrin beta-1 expression. Pharmacological inhibition of either pathway in vivo attenuates liver fibrosis. PAK protein inhibition, in particular, markedly inactivates the pro-fibrotic myofibroblast phenotype, limits scarring from different hepatic insults and represents a new tractable therapeutic target for treating liver fibrosis. PMID:27535340

  2. Vesicle-associated membrane protein 2 mediates trafficking of {alpha}5{beta}1 integrin to the plasma membrane

    SciTech Connect

    Hasan, Nazarul; Hu, Chuan

    2010-01-01

    Integrins are major receptors for cell adhesion to the extracellular matrix (ECM). As transmembrane proteins, the levels of integrins at the plasma membrane or the cell surface are ultimately determined by the balance between two vesicle trafficking events: endocytosis of integrins at the plasma membrane and exocytosis of the vesicles that transport integrins. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, is involved in the trafficking of {alpha}5{beta}1 integrin. VAMP2 was present on vesicles containing endocytosed {beta}1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cell surface {alpha}5{beta}1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of {alpha}5{beta}1, without altering cell surface expression of {alpha}2{beta}1 integrin or {alpha}3{beta}1 integrin. By contrast, silencing of VAMP8, another SNARE protein, had no effect on cell surface expression of the integrins or cell adhesion to fibronectin. In addition, VAMP2-mediated trafficking is involved in cell adhesion to collagen but not to laminin. Consistent with disruption of integrin functions in cell proliferation and survival, VAMP2 silencing diminished proliferation and triggered apoptosis. Collectively, these data indicate that VAMP2 mediates the trafficking of {alpha}5{beta}1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.

  3. Identification of beta1C-2, a novel variant of the integrin beta1 subunit generated by utilization of an alternative splice acceptor site in exon C.

    PubMed Central

    Svineng, G; Fässler, R; Johansson, S

    1998-01-01

    A new splice variant of the human integrin subunit beta1 has been identified and designated beta1C-2. It differs from the previously reported beta1C (in this report designated beta1C-1) by 18 nucleotides, and is generated by splicing from exon 6 to an alternative splice acceptor site within exon C, causing an in-frame deletion of six amino acids of the cytoplasmic region of beta1C-1. The beta1C-2 mRNA is present in several human cell lines and tissues at low levels, similarly to beta1C-1. In peripheral T-lymphocytes, beta1C-2 is the selectively expressed isoform. Neither beta1C-1 nor beta1C-2 mRNA could be detected in mouse tissues, and Southern hybridization of a mouse genomic beta1 clone with a human exon-C-specific probe failed to identify a corresponding mouse exon. The antisense orientation of exon C is highly homologous to an Alu element. Since Alu elements are restricted to primates, the beta1C-1 and beta1C-2 variants of the integrin subunit beta1 are specific for these species. The protein coded for by the beta1C-2 cDNA can be expressed and localized to the surface of beta1 deficient mouse cells. However, while stable transformed clones expressing high levels of the beta1A were commonly found, the beta1C-1 and beta1C-2 expressing clones expressed barely detectable amounts of the beta1 protein. Hence, high levels of beta1C-2 may be incompatible with cell proliferation, as previously suggested for beta1C-1. PMID:9494094

  4. Expression and functional analysis of a cytoplasmic domain variant of the beta 1 integrin subunit.

    PubMed

    Balzac, F; Belkin, A M; Koteliansky, V E; Balabanov, Y V; Altruda, F; Silengo, L; Tarone, G

    1993-04-01

    We have previously described a variant form of the integrin beta 1 subunit (beta 1B)1 characterized by an altered sequence at the cytoplasmic domain. Using polyclonal antibodies to a synthetic peptide corresponding to the unique sequence of the beta 1B, we analyzed the expression of this molecule in human tissues and cultured cells. Western blot analysis showed that the beta 1B is expressed in skin and liver and, in lower amounts, in skeletal and cardiac muscles. The protein was not detectable in brain, kidney, and smooth muscle. In vitro cultured keratinocytes and hepatoma cells are positive, but fibroblasts, endothelial cells, and smooth muscle cells are negative. An astrocytoma cell line derived from immortalized fetal astrocytes was found to express beta 1B. In these cells beta 1B represent integral of 30% of the beta 1 and form heterodimers with alpha 1 and alpha 5 subunits. To investigate the functional properties of beta 1B, the full-length cDNA coding for this molecule was transfected into CHO cells. Stable transfectants were selected and the beta 1B was identified by a mAb that discriminate between the transfected human protein and the endogenous hamster beta 1A. Immunoprecipitation experiments indicated that the beta 1B was exported at the cell surface in association with the endogenous hamster alpha subunits. The alpha 5/beta 1B complex bound to a fibronectin-affinity matrix and was specifically released by RGD-containing peptides. Thus beta 1B and beta 1A are similar as far as the alpha/beta association and fibronectin binding are concerned. The two proteins differ, however, in their subcellular localization. Immunofluorescence studies indicated, in fact, that beta 1B, in contrast to beta 1A, does not localize in focal adhesions. The restricted tissue distribution and the distinct subcellular localization, suggest that beta 1B has unique functional properties.

  5. Expression and functional analysis of a cytoplasmic domain variant of the beta 1 integrin subunit

    PubMed Central

    1993-01-01

    We have previously described a variant form of the integrin beta 1 subunit (beta 1B)1 characterized by an altered sequence at the cytoplasmic domain. Using polyclonal antibodies to a synthetic peptide corresponding to the unique sequence of the beta 1B, we analyzed the expression of this molecule in human tissues and cultured cells. Western blot analysis showed that the beta 1B is expressed in skin and liver and, in lower amounts, in skeletal and cardiac muscles. The protein was not detectable in brain, kidney, and smooth muscle. In vitro cultured keratinocytes and hepatoma cells are positive, but fibroblasts, endothelial cells, and smooth muscle cells are negative. An astrocytoma cell line derived from immortalized fetal astrocytes was found to express beta 1B. In these cells beta 1B represent integral of 30% of the beta 1 and form heterodimers with alpha 1 and alpha 5 subunits. To investigate the functional properties of beta 1B, the full- length cDNA coding for this molecule was transfected into CHO cells. Stable transfectants were selected and the beta 1B was identified by a mAb that discriminate between the transfected human protein and the endogenous hamster beta 1A. Immunoprecipitation experiments indicated that the beta 1B was exported at the cell surface in association with the endogenous hamster alpha subunits. The alpha 5/beta 1B complex bound to a fibronectin-affinity matrix and was specifically released by RGD-containing peptides. Thus beta 1B and beta 1A are similar as far as the alpha/beta association and fibronectin binding are concerned. The two proteins differ, however, in their subcellular localization. Immunofluorescence studies indicated, in fact, that beta 1B, in contrast to beta 1A, does not localize in focal adhesions. The restricted tissue distribution and the distinct subcellular localization, suggest that beta 1B has unique functional properties. PMID:7681433

  6. Role of Integrin-Beta1 in Polycystic Kidney Disease

    DTIC Science & Technology

    2012-04-01

    21702-5012 DISTRIBUTION STATEMENT: Approved for Public Release ; Distribution Unlimited...for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO... Release ; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Increased fibrosis and integrins expression are elevated in in APDKD

  7. Internalization of the alpha 5 beta 1 integrin does not depend on "NPXY" signals.

    PubMed

    Vignoud, L; Usson, Y; Balzac, F; Tarone, G; Block, M R

    1994-03-15

    The alpha 5 beta 1 integrin is a constitutively internalized fibronectin receptor. It contains in the cytoplasmic tail of its beta 1 subunit two NPXY sequences which have been proposed to mediate internalization. Indeed a NPXY motif constitutes the internalization signal for the Low Density Lipoprotein (LDL) and insulin receptors. To learn more about the putative role of the two NPXY sequences in internalization of the alpha 5 beta 1 receptor, we have made and expressed mutants of the human beta 1 subunit in Chinese Hamster Ovary (CHO) cells, in which the two tyrosines of the NPXY motifs were replaced by serine residues. A cytoplasmic variant beta 1B which does not contain any NPXY sequence was also analyzed. Our results indicate that the NPXY mutants and the cytoplasmic variant are still internalized. Thus in the alpha 5 beta 1 receptor, the highly conserved NPXY sequences do not function as internalization motifs.

  8. Sequential regulation of alpha 4 beta 1 and alpha 5 beta 1 integrin avidity by CC chemokines in monocytes: implications for transendothelial chemotaxis

    PubMed Central

    1996-01-01

    Leukocyte emigration possibly requires dynamic regulation of integrin adhesiveness for endothelial and extracellular matrix ligands. Adhesion assays on purified vascular cell adhension molecule (VCAM)-1, fibronectin, and fibronectin fragments revealed distinct kinetic patterns for the regulation of very late antigen (VLA)-4 (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) avidity by the CC chemokines monocyte inflammatory protein (MIP)-1 alpha, RANTES (regulated on activation, normal T expressed and secreted), or monocyte chemoattractant protein (MCP)-1 in monocytes. CC chemokines induced early activation and subsequent deactivation of VLA-4, whereas upregulation of VLA-5 avidity occurred later and persisted. Controlled detachment assays in shear flow suggested that adhesive strength of VLA-4 for VCAM-1 or the 40-kD fragment of fibronectin (FN40) is more rapidly increased and subsequently reduced by MCP-1 than by MIP-1 alpha, and confirmed late and sustained activation of the adhesive strength of VLA-5 for the 120- kD fragment of fibronectin (FN120). Mn2+ or the stimulating beta 1 mAb TS2/16 strongly and stably enhanced monocyte binding to VCAM-1 or fibronectin, and locked beta 1 integrins in a high avidity state, which was not further modulated by CC chemokines. Mn2+ and mAb TS2/16 inhibited CC chemokine-induced transendothelial migration, particularly chemotaxis across stimulated endothelium that involved VLA-4 and VCAM- 1. VLA-4 on Jurkat cells is of constitutively high avidity and interfered with migration across barriers expressing VCAM-1. Low but not high site densities of VCAM-1 or FN40 promoted, while FN120 impaired, beta 1 integrin-dependent monocyte chemotaxis to MCP-1 across filters coated with these substrates. Thus, we show that CC chemokines can differentially and selectively regulate avidity of integrins sharing common beta subunits. Transient activation and deactivation of VLA-4 may serve to facilitate transendothelial diapedesis, whereas late and

  9. Selective, tight-binding inhibitors of integrin alpha4beta1 that inhibit allergic airway responses.

    PubMed

    Lin, K c; Ateeq, H S; Hsiung, S H; Chong, L T; Zimmerman, C N; Castro, A; Lee, W C; Hammond, C E; Kalkunte, S; Chen, L L; Pepinsky, R B; Leone, D R; Sprague, A G; Abraham, W M; Gill, A; Lobb, R R; Adams, S P

    1999-03-11

    Integrin alpha4beta1 mediates leukocyte recruitment, activation, mediator release, and apoptosis inhibition, and it plays a central role in inflammatory pathophysiology. High-affinity, selective inhibitors of alpha4beta1, based on the Leu-Asp-Val (LDV) sequence from the alternatively spliced connecting segment-1 (CS-1) peptide of cellular fibronectin, are described that employ a novel N-terminal peptide "cap" strategy. One inhibitor, BIO-1211, was approximately 10(6)-fold more potent than the starting peptide and exhibited tight-binding properties (koff = 1.4 x 10(-4) s-1, KD = 70 pM), a remarkable finding for a noncovalent, small-molecule inhibitor of a protein receptor. BIO-1211 was also 200-fold selective for the activated form of alpha4beta1, and it stimulated expression of ligand-induced epitopes on the integrin beta1 subunit, a property consistent with occupancy of the receptor's ligand-binding site. Pretreatment of allergic sheep with a 3-mg nebulized dose of BIO-1211 inhibited early and late airway responses following antigen challenge and prevented development of nonspecific airway hyperresponsiveness to carbachol. These results show that highly selective and potent small-molecule antagonists can be identified to integrins with primary specificity for peptide domains other than Arg-Gly-Asp (RGD); they confirm the generality of integrins as small molecule targets; and they validate alpha4beta1 as a therapeutic target for asthma.

  10. Integrin alpha 8 beta 1 promotes attachment, cell spreading, and neurite outgrowth on fibronectin.

    PubMed Central

    Müller, U; Bossy, B; Venstrom, K; Reichardt, L F

    1995-01-01

    The integrin alpha 8 subunit, isolated by low stringency hybridization, is a novel integrin subunit that associates with beta 1. To identify ligands, we have prepared a function-blocking antiserum to the extracellular domain of alpha 8, and we have established by transfection K562 cell lines that stably express alpha 8 beta 1 heterodimers on the cell surface. We demonstrate here by cell adhesion and neurite outgrowth assays that alpha 8 beta 1 is a fibronectin receptor. Studies on fibronectin fragments using RGD peptides as inhibitors show that alpha 8 beta 1 binds to the RGD site of fibronectin. In contrast to the endogenous alpha 5 beta 1 fibronectin receptor in K562 cells, alpha 8 beta 1 not only promotes cell attachment but also extensive cell spreading, suggesting functional differences between the two receptors. In chick embryo fibroblasts, alpha 8 beta 1 is localized to focal adhesions. We conclude that alpha 8 beta 1 is a receptor for fibronectin and can promote attachment, cell spreading, and neurite outgrowth on fibronectin. Images PMID:7626807

  11. Endometrial receptivity: expression of alpha3beta1, alpha4beta1 and alphaVbeta1 endometrial integrins in women with impaired fertility.

    PubMed

    Skrzypczak, J; Mikołajczyk, M; Szymanowski, K

    2001-11-01

    Advances in immunohistochemical methods with the specificity of poly- and monoclonal antibodies allow the description of the endometrial receptivity, which is characterized by the ability of secretion of phase specific proteins and glikoproteins by epithelial and stromal cells. We studied the differences in the expression of alpha3beta1, alpha4beta1 and alphaVbeta1 integrins in endometrium of women with recurrent miscarriages and women with unexplained infertility. The endometrial tissue was collected during hysteroscopy performed between 7th and 9th day after ovulation. The immunohistochemical evaluation of alpha3beta1, alpha4beta1 and alphaVbeta1 integrin expression was determined in all endometrial biopsies. Staining intensity of alpha3beta1 in glandular epithelium and endometrial stroma was similar in both groups. In women with recurrent miscarriages we noted a lower concentrations of the alpha4beta1 and alphaVbeta1 integrins during the midluteal phase than in women with unexplained infertility. Moreover, integrins alpha4beta1 and alphaVbeta1 were expressed more frequently in glandular epithelium and endometrial stroma of women with unexplained infertility than those of women with recurrent miscarriages. However, alphaV(2)1 staining in endometrial stroma was stronger than that of alpha4beta1. It can be concluded, that these integrins may play an important role in the implantation process.

  12. Loss of integrin alpha9beta1 results in defects in proliferation, causing poor re-epithelialization during cutaneous wound healing.

    PubMed

    Singh, Purva; Chen, Chun; Pal-Ghosh, Sonali; Stepp, Mary Ann; Sheppard, Dean; Van De Water, Livingston

    2009-01-01

    The wound microenvironment comprises constituents, such as the extracellular matrix (ECM), that regulate with temporal and spatial precision, the migratory, proliferative, and contractility of wound cells. Prompt closure of the wound is an early and critical phase of healing and beta1 integrins are important in this process. We previously reported a marked increase in integrin alpha9beta1 expression in epidermal keratinocytes in cutaneous and corneal wounds. However, the functional role of keratinocyte alpha9beta1 during re-epithelialization is unknown and analysis has been precluded by the lethal phenotype of integrin alpha9beta1 knockout mice. We now report that in conditional integrin alpha9 knockout (K14-alpha9 null) mice, normal proliferation occurs in epidermal keratinocytes and corneal basal cells. Normal epidermal keratinocyte morphology is also retained. However, corneal basal cell morphology and epithelial thickness are altered, suggesting that loss of integrin alpha9beta1 results in abnormal corneal differentiation. In cutaneous wounds, the number of proliferating epidermal keratinocytes is significantly reduced in K14-alpha9 null mice compared with alpha9(fl/-) mice, but not in Cre (control) mice. The decreased keratinocyte proliferation observed in K14-alpha9 null mice negatively impacts healing, resulting in a thinner migrating epithelium, demonstrating that alpha9beta1 is crucial for efficient and proper re-epithelialization during cutaneous wound healing.

  13. Functional analysis of alpha 1 beta 1 integrin in human natural killer cells.

    PubMed

    Pérez-Villar, J J; Melero, I; Gismondi, A; Santoni, A; López-Botet, M

    1996-09-01

    Upon activation with interleukin (IL)-2 human natural killer (NK) cells acquire on their surface the alpha 1 beta 1 and alpha 2 beta 1 integrins and down-regulate the expression of alpha 6 beta 1. By employing alpha 1 beta 1-specific monoclonal antibody (mAb) HP-2B6, characterized in our laboratory, we examined the functional role of the alpha 1 beta 1 integrin in NK cells. Treatment with HP-2B6 mAb partially interfered with attachment of cultured NK cells to type I collagen, and combined with an anti-alpha 2 beta 1 (TEA 1/41) mAb, it completely abrogated cell adhesion to this extracelular matrix protein. In contrast, NK cell attachment to laminin was completely blocked by the anti-beta 1 LIA 1/2 mAb, but was unaffected by alpha 1 and alpha 2-specific mAb; as alpha 3 beta 1 and alpha 6 beta 1 were undetectable, the data indicate that the alpha 1 beta 1 integrin binding sites for type I collagen and laminin are different. Incubation with anti-alpha 1 HP-2B6 or its F(ab')2 fragments specifically induced a rapid homotypic aggregation of NK cells that was dependent on active metabolism, an intact cytoskeleton and the presence of divalent cations (Ca2+ and Mg2+); homotypic cell adhesion was selectively blocked by anti-CD18, CD11a or CD54 mAb. In addition, stimulation of cultured NK cells with the anti-alpha 1 HP-2B6 enhanced TNF-alpha production and induced tyrosine phosphorylation of a 110-kDa protein. Pretreatment with specific inhibitors of protein tyrosine kinase (PTK) activity (tyrphostin 25 and herbimycin A) completely abrogated the functional effects induced by the anti-alpha 1 HP-2B6 mAb. Our data show that ligation of the alpha 1 beta 1 integrin positively modulates IL-2-activated NK cell function via a PTK-dependent pathway.

  14. Tetraspanin CD151 regulates alpha6beta1 integrin adhesion strengthening

    NASA Technical Reports Server (NTRS)

    Lammerding, Jan; Kazarov, Alexander R.; Huang, Hayden; Lee, Richard T.; Hemler, Martin E.

    2003-01-01

    The tetraspanin CD151 molecule associates specifically with laminin-binding integrins, including alpha6beta1. To probe strength of alpha6beta1-dependent adhesion to laminin-1, defined forces (0-1.5 nN) were applied to magnetic laminin-coated microbeads bound to NIH 3T3 cells. For NIH 3T3 cells bearing wild-type CD151, adhesion strengthening was observed, as bead detachment became more difficult over time. In contrast, mutant CD151 (with the C-terminal region replaced) showed impaired adhesion strengthening. Static cell adhesion to laminin-1, and detachment of beads coated with fibronectin or anti-alpha6 antibody were all unaffected by CD151 mutation. Hence, CD151 plays a key role in selectively strengthening alpha6beta1 integrin-mediated adhesion to laminin-1.

  15. Tetraspanin CD151 regulates alpha6beta1 integrin adhesion strengthening

    NASA Technical Reports Server (NTRS)

    Lammerding, Jan; Kazarov, Alexander R.; Huang, Hayden; Lee, Richard T.; Hemler, Martin E.

    2003-01-01

    The tetraspanin CD151 molecule associates specifically with laminin-binding integrins, including alpha6beta1. To probe strength of alpha6beta1-dependent adhesion to laminin-1, defined forces (0-1.5 nN) were applied to magnetic laminin-coated microbeads bound to NIH 3T3 cells. For NIH 3T3 cells bearing wild-type CD151, adhesion strengthening was observed, as bead detachment became more difficult over time. In contrast, mutant CD151 (with the C-terminal region replaced) showed impaired adhesion strengthening. Static cell adhesion to laminin-1, and detachment of beads coated with fibronectin or anti-alpha6 antibody were all unaffected by CD151 mutation. Hence, CD151 plays a key role in selectively strengthening alpha6beta1 integrin-mediated adhesion to laminin-1.

  16. CD98 modulates integrin beta1 function in polarized epithelial cells.

    PubMed

    Cai, Songmin; Bulus, Nada; Fonseca-Siesser, Priscila M; Chen, Dong; Hanks, Steven K; Pozzi, Ambra; Zent, Roy

    2005-03-01

    The type II transmembrane protein CD98, best known as the heavy chain of the heterodimeric amino acid transporters (HAT), is required for the surface expression and basolateral localization of this transporter complex in polarized epithelial cells. CD98 also interacts with beta1 integrins resulting in an increase in their affinity for ligand. In this study we explored the role of the transmembrane and cytoplasmic domains of CD98 on integrin-dependent cell adhesion and migration in polarized renal epithelial cells. We demonstrate that the transmembrane domain of CD98 was sufficient, whereas the five N-terminal amino acids of this domain were required for CD98 interactions with beta1 integrins. Overexpression of either full-length CD98 or CD98 lacking its cytoplasmic tail increased cell adhesion and migration, whereas deletion of the five N-terminal amino acids of the transmembrane domain of CD98 abrogated this effect. CD98 and mutants that interacted with beta1 integrins increased both focal adhesion formation and FAK and AKT phosphorylation. CD98-induced cell adhesion and migration was inhibited by addition of phosphoinositol 3-OH kinase (PI3-K) inhibitors suggesting these cell functions are PI3-K-dependent. Finally, CD98 and mutants that interacted with beta1, induced marked changes in polarized renal epithelial cell branching morphogenesis in collagen gels. Thus, in polarized renal epithelial cells, CD98 might be viewed as a scaffolding protein that interacts with basolaterally expressed amino acid transporters and beta1 integrins and can alter diverse cellular functions such as amino acid transport as well as cell adhesion, migration and branching morphogenesis.

  17. Novel monoclonal antibody against beta 1 integrin enhances cisplatin efficacy in human lung adenocarcinoma cells.

    PubMed

    Kim, Min-Young; Cho, Woon-Dong; Hong, Kwon Pyo; Choi, Da Bin; Hong, Jeong Won; Kim, Soseul; Moon, Yoo Ri; Son, Seung-Myoung; Lee, Ok-Jun; Lee, Ho-Chang; Song, Hyung Geun

    2016-05-01

    The use of anti-beta 1 integrin monoclonal antibody in lung cancer treatment has proven beneficial. Here, we developed a novel monoclonal antibody (mAb), called P5, by immunizing mice with human peripheral blood mononuclear cells (PBMC). Its anti-tumor effect is now being tested, in a clinical phase III trial, in combinatorial treatments with various chemical drugs. To confirm that P5 indeed binds to beta 1 integrin, cell lysates were immunoprecipitated with commercial anti-beta 1 integrin mAb (TS2/16) and immunoblotted against P5 to reveal a 140 kDa molecular weight band, as expected. Immunoprecipitation with P5 followed by LC/MS protein sequence analysis further verified P5 antigen to be beta 1 integrin. Cisplatin treatment upregulated cell surface expression of beta 1 integrin in A549 cells, while causing inhibition of cell growth. When cells were co-treated with different concentrations of P5 mAb, the cisplatin-mediated inhibitory effect was enhanced in a dose-dependent manner. Our findings show that a combinatorial treatment of P5 mAb and cisplatin in A549 cells resulted in a 30% increase in apoptosis, compared to baseline, and significantly more when compared to either the cisplatin or P5 alone group. The entire peptide sequences in CDR from variable region of Ig heavy and light chain gene for P5 mAb are also disclosed. Together, these results provide evidence of the beneficial effect of P5 mAb in combinatorial treatment of human lung adenocarcinoma.

  18. Contribution of alpha4beta1 integrin to the antiallergic effect of levocabastine.

    PubMed

    Qasem, Ahmed R; Bucolo, Claudio; Baiula, Monica; Spartà, Antonino; Govoni, Paolo; Bedini, Andrea; Fascì, Domenico; Spampinato, Santi

    2008-09-15

    Levocabastine is an antiallergic drug acting as a histamine H1-receptor antagonist. In allergic conjunctivitis (AC), it may also antagonize up-regulation of the intercellular adhesion molecule-1 (ICAM-1) expressed on epithelial conjunctival cells. However, little is known about its effects on eosinophils, important effector cells in AC. The adhesion molecule integrin alpha(4)beta(1) is expressed in eosinophils; it interacts with the vascular cell adhesion molecule-1 (VCAM-1) and fibronectin (FN) in vascular endothelial cells and contributes to eosinophil activation and infiltration in AC. This study provides evidence that in a scintillation proximity assay levocabastine (IC(50) 406 microM), but not the first-generation antihistamine chlorpheniramine, displaced (125)I-FN binding to human integrin alpha(4)beta(1) and, in flow cytometry analysis, levocabastine antagonized the binding of a primary antibody to integrin alpha(4) expressed on the Jurkat cell surface. Levocabastine, but not chlorpheniramine, binds the alpha(4)beta(1) integrin and prevents eosinophil adhesion to VCAM-1, FN or human umbilical vascular endothelial cells (HUVEC) in vitro. Similarly, levocabastine affects alpha(L)beta(2)/ICAM-1-mediated adhesion of Jurkat cells. In a model of AC levocabastine eye drops reduced the clinical aspects of the late-phase reaction and the conjunctival expression of alpha(4)beta(1) integrin by reducing infiltrated eosinophils. We propose that blockade of integrin-mediated cell adhesion might be a target of the antiallergic action of levocabastine and may play a role in preventing eosinophil adhesion and infiltration in AC.

  19. Expression of the VLA beta 1 integrin family in bladder cancer.

    PubMed Central

    Liebert, M.; Washington, R.; Stein, J.; Wedemeyer, G.; Grossman, H. B.

    1994-01-01

    Integrins are a family of transmembrane heterodimers, many of which function as receptors for extracellular matrix molecules and play a role in adherence to and motility on matrix components. Because of these functions, integrins are suspected of participating in metastatic processes. We investigated the expression of beta 1 integrins in human bladder cancer cell lines and tissues. Expression of beta 1 integrins on cultured bladder cancer cell lines was evaluated by flow cytometry, of 8 cell lines tested, alpha 1 was found in 4, alpha 2 and alpha 3 in all 8, alpha 4 in 1, and alpha 5 in 3. These results were in sharp contrast to the expression detected by immunostaining tissues containing normal urothelium and low stage (noninvasive) and high stage (invasive) bladder cancers. All normal urothelial tissues tested expressed alpha 2 and alpha 3 and none expressed alpha 1, alpha 4, or alpha 5. Similarly, a majority (77%) of low stage (noninvasive) bladder cancers stained positively for alpha 3, whereas only 6 of 13 expressed alpha 2 and none expressed alpha 1, alpha 4, or alpha 5. Among invasive bladder cancers, alpha 1 was detected in 7%, alpha 2 in 24%, alpha 3 in 68%, alpha 5 in 10%, and alpha 4 was not found in any samples. These results indicate that integrin expression in cultured human bladder cancer cell lines does not represent expression observed in tissue samples and may reflect adaption to or selection during tissue culture conditions. A progressive loss of alpha 2 expression is seen from normal urothelial cells through invasive bladder cancers. This loss may contribute to an invasive phenotype by a loss of the cell-cell adherence function mediated by the alpha 2 beta 1 and alpha 3 beta 1 integrins. Images Figure 2 Figure 3 PMID:8178925

  20. Transforming growth factor-beta 1 modulates beta 1 and beta 5 integrin receptors and induces the de novo expression of the alpha v beta 6 heterodimer in normal human keratinocytes: implications for wound healing

    PubMed Central

    1995-01-01

    The molecular mechanism underlying the promotion of wound healing by TGF-beta 1 is incompletely understood. We report that TGF-beta 1 regulates the regenerative/migratory phenotype of normal human keratinocytes by modulating their integrin receptor repertoire. In growing keratinocyte colonies but not in fully stratified cultured epidermis, TGF-beta 1: (a) strongly upregulates the expression of the fibronectin receptor alpha 5 beta 1, the vitronectin receptor alpha v beta 5, and the collagen receptor alpha 2 beta 1 by differentially modulating the synthesis of their alpha and beta subunits; (b) downregulates the multifunctional alpha 3 beta 1 heterodimer; (c) induces the de novo expression and surface exposure of the alpha v beta 6 fibronectin receptor; (d) stimulates keratinocyte migration toward fibronectin and vitronectin; (e) induces a marked perturbation of the general mechanism of polarized domain sorting of both beta 1 and beta 4 dimers; and (f) causes a pericellular redistribution of alpha v beta 5. These data suggest that alpha 5 beta 1, alpha v beta 6, and alpha v beta 5, not routinely used by keratinocytes resting on an intact basement membrane, act as "emergency" receptors, and uncover at least one of the molecular mechanisms responsible for the peculiar integrin expression in healing human wounds. Indeed, TGF-beta 1 reproduces the integrin expression pattern of keratinocytes located at the injury site, particularly of cells in the migrating epithelial tongue at the leading edge of the wound. Since these keratinocytes are inhibited in their proliferative capacity, these data might account for the apparent paradox of a TGF-beta 1-dependent stimulation of epidermal wound healing associated with a growth inhibitory effect on epithelial cells. PMID:7537276

  1. Cellular growth and survival are mediated by beta 1 integrins in normal human breast epithelium but not in breast carcinoma

    SciTech Connect

    Howlett, Anthony R; Bailey, Nina; Damsky, Caroline; Petersen, Ole W; Bissell, Mina J

    1994-11-28

    We previously established a rapid three-dimensional assay for discrimination of normal and malignant human breast epithelial cells using a laminin-rich reconstituted basement membrane. In this assay, normal epithelial cells differentiate into well-organized acinar structures whereas tumor cells fail to recapitulate this process and produce large, disordered colonies. The data suggest that breast acinar morphogenesis and differentiation is regulated by cell-extracellular matrix (ECM) interactions and that these interactions are altered in malignancy. Here, we investigated the role of ECM receptors (integrins) in these processes and report on the expression and function of potential laminin receptors in normal and tumorigenic breast epithelial cells. Immmunocytochemical analysis showed that normal and carcinoma cells in a three-dimensional substratum express profiles of integrins similar to normal and malignant breast tissues in situ. Normal cells express {alpha}1, {alpha}2, {alpha}3, {alpha}6, {beta}1 and {beta}4 integrin subunits, whereas breast carcinoma cells show variable losses, disordered expression, or down regulation of these subunits. Function-blocking experiments using inhibitory antiintegrin subunit antibodies showed a >5-fold inhibition of the formation of acinar structures by normal cells in the presence of either anti-{beta}1 or anti-{alpha}3 antibodies, whereas anti-{alpha}2 or -{alpha}6 had little or no effect. In experiments where collagen type I gels were used instead of basement membrane, acinar morphogenesis was blocked by anti-{beta}1 and -{alpha}2 antibodies but not by anti-{alpha}3. These data suggest a specificity of integrin utilization dependent on the ECM ligands encountered by the cell. The interruption of normal acinar morphogenesis by anti-integrin antibodies was associated with an inhibition of cell growth and induction of apoptosis. Function-blocking antibodies had no inhibitory effect on the rate of tumor cell growth, survival or

  2. Laminin and beta1 integrin distribution in the early stages of human kidney development.

    PubMed

    Bernardini, N; Bianchi, F; Dolfi, A

    1999-01-01

    Laminin, an extracellular matrix molecule (EMM) widely expressed in the basal laminae, interacts with specific membrane receptors among which the integrin molecules are the best known. During embryo development laminin is the first synthesized EMM and plays a significant role in the morphogenesis of organs in which epithelial-mesenchymal interactions and branching take place. The present study describes the distribution of laminin and of beta1 integrin receptors during the very early stages of human kidney development. The observations were carried out on paraffin sections of human embryos ranging between the 4th and the 7th gestational week. Laminin was detected within the basement membranes of mesonephric duct, vesicles, glomerular vessels and celomic epithelium. The metanephric anlage reacted with anti-laminin immunoglobulins in the basement membrane underlying the ampullae and in few blastemic cap cells. Low levels of beta1 integrin reactivity were found in both the mesonephric and metanephric structures. This study provides for the first time data about the distribution of laminin and beta1 integrin in the early stages of human renal organogenesis suggesting a key role for these molecules in the epithelial-mesenchymal interactions necessary for kidney development.

  3. Induced keratinocyte hyper-proliferation in alpha2beta1 integrin transgenic mice results in systemic immune cell activation.

    PubMed

    Teige, Ingrid; Bäcklund, Alexandra; Svensson, Lars; Kvist, Peter Helding; Petersen, Thomas Kongstad; Kemp, Kåre

    2010-01-01

    alpha2beta1 integrins are normally confined to the proliferating basal layers of the epidermis. However, during wound healing and in psoriasis, these integrins are expressed on keratinocytes in suprabasal layers correlating with a less differentiated phenotype. Transgenic mice expressing alpha2beta1 integrins under the involucrine promoter have previously been demonstrated, to various degrees, spontaneously develop a skin disorder resembling psoriasis. Herein, we show that a mild epidermal wounding induces a uniform acanthosis together with an influx of immune cells. The disease initiates as a normal wound healing process and is completely restored in wildtype mice by day 14. However, in the integrin transgenic mice a chronic inflammation develops, a process that can be compared to the Koebner phenomenon in psoriatic patients. In this study, we have followed the integrin transgenic mice for five weeks, where substantial keratinocyte hyper-proliferation, inflammatory infiltration and high cytokine levels within the skin can still be observed. In addition, draining lymph nodes were dramatically increased in size and contained highly activated T cells, as well as APCs secreting large amounts of pro-inflammatory cytokines. Furthermore, the systemic immune response was affected with increased spleen size, elevated cytokine levels in the serum and altered lymphocyte trafficking patterns, very much resembling what is seen in psoriasis patients. Finally, CD4(+) T cell depletion was not able to affect the onset or progression of skin inflammation. This suggests that altered keratinocyte differentiation and proliferation can drive a skin inflammation and cause chronic immune cell activation both at a local and systemic level.

  4. Integrin alpha6beta1-laminin interactions regulate early myotome formation in the mouse embryo.

    PubMed

    Bajanca, Fernanda; Luz, Marta; Raymond, Karine; Martins, Gabriel G; Sonnenberg, Arnoud; Tajbakhsh, Shahragim; Buckingham, Margaret; Thorsteinsdóttir, Sólveig

    2006-05-01

    We addressed the potential role of cell-laminin interactions during epaxial myotome formation in the mouse embryo. Assembly of the myotomal laminin matrix occurs as epaxial myogenic precursor cells enter the myotome. Most Myf5-positive and myogenin-negative myogenic precursor cells localise near assembled laminin, while myogenin-expressing cells are located either away from this matrix or in areas where it is being assembled. In Myf5(nlacZ/nlacZ) (Myf5-null) embryos, laminin, collagen type IV and perlecan are present extracellularly near myogenic precursor cells, but do not form a basement membrane and cells are not contained in the myotomal compartment. Unlike wild-type myogenic precursor cells, Myf5-null cells do not express the alpha6beta1 integrin, a laminin receptor, suggesting that integrin alpha6beta1-laminin interactions are required for myotomal laminin matrix assembly. Blocking alpha6beta1-laminin binding in cultured wild-type mouse embryo explants resulted in dispersion of Myf5-positive cells, a phenotype also seen in Myf5(nlacZ/nlacZ) embryos. Furthermore, inhibition of alpha6beta1 resulted in an increase in Myf5 protein and ectopic myogenin expression in dermomyotomal cells, suggesting that alpha6beta1-laminin interactions normally repress myogenesis in the dermomyotome. We conclude that Myf5 is required for maintaining alpha6beta1 expression on myogenic precursor cells, and that alpha6beta1 is necessary for myotomal laminin matrix assembly and cell guidance into the myotome. Engagement of laminin by alpha6beta1 also plays a role in maintaining the undifferentiated state of cells in the dermomyotome prior to their entry into the myotome.

  5. Significance of alpha 9 beta 1 and alpha v beta 6 integrin expression in breast carcinoma.

    PubMed

    Arihiro, K; Kaneko, M; Fujii, S; Inai, K; Yokosaki, Y

    2000-01-01

    Both alpha 9 beta 1 and alpha v beta 6 integrins have been newly identified from the tracheal epithelium of guinea pig. It has been pointed out that alpha 9 beta 1 functions as a receptor for tenascin-C and osteopontin. As for the ligands of alpha v beta 6, fibronectin and tenascin-C have been identified. It has not been ascertained whether alpha 9 beta 1 and alpha v beta 6 are expressed in normal breast tissue, benign breast lesion or breast carcinoma. Immunohistochemical staining for alpha 9 beta 1 and alpha v beta 6 was performed in benign breast lesion and breast carcinoma specimens. Western blotting was carried out on 11 breast carcinoma cases. alpha 9 beta 1 was expressed in the cytoplasm of carcinoma cells in 23 of 90 cases (26%) and alpha v beta 6 in the membrane of carcinoma cells in 16 of 90 cases (18%). However, these findings of alpha 9 beta 1 and alpha v beta 6 did not correlate with any clinicopathological factors including the patients' age, tumor size, histological type of carcinoma, location of carcinoma cells and hormone receptor status. With regard to the histological grade of carcinoma, alpha v beta 6 and alpha 9 beta 1 expression did not statistically correlate, although no expression of alpha v beta 6 was observed in 14 cases of Grade I. On Western-blott analysis strong and weak bands consistent with alpha v beta 6 were noted in the membrane fraction extracted from breast carcinoma cells. On the other hand weak bands consistent with alpha 9 subunit were noted in the whole cell lysates of breast carcinoma cells and very weak or no bands consistent with alpha 9 subunit were noted in the membrane fraction extracted from the breast carcinoma cells. Significance of alpha 9 beta 1 and alpha v beta 6 integrins expression in breast carcinoma was still unknown on clinicopathological examination. The findings of Western blot analysis may indicate that the transportation system of glycoproteins such as integrins to the cell membrane of carcinoma cells is

  6. Wound healing in the alpha2beta1 integrin-deficient mouse: altered keratinocyte biology and dysregulated matrix metalloproteinase expression.

    PubMed

    Grenache, David G; Zhang, Zhonghua; Wells, Laura E; Santoro, Samuel A; Davidson, Jeffrey M; Zutter, Mary M

    2007-02-01

    The alpha2beta1 integrin, a collagen/laminin receptor, is expressed at high level in the basal cell layer of the epidermis. To define the role of the alpha2beta1 integrin in wound healing, wound repair was extensively evaluated in wild-type and alpha2-null mice in vivo. In addition, the impact of alpha2beta1 integrin-deficiency on the function of primary murine keratinocytes in vitro was analyzed. Our in vivo findings demonstrate that genetic deletion of the alpha2beta1 integrin does not significantly alter the rate of re-epithelialization, collagen deposition, or tensile strength during wound closure in mice. In marked contrast to the observed similarities in wound healing, deletion of the alpha2beta1 integrin resulted in a dramatic increase in neoangiogenesis in the wound microenvironment. In contrast to in vivo studies, primary keratinocytes from alpha2-null mice adhered poorly and displayed impaired migration on type I collagen in vitro. We demonstrate that alpha2beta1 integrin-ligation negatively regulates expression of genes including matrix metalloproteinases both in vivo and in vitro. Furthermore, the changes in gene expression could potentially account for relatively normal wound healing in the alpha2-deficient mouse and our recent observation that suggests an antiangiogenic role for the alpha2beta1 integrin in vivo.

  7. Signal-transducing mechanisms involved in activation of the platelet collagen receptor integrin alpha(2)beta(1).

    PubMed

    Jung, S M; Moroi, M

    2000-03-17

    Evidence was obtained about the mechanism responsible for platelet integrin alpha(2)beta activation by determining effects of various inhibitors on soluble collagen binding, a parameter to assess integrin alpha(2)beta(1) activation, in stimulated platelets. Agonists that can also activate platelet glycoprotein IIb/IIIa are able to activate integrin alpha(2)beta(1), but those operating via glycoprotein Ib cannot. Activation of alpha(2)beta(1) induced by low thrombin or collagen-related peptide concentrations was almost completely inhibited by apyrase, and the inhibitors wortmannin, 4-amino-5-(chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, bisindolylmaleimide I, and SQ29548 significantly inhibited it. Activation induced by high thrombin or collagen-related peptide concentrations was far less sensitive to these inhibitors. However, only wortmannin markedly inhibited ADP-induced integrin alpha(2)beta(1) activation, and this was not ADP concentration-dependent. These results suggest that at the low agonist concentrations, the released ADP would be a primary inducer of integrin alpha(2)beta(1) activation, while at the high agonist concentrations, there would be several pathways through which integrin alpha(2)beta(1) activation can be induced. Kinetic analyses revealed that ADP-induced platelets had about the same number of binding sites (B(max)) as thrombin-induced platelets, but their affinity (K(d)) for soluble collagen was 3.7-12.7-fold lower, suggesting that activated integrin alpha(2)beta(1) induced by ADP is different from that induced by thrombin. The data are consistent with an activation mechanism involving released ADP and in which there exists two different states of activated integrin alpha(2)beta(1); these activated forms of integrin alpha(2)beta(1) would have different conformations that determine their ligand affinity.

  8. A 3D structure model of integrin alpha 4 beta 1 complex: I. Construction of a homology model of beta 1 and ligand binding analysis.

    PubMed Central

    You, Tony J; Maxwell, David S; Kogan, Timothy P; Chen, Qi; Li, Jian; Kassir, Jamal; Holland, George W; Dixon, Richard A F

    2002-01-01

    It is well established that integrin alpha 4 beta 1 binds to the vascular cell adhesion molecule (VCAM) and fibronectin and plays an important role in signal transduction. Blocking the binding of VCAM to alpha 4 beta 1 is thought to be a way of controlling a number of disease processes. To better understand how various inhibitors might block the interaction of VCAM and fibronectin with alpha 4 beta 1, we began constructing a structure model for the integrin alpha 4 beta 1 complex. As the first step, we have built a homology model of the beta 1 subunit based on the I domain of the integrin CD11B subunit. The model, including a bound Mg(2+) ion, was optimized through a specially designed relaxation scheme involving restrained minimization and dynamics steps. The native ligand VCAM and two highly active small molecules (TBC772 and TBC3486) shown to inhibit binding of CS-1 and VCAM to alpha 4 beta 1 were docked into the active site of the refined model. Results from the binding analysis fit well with a pharmacophore model that was independently derived from active analog studies. A critical examination of residues in the binding site and analysis of docked ligands that are both potent and selective led to the proposal of a mechanism for beta 1/beta 7 ligand binding selectivity. PMID:11751331

  9. beta 1 integrin inhibition dramatically enhances radiotherapy efficacy in human breast cancer xenografts

    SciTech Connect

    Park, Catherine C.; Park, Catherine C.; Zhang, Hui J.; Yao, Evelyn S.; Park, Chong J.; Bissell, Mina J.

    2008-06-02

    {beta}1 integrin signaling has been shown to mediate cellular resistance to apoptosis after exposure to ionizing radiation (IR). Other signaling molecules that increase resistance include Akt, which promotes cell survival downstream of {beta}1 integrin signaling. We showed previously that {beta}1 integrin inhibitory antibodies, AIIB2, enhance apoptosis and decrease growth in human breast cancer cells in 3 dimensional laminin-rich extracellular matrix (3D lrECM) cultures and in vivo. Here we asked whether AIIB2 could synergize with IR to modify Akt-mediated IR resistance. We used 3D lrECM cultures to test the optimal combination of AIIB2 with IR treatment of two breast cancer cell lines, MCF-7 and HMT3522-T4-2, as well as T4-2 myr-Akt breast cancer colonies or HMT3522-S-1, which form normal organotypic structures in 3D lrECM. Colonies were assayed for apoptosis and {beta}1 integrin/Akt signaling pathways were evaluated using western blot. In addition, mice bearing MCF-7 xenografts were used to validate the findings in 3D lrECM. We report that AIIB2 increased apoptosis optimally post-IR by down regulating Akt in breast cancer colonies in 3D lrECM. In vivo, addition of AIIB2 after IR significantly enhanced tumor growth inhibition and apoptosis compared to either treatment alone. Remarkably, the degree of tumor growth inhibition using AIIB2 plus 2 Gy radiation was similar to that of 8 Gy alone. We showed previously that AIIB2 had no discernible toxicity in mice; here, its addition allowed for a significant reduction in the IR dose that was necessary to achieve comparable growth inhibition and apoptosis in breast cancer xenografts in vivo.

  10. Selective induction of integrin beta1 by hypoxia-inducible factor: implications for wound healing.

    PubMed

    Keely, Simon; Glover, Louise E; MacManus, Christopher F; Campbell, Eric L; Scully, Melanie M; Furuta, Glenn T; Colgan, Sean P

    2009-05-01

    Because of localized vascular damage and increased tissue oxygen demand, wound healing occurs in a relatively hypoxic microenvironment. These features are particularly relevant to wound healing and fibrosis in chronic inflammatory conditions, such as Crohn's disease and ulcerative colitis. In these studies, we sought to identify the contribution of hypoxia to mechanisms of wound repair in a model of the intestinal submucosa. Initial studies revealed that hypoxia promotes wound healing, as modeled by an increase in intestinal fibroblast-mediated collagen gel contraction. Guided by results from transcriptional profiling, we identified the selective induction of fibroblast integrin beta1 (ITGB1) by hypoxia. Further analysis revealed that hypoxia, as well as pharmacological activators of hypoxia-inducible factor (HIF), induce fibroblast beta1 integrin mRNA, protein, and function by as much as 4-fold. Cloning and analysis of the beta1 integrin gene promoter revealed a 10 +/- 0.8-fold increase in promoter activity in response to hypoxia, and subsequent studies identified a functional DNA binding region for HIF in the ITGB1 gene promoter. Mutational analysis of the HIF binding site within the ITGB1 promoter resulted in a significant loss of ITGB1 hypoxia-inducibility. As proof of principle, studies in a murine model of colitis revealed a correlation between colitic disease severity and tissue ITGB1 expression (R(2)=0.80). Taken together, these results demonstrate that hypoxia induces fibroblast ITGB1 expression and function by transcriptional mechanisms dependent on HIF.

  11. A beta 1-integrin receptor for fibronectin in human kidney glomeruli.

    PubMed Central

    Kerjaschki, D.; Ojha, P. P.; Susani, M.; Horvat, R.; Binder, S.; Hovorka, A.; Hillemanns, P.; Pytela, R.

    1989-01-01

    The fibronectin receptor (FNR) is a transmembrane heterodimeric glycoprotein which shares a common beta 1-chain with several other members of the integrin family of adhesion receptors. The authors have prepared a membrane fraction of isolated human glomeruli, from which two proteins (apparent molecular weights 120 kd and 140 kd) bound to a fibronectin-column, and were selectively released by the synthetic peptide Arg-Gly-Asp-Ser. These molecules were labeled in immune overlays by an antibody raised against the FNR from human placenta that recognizes both the FNR-specific a-chain and the group-specific beta 1-integrin chain. In sections of normal human kidneys this antibody labeled predominately the mesangia and the peripheral capillary walls of glomeruli by an immunoperoxidase procedure. Quantitative immunoelectron microscopy, using an indirect immunogold procedure, revealed a preferential localization along the cell membranes of mesangial, epithelial, and endothelial cells that face the mesangial matrix or the glomerular basement membrane (GBM). In kidney biopsies from patients with various glomerular diseases (membranous and other forms of glomerulonephritis, minimal change disease) the distribution was similar to that in normal glomeruli. These findings indicate that a beta 1-integrin-related FNR is present in normal and diseased human glomeruli. Images Figure 1-4 Figure 5 Figure 6-10 Figure 11-16 PMID:2521774

  12. Rapid access of antibodies to alpha5beta1 integrin overexpressed on the luminal surface of tumor blood vessels.

    PubMed

    Magnussen, Anette; Kasman, Ian M; Norberg, Scott; Baluk, Peter; Murray, Richard; McDonald, Donald M

    2005-04-01

    Integrin alpha(5)beta(1) is overexpressed on endothelial cells of tumor vessels and is uniformly and rapidly accessible to antibodies in the bloodstream. Here, we determined whether antibodies rapidly gain access to integrin overexpressed on the abluminal (basolateral) surface of endothelial cells through vascular leakiness or whether the rapid accessibility results instead because the integrin is overexpressed on the luminal (apical) surface of endothelial cells due to loss of cell polarity. Using tumors in RIP-Tag2 transgenic mice as a model, we first compared the binding pattern of intravascular anti-alpha(5)beta(1) integrin antibody with the leakage pattern of nonspecific IgG. The distributions did not match: anti-alpha(5)beta(1) integrin antibody uniformly labeled the tumor vasculature, but IgG was located in patchy sites of leakage. We next injected an antibody to fibrinogen/fibrin, which resulted in patchy labeling of tumors that matched the leakage of IgG and the overall distribution of fibrin in tumors. Similarly, injected antibodies to the basement membrane protein fibronectin, a ligand of alpha(5)beta(1) integrin, or type IV collagen produced patchy sites of leakage instead of uniform labeling of vascular basement membrane. Differences in the kinetics of labeling, which for alpha(5)beta(1) integrin antibody was near maximal by 10 minutes but for the other antibodies gradually increased over 6 hours, indicated differences in accessibility of their respective targets. Isosurface rendering of confocal microscopic images was consistent with antibody binding to alpha(5)beta(1) integrin on the luminal surface of endothelial cells. Together, these findings indicate that the rapid accessibility of alpha(5)beta(1) integrin in RIP-Tag2 tumors results from overexpression of the integrin on the luminal surface of tumor vessels.

  13. Force measurements of the alpha5beta1 integrin-fibronectin interaction.

    PubMed

    Li, Feiya; Redick, Sambra D; Erickson, Harold P; Moy, Vincent T

    2003-02-01

    The interaction of the alpha(5)beta(1) integrin and its ligand, fibronectin (FN), plays a crucial role in the adhesion of cells to the extracellular matrix. An important intrinsic property of the alpha(5)beta(1)/FN interaction is the dynamic response of the complex to a pulling force. We have carried out atomic force microscopy measurements of the interaction between alpha(5)beta(1) and a fibronectin fragment derived from the seventh through tenth type III repeats of FN (i.e., FN7-10) containing both the arg-gly-asp (RGD) sequence and the synergy site. Direct force measurements obtained from an experimental system consisting of an alpha(5)beta(1) expressing K562 cell attached to the atomic force microscopy cantilever and FN7-10 adsorbed on a substrate were used to determine the dynamic response of the alpha(5)beta(1)/FN7-10 complex to a pulling force. The experiments were carried out over a three-orders-of-magnitude change in loading rate and under conditions that allowed for detection of individual alpha(5)beta(1)/FN7-10 interactions. The dynamic rupture force of the alpha(5)beta(1)/FN7-10 complex revealed two regimes of loading: a fast loading regime (>10,000 pN/s) and a slow loading regime (<10,000 pN/s) that characterize the inner and outer activation barriers of the complex, respectively. Activation by TS2/16 antibody increased both the frequency of adhesion and elevated the rupture force of the alpha(5)beta(1)/wild type FN7-10 complex to higher values in the slow loading regime. In experiments carried out with a FN7-10 RGD deleted mutant, the force measurements revealed that both inner and outer activation barriers were suppressed by the mutation. Mutations to the synergy site of FN, however, suppressed only the outer barrier activation of the complex. For both the RGD and synergy deletions, the frequency of adhesion was less than that of the wild type FN7-10, but was increased by integrin activation. The rupture force of these mutants was only slightly less

  14. Skeletal Phenotype of Transgenic Mice Expressing the Beta1 Integrin Cytoplasmic Tail In Osteoblasts

    NASA Technical Reports Server (NTRS)

    Globus, R. K.; vanderMeulen, M. C. H.; Damsky, D.; Kim, J.-B.; Amblard, D.; Amblard, D.; Nishimura, Y.; Almeida, E.; Iwaniec, U. T.; Wronski, T. J.; hide

    2002-01-01

    To define the physiologic role of beta1 integrin in bone formation and mechanical loading, transgenic mice were generated by expressing the cytoplasmic tall and transmembrane domain of Beta1 integrin under the control of the osteocalcin promoter. In cultured cells, this truncated fragment of Beta1 can act as a dominant negative. Previously, the matrix of calvariae was shown to be abnormal in transgenic (TG) compared to wildtype (WT) mice. In this study, we analyzed appendicular bone in TG and WT, male and female mice at 14, 35, 63, 90 and 365 days old (n=8-12/gp). To assess beta1 integrin function in mechanical loading, a pilot study using hindlimb unloading by tail suspension was performed. 35d old TG and WT females were hindlimb unloaded for 4 wks (n=3-5). Body mass, bone mineral content, histomorphometric (distal femur) and biomechanical parameters were analyzed. Statistical significance (P less than.05) was defined by ANOVA using the Tukey-Kramer post-hoc test. We confirmed transgene expression by immunoprecipitating then immunoblotting bone lysates using an antibody against the beta1 tail. Body masses of TG mice at 63, 90 and 365d old were greater (16-25%) than WT. Some TG female mice at 365d appeared obese; mean abdominal fat mass was 415% greater in TG than WT mice. Tibiae were longer (5-7%) in TG than WT mice at 63 and 90d. Tibial mineral mass of 35d males was 7% lower in TG than WT mice, but at 63d was 21% higher. The % osteoblast surface in 35d TG mice was 20% higher than WT, and at 63d was 17% lower, while % osteoclast surface did not differ. In 365d mice, cancellous bone volume (125%) and endocortical mineral apposition rate (40%) were greater in TG than WT males but not females. In WT mice, hindlimb unloading caused a reduction in mineral mass of tibiae (-20%) and lumbar vertebrae (-22%) relative to normally loaded controls. Surprisingly, hindlimb unloading also caused a relative reduction (-13%) in humerus mass. The effects of hindlimb unloading on

  15. Skeletal Phenotype of Transgenic Mice Expressing the Beta1 Integrin Cytoplasmic Tail In Osteoblasts

    NASA Technical Reports Server (NTRS)

    Globus, R. K.; vanderMeulen, M. C. H.; Damsky, D.; Kim, J.-B.; Amblard, D.; Amblard, D.; Nishimura, Y.; Almeida, E.; Iwaniec, U. T.; Wronski, T. J.; Dalton, Bonnie (Technical Monitor)

    2002-01-01

    To define the physiologic role of beta1 integrin in bone formation and mechanical loading, transgenic mice were generated by expressing the cytoplasmic tall and transmembrane domain of Beta1 integrin under the control of the osteocalcin promoter. In cultured cells, this truncated fragment of Beta1 can act as a dominant negative. Previously, the matrix of calvariae was shown to be abnormal in transgenic (TG) compared to wildtype (WT) mice. In this study, we analyzed appendicular bone in TG and WT, male and female mice at 14, 35, 63, 90 and 365 days old (n=8-12/gp). To assess beta1 integrin function in mechanical loading, a pilot study using hindlimb unloading by tail suspension was performed. 35d old TG and WT females were hindlimb unloaded for 4 wks (n=3-5). Body mass, bone mineral content, histomorphometric (distal femur) and biomechanical parameters were analyzed. Statistical significance (P less than.05) was defined by ANOVA using the Tukey-Kramer post-hoc test. We confirmed transgene expression by immunoprecipitating then immunoblotting bone lysates using an antibody against the beta1 tail. Body masses of TG mice at 63, 90 and 365d old were greater (16-25%) than WT. Some TG female mice at 365d appeared obese; mean abdominal fat mass was 415% greater in TG than WT mice. Tibiae were longer (5-7%) in TG than WT mice at 63 and 90d. Tibial mineral mass of 35d males was 7% lower in TG than WT mice, but at 63d was 21% higher. The % osteoblast surface in 35d TG mice was 20% higher than WT, and at 63d was 17% lower, while % osteoclast surface did not differ. In 365d mice, cancellous bone volume (125%) and endocortical mineral apposition rate (40%) were greater in TG than WT males but not females. In WT mice, hindlimb unloading caused a reduction in mineral mass of tibiae (-20%) and lumbar vertebrae (-22%) relative to normally loaded controls. Surprisingly, hindlimb unloading also caused a relative reduction (-13%) in humerus mass. The effects of hindlimb unloading on

  16. Chemokines fail to up-regulate beta 1 integrin-dependent adhesion in human Th2 T lymphocytes.

    PubMed

    Clissi, B; D'Ambrosio, D; Geginat, J; Colantonio, L; Morrot, A; Freshney, N W; Downward, J; Sinigaglia, F; Pardi, R

    2000-03-15

    Th1 and Th2 cells are functionally distinct subsets of CD4+ T lymphocytes whose tissue-specific homing to sites of inflammation is regulated in part by the differential expression of P- and E-selectin ligands and selected chemokine receptors. Here we investigated the expression and function of beta 1 integrins in Th1 and Th2 cells polarized in vitro. Th1 lymphocytes adhere transiently to the extracellular matrix ligands laminin 1 and fibronectin in response to chemokines such as RANTES and stromal cell-derived factor-1, and this process is paralleled by the activation of the Rac1 GTPase and by a rapid burst of actin polymerization. Selective inhibitors of phosphoinositide-3 kinase prevent efficiently all of the above processes, whereas the protein kinase C inhibitor bisindolylmaleimide prevents chemokine-induced adhesion without affecting Rac1 activation and actin polymerization. Notably, chemokine-induced adhesion to beta 1 integrin ligands is markedly reduced in Th2 cells. Such a defect cannot be explained by a reduced sensitivity to chemokine stimulation in this T cell subset, nor by a defective activation of the signaling cascade involving phosphoinositide-3 kinase, Rac1, and actin turnover, as all these processes are activated at comparable levels by chemokines in the two subsets. We propose that reduced beta 1 integrin-mediated adhesion in Th2 cells may restrain their ability to invade and/or reside in sites of chronic inflammation, which are characterized by thickening of basement membranes and extensive fibrosis, requiring efficient interaction with organized extracellular matrices.

  17. Isolation of a highly specific ligand for the alpha 5 beta 1 integrin from a phage display library

    PubMed Central

    1994-01-01

    Our previous studies showed that the alpha 5 beta 1 integrin selects cysteine pair-containing RGD peptides from a phage display library based on a random hexapeptide. We have therefore searched for more selective peptides for this integrin using a larger phage display library, where heptapeptides are flanked by cysteine residues, thus making the inserts potentially cyclic. Most of the phage sequences that bound to alpha 5 beta 1 (69 of 125) contained the RGD motif. Some of the heptapeptides contained an NGR motif. As the NGR sequence occurs in the cell-binding region of the fibronectin molecule, this sequence could contribute to the specific recognition of fibronectin by alpha 5 beta 1. Selection for high affinity peptides for alpha 5 beta 1 surprisingly yielded a sequence RRETAWA that does not bear obvious resemblance to known integrin ligand sequences. The synthetic cyclic peptide GACRRETAWACGA (*CRRETAWAC*) was a potent inhibitor of alpha 5 beta 1-mediated cell attachment to fibronectin. This peptide is nearly specific for the alpha 5 beta 1 integrin, because much higher concentrations were needed to inhibit the alpha v beta 1 integrin, and there was no effect on alpha v beta 3- and alpha v beta 5-mediated cell attachment to vitronectin. The peptide also did not bind to the alpha IIb beta 3 integrin. *CRRETAWAC* appears to interact with the same or an overlapping binding site in alpha 5 beta 1 as RGD, because cell attachment to *CRRETAWAC* coated on plastic was divalent cation dependent and could be blocked by an RGD-containing peptide. These results reveal a novel binding specificity in the alpha 5 beta 1 integrin. PMID:7507494

  18. Synergistic mobilization of hemopoietic progenitor cells using concurrent beta1 and beta2 integrin blockade or beta2-deficient mice.

    PubMed

    Papayannopoulou, T; Priestley, G V; Nakamoto, B; Zafiropoulos, V; Scott, L M; Harlan, J M

    2001-03-01

    The hierarchy of cytoadhesion molecules involved in hematopoietic/stem progenitor cell mobilization has not yet been delineated. Previous studies have suggested an important role for alpha4beta1 integrin in this process. To test whether mobilization involves dynamic interactions of alpha4beta1 with other integrins on hematopoietic cells, especially the beta2 integrins, mice and primates were treated with anti-beta1 or anti-beta2 antibodies alone or in combination. A single injection of anti-alpha4beta1 antibody elicited reproducible mobilization in contrast to other antibodies, and 3 injections yielded higher mobilization efficiency than each of the other antibodies. When the anti-beta2 (anti-CD11a or anti-CD18) or anti-alpha5/beta1 integrin antibody was combined with anti-alpha4, an augmentation in mobilization was seen that was either additive or synergistic, depending on the potency of the antibody used. Synergy between anti-alpha4 and anti-CD18 (beta(2)) antibody blockade was seen in primates and confirmed in anti-alpha4-treated CD18-deficient mice. In the latter, there was a 49-fold increase in mobilization with anti-alpha4, much higher than in littermate control animals, in CD18 hypomorphic mice, or in other strains of mice tested. Data from both the antibody blockade and gene-targeted mice suggest that the cooperativity of alpha4beta1 with beta2 integrins becomes evident when they are concurrently inhibited. It is unclear whether this cooperativity is exerted at the stage of reversible adhesion versus migration, and enhancement of and whether the 2 integrins work in a sequential or parallel manner. Whatever the mechanism, the data provide a novel example of beta1 and beta2 integrin crosstalk in stem/progenitor cell mobilization.

  19. Induction of a laminin isoform and alpha(3)beta(1)-integrin in renal ischemic injury and repair in vivo.

    PubMed

    Zuk, Anna; Matlin, Karl S

    2002-11-01

    Ischemic injury to the kidney, a major cause of acute renal failure, leads to the detachment and loss of numerous tubular epithelial cells. Integrin-laminin interactions may promote regeneration of the damaged epithelium by influencing kidney epithelial cell adhesion and differentiation. Laminins are major structural components of basement membranes. Of the various laminin isoforms, laminin-5 is of particular interest because of its proposed role in the healing of skin wounds. In this study, we investigate the expression of laminin-5 in rat kidney after unilateral ischemia. Using a polyclonal antibody generated against laminin-5, we find that immunostaining is confined to the basement membranes of collecting ducts in the papilla and the major and minor calyces in normal kidney. With injury and regeneration, however, immunostaining becomes much more intense and widespread in basement membranes along the nephron. Immunoblotting of ischemic kidney extracts reveals significantly increased expression of a polypeptide of approximately 220 kDa, possibly corresponding to a precursor of one of the three laminin-5 chains. Immunoblotting and immunostaining also demonstrate significantly increased expression and altered localization of the alpha(3)-integrin subunit, a receptor for laminin-5. These results indicate that there is induction of a laminin isoform, possibly laminin-5, and alpha(3)beta(1)-integrin in the ischemic kidney and may implicate this receptor-ligand combination in the pathogenesis of acute renal failure and/or repair of the injured kidney epithelium.

  20. Expression of estrogen receptor beta increases integrin alpha1 and integrin beta1 levels and enhances adhesion of breast cancer cells.

    PubMed

    Lindberg, Karolina; Ström, Anders; Lock, John G; Gustafsson, Jan-Ake; Haldosén, Lars-Arne; Helguero, Luisa A

    2010-01-01

    Estrogen effects on mammary gland development and differentiation are mediated by two receptors (ERalpha and ERbeta). Estrogen-bound ERalpha induces proliferation of mammary epithelial and cancer cells, while ERbeta is important for maintenance of the differentiated epithelium and inhibits proliferation in different cell systems. In addition, the normal breast contains higher ERbeta levels compared to the early stage breast cancers, suggesting that loss of ERbeta could be important in cancer development. Analysis of ERbeta-/- mice has consistently revealed reduced expression of cell adhesion proteins. As such, ERbeta is a candidate modulator of epithelial homeostasis and metastasis. Consequently, the aim of this study was to analyze estrogenic effects on adhesion of breast cancer cells expressing ERalpha and ERbeta. As ERbeta is widely found in breast cancer but not in cell lines, we used ERalpha positive T47-D and MCF-7 human breast cancer cells to generate cells with inducible ERbeta expression. Furthermore, the colon cancer cell lines SW480 and HT-29 were also used. Integrin alpha1 mRNA and protein levels increased following ERbeta expression. Integrin beta1-the unique partner for integrin alpha1-increased only at the protein level. ERbeta expression enhanced the formation of vinculin containing focal complexes and actin filaments, indicating a more adhesive potential. This was confirmed by adhesion assays where ERbeta increased adhesion to different extracellular matrix proteins, mostly laminin. In addition, ERbeta expression was associated to less cell migration. These results indicate that ERbeta affects integrin expression and clustering and consequently modulates adhesion and migration of breast cancer cells.

  1. Integrin alpha 3 beta 1 participates in the phagocytosis of extracellular matrix molecules by human breast cancer cells.

    PubMed Central

    Coopman, P J; Thomas, D M; Gehlsen, K R; Mueller, S C

    1996-01-01

    The mechanisms and receptors involved in phagocytosis by nonhematopoietic cells are not well understood. The involvement of the alpha 3 beta 1 integrin in phagocytosis of the extracellular matrix by human breast cancer cells was studied. The possible role of this integrin was suggested since alpha 3 and beta 1 but not alpha 2 subunits are concentrated at membrane sites where local degradation of fluorescently labeled gelatin occurs. Strikingly, anti-alpha 3 integrin monoclonal antibodies (mAbs) stimulate the phagocytosis of fluorescently labeled gelatin films, gelatin beads, and Matrigel films in a quantitative phagocytosis assay. Stimulation of the gelatin uptake by the anti-alpha 3 mAb is dose responsive, saturable, and time dependent. Antibodies against other integrin subunits have a lower stimulatory effect (anti-beta 1) or no significant effect (anti-alpha 2, -alpha 5, -alpha 6, and -alpha v) on gelatin phagocytosis. The synthetic HGD-6 human laminin peptide that binds specifically the alpha 3 beta 1 integrin, but not the scrambled HSGD-6 control peptide, also markedly stimulates gelatin uptake in a dose-responsive way. Furthermore, the stimulatory effects of the HGD-6 peptide and the anti-alpha 3 mAb are additive, suggesting that they might promote phagocytosis in different ways. Other laminin (YIGSR, IKVAV) and fibronectin (GRGDS) peptides have no effect on gelatin phagocytosis. Immunofluorescence shows that the alpha 3 and the beta 1, but not the alpha 2 integrin subunit, concentrate into patches on the cell surface after treatment with their respective mAbs. And, both gelatin and the alpha 3 beta 1 but not the alpha 2 beta 1 integrin are cointernalized and routed to acidic vesicles such as lysosomes. In conclusion, we demonstrate that human breast cancer cells locally degrade and phagocytose the extracellular matrix and show for the first time that the alpha 3 beta 1 integrin participates in this phagocytosis. We hypothesize that the anti-alpha 3

  2. Prostaglandin E2 suppresses beta1-integrin expression via E-prostanoid receptor in human monocytes/macrophages.

    PubMed

    Hasegawa, Shunji; Ichiyama, Takashi; Kohno, Fumitaka; Korenaga, Yuno; Ohsaki, Ayami; Hirano, Reiji; Haneda, Yasuhiro; Fukano, Reiji; Furukawa, Susumu

    2010-01-01

    Beta1-integrins mediate cell attachment to different extracellular matrix proteins, intracellular proteins, and intercellular adhesions. Recently, it has been reported that prostaglandin E2 (PGE2) has anti-inflammatory properties such as inhibition of the expression of adhesion molecules or production of chemokines. However, the effect of PGE2 on the expression of beta1-integrin remains unknown. In this study, we investigated the effects of PGE2 on the expression of beta1-integrin in the human monocytic cell line THP-1 and in CD14+ monocytes/macrophages in human peripheral blood. For this, we examined the role of four subtypes of PGE2 receptors and E-prostanoid (EP) receptors on PGE2-mediated inhibition. We found that PGE2 significantly inhibited the expression of beta1-integrin, mainly through EP4 receptors in THP-1 cells and CD14+ monocytes/macrophages in human peripheral blood. We suggest that PGE2 has anti-inflammatory effects, leading to the inhibited expression of beta1-integrin in human monocytes/macrophages, and that the EP4 receptor may play an important role in PGE2-mediated inhibition. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  3. The beta 1 integrin distal promoter is developmentally regulated in transgenic mice.

    PubMed

    Hirsch, E; Balzac, F; Pastore, C; Tarone, G; Silengo, L; Altruda, F

    1993-12-01

    Transgenic mice harbouring 5' flanking sequences of the human beta 1 integrin gene linked to the Escherichia coli lacZ gene have been generated to examine spatial and temporal distribution of the promoter activity during development. Our previous data showed that this regulatory region is composed by two promoters, called distal and proximal, located closely on the human genome. To determine the role of each promoter region during development we generated transgenic mice using these two sequences linked to the lacZ reporter gene. Their analysis shows that these two sequences, as determined by in vitro studies, have different efficiencies in promoting transcription. Actually mice carrying the proximal promoter region exhibit a weak lacZ expression resulting in an undetectable beta-galactosidase activity in both embryonic and adult tissues. On the other hand, transgenic mice carrying the distal promoter express beta-galactosidase at high efficiency during embryonic development. The pattern of transgene expression is consistent with the localization of beta 1 protein on mouse embryos evidenced by immunohistochemistry. Moreover the distal promoter is subjected to a temporal modulation since in adult transgenic mice lacZ expression decreases to a level detected only by RT-PCR analysis. We have determined a similar down-regulation analysing by Northern blot beta 1 mRNA in adult and embryonic organs such as heart and gut.

  4. A human integrin beta 1 subunit with a unique cytoplasmic domain generated by alternative mRNA processing.

    PubMed

    Altruda, F; Cervella, P; Tarone, G; Botta, C; Balzac, F; Stefanuto, G; Silengo, L

    1990-11-15

    The integrin subunit (beta 1) is common to a group of plasma membrane glycoprotein heterodimers that include the fibronectin, laminin and collagen receptors. These receptors span the plasma membrane, providing a transmembrane linkage between the extracellular matrix and the cytoskeleton. Here, we describe a variant of the human beta 1 differing from the previously described beta 1 in the cytoplasmic domain. The variant beta 1 transcript (beta 13'v) is present in different cell types and is synthesized at lower levels compared to the beta 1 mRNA. The cytoplasmic domain of the beta 13'v is characterized by a unique 12-amino acid C-terminal sequence. A Tyr residue present in this region, and known to be phosphorylated in the beta 1, is no longer part of a consensus sequence for phosphorylation by Tyr kinases. The integrin cytoplasmic domain anchors actin fibrils to the plasma membrane by interacting with cytoskeletal proteins such as talin and fibulin. The integrin beta 13'v with the variant cytoplasmic domain is likely to mediate a new type of membrane-cytoskeleton interaction during cell-cell and cell-matrix adhesion. Analysis of genomic clones showed that the new sequences of the variant mRNA are identical to an intron located between the last two exons of the beta 1 gene, indicating that the alternative message is generated either by premature transcription termination or by lack of splicing at this site.

  5. Integrins (alpha7beta1) in muscle function and survival. Disrupted expression in merosin-deficient congenital muscular dystrophy.

    PubMed Central

    Vachon, P H; Xu, H; Liu, L; Loechel, F; Hayashi, Y; Arahata, K; Reed, J C; Wewer, U M; Engvall, E

    1997-01-01

    Mutations in genes coding for dystrophin, for alpha, beta, gamma, and delta-sarcoglycans, or for the alpha2 chain of the basement membrane component merosin (laminin-2/4) cause various forms of muscular dystrophy. Analyses of integrins showed an abnormal expression and localization of alpha7beta1 isoforms in myofibers of merosin-deficient human patients and mice, but not in dystrophin-deficient or sarcoglycan-deficient humans and animals. It was shown previously that skeletal muscle fibers require merosin for survival and function (Vachon, P.H., F. Loechel, H. Xu, U.M. Wewer, and E. Engvall. 1996. J. Cell Biol. 134:1483-1497). Correction of merosin deficiency in vitro through cell transfection with the merosin alpha2 chain restored the normal localization of alpha7beta1D integrins as well as myotube survival. Overexpression of the apoptosis-suppressing molecule Bcl-2 also promoted the survival of merosin-deficient myotubes, but did not restore a normal expression of alpha7beta1D integrins. Blocking of beta1 integrins in normal myotubes induced apoptosis and severely reduced their survival. These findings (a) identify alpha7beta1D integrins as the de facto receptors for merosin in skeletal muscle; (b) indicate a merosin dependence for the accurate expression and membrane localization of alpha7beta1D integrins in myofibers; (c) provide a molecular basis for the critical role of merosin in myofiber survival; and (d) add new insights to the pathogenesis of neuromuscular disorders. PMID:9312189

  6. Galectin-8 binds specific beta1 integrins and induces polarized spreading highlighted by asymmetric lamellipodia in Jurkat T cells.

    PubMed

    Cárcamo, Claudia; Pardo, Evelyn; Oyanadel, Claudia; Bravo-Zehnder, Marcela; Bull, Paulina; Cáceres, Mónica; Martínez, Jorge; Massardo, Loreto; Jacobelli, Sergio; González, Alfonso; Soza, Andrea

    2006-02-15

    Integrin-mediated encounters of T cells with extracellular cues lead these cells to adhere to a variety of substrates and acquire a spread phenotype needed for their tissue incursions. We studied the effects of galectin-8 (Gal-8), a beta-galactoside binding lectin, on Jurkat T cells. Immobilized Gal-8 bound alpha1beta1, alpha3beta1 and alpha5beta1 but not alpha2beta1 and alpha4beta1 and adhered these cells with similar kinetics to immobilized fibronectin (FN). Function-blocking experiments with monoclonal anti-integrin antibodies suggested that alpha5beta1 is the main mediator of cell adhesion to this lectin. Gal-8, but not FN, induced extensive cell spreading frequently leading to a polarized phenotype characterized by an asymmetric lamellipodial protrusion. These morphological changes involved actin cytoskeletal rearrangements controlled by PI3K, Rac-1 and ERK1/2 activity. Gal-8-induced Rac-1 activation and binding to alpha1 and alpha5 integrins have not been described in any other cellular system. Strikingly, Gal-8 was also a strong stimulus on Jurkat cells in suspension, triggering ERK1/2 activation that in most adherent cells is instead dependent on cell attachment. In addition, we found that patients with systemic lupus erythematosus (SLE), a prototypic autoimmune disorder, produce Gal-8 autoantibodies that impede both its binding to integrins and cell adhesion. These are the first function-blocking autoantibodies reported for a member of the galectin family. These results indicate that Gal-8 constitutes a novel extracellular stimulus for T cells, able to bind specific beta1 integrins and to trigger signaling pathways conducive to cell spreading. Gal-8 could modulate a wide range of T cell-driven immune processes that eventually become altered in autoimmune disorders.

  7. Laminin on Toxoplasma gondii mediates parasite binding to the beta 1 integrin receptor alpha 6 beta 1 on human foreskin fibroblasts and Chinese hamster ovary cells.

    PubMed

    Furtado, G C; Cao, Y; Joiner, K A

    1992-11-01

    We investigated the role of parasite-bound laminin and the host cell beta 1 integrin receptors for this extracellular matrix protein in Toxoplasma gondii binding to fibroblasts. Laminin but not fibronectin was detected on extracellular tachyzoites by immunofluorescence and immunoblotting. Binding of parasites to CHO cells was inhibited by polyclonal antibodies to laminin and by a monoclonal antibody directed against the globular carboxyl-terminal portion of the long arm of laminin (at or near the suggested ligand-binding sites for alpha 3 beta 1 and alpha 6 beta 1), but not by a monoclonal antibody directed against the lateral short arms of laminin near the cross region of the molecule. Antibodies to the alpha 6 but not the alpha 2, alpha 3, or alpha 5 chains of the beta 1 family of integrins blocked parasite attachment to human foreskin fibroblasts and CHO cells. Attachment of T. gondii to cells via laminin on the parasite surface and laminin receptors on the mammalian cell is consistent with the capacity of the parasite to invade almost all nucleated cells.

  8. Laminin isoforms and their integrin receptors in glioma cell migration and invasiveness: Evidence for a role of alpha5-laminin(s) and alpha3beta1 integrin.

    PubMed

    Kawataki, Tomoyuki; Yamane, Tetsu; Naganuma, Hirofumi; Rousselle, Patricia; Andurén, Ingegerd; Tryggvason, Karl; Patarroyo, Manuel

    2007-11-01

    Glioma cell infiltration of brain tissue often occurs along the basement membrane (BM) of blood vessels. In the present study we have investigated the role of laminins, major structural components of BMs and strong promoters of cell migration. Immunohistochemical studies of glioma tumor tissue demonstrated expression of alpha2-, alpha3-, alpha4- and alpha5-, but not alpha1-, laminins by the tumor vasculature. In functional assays, alpha3 (Lm-332/laminin-5)- and alpha5 (Lm-511/laminin-10)-laminins strongly promoted migration of all glioma cell lines tested. alpha1-Laminin (Lm-111/laminin-1) displayed lower activity, whereas alpha2 (Lm-211/laminin-2)- and alpha4 (Lm-411/laminin-8)-laminins were practically inactive. Global integrin phenotyping identified alpha3beta1 as the most abundant integrin in all the glioma cell lines, and this laminin-binding integrin exclusively or largely mediate the cell migration. Moreover, pretreatment of U251 glioma cells with blocking antibodies to alpha3beta1 integrin followed by intracerebral injection into nude mice inhibited invasion of the tumor cells into the brain tissue. The cell lines secreted Lm-211, Lm-411 and Lm-511, at different ratios. The results indicate that glioma cells secrete alpha2-, alpha4- and alpha5-laminins and that alpha3- and alpha5-laminins, found in brain vasculature, selectively promote glioma cell migration. They identify alpha3beta1 as the predominant integrin and laminin receptor in glioma cells, and as a brain invasion-mediating integrin.

  9. Cooperative signaling by alpha 5 beta 1 and alpha 4 beta 1 integrins regulates metalloproteinase gene expression in fibroblasts adhering to fibronectin

    PubMed Central

    1995-01-01

    Rabbit synovial fibroblasts (RSF) express basal levels of the metalloproteinases (MMP) collagenase, stromelysin-1 and 92-kD gelatinase when plated on intact fibronectin (FN), but elevated levels when plated on either the central RGD-containing cell-binding region of FN (120FN) or antibody against the alpha 5 beta 1 integrin, suggesting that domains outside 120FN may suppress the induction of MMP (Werb, Z., P. M. Tremble, O. Behrendtsen, E. Crowley, and C.H. Damsky. 1989. J. Cell Biol. 109:877-889). We therefore attempted to reconstitute the basal signaling of intact FN by plating RSF on 120FN together with domains of FN outside this region. Large COOH-terminal fragments containing both the heparin-binding and HICS domains suppressed MMP when combined with 120FN. To map the active sequences, peptides from this region and larger fragments that did, or did not, include the CS-1 portion of IIICS were tested. Only CS-1 peptide, or larger fragments containing CS-1, suppressed MMP expression induced by 120FN. In contrast, peptide V from the heparin-binding region, shown previously to stimulate focal contact formation, further enhanced MMP expression by RSF when present on the substrate with 120FN. RSF expressed alpha 4 beta 1 integrin, the receptor for CS-1, and the anti-alpha 4 mAb blocked the ability of CS-1 to suppress MMP induction by 120FN. These results show that signals modulating MMP expression and focal contact assembly are regulated independently, and that cooperative signaling by alpha 5 beta 1 and alpha 4 beta 1 integrins plays a dominant role in regulating expression of these extracellular matrix-remodeling genes in response to FN. This work demonstrates directly the modular way in which information in the extracellular matrix is detected and processed by cell surface receptors. PMID:7537277

  10. Activation of the alpha 4 beta 1 integrin through the beta 1 subunit induces recognition of the RGDS sequence in fibronectin

    PubMed Central

    1994-01-01

    Lymphocyte attachment to fibronectin is mainly mediated by the interaction of alpha 5 beta 1 and alpha 4 beta 1 integrins with the RGD and CS-1/Hep II sites, respectively. We have recently shown that the anti-beta 1 mAb TS2/16 can convert the partly active alpha 4 beta 1 present on certain hemopoietic cells that recognizes CS-1 but not Hep II, to a high avidity form that binds both ligands. In this report we have studied whether mAb TS2/16 also affects alpha 4 beta 1 ligand specificity. Incubation of the B cell lines Ramos and Daudi (which lack alpha 5 beta 1) with mAb TS2/16 induced specific attachment to an 80-kD fragment which lacks CS-1 and Hep II and contains the RGD sequence. mAbs anti-alpha 4 and the synthetic peptides CS-1 and IDAPS inhibited adhesion to the 80-kD fragment thus implying alpha 4 beta 1 as the receptor for this fragment. Interestingly, the synthetic peptide GRGDSPC and a 15-kD peptic fibronectin fragment containing the RGD sequence also inhibited B cell adhesion to the 80-kD fragment. Because we have previously shown that RGD peptides do not affect the constitutive function of alpha 4 beta 1, we tested whether TS2/16- activated alpha 4 beta 1 acquired the capacity to recognize RGD. Indeed RGD peptides inhibited TS2/16-treated B cell adhesion to a 38-kD fragment containing CS-1 and Hep II but did not affect binding of untreated cells to this fragment. An anti-fibronectin mAb reactive with an epitope on or near the RGD sequence also efficiently inhibited cell adhesion to the 80-kD fragment, indicating that the RGD sequence is a novel adhesive ligand for activated alpha 4 beta 1. These results emphasize the role of alpha 4 beta 1 as a receptor with different ligand specificities according to the activation state, a fact that may be important for lymphocyte migration, localization, and function. PMID:7517944

  11. The alpha2beta1 integrin inhibitor rhodocetin binds to the A-domain of the integrin alpha2 subunit proximal to the collagen-binding site.

    PubMed Central

    Eble, Johannes A; Tuckwell, Danny S

    2003-01-01

    Rhodocetin is a snake venom protein that binds to alpha2beta1 integrin, inhibiting its interaction with its endogenous ligand collagen. We have determined the mechanism by which rhodocetin inhibits the function of alpha2beta1. The interaction of alpha2beta1 with collagen and rhodocetin differed: Ca(2+) ions and slightly acidic pH values increased the binding of alpha2beta1 integrin to rhodocetin in contrast with their attenuating effect on collagen binding, suggesting that rhodocetin preferentially binds to a less active conformation of alpha2beta1 integrin. The alpha2A-domain [von Willebrand factor domain A homology domain (A-domain) of the integrin alpha2 subunit] is the major site for collagen binding to alpha2beta1. Recombinant alpha2A-domain bound rhodocetin, demonstrating that the A-domain is also the rhodocetin-binding domain. Although the interaction of alpha2beta1 with rhodocetin is affected by altering divalent cations, the interaction of the A-domain was divalent-cation-independent. The rhodocetin-binding site on the alpha2A-domain was mapped first by identifying an anti-alpha2 antibody that blocked rhodocetin binding and then mapping the epitope of the antibody using human-mouse alpha2A-domain chimaeras; and secondly, by binding studies with alpha2A-domain, which bear point mutations in the vicinity of the mapped epitope. In this way, the rhodocetin-binding site was identified as the alpha3-alpha4 loop plus adjacent alpha-helices. This region is known to form part of the collagen-binding site, thus attaining a mainly competitive mode of inhibition by rhodocetin. PMID:12871211

  12. A small-molecule inhibitor of integrin alpha2 beta1 introduces a new strategy for antithrombotic therapy.

    PubMed

    Nissinen, L; Pentikäinen, O T; Jouppila, A; Käpylä, J; Ojala, M; Nieminen, J; Lipsanen, A; Lappalainen, H; Eckes, B; Johnson, M S; Lassila, R; Marjamäki, A; Heino, J

    2010-02-01

    Interaction of blood platelets with vascular collagen is an initiating event in haemostasis and thrombus formation. Based on molecular modelling of human integrin alpha2I domain and cell-based screening assays we have developed sulfonamide derivatives, a mechanistically novel class of molecules. These molecules show antiplatelet efficacy by selectively inhibiting alpha2beta1 integrin-mediated collagen binding. One sulfonamide derivative, named BTT-3016, showed inhibitory capacity in several assessments of human platelet interaction with collagen. It inhibited about 90% of the aggregation of gel-filtered magnesium-supplemented platelets and 70% of aggregation in PPACK-anticoagulated platelet-rich plasma when stimulated with collagen but not with ADP. The antiplatelet activity of BTT-3016 was dependent on alpha2beta1 integrin, since in collagen binding test BTT-3016 had no effect on the platelets derived from alpha2 integrin null mice. When tested in an in vivo model in mice, BTT-3016 clearly reduced thrombus formation on the vessel wall after vascular injury. Furthermore, BTT-3016 prolonged tail-bleeding time in a manner comparable to aspirin. We show that new alpha2beta1 inhibitors exert collagen-specific antiplatelet activity and regulate thrombus growth in vivo without compromising primary haemostasis more than aspirin. We suggest that the alpha2beta1 inhibiting strategy could be further developed for the prevention and treatment of arterial thrombosis.

  13. Integrin {beta}1-dependent invasive migration of irradiation-tolerant human lung adenocarcinoma cells in 3D collagen matrix

    SciTech Connect

    Ishihara, Seiichiro; Haga, Hisashi; Yasuda, Motoaki; Mizutani, Takeomi; Kawabata, Kazushige; Shirato, Hiroki; Nishioka, Takeshi

    2010-06-04

    Radiotherapy is one of the effective therapies used for treating various malignant tumors. However, the emergence of tolerant cells after irradiation remains problematic due to their high metastatic ability, sometimes indicative of poor prognosis. In this study, we showed that subcloned human lung adenocarcinoma cells (A549P-3) that are irradiation-tolerant indicate high invasive activity in vitro, and exhibit an integrin {beta}1 activity-dependent migratory pattern. In collagen gel overlay assay, majority of the A549P-3 cells displayed round morphology and low migration activity, whereas a considerable number of A549P-3IR cells surviving irradiation displayed a spindle morphology and high migration rate. Blocking integrin {beta}1 activity reduced the migration rate of A549P-3IR cells and altered the cell morphology allowing them to assume a round shape. These results suggest that the A549P-3 cells surviving irradiation acquire a highly invasive integrin {beta}1-dependent phenotype, and integrin {beta}1 might be a potentially effective therapeutic target in combination with radiotherapy.

  14. Mechanotransduction molecules in the plant gravisensory response: amyloplast/statolith membranes contain a beta 1 integrin-like protein

    NASA Technical Reports Server (NTRS)

    Lynch, T. M.; Lintilhac, P. M.; Domozych, D.

    1998-01-01

    It has been hypothesized that the sedimentation of amyloplasts within root cap cells is the primary event in the plant gravisensory-signal transduction cascade. Statolith sedimentation, with its ability to generate weighty mechanical signals, is a legitimate means for organisms to discriminate the direction of the gravity vector. However, it has been demonstrated that starchless mutants with reduced statolith densities maintain some ability to sense gravity, calling into question the statolith sedimentation hypothesis. Here we report on the presence of a beta 1 integrin-like protein localized inside amyloplasts of tobacco NT-1 suspension culture, callus cells, and whole-root caps. Two different antibodies to the beta 1 integrin, one to the cytoplasmic domain and one to the extracellular domain, localize in the vicinity of the starch grains within amyloplasts of NT-1. Biochemical data reveals a 110-kDa protein immunoprecipitated from membrane fractions of NT-1 suspension culture indicating size homology to known beta 1 integrin in animals. This study provides the first direct evidence for the possibility of integrin-mediated signal transduction in the perception of gravity by higher plants. An integrin-mediated pathway, initiated by starch grain sedimentation within the amyloplast, may provide the signal amplification necessary to explain the gravitropic response in starch-depleted cultivars.

  15. Mechanotransduction molecules in the plant gravisensory response: amyloplast/statolith membranes contain a beta 1 integrin-like protein

    NASA Technical Reports Server (NTRS)

    Lynch, T. M.; Lintilhac, P. M.; Domozych, D.

    1998-01-01

    It has been hypothesized that the sedimentation of amyloplasts within root cap cells is the primary event in the plant gravisensory-signal transduction cascade. Statolith sedimentation, with its ability to generate weighty mechanical signals, is a legitimate means for organisms to discriminate the direction of the gravity vector. However, it has been demonstrated that starchless mutants with reduced statolith densities maintain some ability to sense gravity, calling into question the statolith sedimentation hypothesis. Here we report on the presence of a beta 1 integrin-like protein localized inside amyloplasts of tobacco NT-1 suspension culture, callus cells, and whole-root caps. Two different antibodies to the beta 1 integrin, one to the cytoplasmic domain and one to the extracellular domain, localize in the vicinity of the starch grains within amyloplasts of NT-1. Biochemical data reveals a 110-kDa protein immunoprecipitated from membrane fractions of NT-1 suspension culture indicating size homology to known beta 1 integrin in animals. This study provides the first direct evidence for the possibility of integrin-mediated signal transduction in the perception of gravity by higher plants. An integrin-mediated pathway, initiated by starch grain sedimentation within the amyloplast, may provide the signal amplification necessary to explain the gravitropic response in starch-depleted cultivars.

  16. Reciprocal interactions between Beta1-integrin and epidermal growth factor in three-dimensional basement membrane breast cultures: A different perspective in epithelial biology

    SciTech Connect

    Wang, F.; Weaver, V.M.; Petersen, O.W.; Larabell, C.A.; Dedhar, S.; Briand, P.; Lupu, R.; Bissell, M.J.

    1998-09-30

    Anchorage and growth factor independence are cardinal features of the transformed phenotype. Although it is logical that the two pathways must be coregulated in normal tissues to maintain homeostasis, this has not been demonstrated directly. We showed previously that down-modulation of {beta}1-integrin signaling reverted the malignant behavior of a human breast tumor cell line (T4-2) derived from phenotypically normal cells (HMT-3522) and led to growth arrest in a threedimensional (3D) basement membrane assay in which the cells formed tissue-like acini (14). Here, we show that there is a bidirectional cross-modulation of {beta}1-integrin and epidermal growth factor receptor (EGFR) signaling via the mitogenactivated protein kinase (MAPK) pathway. The reciprocal modulation does not occur in monolayer (2D) cultures. Antibodymediated inhibition of either of these receptors in the tumor cells, or inhibition of MAPK kinase, induced a concomitant downregulation of both receptors, followed by growth-arrest and restoration of normal breast tissue morphogenesis. Crossmodulation and tissue morphogenesis were associated with attenuation of EGF-induced transient MAPK activation. To specifically test EGFR and {beta}1-integrin interdependency, EGFR was overexpressed in nonmalignant cells, leading to disruption of morphogenesis and a compensatory up-regulation of {beta}1-integrin expression, again only in 3D. Our results indicate that when breast cells are spatially organized as a result of contact with basement membrane, the signaling pathways become coupled and bidirectional. They further explain why breast cells fail to differentiate in monolayer cultures in which these events are mostly uncoupled. Moreover, in a subset of tumor cells in which these pathways are misregulated but functional, the cells could be 'normalized' by manipulating either pathway.

  17. Distinct determinants on collagen support alpha 2 beta 1 integrin-mediated platelet adhesion and platelet activation.

    PubMed Central

    Santoro, S A; Walsh, J J; Staatz, W D; Baranski, K J

    1991-01-01

    Recent studies have revealed that the sequence of amino acids asp-gly-glu-ala represents an essential determinant of the site within the alpha 1(I)-CB3 fragment of collagen recognized by the alpha 2 beta 1 integrin cell surface collagen receptor (Staatz et al., 1991). Studies employing chemical modifications of collagen amino acid side chains confirm both the essential nature of the acidic side chains of aspartic acid and glutamic acid residues and the nonessentiality of lysine epsilon-amino groups in supporting adhesion mediated by the alpha 2 beta 1 integrin. The approach also indicates the presence of a distinct determinant on collagen separate from the alpha 2 beta 1 recognition site that contains essential lysine side chains and that is necessary for subsequent interactions with the platelet surface that give rise to collagen-induced platelet activation and secretion. The two-step, two-site model for cellular signaling involving both an integrin and a signal-transducing coreceptor suggested by these data may be common to other integrin-mediated processes. PMID:1809397

  18. Extravillous trophoblast-associated ADAM12 exerts pro-invasive properties, including induction of integrin beta 1-mediated cellular spreading.

    PubMed

    Biadasiewicz, Katarzyna; Fock, Valerie; Dekan, Sabine; Proestling, Katharina; Velicky, Philipp; Haider, Sandra; Knöfler, Martin; Fröhlich, Camilla; Pollheimer, Jürgen

    2014-05-01

    ADAM12, consisting of a membrane-bound (ADAM12L) and a secreted (ADAM12S) form, is expressed exclusively in regenerating and developing tissue as well as in certain cancer types. Strong ADAM12 expression levels have been noticed in the human placenta, and deregulated ADAM12S levels were associated with various pregnancy-related disorders including pre-eclampsia and intrauterine growth restriction. However, the role of ADAM12 in trophoblast motility has not been investigated so far. Hence, the present study aimed to investigate the specific function of the protease by using different primary trophoblast cell models. Immunofluorescence and Western blot analyses of first trimester placental tissue and differentiating primary first trimester cytotrophoblasts (CTBs) indicated strong upregulation of both of the ADAM12 isoforms during extravillous trophoblast differentiation. Functional assays involving short interfering RNA (siRNA)-mediated knockdown studies in primary CTBs and first trimester explant cultures revealed a significant repression of trophoblast motility upon partial loss of ADAM12. Conversely, isoform-specific overexpression in the ADAM12-negative trophoblast cell line SGHPL-5 enhanced the invasive capacity of these cells. We further confirmed proteolytic activity of trophoblast-derived ADAM12S by demonstrating its potential to degrade insulin-like growth factor-binding protein 3. Finally, we suggest that ADAM12S exerts its pro-migratory function in trophoblasts by inducing integrin beta 1-mediated cellular spreading.

  19. The alpha 4 integrin chain is a ligand for alpha 4 beta 7 and alpha 4 beta 1

    PubMed Central

    1995-01-01

    The heterodimeric alpha 4 integrins alpha 4 beta 7 lymphocyte Peyer's patch adhesion molecule ([LPAM]-1) and alpha 4 beta 1 (very late antigen-4) are cell surface adhesion molecules involved in lymphocyte trafficking and lymphocyte-cell and matrix interactions. Known cellular ligands include vascular cell adhesion molecule (VCAM)-1, which binds to alpha 4 beta 1 and alpha 4 beta 7, and the mucosal addressin cell adhesion molecule (MAdCAM)-1, which binds to alpha 4 beta 7. Here we show that the alpha 4 chain of these integrins can itself serve as a ligand. The alpha 4 chain, immunoaffinity purified and immobilized on glass slides, binds thymocytes and T lymphocytes. Binding exhibits divalent cation requirements and temperature sensitivity which are characteristic of integrin-mediated interactions, and is specifically inhibited by anti-alpha 4 integrin antibodies, which exert their effect at the cell surface. Cells expressing exclusively alpha 4 beta 7 (TK-1) or alpha 4 beta 1 (L1-2) both bound avidly, whereas alpha 4-negative cells did not. A soluble 34-kD alpha 4 chain fragment retained binding activity, and it inhibited lymphocyte adhesion to alpha 4 ligands. It has been shown that alpha 4 integrin binding to fibronectin involves an leucine-aspartic acid-valine (LDV) motif in the HepII/IIICS region of fibronectin (CS-1 peptide), and homologous sequences are important in binding to VCAM-1 and MAdCAM-1. Three conserved LDV motifs occur in the extracellular sequence of alpha 4. A synthetic LDV-containing alpha 4- derived oligopeptide supports alpha 4-integrin-dependent lymphocyte adhesion and blocks binding to the 34-kD alpha 4 chain fragment. Our results suggest that alpha 4 beta 7 and alpha 4 beta 1 integrins may be able to bind to the alpha 4 subunit on adjacent cells, providing a novel mechanism for alpha 4 integrin-mediated and activation-regulated lymphocyte interactions during immune responses. PMID:7629498

  20. Alpha1beta1 integrin is crucial for accumulation of epidermal T cells and the development of psoriasis.

    PubMed

    Conrad, Curdin; Boyman, Onur; Tonel, Giulia; Tun-Kyi, Adrian; Laggner, Ute; de Fougerolles, Antonin; Kotelianski, Victor; Gardner, Humphrey; Nestle, Frank O

    2007-07-01

    Psoriasis is a common T cell-mediated autoimmune inflammatory disease. We show that blocking the interaction of alpha1beta1 integrin (VLA-1) with collagen prevented accumulation of epidermal T cells and immunopathology of psoriasis. Alpha1beta1 integrin, a major collagen-binding surface receptor, was exclusively expressed by epidermal but not dermal T cells. Alpha1beta1-positive T cells showed characteristic surface markers of effector memory cells and contained high levels of interferon-gamma but not interleukin-4. Blockade of alpha1beta1 inhibited migration of T cells into the epidermis in a clinically relevant xenotransplantation model. This was paralleled by a complete inhibition of psoriasis development, comparable to that caused by tumor necrosis factor-alpha blockers. These results define a crucial role for alpha1beta1 in controlling the accumulation of epidermal type 1 polarized effector memory T cells in a common human immunopathology and provide the basis for new strategies in psoriasis treatment focusing on T cell-extracellular matrix interactions.

  1. Regulation of the L-type calcium channel by alpha 5beta 1 integrin requires signaling between focal adhesion proteins.

    PubMed

    Wu, X; Davis, G E; Meininger, G A; Wilson, E; Davis, M J

    2001-08-10

    The L-type calcium channel is the major calcium influx pathway in vascular smooth muscle and is regulated by integrin ligands, suggesting an important link between extracellular matrix and vascular tone regulation in tissue injury and remodeling. We examined the role of integrin-linked tyrosine kinases and focal adhesion proteins in regulation of L-type calcium current in single vascular myocytes. Soluble tyrosine kinase inhibitors blocked the increase in current produced by alpha(5) integrin antibody or fibronectin, whereas tyrosine phosphatase inhibition enhanced the effect. Cell dialysis with an antibody to focal adhesion kinase or with FRNK, the C-terminal noncatalytic domain of focal adhesion kinase, produced moderate (24 or 18%, respectively) inhibition of basal current but much greater inhibition (63 or 68%, respectively) of integrin-enhanced current. A c-Src antibody and peptide inhibitors of the Src homology-2 domain or a putative Src tyrosine phosphorylation site on the channel produced similar inhibition. Antibodies to the cytoskeletal proteins paxillin and vinculin, but not alpha-actinin, inhibited integrin-dependent current by 65-80%. Therefore, alpha(5)beta(1) integrin appears to regulate a tyrosine phosphorylation cascade involving Src and various focal adhesion proteins that control the function of the L-type calcium channel. This interaction may represent a novel mechanism for control of calcium influx in vascular smooth muscle and other cell types.

  2. Alpha 1 beta 1 integrin on neural crest cells recognizes some laminin substrata in a Ca(2+)-independent manner

    PubMed Central

    1992-01-01

    Neural crest cells migrate along pathways containing laminin and other extracellular matrix molecules. In the present study, we functionally and biochemically identify an alpha 1 beta 1 integrin heterodimer which bears the HNK-1 epitope on neural crest cells. Using a quantitative cell adhesion assay, we find that this heterodimer mediates attachment to laminin substrata prepared in the presence of Ca2+. Interestingly, neural crest cells bind to laminin-Ca2+ substrata in the presence or absence of divalent cations in the cell attachment medium. In contrast, the attachment of neural crest cells to laminin substrata prepared in the presence of EDTA, heparin, Mg2+, or Mn2+ requires divalent cations. Interactions with these laminin substrata are mediated by a different integrin heterodimer, since antibodies against beta 1 but not alpha 1 integrins inhibit neural crest cell attachment. Thus, the type of laminin substratum appears to dictate the choice of laminin receptor used by neural crest cells. The laminin conformation is determined by the ratio of laminin to Ca2+, though incorporation of heparin during substratum polymerization alters the conformation even in the presence of Ca2+. Once polymerized, the substratum appears stable, not being altered by soaking in either EDTA or divalent cations. Our findings demonstrate: (a) that the alpha 1 beta 1 integrin can bind to some forms of laminin in the absence of soluble divalent cations; (b) that substratum preparation conditions alter the conformation of laminin such that plating laminin in the presence of Ca2+ and/or heparin modulates its configuration; and (c) that neural crest cells utilize different integrins to recognize different laminin conformations. PMID:1280273

  3. Aberrant mucosal mast cell protease expression in the enteric epithelium of nematode-infected mice lacking the integrin alphavbeta6, a transforming growth factor-beta1 activator.

    PubMed

    Knight, Pamela A; Brown, Jeremy K; Wright, Steven H; Thornton, Elisabeth M; Pate, Judith A; Miller, Hugh R P

    2007-10-01

    Infection of mice with the nematode Trichinella spiralis triggers recruitment and differentiation of intraepithelial intestinal mucosal mast cells expressing mouse mast cell protease 1 (Mcpt-1), which contributes to expulsion of the parasite. Expression of Mcpt-1 is transforming growth factor (TGF)-beta1-dependent in vitro. TGF-beta1, which is secreted within tissues as a biologically inactive complex with latency-associated peptide, requires extracellular modification to become functionally active. The integrin-alpha(nu)beta(6) mediates local activation of TGF-beta(1) in association with epithelia. Using T. spiralis-infected beta(6)(-/-) mice, we show accumulation of mucosal mast cells in the lamina propria of the small intestine with minimal recruitment into the epithelial compartment. This was accompanied by a coordinate reduction in expression of both Mcpt-1 and -2 in the jejunum and increased tryptase expression, whereas Mcpt-9 became completely undetectable. In contrast, the cytokine stem cell factor, a regulator of mast cell differentiation and survival, was significantly up-regulated in T. spiralis-infected beta(6)(-/-) mice compared with infected beta(6)(+/+) controls. Despite these changes, beta(6)(-/-) mice still appeared to expel the worms normally. We postulate that compromised TGF-beta(1) activation within the gastrointestinal epithelial compartment is a major, but not the only, contributing factor to the observed changes in mucosal mast cell protease and epithelial cytokine expression in beta(6)(-/-) mice.

  4. Regulation and function of an activation-dependent epitope of the beta 1 integrins in vascular cells after balloon injury in baboon arteries and in vitro.

    PubMed Central

    Koyama, N.; Seki, J.; Vergel, S.; Mattsson, E. J.; Yednock, T.; Kovach, N. L.; Harlan, J. M.; Clowes, A. W.

    1996-01-01

    Migration and proliferation of endothelial cells (ECs) and smooth muscle cells (SMCs) contribute to the response to injury in damaged and atherosclerotic vessels. These events might be regulated by cellular interactions with extracellular matrix through the expression and activation of integrins. To study the functions of beta 1 integrins in the vessel wall, we used monoclonal antibody (MAb) 15/7, which recognizes an activation epitope of beta 1 integrin subunits, and MAb 8A2, which induces a high affinity form of beta 1 integrins recognized by MAb 15/7. Immunohistochemical analyses were done on samples of normal baboon saphenous arteries and from arteries subjected to balloon injury. EC and SMC expressed the activation epitope of beta 1 integrin in uninjured arteries. By contrast, in balloon-injured arteries 6 weeks after injury, regenerating EC did not express the activation epitope, and there was no decrease in the expression of total beta 1 integrin, whereas SMC migrating into the intima exhibited decreased expression of the total and activated beta 1 integrin. Flow cytometer analysis of cultured cells indicated that baboon EC and SMC weakly express the activation epitope of beta 1 integrin. Next, we determined by utilizing MAb 8A2 the effects of increased expression of activation epitope of beta 1 integrin on the functions of SMC and EC. The activation of beta 1 integrins on SMC induced by MAb 8A2 enhanced SMC adhesion and suppressed SMC migration in a Boyden chamber assay. SMC proliferation was inhibited by MAb 8A2 dose-dependently. Similarly, MAb 8A2-induced activation of beta 1 integrins on EC suppressed EC migration into a wound. However, MAb 8A2 did not affect the basic fibroblast growth factor-induced proliferation of EC, although it blocked the decrease in EC number caused by the removal of basic fibroblast growth factor. These results suggest that activation of beta 1 integrins in vascular cells is regulated in a cell-type dependent manner and plays an

  5. Activation of the platelet collagen receptor integrin alpha(2)beta(1): its mechanism and participation in the physiological functions of platelets.

    PubMed

    Jung, S M; Moroi, M

    2000-10-01

    When platelets are stimulated by agonists, integrin alpha(2)beta(1) (GP Ia/IIa), one of the platelet collagen receptors, is activated to forms with high affinities for its ligand collagen. Here we describe our studies to characterize the binding kinetics of the activated integrin forms and the activation mechanism. Under low agonist concentrations, integrin alpha(2)beta(1) is activated through a mechanism involving ADP/ADP receptors; and under high agonist concentrations, multiple signaling pathways are involved in its activation. Such differences in mechanism at low and high agonist concentrations are also suggested in the activation of integrin alpha(IIb)beta(3), the platelet fibrinogen receptor. We describe our flow adhesion studies, from which evidence was obtained about the involvement of integrin alpha(2)beta(1) activation in the physiological function of platelets, adhesion and thrombus formation.

  6. Inhibiting Vimentin or beta 1-integrin Reverts Prostate Tumor Cells in IrECM and Reduces Tumor Growth

    SciTech Connect

    Zhang, Xueping; Fournier, Marcia V.; Ware, Joy L.; Bissell, Mina J.; Zehner, Zendra E.

    2009-07-27

    Prostate epithelial cells grown embedded in laminin-rich extracellular matrix (lrECM) undergo morphological changes that closely resemble their architecture in vivo. In this study, growth characteristics of three human prostate epithelial sublines derived from the same cellular lineage, but displaying different tumorigenic and metastatic properties in vivo, were assessed in three-dimensional (3D) lrECM gels. M12, a highly tumorigenic and metastatic subline, was derived from the parental prostate epithelial P69 cell line by selection in nude mice and found to contain a deletion of 19p-q13.1. The stable reintroduction of an intact human chromosome 19 into M12 resulted in a poorly tumorigenic subline, designated F6. When embedded in lrECM gels, the nontumorigenic P69 line produced acini with clearly defined lumena. Immunostaining with antibodies to {beta}-catenin, E-cadherin or {alpha}6-, {beta}4- and {beta}1-integrins showed polarization typical of glandular epithelium. In contrast, the metastatic M12 subline produced highly disorganized cells with no evidence of polarization. The F6 subline reverted to acini-like structures exhibiting basal polarity marked with integrins. Reducing either vimentin levels via siRNA interference or {beta}1-integrin expression by the addition of the blocking antibody, AIIB2, reorganized the M12 subline into forming polarized acini. The loss of vimentin significantly reduced M12-Vim tumor growth when assessed by subcutaneous injection in athymic mice. Thus, tumorigenicity in vivo correlated with disorganized growth in 3D lrECM gels. These studies suggest that the levels of vimentin and {beta}1-integrin play a key role in the homeostasis of the normal acini in prostate and that their dysregulation may lead to tumorigenesis.

  7. Analysis of the activation state of alpha4beta1 integrin in human B cell lines derived from myeloma, leukemia or lymphoma.

    PubMed

    García-Gila, M; Cabañas, C; García-Pardo, A

    1997-12-01

    Myeloma cells specifically localize in the bone marrow and rarely circulate in blood. To study whether this immobilization could be partially explained by the presence of constitutively activated integrins, particularly alpha4beta1, we used the activation reporter HUTS-21 anti-beta1 mAb. These analyses showed that beta1 integrins on myeloma cells were moderately active and could be upregulated similarly to integrins on lymphoma or leukemia cells. Myeloma cells were also tested for their ability to attach to RGD-containing fibronectin fragments, a property of activated (but not resting) alpha4beta1. Two cell lines adhered to these fragments and this was inhibited by anti-alpha5 but not by anti-alpha4 mAbs. These results show that myeloma cells bear low/moderately active alpha4beta1 and support the notion that multiple interactions contribute to their localization in the bone marrow.

  8. Involvement of integrins alpha(3)beta(1) and alpha(5)beta(1) and glycoprotein IIb in megakaryocyte-induced osteoblast proliferation.

    PubMed

    Lemieux, Justin M; Horowitz, Mark C; Kacena, Melissa A

    2010-04-01

    As the prevalence of osteoporosis is expected to increase over the next few decades, the development of novel therapeutic strategies to combat this disorder becomes clinically imperative. These efforts draw extensively from an expanding body of knowledge pertaining to the physiologic mechanisms of skeletal homeostasis. To this body of knowledge, we contribute that cells of hematopoietic lineage may play a crucial role in balancing osteoblastic bone formation against osteoclastic resorption. Specifically, our laboratory has previously demonstrated that megakaryocytes (MKs) can induce osteoblast (OB) proliferation in vitro, but do so only when direct cell-to-cell contact is permitted. To further investigate the nature of this interaction, we have effectively neutralized several adhesion molecules known to function in the analogous interaction of MKs with another cell type of mesenchymal origin-the fibroblast (FB). Our findings implicate the involvement of fibronectin/RGD-binding integrins including alpha3beta1 (VLA-3) and alpha5beta1 (VLA-5) as well as glycoprotein (gp) IIb (CD41), all of which are known to be expressed on MK membranes. Furthermore, we demonstrate that interleukin (IL)-3 can enhance MK-induced OB activation in vitro, as demonstrated in the MK-FB model system. Taken together, these results suggest that although their physiologic and clinical implications are very different, these two models of hematopoietic-mesenchymal cell activation are mechanistically analogous in several ways.

  9. Endothelial Lu/BCAM glycoproteins are novel ligands for red blood cell alpha4beta1 integrin: role in adhesion of sickle red blood cells to endothelial cells.

    PubMed

    El Nemer, Wassim; Wautier, Marie-Paule; Rahuel, Cécile; Gane, Pierre; Hermand, Patricia; Galactéros, Frédéric; Wautier, Jean-Luc; Cartron, Jean-Pierre; Colin, Yves; Le Van Kim, Caroline

    2007-04-15

    The Lutheran (Lu) blood group and basal cell adhesion molecule (BCAM) antigens are both carried by 2 glycoprotein isoforms of the immunoglobulin superfamily representing receptors for the laminin alpha(5) chain. In addition to red blood cells, Lu/BCAM proteins are highly expressed in endothelial cells. Abnormal adhesion of red blood cells to the endothelium could potentially contribute to the vaso-occlusive episodes in sickle cell disease. Considering the presence of integrin consensus-binding sites in Lu/BCAM proteins, we investigated their potential interaction with integrin alpha(4)beta(1), the unique integrin expressed on immature circulating sickle red cells. Using cell adhesion assays under static and flow conditions, we demonstrated that integrin alpha(4)beta(1) expressed on transfected cells bound to chimeric Lu-Fc protein. We showed that epinephrine-stimulated sickle cells, but not control red cells, adhered to Lu-Fc via integrin alpha(4)beta(1) under flow conditions. Antibody-mediated activation of integrin alpha(4)beta(1) induced adhesion of sickle red cells to primary human umbilical vein endothelial cells; this adhesion was inhibited by soluble Lu-Fc and vascular cell adhesion molecule-1 (VCAM-1)-Fc proteins. This novel interaction between integrin alpha(4)beta(1) in sickle red cells and endothelial Lu/BCAM proteins could participate in sickle cell adhesion to endothelium and potentially play a role in vaso-occlusive episodes.

  10. Expression of beta 1B integrin isoform in CHO cells results in a dominant negative effect on cell adhesion and motility.

    PubMed

    Balzac, F; Retta, S F; Albini, A; Melchiorri, A; Koteliansky, V E; Geuna, M; Silengo, L; Tarone, G

    1994-10-01

    The integrin subunit beta 1B, a beta 1 isoform with a unique sequence at the cytoplasmic domain, forms heterodimers with integrin alpha chains and binds fibronectin, but it does not localize to focal adhesion sites (Balzac, F., A. Belkin, V. Koteliansky, Y. Balabanow, F. Altruda, L. Silengo, and G. Tarone. 1993. J. Cell Biol. 121:171-178). Here we analyze the functional properties of human beta 1B by expressing it in hamster CHO cells. When stimulated by specific antibodies, beta 1B does not trigger tyrosine phosphorylation of a 125-kD cytosolic protein, an intracellular signalling pathway that is activated both by the endogenous hamster or the transfected human beta 1A. Moreover, expression of beta 1B results in reduced spreading on fibronectin and laminin, but not on vitronectin. Expression of beta 1B also results in severe reduction of cell motility in the Boyden chamber assay. Reduced cell spreading and motility could not be accounted for by preferential association of beta 1B with a given integrin alpha subunit. These data, together with our previous results, indicate that beta 1B interferes with beta 1A function when expressed in CHO cells resulting in a dominant negative effect on cell adhesion and migration.

  11. Expression of beta 1B integrin isoform in CHO cells results in a dominant negative effect on cell adhesion and motility

    PubMed Central

    1994-01-01

    The integrin subunit beta 1B, a beta 1 isoform with a unique sequence at the cytoplasmic domain, forms heterodimers with integrin alpha chains and binds fibronectin, but it does not localize to focal adhesion sites (Balzac, F., A. Belkin, V. Koteliansky, Y. Balabanow, F. Altruda, L. Silengo, and G. Tarone. 1993. J. Cell Biol. 121:171-178). Here we analyze the functional properties of human beta 1B by expressing it in hamster CHO cells. When stimulated by specific antibodies, beta 1B does not trigger tyrosine phosphorylation of a 125- kD cytosolic protein, an intracellular signalling pathway that is activated both by the endogenous hamster or the transfected human beta 1A. Moreover, expression of beta 1B results in reduced spreading on fibronectin and laminin, but not on vitronectin. Expression of beta 1B also results in severe reduction of cell motility in the Boyden chamber assay. Reduced cell spreading and motility could not be accounted for by preferential association of beta 1B with a given integrin alpha subunit. These data, together with our previous results, indicate that beta 1B interferes with beta 1A function when expressed in CHO cells resulting in a dominant negative effect on cell adhesion and migration. PMID:7523423

  12. Integrin expression in stem cells from bone marrow and adipose tissue during chondrogenic differentiation.

    PubMed

    Goessler, Ulrich Reinhart; Bugert, Peter; Bieback, Karen; Stern-Straeter, Jens; Bran, Gregor; Hörmann, Karl; Riedel, Frank

    2008-03-01

    The use of adult mesenchymal stem cells (MSC) in cartilage tissue engineering offers new perspectives in the generation of transplants for reconstructive surgery. The extracelular matrix (ECM) plays a key role in modulating the function and phenotype of the embedded cells and contains the integrins as adhesion receptors mediating cell-cell and cell-matrix interactions. In our study, characteristic changes in integrin expression during the course of chondrogenic differentiation of MSC from bone marrow and adipose tissue were compared. MSC were isolated from bone marrow biopsies and adipose tissue. During cell culture, chondrogenic differentiation was performed. The expression of integrins and their signaling components were analysed with microarray and immunohistochemistry in freshly isolated MSC and after chondrogenic differentiation. The fibronectin receptor (integrin alpha5beta1) was expressed by undifferentiated MSC, and expression rose during chondrogenic differentiation in both types of MSC. The components of the vitronectin/osteopontin receptors (alphavbeta5) were not expressed by freshly isolated MSC, and expression rose with ongoing differentiation. Receptors for the collagens (alpha1beta1, alpha2beta1, alpha3beta1) were weakly expressed by undifferentiated MSC and were activated during differentiation. Intracellular signaling components integrin-linked kinase (ILK) and CD47 showed increased expression with ongoing differentiation. For all integrins, no significant differences were be found in the 2 types of MSC. Integrin-mediated signaling appeared to play an important role in the generation and maintenance of the chondrocytic phenotype during chondrogenic differentiation. Particularly, the receptors for fibronectin, vitronectin, osteopontin and the collagens may be involved in the generation of the ECM. Intracellularly, their signals might be transduced by ILK and CD47. To fully harness the potential of these cells, future studies should be directed to

  13. Analysis of swine beta1 integrin (CD29) epitopes through monoclonal antibodies developed using two immunization strategies.

    PubMed

    Paños, G; Moreno, A; Jiménez-Marín, A; Garrido, J J; Martin de la Mulas, J; Ordás, J; Llanes, D

    2004-10-01

    This report describes the production and characterization of monoclonal antibodies (MAbs) to swine beta1 integrin subunit (CD29) using two different immunization strategies. MAb GP4B4 was developed from a mouse immunized with porcine peripheral blood mononuclear cells (PBMC), while MAbs GP1A5, GP1A6, and GP4A1 were produced by immunizing mice with a porcine CD29 recombinant protein (rpCD29) developed in our laboratory, which includes the ligand binding site. GP4B4 and UCP1D2 (specific to porcine CD29) immunoprecipitated two bands of approximately 115 and 150 kDa under reducing conditions. The molecule recognized by these two antibodies was studied using flow cytometry and was found in practically all cells studied, displaying a similar reaction pattern. Western blot assays performed with rpCD29 indicated that MAbs GP1A5, GP1A6, and GP4A1 recognized the 30-kDa band for this recombinant protein, confirming their specificity for the beta1, integrin subunit. Immunohistochemical analyses of these MAbs disclosed a morphological pattern associated with smooth muscle, epithelium, and myeloid cells, as expected in MAbs recognizing CD29. This MAb panel could be useful as a general anti-CD29 reagent and would allow further research into this important integrin in swine.

  14. Skeletal phenotype of growing transgenic mice that express a function-perturbing form of beta1 integrin in osteoblasts

    NASA Technical Reports Server (NTRS)

    Globus, R. K.; Amblard, D.; Nishimura, Y.; Iwaniec, U. T.; Kim, J-B; Almeida, E. A. C.; Damsky, C. D.; Wronski, T. J.; van der Meulen, M. C. H.

    2005-01-01

    Skeletal modeling entails the deposition of large amounts of extracellular matrix (ECM) to form structures tailored to withstand increasing mechanical loads during rapid growth. Specific ECM molecules bind to integrin receptors on the cell surface, thereby triggering a cascade of signaling events that affect critical cell functions. To evaluate the role of integrins during skeletal growth, transgenic mice were engineered to express a function-perturbing fragment of beta1 integrin consisting of the transmembrane domain and cytoplasmic tail under the control of the osteocalcin promoter (TG mice). Thus, transgene expression was targeted to mature cells of the osteoblast lineage, and herein we show that cultured cells resembling osteocytes from 90-day-old TG mice display impaired adhesion to collagen I, a ligand for beta1 integrin. To determine the influence of beta1 integrin on bones that are responsible for providing structural support during periods of rapid growth, we examined the phenotype of the appendicular skeleton in TG mice compared to wild type (WT) mice. According to radiographs, bones from mice of both genotypes between 14 and 90 days of age appeared similar in gross structure and density, although proximal tibiae from 35-90 days old TG mice were less curved than those of WT mice (72-92% TG/WT). Although there were only mild and transient differences in absolute bone mass and strength, once normalized to body mass, the tibial dry mass (79.1% TG/WT females), ash mass (78.5% TG/WT females), and femoral strength in torsion (71.6% TG/WT females) were reduced in TG mice compared to WT mice at 90 days of age. Similar effects of genotype on bone mass and curvature were observed in 1-year-old retired breeders, indicating that these phenotypic differences between TG and WT mice were stable well into adulthood. Effects of genotype on histomorphometric indices of cancellous bone turnover were minimal and evident only transiently during growth, but when present they

  15. Skeletal phenotype of growing transgenic mice that express a function-perturbing form of beta1 integrin in osteoblasts

    NASA Technical Reports Server (NTRS)

    Globus, R. K.; Amblard, D.; Nishimura, Y.; Iwaniec, U. T.; Kim, J-B; Almeida, E. A. C.; Damsky, C. D.; Wronski, T. J.; van der Meulen, M. C. H.

    2005-01-01

    Skeletal modeling entails the deposition of large amounts of extracellular matrix (ECM) to form structures tailored to withstand increasing mechanical loads during rapid growth. Specific ECM molecules bind to integrin receptors on the cell surface, thereby triggering a cascade of signaling events that affect critical cell functions. To evaluate the role of integrins during skeletal growth, transgenic mice were engineered to express a function-perturbing fragment of beta1 integrin consisting of the transmembrane domain and cytoplasmic tail under the control of the osteocalcin promoter (TG mice). Thus, transgene expression was targeted to mature cells of the osteoblast lineage, and herein we show that cultured cells resembling osteocytes from 90-day-old TG mice display impaired adhesion to collagen I, a ligand for beta1 integrin. To determine the influence of beta1 integrin on bones that are responsible for providing structural support during periods of rapid growth, we examined the phenotype of the appendicular skeleton in TG mice compared to wild type (WT) mice. According to radiographs, bones from mice of both genotypes between 14 and 90 days of age appeared similar in gross structure and density, although proximal tibiae from 35-90 days old TG mice were less curved than those of WT mice (72-92% TG/WT). Although there were only mild and transient differences in absolute bone mass and strength, once normalized to body mass, the tibial dry mass (79.1% TG/WT females), ash mass (78.5% TG/WT females), and femoral strength in torsion (71.6% TG/WT females) were reduced in TG mice compared to WT mice at 90 days of age. Similar effects of genotype on bone mass and curvature were observed in 1-year-old retired breeders, indicating that these phenotypic differences between TG and WT mice were stable well into adulthood. Effects of genotype on histomorphometric indices of cancellous bone turnover were minimal and evident only transiently during growth, but when present they

  16. The intermediate filament protein vimentin binds specifically to a recombinant integrin {alpha}2/{beta}1 cytoplasmic tail complex and co-localizes with native {alpha}2/{beta}1 in endothelial cell focal adhesions

    SciTech Connect

    Kreis, Stephanie; Schoenfeld, Hans-Joachim; Melchior, Chantal; Steiner, Beat; Kieffer, Nelly . E-mail: kieffer@cu.lu

    2005-04-15

    Integrin receptors are crucial players in cell adhesion and migration. Identification and characterization of cellular proteins that interact with their short {alpha} and {beta} cytoplasmic tails will help to elucidate the molecular mechanisms by which integrins mediate bi-directional signaling across the plasma membrane. Integrin {alpha}2{beta}1 is a major collagen receptor but to date, only few proteins have been shown to interact with the {alpha}2 cytoplasmic tail or with the {alpha}2{beta}1 complex. In order to identify novel binding partners of a {alpha}2{beta}1cytoplasmic domain complex, we have generated recombinant GST-fusion proteins, incorporating the leucine zipper heterodimerization cassettes of Jun and Fos. To ascertain proper functionality of the recombinant proteins, interaction with natural binding partners was tested. GST-{alpha}2 and GST-Jun {alpha}2 bound His-tagged calreticulin while GST-{beta}1 and GST-Fos {beta}1 proteins bound talin. In screening assays for novel binding partners, the immobilized GST-Jun {alpha}2/GST-Fos {beta}1 heterodimeric complex, but not the single subunits, interacted specifically with endothelial cell-derived vimentin. Vimentin, an abundant intermediate filament protein, has previously been shown to co-localize with {alpha}v{beta}3-positive focal contacts. Here, we provide evidence that this interaction also occurs with {alpha}2{beta}1-enriched focal adhesions and we further show that this association is lost after prolonged adhesion of endothelial cells to collagen.

  17. beta1-integrin mediates asbestos-induced phosphorylation of AKT and ERK1/2 in a rat pleural mesothelial cell line.

    PubMed

    Berken, Antje; Abel, Josef; Unfried, Klaus

    2003-11-20

    Integrin-mediated signalling has been implicated in asbestos-induced carcinogenesis. In studies here, we examined signal transduction events associated with integrin-directed cell reactions triggered by crocidolite asbestos in the pleural mesothelial cell line 4/4 RM-4. Crocidolite fibres induced a significant time- and dose-dependent activation of the extracellular-signal-regulated kinases ERK1 and ERK2. ERK activation was specifically inhibited by integrin-blocking agents, that are integrin-binding peptides containing the sequence arginine-glycine-aspartic acid (RGD), and monoclonal antibodies against the integrin beta1-chain. Integrin-dependent activation of ERK1/2 in response to asbestos appeared to be independent of focal adhesion kinase pp125FAK (FAK) since FAK autophosphorylation remained unaffected in crocidolite-exposed mesothelial cells. Instead, we observed striking similarities in the kinetics of asbestos-induced ERK1/2 responses and phosphorylation of protein kinase B (AKT) at serine 473, a possible target residue for integrin-linked kinase. As with ERK activation, asbestos-induced AKT stimulation was significantly blocked by both the RGD-peptide and the beta1-integrin antibodies. These studies are the first to establish that in mesothelial cells ERK1/2 and AKT are simultaneously phosphorylated upon asbestos exposure in a beta1-integrin-dependent manner.

  18. A novel integrin {alpha}5{beta}1 antagonistic peptide, A5-1, screened by Protein Chip system as a potent angiogenesis inhibitor

    SciTech Connect

    Kim, Eung-Yoon; Bang, Ji Young; Chang, Soo-Ik; Kang, In-Cheol

    2008-12-26

    Integrin {alpha}5{beta}1 immobilized on a ProteoChip was used to screen new antagonistic peptides from multiple hexapeptide sub-libraries of the positional scanning synthetic peptide combinatorial library (PS-SPCL). The integrin {alpha}5{beta}1-Fibronectin interaction was demonstrated on the chip. A novel peptide ligand, A5-1 (VILVLF), with high affinity to integrin {alpha}5{beta}1 was identified from the hexapeptide libraries with this chip-based screening method on the basis of a competitive inhibition assay. A5-1 inhibits the integrin-fibronectin interaction in a dose-dependent manner (IC{sub 50}; 1.56 {+-} 0.28 {mu}M. In addition, it inhibits human umbilical vein endothelial cell proliferation, migration, adhesion, tubular network formation, and bFGF-induced neovascularization in a chick chorioallantoic membrane. These results suggest that A5-1 will be a potent inhibitor of neovascularization.

  19. Central roles of alpha5beta1 integrin and fibronectin in vascular development in mouse embryos and embryoid bodies.

    PubMed

    Francis, Sheila E; Goh, Keow Lin; Hodivala-Dilke, Kairbaan; Bader, Bernhard L; Stark, Margaret; Davidson, Duncan; Hynes, Richard O

    2002-06-01

    Vascular development and maturation are dependent on the interactions of endothelial cell integrins with surrounding extracellular matrix. Previous investigations of the primacy of certain integrins in vascular development have not addressed whether this could also be a secondary effect due to poor embryonic nutrition. Here, we show that the alpha5 integrin subunit and fibronectin have critical roles in blood vessel development in mouse embryos and in embryoid bodies (EBs) differentiated from embryonic stem cells (a situation in which there is no nutritional deficit caused by the mutations). In contrast, vascular development in vivo and in vitro is not strongly dependent on alpha(v) or beta3 integrin subunits. In mouse embryos lacking alpha5 integrin, greatly distended blood vessels are seen in the vitelline yolk sac and in the embryo itself. Additionally, overall blood vessel pattern complexity is reduced in alpha5-null tissues. This defective vascular phenotype is correlated with a decrease in the ligand for alpha5 integrin, fibronectin (FN), in the endothelial basement membranes. A striking and significant reduction in early capillary plexus formation and maturation was apparent in EBs formed from embryonic stem cells lacking alpha5 integrin or FN compared with wild-type EBs or EBs lacking alpha(v) or beta3 integrin subunits. Vessel phenotype could be partially restored to FN-null EBs by the addition of whole FN to the culture system. These findings confirm a clear role for alpha5 and FN in early blood vessel development not dependent on embryo nutrition or alpha(v) or beta3 integrin subunits. Thus, successful early vasculogenesis and angiogenesis require alpha5-FN interactions.

  20. Allosteric beta1 integrin antibodies that stabilize the low affinity state by preventing the swing-out of the hybrid domain.

    PubMed

    Luo, Bing-Hao; Strokovich, Konstantin; Walz, Thomas; Springer, Timothy A; Takagi, Junichi

    2004-06-25

    The ligand binding function of integrins can be modulated by various monoclonal antibodies by both direct and indirect mechanisms. We have characterized an anti-beta(1) antibody, SG/19, that had been reported to inhibit the function of the beta(1) integrin on the cell surface. SG/19 recognized the wild type beta(1) subunit that exists in a conformational equilibrium between the high and low affinity states but bound poorly to a mutant beta(1) integrin that had been locked in a high affinity state. Epitope mapping of SG/19 revealed that Thr(82) in the beta(1) subunit, located at the outer face of the boundary between the I-like and hybrid domains, was the key binding determinant for this antibody. Direct visualization of the alpha (5)beta(1) headpiece fragment in complex with SG/19 Fab with electron microscopy confirmed the location of the binding surface and showed that the ligand binding site is not occluded by the bound Fab. Surface plasmon resonance showed that alpha (5)beta(1) integrin bound by SG/19 maintained a low affinity toward its physiological ligand fibronectin (Fn) whereas binding by function-blocking anti-alpha(5) antibodies resulted in a complete loss of fibronectin binding. Thus a class of the anti-beta antibodies represented by SG/19 attenuate the ligand binding function by restricting the conformational shift to the high affinity state involving the swing-out of the hybrid domain without directly interfering with ligand docking.

  1. Neisseria meningitidis adhesin NadA targets beta1 integrins: functional similarity to Yersinia invasin.

    PubMed

    Nägele, Virginie; Heesemann, Jürgen; Schielke, Stephanie; Jiménez-Soto, Luisa F; Kurzai, Oliver; Ackermann, Nikolaus

    2011-06-10

    Meningococci are facultative-pathogenic bacteria endowed with a set of adhesins allowing colonization of the human upper respiratory tract, leading to fulminant meningitis and septicemia. The Neisseria adhesin NadA was identified in about 50% of N. meningitidis isolates and is closely related to the Yersinia adhesin YadA, the prototype of the oligomeric coiled-coil adhesin (Oca) family. NadA is known to be involved in cell adhesion, invasion, and induction of proinflammatory cytokines. Because of the enormous diversity of neisserial cell adhesins the analysis of the specific contribution of NadA in meningococcal host interactions is limited. Therefore, we used a non-invasive Y. enterocolitica mutant as carrier to study the role of NadA in host cell interaction. NadA was shown to be efficiently produced and localized in its oligomeric form on the bacterial surface of Y. enterocolitica. Additionally, NadA mediated a β1 integrin-dependent adherence with subsequent internalization of yersiniae by a β1 integrin-positive cell line. Using recombinant NadA(24-210) protein and human and murine β1 integrin-expressing cell lines we could demonstrate the role of the β1 integrin subunit as putative receptor for NadA. Subsequent inhibition assays revealed specific interaction of NadA(24-210) with the human β1 integrin subunit. Cumulatively, these results indicate that Y. enterocolitica is a suitable toolbox system for analysis of the adhesive properties of NadA, revealing strong evidence that β1 integrins are important receptors for NadA. Thus, this study demonstrated for the first time a direct interaction between the Oca-family member NadA and human β1 integrins.

  2. Integrin-extracellular matrix interactions in connective tissue remodeling and osteoblast differentiation

    NASA Technical Reports Server (NTRS)

    Globus, R. K.; Moursi, A.; Zimmerman, D.; Lull, J.; Damsky, C.

    1995-01-01

    The differentiaton of bone cells is a complex multistep process. Bone is somewhat unusual in that it is very actively and continually remodeled in the adult and that maintenance of its mass in the mature organism is exquisitely sensitive to mechanical as well as chemical signals. Bone is also unique because it consists of a very large amount of extracellular matrix (ECM) that is mineralized. The integrin family of ECM receptors has been shown to play an important role in tissue morphogenesis in several systems. Our studies on the regulation of matrix remodeling enzymes by integrins in rabbit synovial fibroblasts show that two b1 integrin fibronectin (FN) receptor complexes (alpha 5 beta 1 and alpha 4 beta 1) cooperate in detecting subtle changes in the composition of the ECM. As a result of signal transduction by these integrins, the levels of mRNA and protein for several members of the metalloproteinase family are regulated in these cells. We have also used antibody and RGD peptide perturbation studies to determine the significance of cell/ECM interactions to normal osteogenesis. We found that interactions between the cell binding domain of FN and integrins are required for both normal morphogenesis and gene expression in cultured osteoblasts that differentiate to form bone-like tissue in culture. These data lead us to propose that beta 1 integrins play an important role in osteoblast differentiation as well as in bone remodeling.

  3. Integrin-extracellular matrix interactions in connective tissue remodeling and osteoblast differentiation

    NASA Technical Reports Server (NTRS)

    Globus, R. K.; Moursi, A.; Zimmerman, D.; Lull, J.; Damsky, C.

    1995-01-01

    The differentiaton of bone cells is a complex multistep process. Bone is somewhat unusual in that it is very actively and continually remodeled in the adult and that maintenance of its mass in the mature organism is exquisitely sensitive to mechanical as well as chemical signals. Bone is also unique because it consists of a very large amount of extracellular matrix (ECM) that is mineralized. The integrin family of ECM receptors has been shown to play an important role in tissue morphogenesis in several systems. Our studies on the regulation of matrix remodeling enzymes by integrins in rabbit synovial fibroblasts show that two b1 integrin fibronectin (FN) receptor complexes (alpha 5 beta 1 and alpha 4 beta 1) cooperate in detecting subtle changes in the composition of the ECM. As a result of signal transduction by these integrins, the levels of mRNA and protein for several members of the metalloproteinase family are regulated in these cells. We have also used antibody and RGD peptide perturbation studies to determine the significance of cell/ECM interactions to normal osteogenesis. We found that interactions between the cell binding domain of FN and integrins are required for both normal morphogenesis and gene expression in cultured osteoblasts that differentiate to form bone-like tissue in culture. These data lead us to propose that beta 1 integrins play an important role in osteoblast differentiation as well as in bone remodeling.

  4. Endorepellin causes endothelial cell disassembly of actin cytoskeleton and focal adhesions through alpha2beta1 integrin.

    PubMed

    Bix, Gregory; Fu, Jian; Gonzalez, Eva M; Macro, Laura; Barker, Amy; Campbell, Shelly; Zutter, Mary M; Santoro, Samuel A; Kim, Jiyeun K; Höök, Magnus; Reed, Charles C; Iozzo, Renato V

    2004-07-05

    Endorepellin, the COOH-terminal domain of the heparan sulfate proteoglycan perlecan, inhibits several aspects of angiogenesis. We provide evidence for a novel biological axis that links a soluble fragment of perlecan protein core to the major cell surface receptor for collagen I, alpha2beta1 integrin, and provide an initial investigation of the intracellular signaling events that lead to endorepellin antiangiogenic activity. The interaction between endorepellin and alpha2beta1 integrin triggers a unique signaling pathway that causes an increase in the second messenger cAMP; activation of two proximal kinases, protein kinase A and focal adhesion kinase; transient activation of p38 mitogen-activated protein kinase and heat shock protein 27, followed by a rapid down-regulation of the latter two proteins; and ultimately disassembly of actin stress fibers and focal adhesions. The end result is a profound block of endothelial cell migration and angiogenesis. Because perlecan is present in both endothelial and smooth muscle cell basement membranes, proteolytic activity during the initial stages of angiogenesis could liberate antiangiogenic fragments from blood vessels' walls, including endorepellin.

  5. Beta1B integrin interferes with matrix assembly but not with confluent monolayer polarity, and alters some morphogenetic properties of FRT epithelial cells.

    PubMed

    Calí, G; Retta, S F; Negri, R; Damiano, I; Gentile, R; Tarone, G; Nitsch, L; Garbi, C

    1998-02-01

    Beta1B is a beta1 integrin splice variant that differs from the ubiquitous beta1A in the terminal portion of the cytosolic tail. The expression of this variant in CHO cells results in reduced fibroblast adhesion and motility (Balzac, E et al., J. Cell Biol. 127, 557-565 (1994)). We have evaluated the phenotypic changes induced by the expression of beta1B in the FRT epithelial cell line. Stable transfectants of FRT cells expressing beta1B or beta1A human integrins were obtained. The transfected integrins associated with the endogenous alpha subunits and were delivered to the plasma membrane. Beta1B expressing cells attached less efficiently and spread less on fibronectin, laminin or type IV collagen coated dishes. A great reduction of fibronectin fibrils associated to the basal membrane of non-confluent beta1B transfected cells was observed. This was paralleled by the disappearance of microfilament bundles and loss of basally located focal adhesions. On the contrary, upon beta1A transfection, a higher amount of fibronectin fibrils, together with microfilament bundles and focal adhesions, was observed. Expression of beta1B did not significantly modify the ability to manifest the polarized phenotype when cells were grown to confluence on filters in two-chamber-systems. Beta1B-transfected cells showed reduced motile properties when embedded as aggregates in type I collagen gels. Moreover, formation of polarized cysts in suspension culture was impaired. The results show that beta1B, by interfering with focal adhesion organization, microfilament and fibronectin assembly, cell spreading and migration, affects some morphogenetic properties of FRT epithelial cells.

  6. Enhancement of activated beta1-integrin expression by prostaglandin E2 via EP receptors in isolated human coronary arterial endothelial cells: implication for the treatment of Kawasaki disease.

    PubMed

    Kajimoto, M; Ichiyama, T; Ueno, Y; Shiraishi, M; Hasegawa, M; Furukawa, S

    2009-04-01

    Plasma prostaglandin E(2) (PGE(2)) levels are markedly elevated in acute Kawasaki disease (KD). We evaluated the function of the EP receptors in the expression of activated beta(1)-integrin stimulated by PGE(2) in human coronary arterial endothelial cells (HCAEC). We determined the mRNA expression of the PGE(2) receptors, EP receptors (EP(1-4)) in HCAEC by RT-PCR and protein expression by Western blotting. We evaluated the function of the EP receptors in the expression of activated beta(1)-integrin stimulated by PGE(2) in HCAEC, using antagonists and agonists of the EP receptors, by flow cytometry. RT-PCR revealed mRNAs for all four EP receptors in HCAEC. Western blotting demonstrated EP(1), EP(2) and EP(3) expression in HCAEC. The EP(2) and EP(3) agonists enhanced the expression of activated beta(1)-integrin in HCAEC. The potency of the EP(2) agonist was significantly greater than that of the EP(3) agonist. Pretreatment with the EP(1), EP(2) and EP(3) antagonists inhibited the expression of activated beta(1)-integrin induced by PGE(2) in HCAEC. The potency of the EP(2) antagonist was significantly greater than that of the EP(1) and EP(3) antagonists. Our results suggest that PGE(2) mainly induces the activation of beta(1)-integrins via the EP(2) receptor in HCAEC. Our results further suggest that the EP(2) antagonist modulates the inflammatory response during KD vasculitis.

  7. Are integrin alpha(2)beta(1), glycoprotein Ib and vWf levels correlated with their contributions to platelet adhesion on collagen under high-shear flow?

    PubMed

    Jung, Stephanie M; Sonoda, Mamiko; Tsuji, Kayoko; Jimi, Atsuo; Nomura, Shosaku; Kanaji, Taisuke; Moroi, Masaaki

    2010-01-01

    Platelets in flowing blood at high-shear stress are recruited to exposed subendothelial collagen of injured vessels by GPIb-von Willebrand factor (vWf) and integrin alpha(2)beta(1) (alpha(2)beta(1))-collagen interactions. Platelet adhesion to type I collagen depends mainly on the alpha(2)beta(1)-collagen interaction and that to type III collagen depends on the GPIb-vWf interaction due to vWf's weak affinity for type I collagen. Contributions of these two interactions would differ depending on expressions of alpha(2)beta(1), vWf, or GPIb. We quantitated platelet adhesion to low- and high-density collagen under high-shear flow conditions in the presence of anti-alpha(2)beta(1) (Gi9) and anti-GPIb (NNKY5-5) antibodies to determine if their inhibitory effects were correlated with the amounts of alpha(2)beta(1), GPIb and vWf. Gi9 inhibition of adhesion to type I collagen was decreased in platelets with more integrin alpha(2)beta(1). Gi9 and NNKY5-5 are more inhibitory against adhesion to low-density type III and I, respectively. Higher alpha(2)beta(1) expression decreases adhesion to low-density type III and increases Gi9 inhibition of adhesion to high-density type III, suggesting crosstalk between the alpha(2)beta(1)-collagen and GPIb-vWf interactions in adhesion to type III. Integrin alpha(2)beta(1)-collagen and GPIb-vWf interactions both contribute to platelet adhesion to collagen under high-shear flow. In adhesion under high-shear stress, the two interactions would compensate for each other, when there is a deficiency in one or the other. The alpha(2)beta(1)-collagen interaction was also suggested to have an inhibitory effect on platelet adhesion to type III collagen, through a yet undefined mechanism.

  8. The role of alpha3beta1 integrin in determining the supramolecular organization of laminin-5 in the extracellular matrix of keratinocytes.

    PubMed

    deHart, Gregory W; Healy, Kevin E; Jones, Jonathan C R

    2003-02-01

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

  9. Integrin alpha5beta1 and ADAM-17 interact in vitro and co-localize in migrating HeLa cells.

    PubMed

    Bax, Daniel V; Messent, Anthea J; Tart, Jonathan; van Hoang, Mien; Kott, Jane; Maciewicz, Rose A; Humphries, Martin J

    2004-05-21

    Tumor necrosis factor (TNF) alpha-converting enzyme (TACE/ADAM-17) has diverse roles in the proteolytic processing of cell surface molecules and, due to its ability to process TNFalpha, is a validated therapeutic target for anti-inflammatory therapies. Unlike a number of other ADAM proteins, which interact with integrin receptors via their disintegrin domains, there is currently no evidence for an ADAM-17-integrin association. By analyzing the adhesion of a series of cell lines with recombinant fragments of the extracellular domain of ADAM-17, we now demonstrate a functional interaction between ADAM-17 and alpha(5)beta(1) integrin in a trans orientation. Because ADAM-17-mediated adhesion was sensitive to RGD peptides and EDTA, and the integrin-binding site within ADAM-17 was narrowed down to the disintegrin/cysteine-rich region, the two molecules appear to have a ligand-receptor relationship mediated by the alpha(5)beta(1) ligand binding pocket. Intriguingly, ADAM-17 and alpha(5)beta(1) were found to co-localize in both membrane ruffles and focal adhesions in HeLa cells. When confluent HeLa cell monolayers were wounded, ADAM-17 and alpha(5)beta(1) redistributed to the leading edge and co-localized, which is suggestive of a cis orientation. We postulate that the interaction of ADAM-17 with alpha(5)beta(1) may target or modulate its metalloproteolytic activity.

  10. The integrin alpha 9 beta 1 mediates cell attachment to a non-RGD site in the third fibronectin type III repeat of tenascin.

    PubMed

    Yokosaki, Y; Palmer, E L; Prieto, A L; Crossin, K L; Bourdon, M A; Pytela, R; Sheppard, D

    1994-10-28

    We have previously reported the sequence of the integrin alpha 9 subunit, a partner of the beta 1 subunit that is expressed in basal keratinocytes, hepatocytes, airway epithelial cells, and smooth and skeletal muscle. In the present study, we have stably expressed alpha 9 beta 1 on the surface of the human embryonic kidney cell line 293 and the human colon carcinoma cell line SW480 and used these transfected cells lines to identify ligand(s) for this integrin. Transfected cells did not appear to utilize alpha 9 beta 1 for attachment to the extracellular matrix proteins fibronectin, laminin, vitronectin, fibrinogen, thrombospondin, or type I or IV collagen. However, in contrast to mock transfectants, both 293 cells and SW480 cells expressing alpha 9 beta 1 adhered to intact chicken tenascin. By utilizing a variety of recombinant fragments of tenascin, we were able to localize the binding site for alpha 9 beta 1 to the third type III repeat. This repeat contains the arginine-glycine-aspartic acid (RGD) tripeptide that has been shown to serve as a binding site in tenascin for alpha v-integrins. However, the RGD site does not appear to be the binding site for alpha 9 beta 1, as the attachment of alpha 9 transfectants to this fragment was not inhibited by RGD peptide, nor by changing the RGD site to RAD or RAA.

  11. Highly Potent, Water Soluble Benzimidazole Antagonist for Activated (alpha)4(beta)1 Integrin

    SciTech Connect

    Carpenter, R D; Andrei, M; Lau, E Y; Lightstone, F C; Liu, R; Lam, K S; Kurth, M J

    2007-08-29

    The cell surface receptor {alpha}{sub 4}{beta}{sub 1} integrin, activated constitutively in lymphoma, can be targeted with the bisaryl urea peptidomimetic antagonist 1 (LLP2A). However, concerns on its preliminary pharmacokinetic (PK) profile provided an impetus to change the pharmacophore from a bisaryl urea to a 2-arylaminobenzimidazole moiety resulting in improved solubility while maintaining picomolar potency [5 (KLCA4); IC{sub 50} = 305 pM]. With exceptional solubility, this finding has potential for improving PK to help diagnose and treat lymphomas.

  12. Laminins 2 (alpha2beta1gamma1, Lm-211) and 8 (alpha4beta1gamma1, Lm-411) are synthesized and secreted by tooth pulp fibroblasts and differentially promote neurite outgrowth from trigeminal ganglion sensory neurons.

    PubMed

    Fried, Kaj; Sime, Wondossen; Lillesaar, Christina; Virtanen, Ismo; Tryggvasson, Karl; Patarroyo, Manuel

    2005-07-15

    The tooth pulp innervation originates from the trigeminal ganglion (TG) and represents an illustrative example of tissue targeting by sensory nerves. Pulpal fibroblasts strongly promote neurite outgrowth from TG neurons in vitro. In the present study, we have investigated the possible participation of laminins (LNs), potent neuritogenic extracellular matrix components. Immunohistochemistry of human tooth pulp demonstrated expression of LN alpha1, alpha2, alpha4, alpha5, beta1 and gamma1, and laminin-binding integrin alpha3, alpha6, beta1 and beta4 chains in nerves. Though faintly stained for laminins in situ, pulpal fibroblasts reacted, once cultured and permeabilized, with antibodies to LN alpha2, alpha4, beta1 and gamma1 chains by flow cytometry. The cells also expressed the corresponding mRNAs and were able to assemble and secrete LN-2 (alpha2beta1gamma1, Lm-211) and LN-8 (alpha4beta1gamma1, Lm-411). LN-8 displayed a chondroitin sulphate (CS) modification in its alpha4 chain. In functional assays, mouse LN-1 (alpha1beta1gamma1, Lm-111) and recombinant human (rh) LN-8, but not native or rhLN-2, strongly promoted neurite outgrowth from TG neurons, mimicking the effect of cultured pulp fibroblast. Altogether, the results indicate that LN-2 and LN-8 are synthesized by tooth pulp fibroblasts and differentially promote neurite outgrowth from TG neurons. LN-8 may contribute to sensory innervation of teeth and other tissues during development and/or regeneration.

  13. Focal Adhesion Kinase Regulates Fibroblast Migration via Integrin beta-1 and Plays a Central Role in Fibrosis.

    PubMed

    Zhao, Xue-Ke; Cheng, Yiju; Liang Cheng, Ming; Yu, Lei; Mu, Mao; Li, Hong; Liu, Yang; Zhang, Baofang; Yao, Yumei; Guo, Hui; Wang, Rong; Zhang, Quan

    2016-01-14

    Lung fibrosis is a major medical problem for the aging population worldwide. Fibroblast migration plays an important role in fibrosis. Focal Adhesion Kinase (FAK) senses the extracellular stimuli and initiates signaling cascades that promote cell migration. This study first examined the dose and time responses of FAK activation in human lung fibroblasts treated with platelet derived growth factor BB (PDGF-BB). The data indicate that FAK is directly recruited by integrin β1 and the subsequent FAK activation is required for fibroblast migration on fibronectin. In addition, the study has identified that α5β1 and α4β1 are the major integrins for FAK-mediated fibroblast migration on fibronect. In contrast, integrins αvβ3, αvβ6, and αvβ8 play a minor but distinct role in fibroblast migration on fibronectin. FAK inhibitor significantly reduces PDGF-BB stimulated fibroblast migration. Importantly, FAK inhibitor protects bleomycin-induced lung fibrosis in mice. FAK inhibitor blocks FAK activation and significantly reduces signaling cascade of fibroblast migration in bleomycin-challenged mice. Furthermore, FAK inhibitor decreases lung fibrotic score, collagen accumulation, fibronectin production, and myofibroblast differentiation in in bleomycin-challenged mice. These data demonstrate that FAK mediates fibroblast migration mainly via integrin β1. Furthermore, the findings suggest that targeting FAK signaling is an effective therapeutic strategy against fibrosis.

  14. Relationship among follicular apoptosis, integrin beta1 and collagen type IV during early ovarian regression in the teleost Prochilodus argenteus after induced spawning.

    PubMed

    Santos, H B; Sato, Y; Moro, L; Bazzoli, N; Rizzo, E

    2008-04-01

    Early ovarian regression was analyzed in the neotropical freshwater teleost, curimatã-pacu (Prochilodus argenteus), in order to evaluate follicular apoptosis, basement membrane morphology, and integrin beta1 and collagen type IV immunostainning in postovulatory follicles. Mature females were induced to spawn by using carp pituitary extract for study of ovarian regression up to 5 days post-spawning. Morphometric analyses showed that the postovulatory follicle area decreased progressively after spawning and was coupled to the gonadosomatic index (r=0.92). During ovarian regression, follicular cells detached from the neighboring cells and basement membrane and then died by apoptosis. The follicular basement membrane became thicker and diffuse and was breached during regression of the postovulatory follicles. Follicular apoptosis was detected by TUNEL, histology, and electron microscopy. The ladder pattern of apoptotic DNA was revealed by agarose gel electrophoresis. The apoptotic index for the follicular cells increased until 3 days post-spawning and decreased thereafter. Immunohistochemistry reactions detected caspase 3, integrin beta1, and collagen type IV in the follicular layer of the postovulatory follicles. Labeling for integrin beta1 and collagen type IV decreased significantly, whereas a peak in cell death occurred 3 days post-spawning. At 4-5 days post-spawning, the connective theca was more thickened and vascularized. Simultaneously, granulocytes migrated toward the follicular lumen. Thus, follicular apoptosis contributes to early ovarian regression in P. argenteus. Additionally, our findings suggest integrin beta1 and collagen type IV as possible survival factors for follicular cells in teleost ovary.

  15. Proteolysis of integrin alpha5 and beta1 subunits involved in retinoic acid-induced apoptosis in human hepatoma Hep3B cells.

    PubMed

    Hsu, S L; Cheng, C C; Shi, Y R; Chiang, C W

    2001-06-26

    Our previous report demonstrated that all-trans-retinoic acid (ATRA) induces detachment and death under serum starvation in several human tumor cell lines. In this study, we examined the influence of cell-extracellular matrix interaction on the ability of ATRA to induce apoptosis. Plating of human hepatoma Hep3B cells onto poly-hydroxyethylmethacrylate-coated plates in the absence of serum resulted in the acceleration of ATRA-induced apoptosis. In contrast, ATRA-induced apoptosis was significantly suppressed by plating cells onto Matrigel-coated plates but not suppressed by culturing onto collagen-, laminin-, vitronectin-, or fibronectin-coated plates. Exogenously added soluble collagen, laminin, fibronectin, vitronectin or Matrigel failed to suppress ATRA-induced apoptosis. Results from the adhesion assay indicated that the cell attachment to fibronectin was significantly inhibited by ATRA. Treatment with perturbing antibody against integrin alpha5 or beta1 subunits resulted in promotion of ATRA-induced apoptosis. Moreover, the proteolytic cleavage of alpha5beta1 integrin and focal adhesion kinase (FAK) proteins is linked to the early phase of the ATRA-induced apoptotic process. Furthermore, ATRA-induced detachment, death, and cleavage of alpha5beta1 integrin and FAK were drastically suppressed by plating cells onto Matrigel-coated plates. These findings provide evidence that abrogation of cell adhesion, through proteolysis of alpha5beta1 integrin and FAK, is closely linked to ATRA-induced apoptosis in Hep3B cells.

  16. Plumieribetin, a fish lectin homologous to mannose-binding B-type lectins, inhibits the collagen-binding alpha1beta1 integrin.

    PubMed

    de Santana Evangelista, Karla; Andrich, Filipe; Figueiredo de Rezende, Flávia; Niland, Stephan; Cordeiro, Marta N; Horlacher, Tim; Castelli, Riccardo; Schmidt-Hederich, Alletta; Seeberger, Peter H; Sanchez, Eladio F; Richardson, Michael; Gomes de Figueiredo, Suely; Eble, Johannes A

    2009-12-11

    Recently, a few fish proteins have been described with a high homology to B-type lectins of monocotyledonous plants. Because of their mannose binding activity, they have been ascribed a role in innate immunity. By screening various fish venoms for their integrin inhibitory activity, we isolated a homologous protein from the fin stings and skin mucus of the scorpionfish (Scorpaena plumieri). This protein inhibits alpha1beta1 integrin binding to basement membrane collagen IV. By protein chemical and spectroscopic means, we demonstrated that this fish protein, called plumieribetin, is a homotetramer and contains a high content of anti-parallel beta strands, similar to the mannose-binding monocot B-lectins. It lacks both N-linked glycoconjugates and common O-glycan motifs. Despite its B-lectin-like structure, plumieribetin binds to alpha1beta1 integrin irrespective of N-glycosylation, suggesting a direct protein-protein interaction. This interaction is independent of divalent cations. On the cellular level, plumieribetin failed to completely detach hepatocarcinoma HepG2 cells and primary arterial smooth muscle cells from the collagen IV fragment CB3. However, plumieribetin weakened the cell-collagen contacts, reduced cell spreading, and altered the actin cytoskeleton, after the compensating alpha2beta1 integrin was blocked. The integrin inhibiting effect of plumieribetin adds a new function to the B-lectin family, which is known for pathogen defense.

  17. Differential pattern of integrin receptor expression in differentiated and anaplastic thyroid cancer cell lines.

    PubMed

    Hoffmann, S; Maschuw, K; Hassan, I; Reckzeh, B; Wunderlich, A; Lingelbach, S; Zielke, A

    2005-09-01

    Adhesion of tumor cells to the extracellular matrix (ECM) is a crucial step for the development of metastatic disease and is mediated by specific integrin receptor molecules (IRM). The pattern of metastatic spread differs substantially among the various histotypes of thyroid cancer (TC). However, IRM have only occasionally been characterized in TC until now. IRM expression was investigated in 10 differentiated (FTC133, 236, 238, HTC, HTC TSHr, XTC, PTC4.0/4.2, TPC1, Kat5) and two anaplastic TC cell lines (ATC, C643, Hth74), primary cultures of normal thyroid tissue (Thy1,3), and thyroid cancer specimens (TCS). Expression of 16 IRM (beta1-4, beta7, alpha1-6, alphaV, alphaIIb, alphaL, alphaM, alphaX) and of four IRM heterodimers (alpha2beta1, alpha5beta1, alphaVbeta3, alphaVbeta5), was analyzed by fluorescent-activated cell sorter (FACS) and immunohistochemical staining. Thyroid tumor cell adhesion to ECM proteins and their IRM expression in response to thyrotropin (TSH) was assessed. Follicular TC cell lines presented high levels of integrins alpha2, alpha3, alpha5, beta1, beta3 and low levels of alpha1, whereas papillary lines expressed a heterogenous pattern of IRM, dominated by alpha5 and beta1. ATC mainly displayed integrins alpha2, alpha3, alpha5, alpha6, beta1 and low levels of alpha1, alpha4 and alphaV. Integrin heterodimers correlated with monomer expression. Evaluation of TCS largely confirmed these results with few exceptions, namely alpha4, alpha6, and beta3. The ability of TC cell lines to adhere to purified ECM proteins correlated with IRM expression. TSH induced TC cell adhesion in a dose-dependent fashion, despite an unchanged array of IRM expression or level of a particular IRM. Thyroid carcinoma cell lines of different histogenetic background display profoundly different patterns of IRM expression that appear to correlate with tumor aggressiveness. In vitro adhesion to ECM proteins and IRM expression concur. Finally, TSH-stimulated adhesion of

  18. Leishmania infection modulates beta-1 integrin activation and alters the kinetics of monocyte spreading over fibronectin

    PubMed Central

    Figueira, Cláudio Pereira; Carvalhal, Djalma Gomes Ferrão; Almeida, Rafaela Andrade; Hermida, Micely d’ El-Rei; Touchard, Dominique; Robert, Phillipe; Pierres, Anne; Bongrand, Pierre; dos-Santos, Washington LC

    2015-01-01

    Contact with Leishmania leads to a decreases in mononuclear phagocyte adherence to connective tissue. In this work, we studied the early stages of bond formation between VLA4 and fibronectin, measured the kinetics of membrane alignment and the monocyte cytoplasm spreading area over a fibronectin-coated surface, and studied the expression of high affinity integrin epitope in uninfected and Leishmania-infected human monocytes. Our results show that the initial VLA4-mediated interaction of Leishmania-infected monocyte with a fibronectin-coated surface is preserved, however, the later stage, leukocyte spreading over the substrate is abrogated in Leishmania-infected cells. The median of spreading area was 72 [55–89] μm2 for uninfected and 41 [34–51] μm2 for Leishmania-infected monocyte. This cytoplasm spread was inhibited using an anti-VLA4 blocking antibody. After the initial contact with the fibronectrin-coated surface, uninfected monocyte quickly spread the cytoplasm at a 15 μm2 s−1 ratio whilst Leishmania-infected monocytes only made small contacts at a 5.5 μm2 s−1 ratio. The expression of high affinity epitope by VLA4 (from 39 ± 21% to 14 ± 3%); and LFA1 (from 37 ± 32% to 18 ± 16%) molecules was reduced in Leishmania-infected monocytes. These changes in phagocyte function may be important for parasite dissemination and distribution of lesions in leishmaniasis. PMID:26249106

  19. Leishmania infection modulates beta-1 integrin activation and alters the kinetics of monocyte spreading over fibronectin.

    PubMed

    Figueira, Cláudio Pereira; Carvalhal, Djalma Gomes Ferrão; Almeida, Rafaela Andrade; Hermida, Micely d' El-Rei; Touchard, Dominique; Robert, Phillipe; Pierres, Anne; Bongrand, Pierre; dos-Santos, Washington L C

    2015-08-07

    Contact with Leishmania leads to a decreases in mononuclear phagocyte adherence to connective tissue. In this work, we studied the early stages of bond formation between VLA4 and fibronectin, measured the kinetics of membrane alignment and the monocyte cytoplasm spreading area over a fibronectin-coated surface, and studied the expression of high affinity integrin epitope in uninfected and Leishmania-infected human monocytes. Our results show that the initial VLA4-mediated interaction of Leishmania-infected monocyte with a fibronectin-coated surface is preserved, however, the later stage, leukocyte spreading over the substrate is abrogated in Leishmania-infected cells. The median of spreading area was 72 [55-89] μm(2) for uninfected and 41 [34-51] μm(2) for Leishmania-infected monocyte. This cytoplasm spread was inhibited using an anti-VLA4 blocking antibody. After the initial contact with the fibronectrin-coated surface, uninfected monocyte quickly spread the cytoplasm at a 15 μm(2) s(-1) ratio whilst Leishmania-infected monocytes only made small contacts at a 5.5 μm(2) s(-1) ratio. The expression of high affinity epitope by VLA4 (from 39 ± 21% to 14 ± 3%); and LFA1 (from 37 ± 32% to 18 ± 16%) molecules was reduced in Leishmania-infected monocytes. These changes in phagocyte function may be important for parasite dissemination and distribution of lesions in leishmaniasis.

  20. Modeled Microgravity Disrupts Collagen I/Integrin Signaling During Osteoblastic Differentiation of Human Mesenchymal Stem Cells

    NASA Technical Reports Server (NTRS)

    Meyers, Valerie E.; Zayzafoon, Majd; Gonda, Steven R.; Gathings, William E.; McDonald, Jay M.

    2004-01-01

    Spaceflight leads to reduced bone mineral density in weight bearing bones that is primarily attributed to a reduction in bone formation. We have previously demonstrated severely reduced osteoblastogenesis of human mesenchymal stem cells (hMSC) following seven days culture in modeled microgravity. One potential mechanism for reduced osteoblastic differentiation is disruption of type I collagen-integrin interactions and reduced integrin signaling. Integrins are heterodimeric transmembrane receptors that bind extracellular matrix proteins and produce signals essential for proper cellular function, survival, and differentiation. Therefore, we investigated the effects of modeled microgravity on integrin expression and function in hMSC. We demonstrate that seven days of culture in modeled microgravity leads to reduced expression of the extracellular matrix protein, type I collagen (Col I). Conversely, modeled microgravity consistently increases Col I-specific alpha2 and beta1 integrin protein expression. Despite this increase in integrin sub-unit expression, autophosphorylation of adhesion-dependent kinases, focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2), is significantly reduced. Activation of Akt is unaffected by the reduction in FAK activation. However, reduced downstream signaling via the Ras-MAPK pathway is evidenced by a reduction in Ras and ERK activation. Taken together, our findings indicate that modeled microgravity decreases integrin/MAPK signaling, which likely contributes to the observed reduction in osteoblastogenesis.

  1. Modeled microgravity disrupts collagen I/integrin signaling during osteoblastic differentiation of human mesenchymal stem cells.

    PubMed

    Meyers, Valerie E; Zayzafoon, Maid; Gonda, Steve R; Gathings, William E; McDonald, Jay M

    2004-11-01

    Spaceflight leads to reduced bone mineral density in weight bearing bones that is primarily attributed to a reduction in bone formation. We have previously demonstrated severely reduced osteoblastogenesis of human mesenchymal stem cells (hMSC) following 7 days culture in modeled microgravity (MMG). One potential mechanism for reduced osteoblastic differentiation is disruption of type I collagen (Col I)-integrin interactions and reduced integrin signaling. Integrins are heterodimeric transmembrane receptors that bind extracellular matrix (ECM) proteins and produce signals essential for proper cellular function, survival, and differentiation. Therefore, we investigated the effects of MMG on integrin expression and function in hMSC. We demonstrate that 7 days of culture in MMG leads to reduced expression of the ECM protein, Col I. Conversely, MMG consistently increases Col I-specific alpha2 and beta1 integrin protein expression. Despite this increase in integrin subunit expression, autophosphorylation of adhesion-dependent kinases, focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2), is significantly reduced. Activation of Akt protein kinase (Akt) is unaffected by the reduction in FAK activation. However, reduced downstream signaling via the Ras-mitogen activated protein kinase (MAPK) pathway is evidenced by a reduction in Ras and extracellular signal-related protein kinase (ERK) activation. Taken together, our findings indicate that MMG decreases integrin/MAPK signaling, which likely contributes to the observed reduction in osteoblastogenesis.

  2. Beta ig-h3 promotes renal proximal tubular epithelial cell adhesion, migration and proliferation through the interaction with alpha3beta1 integrin.

    PubMed

    Park, Sun-Woo; Bae, Jong-Sup; Kim, Ki-San; Park, Sun-Hee; Lee, Byung-Heon; Choi, Je-Yong; Park, Jae-Yong; Ha, Sung-Woo; Kim, Yong-Lim; Kwon, Tae-Hwan; Kim, In-San; Park, Rang-Woon

    2004-06-30

    Betaig-h3 (betaig-h3) is a secretory protein composed of fasciclin I-like repeats containing sequences that allows binding of integrins and glycosaminoglycans in vivo. Expression of betaig-h3 is responsive to TGF-Beta and the protein is found to be associated with extracellular matrix (ECM) molecules, implicating betaig-h3 as an ECM adhesive protein of developmental processes. We previously observed predominant expression of betaig-h3 expression in the basement membrane of proximal tubules of kidney. In this study, the physiological relevance of such localized expression of betaig-h3 was examined in the renal proximal tubular epithelial cells (RPTEC). RPTEC constitutively expressed betaig-h3 and the expression was dramatically induced by exogenous TGF-Beta1 treatment. betaig-h3 and its second and fourth FAS1 domain were able to mediate RPTEC adhesion, spreading and migration. Two known alpha3beta1 integrin-interaction motifs including aspartatic acid and isoleucine residues, NKDIL and EPDIM in betaig-h3 were responsible to mediate RPTEC adhesion, spreading, and migration. By using specific antibodies against integrins, we confirmed that alpha3beta1 integrin mediates the adhesion and migration of RPTECs on betaig-h3. In addition, it also enhanced proliferation of RPTECs through NKDIL and EPDIM. These results indicate that betaig-h3 mediates adhesion, spreading, migration and proliferation of RPTECs through the interaction with alpha3beta1 integrin and is intimately involved in the maintenance and the regeneration of renal proximal tubular epithelium.

  3. A monoclonal antibody to beta 1 integrin (CD29) stimulates VLA- dependent adherence of leukocytes to human umbilical vein endothelial cells and matrix components

    PubMed Central

    1992-01-01

    The leukocyte beta 1 integrin receptor very late activation antigen-4 (VLA-4) (alpha 4 beta 1, CD49d/CD29) binds to vascular cell adhesion molecule-1 (VCAM-1) expressed on cytokine-activated endothelium. A mAb designated 8A2 was identified that stimulated the binding of U937 cells to CHO cells transfected with VCAM-1 cDNA but not endothelial-leukocyte adhesion molecule or CD4 cDNA. mAb 8A2 also rapidly stimulated the adherence of peripheral blood lymphocytes (PBLs) to VCAM-1-transfected CHO cells or recombinant human tumor necrosis factor-treated human umbilical vein endothelial cells. mAb 8A2-stimulated binding of PBL was inhibited by mAbs to VLA-4 or VCAM-1. Surface expression of VLA-4 was not altered by mAb 8A2 treatment and monovalent Fab fragments of mAb 8A2 were active. Immunoprecipitation studies reveal that mAb 8A2 recognizes beta 1-subunit (CD29) of integrin receptors. In contrast to mAbs directed to VLA-4 alpha-subunit (alpha 4, CD49d), mAb 8A2 did not induce homotypic aggregation of PBL. Additionally, mAb 8A2 stimulated adherence of PBL and hematopoietic cell lines to purified matrix components laminin and fibronectin. This binding was blocked by mAbs to the VLA alpha-subunits alpha 6 (CD49f), or alpha 5 (CD49e) and alpha 4 (CD49d), respectively. We conclude that mAb 8A2 modulates the affinity of VLA-4 and other leukocyte beta 1 integrins, and should prove useful in studying the regulation of beta 1 integrin function. PMID:1370496

  4. The role of alpha 6 beta 1 integrin and EGF in normal and malignant acinar morphogenesis of human prostatic epithelial cells.

    PubMed

    Bello-DeOcampo, D; Kleinman, H K; Webber, M M

    2001-09-01

    Complex multiple interactions between cells and extracellular matrix occur during acinar morphogenesis involving integrin receptors and growth factors. Changes in these interactions occur during carcinogenesis as cells progress from a normal to a malignant, invasive phenotype. We have developed human prostatic epithelial cell lines of the same lineage, which represent multiple steps in carcinogenesis, similar to prostatic intraepithelial neoplasia and subsequent tumor progression. The non-tumorigenic, RWPE-1 and the tumorigenic WPE1-NB27 and WPE1-NB26 cell lines were used to examine their ability to undergo acinar morphogenesis in a 3-D cell culture model and its relationship to invasion, integrin expression and EGF presence. An inverse relationship between the degree of acinar formation and invasive ability was observed. The non-tumorigenic, non-invasive RWPE-1 and the low tumorigenic, low invasive, WPE1-NB27 cells show high and decreased acinar forming ability, respectively, while the more invasive WPE1-NB26 cells show a loss of acinar formation. While RWPE-1 acini show basal expression of alpha 6 beta 1 integrin, which correlates with their ability to polarize and form acini, WPE1-NB27 cells lack alpha 6 but show basal, but weaker expression of beta 1 integrin. WPE1-NB26 cells show loss alpha 6 and abnormal, diffused beta 1 integrin expression. A dose-dependent decrease in acinar formation was observed in RWPE-1 cells when cell proliferation was induced by EGF. Anti-functional antibody to EGF caused an increase in acinar formation in RWPE-1 cells. These results suggest that malignant cells lose the ability to undergo acinar morphogenesis and that the degree of this loss appears to be related to invasive ability, EGF levels and alterations in laminin-specific integrin expression. This model system mimics different steps in prostate carcinogenesis and has applications in the secondary and tertiary prevention of prostate cancer.

  5. Mapping regions of the beta1 integrin cytoplasmic domain involved in migration and survival in primary oligodendrocyte precursors using cell-permeable homeopeptides.

    PubMed

    Buttery, P C; Mallawaarachchi, C M; Milner, R; Doherty, P; ffrench-Constant, C

    1999-05-27

    The mapping of regions within integrin cytoplasmic domains responsible for the different effects on cell behaviour is an important part of an analysis of integrin-mediated signalling. In order to facilitate this analysis in primary cells, we have used cell-permeable homeopeptides to deliver sequences mimicking parts of the integrin beta1 cytoplasmic domain into the cell. In a study using oligodendrocyte precursors, the cells that give rise to myelin-forming oligodendrocytes during CNS development, we show that these peptides can be used to manipulate beta1 integrin signalling and that the regions of the cytoplasmic domain involved in migration and survival are distinct. Peptides mimicking the N-terminal portion of the cytoplasmic domain previously implicated in binding to Focal Adhesion Kinase (FAK) induce apoptosis, while peptides mimicking more C-terminal sequences do not cause cell death. In contrast they show that the NPIY sequence, the N-terminal one of two NPXY motifs previously implicated in signalling, is involved in migration. Peptides containing this sequence promote migration while alteration of NPIY to NPIA makes the peptide inhibitory to migration. Our results show that these peptides represent a novel approach to integrin signalling that allow rapid definition of critical cytoplasmic sequences in primary cells.

  6. Integrin {alpha}{beta}1, {alpha}{sub v}{beta}, {alpha}{sub 6}{beta} effectors p130Cas, Src and talin regulate carcinoma invasion and chemoresistance

    SciTech Connect

    Sansing, Hope A.; Sarkeshik, Ali; Yates, John R.; Patel, Vyomesh; Gutkind, J. Silvio; Yamada, Kenneth M.; Berrier, Allison L.

    2011-03-11

    Research highlights: {yields} Proteomics of clustered integrin {alpha}{beta}1, {alpha}{sub v}{beta}, {alpha}{sub 6}{beta} receptors in oral carcinoma. {yields} p130Cas, Dek, Src and talin regulate oral carcinoma invasion. {yields} p130Cas, talin, Src and zyxin regulate oral carcinoma resistance to cisplatin. -- Abstract: Ligand engagement by integrins induces receptor clustering and formation of complexes at the integrin cytoplasmic face that controls cell signaling and cytoskeletal dynamics critical for adhesion-dependent processes. This study searches for a subset of integrin effectors that coordinates both tumor cell invasion and resistance to the chemotherapeutic drug cisplatin in oral carcinomas. Candidate integrin effectors were identified in a proteomics screen of proteins recruited to clustered integrin {alpha}{beta}1, {alpha}{sub v}{beta} or {alpha}{sub 6}{beta} receptors in oral carcinomas. Proteins with diverse functions including microtubule and actin binding proteins, and factors involved in trafficking, transcription and translation were identified in oral carcinoma integrin complexes. Knockdown of effectors in the oral carcinoma HN12 cells revealed that p130Cas, Dek, Src and talin were required for invasion through Matrigel. Disruption of talin or p130Cas by RNA interference increased resistance to cisplatin, whereas targeting Dek, Src or zyxin reduced HN12 resistance to cisplatin. Analysis of the spreading of HN12 cells on collagen I and laminin I revealed that a decrease in p130Cas or talin expression inhibited spreading on both matrices. Interestingly, a reduction in zyxin expression enhanced spreading on laminin I and inhibited spreading on collagen I. Reduction of Dek, Src, talin or zyxin expression reduced HN12 proliferation by 30%. Proliferation was not affected by a reduction in p130Cas expression. We conclude that p130Cas, Src and talin function in both oral carcinoma invasion and resistance to cisplatin.

  7. Integrin alpha 2 beta 1-independent activation of platelets by simple collagen-like peptides: collagen tertiary (triple-helical) and quaternary (polymeric) structures are sufficient alone for alpha 2 beta 1-independent platelet reactivity.

    PubMed Central

    Morton, L F; Hargreaves, P G; Farndale, R W; Young, R D; Barnes, M J

    1995-01-01

    The platelet reactivities of two simple collagen-like synthetic peptides, Gly-Lys-Hyp-(Gly-Pro-Hyp)10-Gly-Lys-Hyp-Gly and Gly-Cys-Hyp-(Gly-Pro-Hyp)10-Gly-Cys-Hyp-Gly, were investigated. Both peptides adopted a stable triple-helical conformation in solution. Following cross-linking, both peptides proved to be highly platelet-aggregatory, more active than collagen fibres, inducing aggregation at concentrations as low as 20 ng/ml. These peptides formed microaggregates in solution, and cross-linking was thought to stabilize these structures, allowing expression of their platelet reactivity at 37 degrees C. Like collagen fibres, the peptides caused platelet secretion and release of arachidonate from platelet membrane lipids as well as activation of integrin alpha IIb beta 3 culminating in aggregation. Monoclonal antibodies directed against the integrin alpha 2 beta 1 failed to prevent aggregation release of arachidonate or platelet adhesion to the peptides. Our results indicate that collagen can activate platelets by a mechanism that is independent of integrin alpha 2 beta 1 and for which collagen tertiary and quaternary structures are sufficient alone for activity without the involvement of highly specific cell-recognition sequences. Images Figure 5 PMID:7534064

  8. Junctional adhesion molecule (JAM)-B supports lymphocyte rolling and adhesion through interaction with alpha4beta1 integrin.

    PubMed

    Ludwig, Ralf J; Hardt, Katja; Hatting, Max; Bistrian, Roxana; Diehl, Sandra; Radeke, Heinfried H; Podda, Maurizio; Schön, Michael P; Kaufmann, Roland; Henschler, Reinhard; Pfeilschifter, Josef M; Santoso, Sentot; Boehncke, Wolf-Henning

    2009-10-01

    Junctional adhesion molecule-A (JAM-A), JAM-B and JAM-C have been implicated in leucocyte transmigration. As JAM-B binds to very late activation antigen (VLA)-4, a leucocyte integrin that contributes to rolling and firm adhesion of lymphocytes to endothelial cells through binding to vascular cell adhesion molecule (VCAM)-1, we hypothesized that JAM-B is also involved in leucocyte rolling and firm adhesion. To test this hypothesis, intravital microscopy of murine skin microvasculature was performed. Rolling interactions of murine leucocytes were significantly affected by blockade of JAM-B [which reduced rolling interactions from 9.1 +/- 2.6% to 3.2 +/- 1.2% (mean +/- standard deviation)]. To identify putative ligands, T lymphocytes were perfused over JAM-B-coated slides in a dynamic flow chamber system. JAM-B-dependent rolling and sticking interactions were observed at low shear stress [0.3 dyn/cm(2): 220 +/- 71 (mean +/- standard deviation) versus 165 +/- 88 rolling (P < 0.001; Mann-Whitney rank sum test) and 2.6 +/- 1.3 versus 1.0 +/- 0.7 sticking cells/mm(2)/min (P = 0.026; Mann-Whitney rank sum test) on JAM-B- compared with baseline], but not at higher shear forces (1.0 dyn/cm(2)). As demonstrated by antibody blocking experiments, JAM-B-mediated rolling and sticking of T lymphocytes was dependent on alpha4 and beta1 integrin, but not JAM-C expression. To investigate whether JAM-B-mediated leucocyte-endothelium interactions are involved in a disease-relevant in vivo model, adoptive transfer experiments in 2,4,-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity reactions were performed in mice in the absence or in the presence of a function-blocking JAM-B antibody. In this model, JAM-B blockade during the sensitization phase impaired the generation of the immune response to DNFB, which was assessed as the increase in ear swelling in untreated, DNFB-challenged mice, by close to 40% [P = 0.037; analysis of variance (anova)]. Overall, JAM-B appears to

  9. Recognition of the cellular beta1-chain integrin by the bacterial AfaD invasin is implicated in the internalization of afa-expressing pathogenic Escherichia coli strains.

    PubMed

    Plançon, Laure; Du Merle, Laurence; Le Friec, Sandrine; Gounon, Pierre; Jouve, Mabel; Guignot, Julie; Servin, Alain; Le Bouguénec, Chantal

    2003-10-01

    The afa operons from Escherichia coli associated with extra-intestinal and intestinal infections have been characterized and the AfaD protein has been shown to be involved in the low internalization of laboratory strains expressing the afa-3 operon. The aim of this study was to determine the role of the AfaD invasin during the interaction of pathogenic E. coli with epithelial cells. We show that AfaD is implicated in the entry of a clinical isolate into both HeLa and undifferentiated Caco-2 cells. Once in the cytoplasm of these cells, the bacteria formed inclusions in which they were able to survive for at least 72 h. Internalization assays using polystyrene beads coated with His6-tagged purified AfaD (rAfaD) demonstrated that this invasin mediates entry into cells derived from various tissues (intestine and urothelium) that are targets for afa-positive strains. Consistent with the previous observation that an antibody blockade involving anti-alpha5beta1 integrin abolishes bacterial internalization, we show here that the entry of rAfaD-coated beads was dependent on the production and accessibility of beta1 integrins on the cells. The AfaD proteins belong to a family of invasins that are at least 45% identical. Despite their differences, the recombinant rAfaD-III and rAfaD-VIII proteins both bound to beta1 integrins. Our results suggest that beta1 integrin is a common receptor for AfaD invasins and that additional AfaD-type-specific receptors exist.

  10. Possible role of Malassezia furfur in psoriasis: modulation of TGF-beta1, integrin, and HSP70 expression in human keratinocytes and in the skin of psoriasis-affected patients.

    PubMed

    Baroni, Adone; Paoletti, Iole; Ruocco, Eleonora; Agozzino, Marina; Tufano, Maria Antonietta; Donnarumma, Giovanna

    2004-01-01

    Psoriasis is a disease characterized by an abnormal pattern of keratinocyte growth and differentiation. Malassezia furfur forms part of the normal human skin flora. It may also be involved in the pathogenesis of psoriasis. To define the role of M. furfur in the pathogenesis of psoriasis, we investigated how M. furfur regulates molecules involved in cell migration and proliferation. The experiments were performed using human keratinocytes and skin biopsies from M. furfur-positive and -negative psoriasis-affected patients. In addition, we examined the signal transduction mechanisms involved. Western blot analysis was performed on human keratinocytes lysates treated or untreated with M. furfur and on biopsies from healthy and psoriasis patients. Signal transduction mechanisms involved were evaluated by electrophoretic mobility shift assay using the AP-1 inhibitor curcumin. We found that M. furfur up-regulates transforming growth factor-beta1 (TGF-beta1), integrin chain, and HSP70 expression in human keratinocytes via AP-1-dependent mechanism. In the biopsies of M. furfur-positive psoriasis-affected patients, an increase in TGF-beta1, integrin chains, and HSP70 expression was found. Our data suggest that M. furfur can induce the overproduction of molecules involved in cell migration and hyperproliferation, thereby favoring the exacerbation of psoriasis.

  11. Alphavbeta integrins play an essential role in BMP-2 induction of osteoblast differentiation.

    PubMed

    Lai, Chung-Fang; Cheng, Su-Li

    2005-02-01

    Both integrins and BMP-2 exert similar effects on osteoblasts. We examined the relationship between the alphav-containing integrins (alphavbeta) and BMP-2 in osteoblast function. BMP-2 stimulates alphavbeta expression. BMP-2 receptors co-localize/overlap with alphavbeta integrins, and the intact function of alphavbeta is essential in BMP-2 activity. Bone morphogenetic protein (BMP)-2 not only induces osteoblast differentiation and bone matrix mineralization, but also stimulates osteoblast migration on and adhesion to bone matrix proteins. The alphavbeta- and beta1- (alphabeta1) containing integrins mediate osteoblast interaction with many bone matrix proteins and play important roles in osteoblast adhesion, migration, and differentiation. Because alphavbeta integrins and BMP-2 share common effects on osteoblasts, we analyzed their relationship in osteoblast function. The effects of BMP-2 on integrin expression were determined by surface labeling/immunoprecipitation and cell adhesion to matrix proteins. Confocal analysis of the immunostained cells and co-immunoprecipitation of cell extracts were used to study the spatial relationship between integrins and BMP-2 receptors. A function-blocking anti-alphavbeta integrin antibody (L230) was employed to investigate the roles of alphavbeta integrins in BMP-2 function. Human osteoblasts (HOBs) express alphabeta1, alphavbeta3, alphavbeta5, alphavbeta6, and alphavbeta8 integrins at focal adhesion sites. BMP-2 increases the levels of these integrins on osteoblast surface and enhances HOB adhesion to osteopontin and vitronectin. Immunoprecipitation and immunostaining analyses show that BMP-2 receptors co-localize or overlap with alphavbeta and alphabeta1 integrins. Incubation of HOBs with L230 abolishes the antiproliferative effect of BMP-2 and reduces the capacity of BMP-2 to stimulate alkaline phosphatase activity and the expression of osteocalcin, osteopontin, and bone sialoprotein. Furthermore, L230 prevents BMP-2 induction

  12. TAT-Hsp27 promotes adhesion and migration of murine dental papilla-derived MDPC-23 cells through beta1 integrin-mediated signaling.

    PubMed

    Park, Jong-Hwan; Yoon, Ji-Hye; Lim, Young-Sin; Hwang, Ho-Keel; Kim, Soo-A; Ahn, Sang-Gun; Yoon, Jung-Hoon

    2010-09-01

    Odontoblasts are involved in tooth repair and regeneration as well as dentin formation. The aim of this study was to examine whether delivery of heat shock protein 27 (Hsp27) into cells using a TAT fusion protein system (TAT-Hsp27) enhances adhesion and migration of murine dental papilla-derived MDPC-23 cells. Hsp27 was delivered into cells by the TAT-fusion protein system. To examine whether TAT-Hsp27 affects the viability of MDPC-23 cells, MTT assay was performed. The effect of TAT-Hsp27 on adhesion and migration of MDPC-23 cells was determined using type I collagen-coated plates and a commercial kit, respectively. In addition, a precise molecular mechanism was examined by Western blot analysis and focal adhesion activity. TAT-fusion protein system delivered Hsp27 into cells successfully. Transduction of TAT-Hsp27 induced adhesion and migration of MDPC-23 cells in a dose-dependent manner. Moreover, transduction of TAT-Hsp27 increased the protein expression of beta1 integrin and focal adhesion formation, and induced phosphorylation of FAK and ERK. TAT-Hsp27-induced migration of MDPC-23 cells was restored by treatment of anti-beta1 integrin antibody. These findings suggest that TAT-Hsp27 promotes adhesion and migration of MDPC-23 cells via beta1 integrin-mediated signaling and is a promising candidate for therapeutic application of dental pulp regeneration.

  13. Endothelial-monocyte activating polypeptide II alters fibronectin based endothelial cell adhesion and matrix assembly via alpha5 beta1 integrin

    SciTech Connect

    Schwarz, Margaret A. . E-mail: m.schwarz@umdnj.edu; Zheng, Hiahua; Liu, Jie; Corbett, Siobhan; Schwarz, Roderich E.

    2005-12-10

    Mature Endothelial-Monocyte Activating Polypeptide (mEMAP) II functions as a potent antiangiogenic peptide. Although the anti-tumor effect of mEMAP II has been described, little is known regarding its mechanism of action. Observations that mEMAP II induced apoptosis only in a subset of migrating and proliferating endothelial cells (EC) suggests a targeted effect on cells engaged in angiogenic activities which are known to rely upon cell adhesion and migration. Indeed, we demonstrate that mEMAP II inhibited fibronectin (FN) dependent microvascular EC (MEC) adhesion and spreading and we show that this depends upon the alpha5 beta1 integrin. Immunofluorescence analysis demonstrated that mEMAP II-dependent blockade of FN-alpha5 beta1 interactions was associated with disassembly of both actin stress fiber networks and FN matrix. These findings suggest that mEMAP II blocks MEC adhesion and spreading on fibronectin, via a direct interaction with the integrin alpha5 beta1, thus implicating that alpha5 integrin may be a mediator of mEMAP II's antiangiogenic function.

  14. The PPFLMLLKGSTR motif in globular domain 3 of the human laminin-5 {alpha}3 chain is crucial for integrin {alpha}3{beta}1 binding and cell adhesion

    SciTech Connect

    Kim, Jin-Man; Park, Won Ho; Min, Byung-Moo . E-mail: bmmin@snu.ac.kr

    2005-03-10

    Laminin-5 regulates various cellular functions, including cell adhesion, spreading, and motility. Here, we expressed the five human laminin {alpha}3 chain globular (LG) domains as monomeric, soluble fusion proteins, and examined their biological functions and signaling. Recombinant LG3 (rLG3) protein, unlike rLG1, rLG2, rLG4, and rLG5, played roles in cell adhesion, spreading, and integrin {alpha}3{beta}1 binding. More significantly, we identified a novel motif (PPFLMLLKGSTR) in the LG3 domain that is crucial for these responses. Studies with the synthetic peptides delineated the PPFLMLLKGSTR peptide within LG3 domain as a major site for both integrin {alpha}3{beta}1 binding and cell adhesion. Substitution mutation experiments suggest that the Arg residue is important for these activities. rLG3 protein- and PPFLMLLKGSTR peptide-induced keratinocyte adhesion triggered cell signaling through FAK phosphorylation at tyrosine-397 and -577. To our knowledge, this is the first report demonstrating that the PPFLMLLKGSTR peptide within the LG3 domain is a novel motif that is capable of supporting integrin {alpha}3{beta}1-dependent cell adhesion and spreading.

  15. In vitro analysis of integrin expression during chondrogenic differentiation of mesenchymal stem cells and chondrocytes upon dedifferentiation in cell culture.

    PubMed

    Goessler, Ulrich Reinhart; Bieback, Karen; Bugert, Peter; Heller, Tobias; Sadick, Haneen; Hörmann, Karl; Riedel, Frank

    2006-02-01

    Tissue engineering represents a promising method for generating chondrogenic grafts for reconstructive surgery. In cultured chondrocytes, the dedifferentiation of cells seems unavoidable for multiplication. Stem cells, however, displaying unlimited self-renewal and the capacity to differentiate towards chondrocytes, might be usable after further characterization. As the interactions between the extracellular matrix and the cellular compartment can alter the cellular behaviour, we investigated the expression of integrins using microarray analysis during chondrogenic differentiation of human mesenchymal stem cells (MSC) in comparison with de-differentiating human chondrocytes (HC) harvested during septoplasty. During chondrogenic differentiation of MSC, the fibronectin-receptor (Integrin beta1alpha5), fibronectin and the GPIIb/IIIa-receptor were downregulated. The components of the vitronectin-receptor (Integrin alphavbeta3) and CD47 were constantly expressed and ILK was downregulated. Vitronectin and osteopontin were not expressed by the cells. In HC, Integrin beta1alpha5 in conjunction with the ligand fibronectin were upregulated during dedifferentiation, Integrin alphavbeta3 as well as the GBIIb/IIIa-receptor were activated on day 21 but neither vitronectin nor osteopontin were expressed by the cells. The integrins, beta2, beta4, beta6, beta8 and alpha2, alpha4, alpha6, alpha7, alpha11, were not expressed at any time. ILK, CD47, and ICAP were activated with ongoing dedifferentiation. In conclusion, a candidate for signal-transmission is the fibronectin receptor (integrin alpha5beta1) in conjunction with its ligand fibronectin. Other receptors, e.g. for vitronectin and osteopontin (alphavbeta3), or their ligands do not seem to be involved in signal transmission for dedifferentiation. The GPIIb/IIIa-receptor might assist the process of dedifferentiation. Intracellularly, ILK, ICAP1 and CD47 might be involved in the transduction of integrin-dependent signals.

  16. Arg-Tyr-Asp (RYD) and Arg-Cys-Asp (RCD) motifs in dendroaspin promote selective inhibition of beta1 and beta3 integrins.

    PubMed

    Wattam, B; Shang, D; Rahman, S; Egglezou, S; Scully, M; Kakkar, V; Lu, X

    2001-05-15

    Arg-Gly-Asp (RGD) is a unique minimal integrin-binding sequence that is found within several glycoprotein ligands. This sequence has also been found in snake-venom anti-platelet proteins, including the disintegrins and dendroaspin, a natural variant of short-chain neurotoxins isolated from the venom of Dendroaspis jamesonii. In the present study, the motifs RYD and RCD were introduced into the dendroaspin scaffold to replace RGD. Both motifs in dendroaspin caused inhibition of ADP-induced platelet aggregation with IC(50) values of 200 and 300 nM respectively, similar to that of the wild-type RGD motif (170 nM). In comparison with wild-type dendroaspin, both RYD- and RCD-containing dendroaspins were more selective in the inhibition of the adhesion of K562 cells to laminin rather than to fibrinogen and fibronectin, even though they were 10-30-fold less potent at inhibiting K562 cell (containing alpha(5)beta(1) integrin) adhesion to laminin compared with wild-type. Interestingly, the RYD motif produced a similar IC(50) value to the RGD motif at inhibiting A375-SM cell (beta(3) integrin) adhesion to collagen, whereas the RCD motif was approx. 2-6-fold less potent compared with either RGD or RYD. These findings show that the selectivity of dendroaspin binding to beta(1) and beta(3) integrins can be modulated by the introduction of alternative cell recognition sequences.

  17. Beta-1 integrin-mediated adhesion may be initiated by multiple incomplete bonds, thus accounting for the functional importance of receptor clustering.

    PubMed

    Vitte, Joana; Benoliel, Anne-Marie; Eymeric, Philippe; Bongrand, Pierre; Pierres, Anne

    2004-06-01

    The regulation of cell integrin receptors involves modulation of membrane expression, shift between different affinity states, and topographical redistribution on the cell membrane. Here we attempted to assess quantitatively the functional importance of receptor clustering. We studied beta-1 integrin-mediated attachment of THP-1 cells to fibronectin-coated surfaces under low shear flow. Cells displayed multiple binding events with a half-life of the order of 1 s. The duration of binding events after the first second after arrest was quantitatively accounted for by a model assuming the existence of a short-time intermediate binding state with 3.6 s(-1) dissociation rate and 1.3 s(-1) transition frequency toward a more stable state. Cell binding to surfaces coated with lower fibronectin densities was concluded to be mediated by single molecular interactions, whereas multiple bonds were formed <1 s after contact with higher fibronectin surface densities. Cell treatment with microfilament inhibitors or a neutral antiintegrin antibody decreased bond number without changing aforementioned kinetic parameters whereas a function enhancing antibody increased the rate of bond formation and/or the lifetime of intermediate state. Receptor aggregation was induced by treating cells with neutral antiintegrin antibody and antiimmunoglobulin antibodies. A semiquantitative confocal microscopy study suggested that this treatment increased between 40% and 100% the average number of integrin receptors located in a volume of approximately 0.045 microm(3) surrounding each integrin. This aggregation induced up to 2.7-fold increase of the average number of bonds. Flow cytometric analysis of fluorescent ligand binding showed that THP-1 cells displayed low-affinity beta-1 integrins with a dissociation constant in the micromolar range. It is concluded that the initial step of cell adhesion was mediated by multiple incomplete bonds rather than a single equilibrium-state ligand receptor

  18. Fibronectin type III5 repeat contains a novel cell adhesion sequence, KLDAPT, which binds activated alpha4beta1 and alpha4beta7 integrins.

    PubMed

    Moyano, J V; Carnemolla, B; Domínguez-Jiménez, C; García-Gila, M; Albar, J P; Sánchez-Aparicio, P; Leprini, A; Querzé, G; Zardi, L; Garcia-Pardo, A

    1997-10-03

    The region of fibronectin encompassing type III repeats 4-6 contains a low affinity heparin binding domain, but its physiological significance is not clear. We have studied whether this domain is able to interact with cells as already shown for other heparin binding domains of fibronectin. A computer search based on homologies with known active sites in fibronectin revealed the sequence KLDAPT located in FN-III5. A synthetic peptide containing this sequence induced lymphoid cell adhesion upon treatment with the activating anti-beta1 monoclonal antibody (mAb) TS2/16 or with Mn2+, indicating that KLDAPT was binding to an integrin. A recombinant fragment containing repeat III5 (FN-III5) also mediated adhesion of TS2/16/Mn2+-treated cells while the FN-III6 fragment did not. Soluble KLDAPT peptide inhibited cell adhesion to FN-III5 as well as to a 38-kDa fibronectin fragment and VCAM-1, two previously known ligands for alpha4beta1 integrin. KLDAPT also competed with the binding of soluble alkaline phosphatase-coupled VCAM-Ig to Mn2+-treated alpha4beta1. Furthermore, mAbs anti-alpha4 and anti-alpha4beta7, but not mAbs to other integrins, inhibited cell adhesion to FN-III5 and KLDAPT. These results therefore establish a cell adhesive function for the FN-III5 repeat and show that KLDAPT is a novel fibronectin ligand for activated alpha4 integrins.

  19. Integrin alpha1beta1 controls reactive oxygen species synthesis by negatively regulating epidermal growth factor receptor-mediated Rac activation.

    PubMed

    Chen, Xiwu; Abair, Tristin D; Ibanez, Maria R; Su, Yan; Frey, Mark R; Dise, Rebecca S; Polk, D Brent; Singh, Amar B; Harris, Raymond C; Zent, Roy; Pozzi, Ambra

    2007-05-01

    Integrins control many cell functions, including generation of reactive oxygen species (ROS) and regulation of collagen synthesis. Mesangial cells, found in the glomerulus of the kidney, are able to produce large amounts of ROS via the NADPH oxidase. We previously demonstrated that integrin alpha1-null mice develop worse fibrosis than wild-type mice following glomerular injury and this is due, in part, to excessive ROS production by alpha1-null mesangial cells. In the present studies, we describe the mechanism whereby integrin alpha1-null mesangial cells produce excessive ROS. Integrin alpha1-null mesangial cells have constitutively increased basal levels of activated Rac1, which result in its increased translocation to the cell membrane, excessive ROS production, and consequent collagen IV deposition. Basal Rac1 activation is a direct consequence of ligand-independent increased epidermal growth factor receptor (EGFR) phosphorylation in alpha1-null mesangial cells. Thus, our study demonstrates that integrin alpha1beta1-EGFR cross talk is a key step in negatively regulating Rac1 activation, ROS production, and excessive collagen synthesis, which is a hallmark of diseases characterized by irreversible fibrosis.

  20. Beta 1-integrin-c-Met cooperation reveals an inside-in survival signalling on autophagy-related endomembranes.

    PubMed

    Barrow-McGee, Rachel; Kishi, Naoki; Joffre, Carine; Ménard, Ludovic; Hervieu, Alexia; Bakhouche, Bakhouche A; Noval, Alejandro J; Mai, Anja; Guzmán, Camilo; Robert-Masson, Luisa; Iturrioz, Xavier; Hulit, James; Brennan, Caroline H; Hart, Ian R; Parker, Peter J; Ivaska, Johanna; Kermorgant, Stéphanie

    2016-06-23

    Receptor tyrosine kinases (RTKs) and integrins cooperate to stimulate cell migration and tumour metastasis. Here we report that an integrin influences signalling of an RTK, c-Met, from inside the cell, to promote anchorage-independent cell survival. Thus, c-Met and β1-integrin co-internalize and become progressively recruited on LC3B-positive 'autophagy-related endomembranes' (ARE). In cells growing in suspension, β1-integrin promotes sustained c-Met-dependent ERK1/2 phosphorylation on ARE. This signalling is dependent on ATG5 and Beclin1 but not on ATG13, suggesting ARE belong to a non-canonical autophagy pathway. This β1-integrin-dependent c-Met-sustained signalling on ARE supports anchorage-independent cell survival and growth, tumorigenesis, invasion and lung colonization in vivo. RTK-integrin cooperation has been assumed to occur at the plasma membrane requiring integrin 'inside-out' or 'outside-in' signalling. Our results report a novel mode of integrin-RTK cooperation, which we term 'inside-in signalling'. Targeting integrin signalling in addition to adhesion may have relevance for cancer therapy.

  1. Decrease in fibronectin occurs coincident with the increased expression of its integrin receptor alpha5beta1 in stress-deprived ligaments.

    PubMed Central

    AbiEzzi, S. S.; Foulk, R. A.; Harwood, F. L.; Akeson, W. H.; Amiel, D.

    1997-01-01

    Stress deprivation secondary to immobilization leads to atrophic changes in periarticular soft tissues. The changes in ligaments include a disorganization of collagen and cellular ultrastructure with varied biochemical alterations resulting in a functionally weaker tissue. This study tests the hypothesis that alterations in fibronectin (Fn) and the expression of its integrin receptor alpha5beta1 in ligament fibroblasts accompany the extracellular matrix remodeling which occurs in stress-deprived knee ligaments. The left knees of eighteen New Zealand white rabbits were surgically immobilized in acute flexion. Fibroblasts within three nine week and three twelve week stress-deprived anterior cruciate ligaments (ACLs) and medial collateral ligaments (MCLs) demonstrated markedly increased immunostaining for the beta1 and alpha5 integrin subunits, as compared to fibroblasts in the contralateral unoperated control ligaments. The effects of stress deprivation on the concentration of Fn was measured by competitive ELISA on the remaining twelve rabbits. Decreases in Fn of 54.0 percent and 63.7 percent occurred in the ACL after nine and twelve weeks of stress deprivation when compared to contralateral controls. The MCL had less of a decrease, losing 37.7 percent and 41.7 percent at nine and twelve weeks, respectively. These results suggest an important role for the Fn-specific integrin receptor alpha5beta1 in remodeling stress-deprived periarticular ligamentous tissue, and the importance of maintaining normal stresses on periarticular ligaments to prevent the degradation of extracellular matrix components such as Fn. Images Figure 1 Figure 2 Figure 3 PMID:9234981

  2. Coordinate role for cell surface chondroitin sulfate proteoglycan and alpha 4 beta 1 integrin in mediating melanoma cell adhesion to fibronectin

    PubMed Central

    1992-01-01

    Cellular recognition and adhesion to the extracellular matrix (ECM) has a complex molecular basis, involving both integrins and cell surface proteoglycans (PG). The current studies have used specific inhibitors of chondroitin sulfate proteoglycan (CSPG) synthesis along with anti- alpha 4 integrin subunit monoclonal antibodies to demonstrate that human melanoma cell adhesion to an A-chain derived, 33-kD carboxyl- terminal heparin binding fragment of human plasma fibronectin (FN) involves both cell surface CSPG and alpha 4 beta 1 integrin. A direct role for cell surface CSPG in mediating melanoma cell adhesion to this FN fragment was demonstrated by the identification of a cationic synthetic peptide, termed FN-C/H-III, within the fragment. FN-C/H-III is located close to the amino terminal end of the fragment, representing residues #1721-1736 of intact FN. FN-C/H-III binds CSPG directly, can inhibit CSPG binding to the fragment, and promotes melanoma cell adhesion by a CSPG-dependent, alpha 4 beta 1 integrin- independent mechanism. A scrambled version of FN-C/H-III does not inhibit CSPG binding or cell adhesion to the fragment or to FN-C/H-III, indicating that the primary sequence of FN-C/H-III is important for its biological properties. Previous studies have identified three other synthetic peptides from within this 33-kD FN fragment that promote cell adhesion by an arginyl-glycyl-aspartic acid (RGD) independent mechanism. Two of these synthetic peptides (FN-C/H-I and FN-C/H-II) bind heparin and promote cell adhesion, implicating cell surface PG in mediating cellular recognition of these two peptides. Additionally, a third synthetic peptide, CS1, is located in close proximity to FN-C/H-I and FN-C/H-II and it promotes cell adhesion by an alpha 4 beta 1 integrin-dependent mechanism. In contrast to FN-C/H-III, cellular recognition of these three peptides involved contributions from both CSPG and alpha 4 integrin subunits. Of particular importance are observations

  3. Dystrophin Dp71f associates with the beta1-integrin adhesion complex to modulate PC12 cell adhesion

    PubMed Central

    Cerna, Joel; Cerecedo, Doris; Ortega, Arturo; García-Sierra, Francisco; Centeno, Federico; Garrido, Efrain; Mornet, Dominique; Cisneros, Bulmaro

    2006-01-01

    Dystrophin Dp71 is the main product of the Duchenne muscular dystrophy gene in the brain; however, its function is unknown. To study the role of Dp71 in neuronal cells, we previously generated by antisense treatment PC12 neuronal cell clones with decreased Dp71 expression (antisense-Dp71 cells). PC12 cells express two different splicing isoforms of Dp71, a cytoplasmic variant called Dp71f and a nuclear isoform called Dp71d. We previously reported that antisense-Dp71 cells display deficient adhesion to substrate and reduced immunostaining of β1-integrin in the cell area contacting the substrate. In this study, we isolated additional antisenseDp71 clones to analyze in detail the potential involvement of Dp71f isoform with the β1-integrin adhesion system of PC12 cells. Immunofluorescence analyses as well as immunoprecipitation assays demonstrated that the PC12 cell β1-integrin adhesion complex is composed of β1-integrin, talin, paxillin, α-actinin, FAK and actin. In addition, our results showed that Dp71f associates with most of the β1-integrin complex components (β1-integrin, FAK, α-actinin, talin and actin). In the antisense-Dp71 cells, the deficiency of Dp71 provokes a significant reduction of the β1-integrin adhesion complex and, consequently, the deficient adhesion of these cells to laminin. In vitro binding experiments confirmed the interaction of Dp71f with FAK and β1-integrin. Our data indicate that Dp71f is a structural component of the β1-integrin adhesion complex of PC12 cells that modulates PC12 cell adhesion by conferring proper complex assembly and/or maintenance. PMID:16935300

  4. Surface densities of ephrin-B1 determine EphB1-coupled activation of cell attachment through alphavbeta3 and alpha5beta1 integrins.

    PubMed Central

    Huynh-Do, U; Stein, E; Lane, A A; Liu, H; Cerretti, D P; Daniel, T O

    1999-01-01

    Receptors of the Eph family and their ligands (ephrins) mediate developmental vascular assembly and direct axonal guidance. Migrating cell processes identify appropriate targets within migratory fields based on topographically displayed ephrin gradients. Here, EphB1 regulated cell attachment by discriminating the density at which ephrin-B1 was displayed on a reconstituted surface. EphB1-ephrin-B1 engagement did not promote cell attachment through mechanical tethering, but did activate integrin-mediated attachment. In endothelial cells, attachment to RGD peptides or fibrinogen was mediated through alphavbeta3 integrin. EphB1 transfection conferred ephrin-B1-responsive activation of alpha5beta1 integrin-mediated cell attachment in human embryonic kidney cells. Activation-competent but signaling-defective EphB1 point mutants failed to stimulate ephrin-B1 dependent attachment. These findings lead us to propose that EphB1 functions as a 'ligand density sensor' to signal integrin-mediated cell-matrix attachment. PMID:10205170

  5. Bone regeneration using an alpha 2 beta 1 integrin-specific hydrogel as a BMP-2 delivery vehicle.

    PubMed

    Shekaran, Asha; García, José R; Clark, Amy Y; Kavanaugh, Taylor E; Lin, Angela S; Guldberg, Robert E; García, Andrés J

    2014-07-01

    Non-healing bone defects present tremendous socioeconomic costs. Although successful in some clinical settings, bone morphogenetic protein (BMP) therapies require supraphysiological dose delivery for bone repair, raising treatment costs and risks of complications. We engineered a protease-degradable poly(ethylene glycol) (PEG) synthetic hydrogel functionalized with a triple helical, α2β1 integrin-specific peptide (GFOGER) as a BMP-2 delivery vehicle. GFOGER-functionalized hydrogels lacking BMP-2 directed human stem cell differentiation and produced significant enhancements in bone repair within a critical-sized bone defect compared to RGD hydrogels or empty defects. GFOGER functionalization was crucial to the BMP-2-dependent healing response. Importantly, these engineered hydrogels outperformed the current clinical carrier in repairing non-healing bone defects at low BMP-2 doses. GFOGER hydrogels provided sustained in vivo release of encapsulated BMP-2, increased osteoprogenitor localization in the defect site, enhanced bone formation and induced defect bridging and mechanically robust healing at low BMP-2 doses which stimulated almost no bone regeneration when delivered from collagen sponges. These findings demonstrate that GFOGER hydrogels promote bone regeneration in challenging defects with low delivered BMP-2 doses and represent an effective delivery vehicle for protein therapeutics with translational potential.

  6. Selection of a 2-azabicyclo[2.2.2]octane-based alpha4beta1 integrin antagonist as an inhaled anti-asthmatic agent.

    PubMed

    Lawson, Edward C; Santulli, Rosemary J; Dyatkin, Alexey B; Ballentine, Scott A; Abraham, William M; Rudman, Sandra; Page, Clive P; de Garavilla, Lawrence; Damiano, Bruce P; Kinney, William A; Maryanoff, Bruce E

    2006-06-15

    The alpha4beta1 integrin, expressed on eosinophils and neutrophils, induces inflammation in the lung by facilitating cellular infiltration and activation. From a number of potent alpha4beta1 antagonists that we evaluated for safety and efficacy, 1 was selected as a lead candidate for anti-asthma therapy by the inhalation route. We devised an optimized stereoselective synthesis to facilitate the preparation of a sufficiently large quantity of 1 for assessment in vivo. Administration of 1 to allergen-sensitive sheep by inhalation blocked the late-phase response of asthma and abolished airway hyper-responsiveness at 24h following the antigen challenge. Additionally, the recruitment of inflammatory cells into the lungs was inhibited. Administration of 1 to ovalbumin-sensitized guinea pigs intraperitoneally blocked airway resistance and inhibited the recruitment of inflammatory cells.

  7. Cyclooxygenase-2 enhances alpha2beta1 integrin expression and cell migration via EP1 dependent signaling pathway in human chondrosarcoma cells.

    PubMed

    Liu, Ju-Fang; Fong, Yi-Chin; Chang, Chih-Shiang; Huang, Chun-Yin; Chen, Hsien-Te; Yang, Wei-Hung; Hsu, Chin-Jung; Jeng, Long-Bin; Chen, Chih-Yi; Tang, Chih-Hsin

    2010-02-23

    Cyclooxygenase (COX)-2, the inducible isoform of prostaglandin (PG) synthase, has been implicated in tumor metastasis. Interaction of COX-2 with its specific EP receptors on the surface of cancer cells has been reported to induce cancer invasion. However, the effects of COX-2 on migration activity in human chondrosarcoma cells are mostly unknown. In this study, we examined whether COX-2 and EP interaction are involved in metastasis of human chondrosarcoma. We found that over-expression of COX-2 or exogenous PGE2 increased the migration of human chondrosarcoma cells. We also found that human chondrosarcoma tissues and chondrosarcoma cell lines had significant expression of the COX-2 which was higher than that in normal cartilage. By using pharmacological inhibitors or activators or genetic inhibition by the EP receptors, we discovered that the EP1 receptor but not other PGE receptors is involved in PGE2-mediated cell migration and alpha2beta1 integrin expression. Furthermore, we found that human chondrosarcoma tissues expressed a higher level of EP1 receptor than normal cartilage. PGE2-mediated migration and integrin up-regulation were attenuated by phospholipase C (PLC), protein kinase C (PKC) and c-Src inhibitor. Activation of the PLCbeta, PKCalpha, c-Src and NF-kappaB signaling pathway after PGE2 treatment was demonstrated, and PGE2-induced expression of integrin and migration activity were inhibited by the specific inhibitor, siRNA and mutants of PLC, PKC, c-Src and NF-kappaB cascades. Our results indicated that PGE2 enhances the migration of chondrosarcoma cells by increasing alpha2beta1 integrin expression through the EP1/PLC/PKCalpha/c-Src/NF-kappaB signal transduction pathway.

  8. Small GTPase Rho signaling is involved in {beta}1 integrin-mediated up-regulation of intercellular adhesion molecule 1 and receptor activator of nuclear factor {kappa}B ligand on osteoblasts and osteoclast maturation

    SciTech Connect

    Hirai, Fumihiko; Nakayamada, Shingo; Okada, Yosuke; Saito, Kazuyoshi; Kurose, Hitoshi; Mogami, Akira; Tanaka, Yoshiya . E-mail: tanaka@med.uoeh-u.ac.jp

    2007-04-27

    We assessed the characteristics of human osteoblasts, focusing on small GTPase Rho signaling. {beta}1 Integrin were highly expressed on osteoblasts. Engagement of {beta}1 integrins by type I collagen augmented expression of intercellular adhesion molecule 1 (ICAM-1) and receptor activator of nuclear factor {kappa}B ligand (RANKL) on osteoblasts. Rho was activated by {beta}1 stimulation in osteoblasts. {beta}1 Integrin-induced up-regulation of ICAM-1 and RANKL was inhibited by transfection with adenoviruses encoding C3 transferase or pretreated with Y-27632, specific Rho and Rho-kinase inhibitors. Engagement of {beta}1 integrin on osteoblasts induced formation of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNC) in a coculture system of osteoblasts and peripheral monocytes, but this action was completely abrogated by transfection of C3 transferase. Our results indicate the direct involvement of Rho-mediated signaling in {beta}1 integrin-induced up-regulation of ICAM-1 and RANKL and RANKL-dependent osteoclast maturation. Thus, Rho-mediated signaling in osteoblasts seems to introduce major biases to bone resorption.

  9. Retinoic acid receptor gamma impacts cellular adhesion, Alpha5Beta1 integrin expression and proliferation in K562 cells.

    PubMed

    Kelley, Melissa D; Phomakay, Raynin; Lee, Madison; Niedzwiedz, Victoria; Mayo, Rachel

    2017-01-01

    The interplay between cellular adhesion and proliferation is complex; however, integrins, particularly the α5β1 subset, play a pivotal role in orchestrating critical cellular signals that culminate in cellular adhesion and growth. Retinoids modify the expression of a variety of adhesive/proliferative signaling proteins including α5β1 integrins; however, the role of specific retinoic acid receptors involved in these processes has not been elucidated. In this study, the effect of all-trans-retinoic acid receptor (RAR) agonists on K562 cellular adhesion, proliferation, and α5β1 integrin cell surface expression was investigated. RARγ agonist exposure increased K562 cellular adhesion to RGD containing extracellular matrix proteins fibronectin and FN-120 in a time- and concentration dependent manner, while RARα or RARβ agonist treatment had no effect on cellular adhesion. Due to the novel RARγ- dependent cellular adhesion response exhibited by K562 cells, we examined α5 and β1 integrin subunit expression when K562 cells were exposed to retinoid agonists or vehicle for 24, 48, 72 or 96 hours. Our data demonstrates no differences in K562 cell surface expression of the α5 integrin subunit when cells were exposed to RARα, RARβ, or RARγ agonists for all time points tested. In contrast, RARγ agonist exposure resulted in an increase in cell surface β1 integrin subunit expression within 48 hours that was sustained at 72 and 96 hours. Finally, we demonstrate that while exposure to RARα or RARβ agonists have no effect on K562 cellular proliferation, the RARγ agonist significantly dampens K562 cellular proliferation levels in a time- and concentration- dependent manner. Our study is the first to report that treatment with a RARγ specific agonist augments cellular adhesion to α5β1 integrin substrates, increases cell surface levels of the β1 integrin subunit, and dampens cellular proliferation in a time and concentration dependent manner in a human

  10. Aggretin Venom Polypeptide as a Novel Anti-angiogenesis Agent by Targeting Integrin alpha2beta1

    PubMed Central

    Chung, Ching Hu; Chang, Chien Hsin; Hsu, Chun Chieh; Lin, Kung Tin; Peng, Hui Chin; Huang, Tur Fu

    2017-01-01

    VEGF and VEGFR antibodies have been used as a therapeutic strategy to inhibit angiogenesis in many diseases; however, frequent and repeated administration of these antibodies to patients induces immunogenicity. In previous studies, we demonstrated that aggretin, a heterodimeric snake venom C-type lectin, exhibits pro-angiogenic activities via integrin α2β1 ligation. We hypothesised that small-mass aggretin fragments may bind integrin α2β1 and act as antagonists of angiogenesis. In this study, the anti-angiogenic efficacy of a synthesised aggretin α-chain C-terminus (AACT, residue 106–136) was evaluated in both in vitro and in vivo angiogenesis models. The AACT demonstrated inhibitory effects on collagen-induced platelet aggregation and HUVEC adhesion to immobilised collagen. These results indicated that AACT may block integrin α2β1−collagen interaction. AACT also inhibited HUVEC migration and tube formation. Aortic ring sprouting and Matrigel implant models demonstrated that AACT markedly inhibited VEGF-induced neovascularisation. In addition, induction of FAK/PI3K/ERK1/2 tyrosine phosphorylation and talin 1/2 associated with integrin β1 which are induced by VEGF were blocked by AACT. Similarly, tyrosine phosphorylation of VEFGR2 and ERK1/2 induced by VEGF was diminished in integrin α2-silenced endothelial cells. Our results demonstrate that AACT is a potential therapeutic candidate for angiogenesis related-diseases via integrin α2β1 blockade. PMID:28252668

  11. JAM-L-mediated leukocyte adhesion to endothelial cells is regulated in cis by alpha4beta1 integrin activation.

    PubMed

    Luissint, Anny-Claude; Lutz, Pierre G; Calderwood, David A; Couraud, Pierre-Olivier; Bourdoulous, Sandrine

    2008-12-15

    Junctional adhesion molecules (JAMs) are endothelial and epithelial adhesion molecules involved in the recruitment of circulating leukocytes to inflammatory sites. We show here that JAM-L, a protein related to the JAM family, is restricted to leukocytes and promotes their adhesion to endothelial cells. Cis dimerization of JAM-L is required to engage in heterophilic interactions with its cognate counter-receptor CAR (coxsackie and adenovirus receptor). Interestingly, JAM-L expressed on neutrophils binds CAR independently of integrin activation. However, on resting monocytes and T lymphocytes, which express the integrin VLA-4, JAM-L molecules engage in complexes with VLA-4 and mainly accumulate in their monomeric form. Integrin activation is required for the dissociation of JAM-L-VLA-4 complexes and the accumulation of functional JAM-L dimers, which indicates that the leukocyte integrin VLA-4 controls JAM-L function in cis by controlling its dimerization state. This provides a mechanism through which VLA-4 and JAM-L functions are coordinately regulated, allowing JAM-L to strengthen integrin-dependent adhesion of leukocytes to endothelial cells.

  12. Design, synthesis, and analysis of a polyethelene glycol-modified (PEGylated) small molecule inhibitor of integrin {alpha}4{beta}1 with improved pharmaceutical properties.

    PubMed

    Pepinsky, R B; Lee, W-C; Cornebise, M; Gill, A; Wortham, K; Chen, L L; Leone, D R; Giza, K; Dolinski, B M; Perper, S; Nickerson-Nutter, C; Lepage, D; Chakraborty, A; Whalley, E T; Petter, R C; Adams, S P; Lobb, R R; Scott, D M

    2005-02-01

    Integrin alpha4beta1 plays an important role in inflammatory processes by regulating the migration of leukocytes into inflamed tissues. Previously, we identified BIO5192 [2(S)-{[1-(3,5-dichloro-benzenesulfonyl)-pyrrolidine-2(S)-carbonyl]-amino}-4-[4-methyl-2(S)-(methyl-{2-[4-(3-o-tolyl-ureido)-phenyl]-acetyl}-amino)-pentanoylamino]-butyric acid], a highly selective and potent (K(D) of 9 pM) small molecule inhibitor of alpha4beta1. Although BIO5192 is efficacious in various animal models of inflammatory disease, high doses and daily treatment of the compound are needed to achieve a therapeutic effect because of its relatively short serum half-life. To address this issue, polyethylene glycol modification (PEGylation) was used as an approach to improve systemic exposure. BIO5192 was PEGylated by a targeted approach in which derivatizable amino groups were incorporated into the molecule. Two sites were identified that could be modified, and from these, five PEGylated compounds were synthesized and characterized. One compound, 2a-PEG (K(D) of 19 pM), was selected for in vivo studies. The pharmacokinetic and pharmacodynamic properties of 2a-PEG were dramatically improved relative to the unmodified compound. The PEGylated compound was efficacious in a rat model of experimental autoimmune encephalomyelitis at a 30-fold lower molar dose than the parent compound and required only a once-a-week dosing regimen compared with a daily treatment for BIO5192. Compound 2a-PEG was highly selective for alpha4beta1. These studies demonstrate the feasibility of PEGylation of alpha4beta1-targeted small molecules with retention of activity in vitro and in vivo. 2a-PEG, and related compounds, will be valuable reagents for assessing alpha4beta1 biology and may provide a new therapeutic approach to treatment of human inflammatory diseases.

  13. Collagen-mimetic peptides mediate flow-dependent thrombus formation by high- or low-affinity binding of integrin alpha2beta1 and glycoprotein VI.

    PubMed

    Munnix, I C A; Gilio, K; Siljander, P R M; Raynal, N; Feijge, M A H; Hackeng, T M; Deckmyn, H; Smethurst, P A; Farndale, R W; Heemskerk, J W M

    2008-12-01

    Collagen acts as a potent surface for platelet adhesion and thrombus formation under conditions of blood flow. Studies using collagen-derived triple-helical peptides have identified the GXX'GER motif as an adhesive ligand for platelet integrin alpha2beta1, and (GPO)(n) as a binding sequence for the signaling collagen receptor, glycoprotein VI (GPVI). The potency was investigated of triple-helical peptides, consisting of GXX'GER sequences within (GPO)(n) or (GPP)(n) motifs, to support flow-dependent thrombus formation. At a high-shear rate, immobilized peptides containing both the high-affinity alpha2beta1-binding motif GFOGER and the (GPO)(n) motif supported platelet aggregation and procoagulant activity, even in the absence of von Willebrand factor (VWF). With peptides containing only one of these motifs, co-immobilized VWF was needed for thrombus formation. The (GPO)(n) but not the (GPP)(n) sequence induced GPVI-dependent platelet aggregation and procoagulant activity. Peptides with intermediate affinity (GLSGER, GMOGER) or low-affinity (GASGER, GAOGER) alpha2beta1-binding motifs formed procoagulant thrombi only if both (GPO)(n) and VWF were present. At a low-shear rate, immobilized peptides with high- or low-affinity alpha2beta1-binding motifs mediated formation of thrombi with procoagulant platelets only in combination with (GPO)(n). Triple-helical peptides with specific receptor-binding motifs mimic the properties of native collagen I in thrombus formation by binding to both platelet collagen receptors. At a high-shear rate, either GPIb or high-affinity (but not low-affinity) GXX'GER mediates GPVI-dependent formation of procoagulant thrombi. By extension, high-affinity binding for alpha2beta1 can control the overall platelet-adhesive activity of native collagens.

  14. Beta1 integrin inhibits apoptosis induced by cyclic stretch in annulus fibrosus cells via ERK1/2 MAPK pathway.

    PubMed

    Zhang, Kai; Ding, Wei; Sun, Wei; Sun, Xiao-jiang; Xie, You-zhuan; Zhao, Chang-qing; Zhao, Jie

    2016-01-01

    Low back pain is associated with intervertebral disc degeneration (IVDD) due to cellular loss through apoptosis. Mechanical factors play an important role in maintaining the survival of the annulus fibrosus (AF) cells and the deposition of extracellular matrix. However, the mechanisms that excessive mechanical forces lead to AF cell apoptosis are not clear. The present study was to look for how AF cells sense mechanical changes. In vivo experiments, the involvement of mechanoreceptors in apoptosis was examined by RT-PCR and/or immunoblotting in the lumbar spine of rats subjected to unbalanced dynamic and static forces. In vitro experiments, we investigated apoptotic signaling pathways in untransfected and transfected AF cells with the lentivirus vector for rat β1 integrin overexpression after cyclic stretch. Apoptosis in AF cells was assessed using flow cytometry, Hoechst 33258 nuclear staining. Western blotting was used to analyze expression of β1 integrin and caspase-3 and ERK1/2 MAPK signaling molecules. In the rat IVDD model, unbalanced dynamic and static forces induced apoptosis of disc cells, which corresponded to decreased expression of β1 integrin. Cyclic stretch-induced apoptosis in rat AF cells correlated with the activation of caspase-3 and with decreased levels of β1 integrin and the phosphorylation levels of ERK1/2 activation level. However, the overexpression of β1 integrin in AF cells ameliorated cyclic stretch-induced apoptosis and decreased caspase-3 activation. Furthermore, ERK1/2-specific inhibitor promotes apoptosis in vector β1-infected AF cells. These results suggest that the disruption of β1 integrin signaling may underlie disc cell apoptosis induced by mechanical stress. Further work is necessary to fully elucidate the pathophysiological mechanisms that underlie IVDD caused by unbalanced dynamic and static forces.

  15. Mobility of integrin alpha5beta1 measured on the isolated ventral membranes of human skin fibroblasts.

    PubMed

    Hirata, Hiroaki; Ohki, Kazuo; Miyata, Hidetake

    2005-05-25

    We have measured the lateral mobility of individual alpha5 integrin molecules in ventral plasma membranes of fibroblasts, which were prepared by removal of apical surfaces and nuclei followed by elimination of actin filaments with gelsolin, an actin-severing protein. The cytoplasmic domain of individual integrin molecules was tagged with 100 nm fluorescent polystyrene bead, and motion of the bead was observed and video-recorded. Position of the bead in each frame was determined from the centroid of the fluorescence image, from which plots of the mean-square displacement against time intervals were derived. Within short intervals of time (<100 ms) the mean-square displacement was proportional to the time interval, and the averaged translational diffusion coefficient of (5.3+/-4.4) x 10(-10) cm2/s was obtained with a broad distribution of (1.3-20) x 10(-10) cm2/s. The broad distribution might reflect the oligomerized state of integrin. The largest diffusion coefficient was comparable to that of lipid molecules previously measured in cells and probably represented the diffusion of a single integrin molecule in the presence of little interference of actin cytoskeleton or extracellular matrix. In longer time intervals (>100 ms) the motion of the bead was confined in an area, the average diameter of which was 410+/-160 nm. This was similar to the values described in previous reports, in which the motion of other membrane receptors labeled on their extracellular domain was measured in living cells.

  16. Identification of putative ligand-binding sites of the integrin alpha 4 beta 1 (VLA-4, CD49d/CD29)

    PubMed Central

    Kamata, T; Puzon, W; Takada, Y

    1995-01-01

    Integrin alpha 4 beta 1 recognizes both fibronectin (CS-1 sequence) and vascular cell adhesion molecule-1 (VCAM-1). To localize the ligand-binding sites of alpha 4, we located the epitopes for function-blocking anti-alpha 4 monoclonal antibodies (mAbs), including those that recognize previously described (but not yet physically localized) functional epitopes (A, B1, B2 and C) using interspecies alpha 4 chimeras expressed in mammalian cells. Epitopes B1 and B2 were associated with ligand binding, and epitopes A and B2 with homotypic cellular aggregation. mAbs P4C2 (epitope B2), 20E4 and PS/2 were mapped within residues 108-182; mAbs HP2/1 (epitope B1), SG/73 and R1-2 within residues 195-268; mAbs HP1/3 (epitope A) and P4G9 within residues 1-52; and B5G10 (epitope C) within residues 269-548. The data suggest that residues 108-268, which do not include bivalent-cation-binding motifs, are related to VCAM-1 and CS-1 binding, and more N-terminal portions of alpha 4 (residues 1 and 52 and 108-182) to homotypic aggregation. Since mAbs PS/2 and HP2/1 block alpha 4 beta 7 binding to mucosal addressin cell adhesion molecule-1 (MAdCAM-1), the MAdCAM-1-binding site is close to, or overlapping with, VCAM-1- and CS-1-binding sites. The role of Asp-130 of beta 1 in the binding to VCAM-1 and CS-1 peptide was examined. Chinese hamster ovary (CHO) cells expressing beta 1 (D130A) (Asp-130 to Ala mutant of beta 1) and alpha 4 showed much less binding to both ligands than CHO cells expressing wild-type beta 1 and alpha 4 [a dominant negative effects of beta 1 (D130A)], suggesting that Asp-130 of beta 1 is critical for binding to both ligands and that the two ligand share common binding mechanisms [corrected]. Images Figure 2 Figure 3 Figure 5 PMID:7531439

  17. Human macrophage differentiation involves an interaction between integrins and fibronectin

    SciTech Connect

    Laouar, A.; Chubb, C.B.H.; Collart, F.; Huberman, E.

    1997-03-14

    The authors have examined the role of integrins and extracellular matrix (ECM) proteins in macrophage differentiation of (1) human HL-60 myeloid leukemia cells induced by phorbol 12-myristate 13-acetate (PMA) and (2) human peripheral blood monocytes induced by either PMA or macrophage-colony stimulating factor (M-CSF). Increased {beta}{sub 1} integrin and fibronectin (FN) gene expression was observed in PMA-treated HL-60 cells and PMA- or M-CSF-treated monocytes, even at a time preceding the manifestation of macrophage markers. Treated HL-60 cells and monocytes also released and deposited FN on the culture dishes. An HL-60 cell variant, HL-525, which is deficient in protein kinase C {beta} (PKC{beta}) and resistant to PMA-induced differentiation, failed to express FN after PMA treatment. Restoration of PKC{beta} resulted in PMA-induced FN gene expression and macrophage differentiation. The macrophage phenotype induced in HL-60 cells or monocytes was attenuated by anti-{beta}{sub 1} integrin or anti-FN MAbs. The authors suggest that macrophage differentiation involves activation of PKC and expression of specific integrins and ECM proteins. The stimulated cells, through their integrins, attach and spread on these substrates by binding to the deposited ECM proteins. This attachment and spreading in turn, through integrin signaling, leads to the macrophage phenotype.

  18. Extracellular Matrix and Integrins in Embryonic Stem Cell Differentiation

    PubMed Central

    Wang, Han; Luo, Xie; Leighton, Jake

    2015-01-01

    Embryonic stem cells (ESCs) are pluripotent cells with great therapeutic potentials. The in vitro differentiation of ESC was designed by recapitulating embryogenesis. Significant progress has been made to improve the in vitro differentiation protocols by toning soluble maintenance factors. However, more robust methods for lineage-specific differentiation and maturation are still under development. Considering the complexity of in vivo embryogenesis environment, extracellular matrix (ECM) cues should be considered besides growth factor cues. ECM proteins bind to cells and act as ligands of integrin receptors on cell surfaces. Here, we summarize the role of the ECM and integrins in the formation of three germ layer progenies. Various ECM–integrin interactions were found, facilitating differentiation toward definitive endoderm, hepatocyte-like cells, pancreatic beta cells, early mesodermal progenitors, cardiomyocytes, neuroectoderm lineages, and epidermal cells, such as keratinocytes and melanocytes. In the future, ECM combinations for the optimal ESC differentiation environment will require substantial study. PMID:26462244

  19. Microvessel vascular smooth muscle cells contribute to collagen type I deposition through ERK1/2 MAP kinase, alphavbeta3-integrin, and TGF-beta1 in response to ANG II and high glucose.

    PubMed

    Belmadani, Souad; Zerfaoui, Mourad; Boulares, Hamid A; Palen, Desiree I; Matrougui, Khalid

    2008-07-01

    This study determines that vascular smooth muscle cell (VSMC) signaling through extracellular signal-regulated kinase (ERK) 1/2-mitogen-activated protein (MAP) kinase, alphavbeta(3)-integrin, and transforming growth factor (TGF)-beta1 dictates collagen type I network induction in mesenteric resistance arteries (MRA) from type 1 diabetic (streptozotocin) or hypertensive (HT; ANG II) mice. Isolated MRA were subjected to a pressure-passive-diameter relationship. To delineate cell types and mechanisms, cultured VSMC were prepared from MRA and stimulated with ANG II (100 nM) and high glucose (HG, 22 mM). Pressure-passive-diameter relationship reduction was associated with increased collagen type I deposition in MRA from HT and diabetic mice compared with control. Treatment of HT and diabetic mice with neutralizing TGF-beta1 antibody reduced MRA stiffness and collagen type I deposition. Cultured VSMC stimulated with HG or ANG II for 5 min increased ERK1/2-MAP kinase phosphorylation, whereas a 48-h stimulation induced latent TGF-beta1, alphavbeta(3)-integrin, and collagen type 1 release in the conditioned media. TGF-beta1 bioactivity and Smad2 phosphorylation were alphavbeta(3)-integrin-dependent, since beta(3)-integrin antibody and alphavbeta(3)-integrin inhibitor (SB-223245, 10 microM) significantly prevented TGF-beta1 bioactivity and Smad2 phosphorylation. Pretreatment of VSMC with ERK1/2-MAP kinase inhibitor (U-0126, 1 microM) reduced alphavbeta(3)-integrin, TGF-beta1, and collagen type 1 content. Additionally, alphavbeta(3)-integrin antibody, SB-223245, TGF-beta1-small-intefering RNA (siRNA), and Smad2-siRNA (40 nM) prevented collagen type I network formation in response to ANG II and HG. Together, these data provide evidence that resistance artery fibrosis in type 1 diabetes and hypertension is a consequence of abnormal collagen type I release by VSMC and involves ERK1/2, alphavbeta(3)-integrin, and TGF-beta1 signaling. This pathway could be a potential target for

  20. Coordinate expression of beta1 integrins and their regulator, TGF beta2 at the floor plate of the medulla oblongata is correlated with the crossing of the fibers of olivocerebellar projection in mice.

    PubMed

    Ohyama, Kyoji; Kawamura, Koki

    2002-01-31

    During embryonic day 11 (E11) to E16, contact-dependent interacting molecules beta1 integrins and their putative regulator TGF beta2 are coordinately expressed at the floor plate in the caudal part of mouse myelencephalon. Their expression disappears at E18. Consistent with the peak of their expression (E13-E16), olivocerebellar fibers primarily cross the floor plate. These data indicate that spatiotemporal expression of beta1 integrins and TGF beta2 is correlated with the crossing of olivocerebellar fibers.

  1. Monoclonal antibodies identify residues 199-216 of the integrin alpha2 vWFA domain as a functionally important region within alpha2beta1.

    PubMed Central

    Tuckwell, D S; Smith, L; Korda, M; Askari, J A; Santoso, S; Barnes, M J; Farndale, R W; Humphries, M J

    2000-01-01

    Integrin alpha2beta1 is the major receptor for collagens in the human body, and the collagen-binding site on the alpha2 subunit von Willebrand factor A-type domain (vWFA domain) is now well defined. However, the biologically important conformational changes that are associated with collagen binding, and the means by which the vWFA domain is integrated into the whole integrin are not completely understood. We have raised monoclonal antibodies against recombinant alpha2 vWFA domain for use as probes of function. Three antibodies, JA202, JA215 and JA218, inhibited binding to collagen, collagen I C-propeptide and E-cadherin, demonstrating that their function is important for structurally diverse alpha2beta1 ligands. Cross-blocking studies grouped the epitopes into two clusters: (I) JA202, the inhibitory antibody, Gi9, and a non-inhibitory antibody, JA208; (II) JA215 and JA218. Both clusters were sensitive to events at the collagen binding site, as binding of Gi9, JA202, JA215 and JA218 were inhibited by collagen peptide, JA208 binding was enhanced by collagen peptide, and binding of JA202 was decreased after mutagenesis of the cation-binding residue Thr(221) to alanine. Binding of cluster I antibodies was inhibited by the anti-functional anti-beta1 antibody Mab13, and binding of Gi9 and JA218 to alpha2beta1 was inhibited by substituting Mn(2+) for Mg(2+), demonstrating that these antibodies were sensitive to changes initiated outside the vWFA domain. Mapping of epitopes showed that JA202 and Gi9 bound between residues 212-216, while JA208 bound between residues 199-216. We have therefore identified two epitope clusters with novel properties; i.e. they are intimately associated with the collagen-binding site, responsive to conformational changes at the collagen-binding site and sensitive to events initiated outside the vWFA domain. PMID:10947963

  2. Role of integrin subunits in mesenchymal stem cell differentiation and osteoblast maturation on graphitic carbon-coated microstructured surfaces.

    PubMed

    Olivares-Navarrete, Rene; Rodil, Sandra E; Hyzy, Sharon L; Dunn, Ginger R; Almaguer-Flores, Argelia; Schwartz, Zvi; Boyan, Barbara D

    2015-05-01

    Surface roughness, topography, chemistry, and energy promote osteoblast differentiation and increase osteogenic local factor production in vitro and bone-to-implant contact in vivo, but the mechanisms involved are not well understood. Knockdown of integrin heterodimer alpha2beta1 (α2β1) blocks the osteogenic effects of the surface, suggesting signaling by this integrin homodimer is required. The purpose of the present study was to separate effects of surface chemistry and surface structure on integrin expression by coating smooth or rough titanium (Ti) substrates with graphitic carbon, retaining surface morphology but altering surface chemistry. Ti surfaces (smooth [Ra < 0.4 μm], rough [Ra ≥ 3.4 μm]) were sputter-coated using a magnetron sputtering system with an ultrapure graphite target, producing a graphitic carbon thin film. Human mesenchymal stem cells and MG63 osteoblast-like cells had higher mRNA for integrin subunits α1, α2, αv, and β1 on rough surfaces in comparison to smooth, and integrin αv on graphitic-carbon-coated rough surfaces in comparison to Ti. Osteogenic differentiation was greater on rough surfaces in comparison to smooth, regardless of chemistry. Silencing integrins β1, α1, or α2 decreased osteoblast maturation on rough surfaces independent of surface chemistry. Silencing integrin αv decreased maturation only on graphitic carbon-coated surfaces, not on Ti. These results suggest a major role of the integrin β1 subunit in roughness recognition, and that integrin alpha subunits play a major role in surface chemistry recognition. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Role of Integrin Subunits in Mesenchymal Stem Cell Differentiation and Osteoblast Maturation on Graphitic Carbon-coated Microstructured Surfaces

    PubMed Central

    Olivares-Navarrete, Rene; Rodil, Sandra E.; Hyzy, Sharon L.; Dunn, Ginger R.; Almaguer-Flores, Argelia; Schwartz, Zvi; Boyan, Barbara D.

    2015-01-01

    Surface roughness, topography, chemistry, and energy promote osteoblast differentiation and increase osteogenic local factor production in vitro and bone-to-implant contact in vivo, but the mechanisms involved are not well understood. Knockdown of integrin heterodimer alpha2beta1 (α2β1) blocks the osteogenic effects of the surface, suggesting signaling by this integrin homodimer is required. The purpose of the present study was to separate effects of surface chemistry and surface structure on integrin expression by coating smooth or rough titanium (Ti) substrates with graphitic carbon, retaining surface morphology but altering surface chemistry. Ti surfaces (smooth [Ra<0.4μm], rough [Ra≥3.4μm]) were sputter-coated using a magnetron sputtering system with an ultrapure graphite target, producing a graphitic carbon thin film. Human mesenchymal stem cells and MG63 osteoblast-like cells had higher mRNA for integrin subunits α1, α2, αv, and β1 on rough surfaces in comparison to smooth, and integrin αv on graphitic-carbon-coated rough surfaces in comparison to Ti. Osteogenic differentiation was greater on rough surfaces in comparison to smooth, regardless of chemistry. Silencing integrins β1, α1, or α2 decreased osteoblast maturation on rough surfaces independent of surface chemistry. Silencing integrin αv decreased maturation only on graphitic carbon-coated surfaces, not on Ti. These results suggest a major role of the integrin β1 subunit in roughness recognition, and that integrin alpha subunits play a major role in surface chemistry recognition. PMID:25770999

  4. Pak1 regulates branching morphogenesis in 3D MDCK cell culture by a PIX and beta1-integrin-dependent mechanism.

    PubMed

    Hunter, Michael P; Zegers, Mirjam M

    2010-07-01

    Branching morphogenesis is a fundamental process in the development of the kidney. This process gives rise to a network of ducts, which form the collecting system. Defective branching can lead to a multitude of kidney disorders including agenesis and reduced nephron number. The formation of branching tubules involves changes in cell shape, cell motility, and reorganization of the cytoskeleton. However, the exact intracellular mechanisms involved are far from understood. We have used the three-dimensional (3D) Madin-Darby canine kidney (MDCK) cell culture system to study how p21-activated kinase 1 (Pak1), which is an important regulator of the cytoskeleton, modulates branching. Our data reveal that Pak1 plays a crucial role in regulating branching morphogenesis. Expression of a dominant-negative Pak1 mutant (DN-Pak1) in MDCK cysts resulted in the spontaneous formation of extensions and branching tubules. Cellular contractility and levels of phosphorylated myosin light chain (pMLC) were increased in DN-Pak1 cells in collagen. Expression of a DN-Pak1 mutant that does not bind to PIX (DN-Pak1-DeltaPIX) failed to form extensions in collagen and did not have increased contractility. This shows that the DN-Pak1 mutant requires PIX binding to generate extensions and increased contractility in 3D culture. Furthermore, a beta1-integrin function-blocking antibody (AIIB2) inhibited the formation of branches and blocked the increased contractility in DN-Pak1 cysts. Taken together, our work shows that DN-Pak1-induced branching morphogenesis requires PIX binding and beta1-integrin signaling.

  5. Bifunctional peptides derived from homologous loop regions in the laminin alpha chain LG4 modules interact with both alpha 2 beta 1 integrin and syndecan-2.

    PubMed

    Yokoyama, Fumiharu; Suzuki, Nobuharu; Kadoya, Yuichi; Utani, Atsushi; Nakatsuka, Hiroko; Nishi, Norio; Haruki, Masahiro; Kleinman, Hynda K; Nomizu, Motoyoshi

    2005-07-19

    Laminin alpha chains show diverse biological functions in a chain-specific fashion. The laminin G-like modules (LG modules) of the laminin alpha chains consist of a 14-stranded beta-sheet sandwich structure with biologically active sequences found in the connecting loops. Previously, we reported that connecting loop regions between beta-strands E and F in the mouse laminin alpha chain LG4 modules exhibited chain-specific activities. In this study, we focus on the homologous loop regions in human laminin alpha chain LG4 modules using five synthetic peptides (hEF-1-hEF-5). These homologous peptides induced chain-specific cellular responses in various cell types. Next, to examine the dual-receptor recognition model, we synthesized chimeras (cEF13A-cEF13E) derived from peptides hEF-1 and hEF-3. All of the chimeric peptides promoted fibroblast attachment as well as the parental peptides. Attachment of fibroblasts to cEF13A and cEF13B was inhibited by anti-integrin alpha2 and beta1 antibodies and by heparin, while cell adhesion to cEF13C, cEF13D, and cEF13E was blocked only by heparin. Actin organization of fibroblasts on cEF13C was not different from that on hEF-3, but cEF13B induced membrane ruffling at the tips of the actin stress fibers. These results suggest that cEF13B had bifunctional effects on cellular behaviors through alpha2beta1 integrin and heparin/heparan sulfate proteoglycan. We conclude that the approach utilizing chimeric peptides is useful for examining cellular mechanisms in dual-receptor systems.

  6. Fps/Fes protein-tyrosine kinase regulates mast cell adhesion and migration downstream of Kit and beta1 integrin receptors.

    PubMed

    Smith, Julie A; Samayawardhena, Lionel A; Craig, Andrew W B

    2010-03-01

    Activation of Kit receptor protein-tyrosine kinase (PTK) by its ligand Stem Cell Factor (SCF) is required for the development of mast cells, and for the regulation of mast cell proliferation, migration and modulation of inflammatory mediator release. Recent studies have implicated the non-receptor PTK Fps/Fes (hereafter referred to as Fes) in signaling downstream of oncogenic Kit, however, the potential role of Fes in regulating Kit signaling is not well defined. In this study, we show that SCF induces transient tyrosine phosphorylation of wild-type Fes as well as kinase-dead Fes in bone marrow-derived mast cells (BMMCs). The latter finding implicates an upstream kinase acting on Fes, which we identified as Fyn PTK. SCF treatment of BMMCs promoted recruitment of Fes to Kit, potentially via direct interaction of the Fes SH2 domain with phosphorylated Kit. While Fes was not required for SCF-induced signaling to Akt and Erk kinases, Fes-deficient (fes-/-) BMMCs displayed a defect in sustained p38 kinase activation, compared to control cells. SCF-treated Fes-deficient BMMCs also displayed elevated beta1 integrin-mediated cell adhesion and spreading on fibronectin, compared to control cells, and a reduction in cell polarization at later times of SCF treatment. Restoring Fes expression in fes-/- BMMCs by retroviral transduction was sufficient to rescue cell spreading and polarization defects. Interestingly, SCF-induced chemotaxis of BMMCs was also defective in Fes-deficient BMMCs, and restored in Fes-rescue BMMCs. Overall, these results implicate Fes in regulating cross-talk between Kit and beta1 integrins to promote cytoskeletal reorganization and motility of mast cells.

  7. Catalytic activity of nuclear PLC-beta(1) is required for its signalling function during C2C12 differentiation.

    PubMed

    Ramazzotti, Giulia; Faenza, Irene; Gaboardi, Gian Carlo; Piazzi, Manuela; Bavelloni, Alberto; Fiume, Roberta; Manzoli, Lucia; Martelli, Alberto M; Cocco, Lucio

    2008-11-01

    Here we report that PLC-beta(1) catalytic activity plays a role in the increase of cyclin D3 levels and induces the differentiation of C2C12 skeletal muscle cells. PLC-beta(1) mutational analysis revealed the importance of His(331) and His(378) for the catalysis. The expression of PLC-beta(1) and cyclin D3 proteins is highly induced during the process of skeletal myoblast differentiation. We have previously shown that PLC-beta(1) activates cyclin D3 promoter during the differentiation of myoblasts to myotubes, indicating that PLC-beta(1) is a crucial regulator of the mouse cyclin D3 gene. We show that after insulin treatment cyclin D3 mRNA levels are lower in cells overexpressing the PLC-beta(1) catalytically inactive form in comparison to wild type cells. We describe a novel signalling pathway elicited by PLC-beta(1) that modulates AP-1 activity. Gel mobility shift assay and supershift performed with specific antibodies indicate that the c-jun binding site is located in a cyclin D3 promoter region specifically regulated by PLC-beta(1) and that c-Jun binding activity is significantly increased by insulin and PLC-beta(1) overexpression. Mutation of AP-1 site decreased the basal cyclin D3 promoter activity and eliminated its induction by insulin and PLC-beta(1). These results hint at the fact that PLC-beta(1) catalytic activity signals a c-jun/AP-1 target gene, i.e. cyclin D3, during myogenic differentiation.

  8. Polarized entry of uropathogenic Afa/Dr diffusely adhering Escherichia coli strain IH11128 into human epithelial cells: evidence for alpha5beta1 integrin recognition and subsequent internalization through a pathway involving caveolae and dynamic unstable microtubules.

    PubMed

    Guignot, J; Bernet-Camard, M F; Poüs, C; Plançon, L; Le Bouguenec, C; Servin, A L

    2001-03-01

    Afa/Dr diffusely adhering Escherichia coli strain IH11128 bacteria basolaterally entered polarized epithelial cells by a CD55- and CD66e-independent mechanism through interaction with the alpha5beta1 integrin and a pathway involving caveolae and dynamic microtubules (MTs). IH11128 invasion within HeLa cells was dramatically decreased after the cells were treated with the cholesterol-extracting drug methyl-beta-cyclodextrin or the caveola-disrupting drug filipin. Disassembly of the dynamically unstable MT network by the compound 201-F resulted in a total abolition of IH11128 entry. In apically infected polarized fully differentiated Caco-2/TC7 cells, no IH11128 entry was observed. The entry of bacteria into apically IH11128-infected fully differentiated Caco-2/TC7 cells was greatly enhanced by treating cells with Ca2+-free medium supplemented with EGTA, a procedure that disrupts intercellular junctions and thus exposes the basolateral surface to bacteria. Basally infected fully differentiated polarized Caco-2/TC7 cells grown on inverted inserts mounted in chamber culture showed a highly significant level of intracellular IH11128 bacteria compared with cells subjected to the apical route of infection. No expression of CD55 and CD66e, the receptors for the Afa/Dr adhesins, was found at the basolateral domains of these cells. Consistent with the hypothesis that a cell-to-cell adhesion molecule acts as a receptor for polarized IH11128 entry, an antibody blockade using anti-alpha5beta1 integrin polyclonal antibody completely abolished bacterial entry. Experiments conducted with the laboratory strain E. coli K-12 EC901 carrying the recombinant plasmid pBJN406, which expresses Dr hemagglutinin, demonstrated that the dra operon is involved in polarized entry of IH11128 bacteria. Examined as a function of cell differentiation, the number of internalized bacteria decreased dramatically beyond cell confluency. Surviving intracellular IH11128 bacteria residing intracellularly

  9. Monocyte adhesion to endothelium in simian immunodeficiency virus-induced AIDS encephalitis is mediated by vascular cell adhesion molecule-1/alpha 4 beta 1 integrin interactions.

    PubMed Central

    Sasseville, V. G.; Newman, W.; Brodie, S. J.; Hesterberg, P.; Pauley, D.; Ringler, D. J.

    1994-01-01

    Because the mechanisms associated with recruitment of monocytes to brain in AIDS encephalitis are unknown, we used tissues from rhesus monkeys infected with simian immunodeficiency virus (SIV) to examine the relative contributions of various adhesion pathways in mediating monocyte adhesion to endothelium from encephalitic brain. Using a modified Stamper and Woodruff tissue adhesion assay, we found that the human monocytic cell lines, THP-1 and U937, and the B cell line, Ramos, preferentially bound to brain vessels from monkeys with AIDS encephalitis. Using a combined tissue adhesion/immunohistochemistry approach, these cells only bound to vessels expressing vascular cell adhesion molecule-1 (VCAM-1). Furthermore, pretreatment of tissues with antibodies to VCAM-1 or cell lines with antibodies to VLA-4 (CD49d) inhibited adhesion by more than 70%. Intercellular adhesion molecule-1 (ICAM-1)/beta 2 integrin interactions were not significant in mediating cell adhesion to the vasculature in encephalitic simian brain using a cell line (JY) capable of binding rhesus monkey ICAM-1. In addition, selectin-mediated interactions did not significantly contribute to cell binding to encephalitic brain as there was no immunohistochemical expression of E-selectin and P-selectin in either normal or encephalitic brain, nor was there a demonstrable adhesive effect from L-selectin using L-selectin-transfected 300.19 cells on simian encephalitic brain. These results demonstrate that using the tissue adhesion assay, THP-1, U937, and Ramos cells bind to vessels in brain from animals with AIDS encephalitis using VCAM-1/alpha 4 beta 1 integrin interactions and suggest that VCAM-1 and VLA-4 may be integral for monocyte recruitment to the central nervous system during the development of AIDS encephalitis. Images Figure 1 PMID:7507300

  10. Extracellular matrix proteoglycan decorin-mediated myogenic satellite cell responsiveness to transforming growth factor-beta1 during cell proliferation and differentiation Decorin and transforming growth factor-beta1 in satellite cells.

    PubMed

    Li, Xuehui; McFarland, Douglas C; Velleman, Sandra G

    2008-10-01

    Transforming growth factor-beta1 (TGF-beta1) is a potent inhibitor of muscle cell proliferation and differentiation. Decorin, a small proteoglycan in the extracellular matrix, binds to TGF-beta1 and modulates the activity of TGF-beta1 during muscle cell growth and development. However, its interaction with TGF-beta1 and involvement in myogenesis is not well characterized. In the present study, chicken myogenic satellite cells, myogenic precursors for muscle growth and repair, were isolated from the pectoralis major muscle and used to investigate the biological function of TGF-beta1 and decorin during myogenesis. The over-expression of decorin in satellite cells significantly increased cell proliferation, compared to the control cells. Consistent with this result, reducing decorin expression decreased cell proliferation, which suggests a decorin-mediated mechanism is involved in the regulation of myogenic satellite cell proliferation. Satellite cells over-expressing decorin were less sensitive to TGF-beta1 during proliferation, which indicates that decorin may sequester TGF-beta1 leading to increased proliferation. During satellite cell differentiation, the over-expression of decorin induced differentiation by increasing the muscle specific creatine kinase concentration. However, the addition of TGF-beta1 diminished decorin-mediated cell responsiveness to TGF-beta1 during differentiation. Taken together, these results suggest that decorin induces myogenic satellite cell proliferation and differentiation by regulating cellular responsiveness to TGF-beta1. An alternative TGF-beta1-independent pathway may be involved in the regulation of satellite cells by decorin.

  11. PED/PEA-15 induces autophagy and mediates TGF-beta1 effect on muscle cell differentiation.

    PubMed

    Iovino, S; Oriente, F; Botta, G; Cabaro, S; Iovane, V; Paciello, O; Viggiano, D; Perruolo, G; Formisano, P; Beguinot, F

    2012-07-01

    TGF-beta1 has been shown to induce autophagy in certain cells but whether and how this action is exerted in muscle and whether this activity relates to TGF-beta1 control of muscle cell differentiation remains unknown. Here, we show that expression of the autophagy-promoting protein phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15) progressively declines during L6 and C2C12 skeletal muscle cell differentiation. PED/PEA-15 underwent rapid induction upon TGF-beta1 exposure of L6 and C2C12 myoblasts, accompanied by impaired differentiation into mature myotubes. TGF-beta1 also induced autophagy in the L6 and C2C12 cells through a PP2A/FoxO1-mediated mechanism. Both the TGF-beta1 effect on differentiation and that on autophagy were blocked by specific PED/PEA-15 ShRNAs. Myoblasts stably overexpressing PED/PEA-15 did not differentiate and showed markedly enhanced autophagy. In these same cells, the autophagy inhibitor 3-methyladenine rescued TGF-beta1 effect on both autophagy and myogenesis, indicating that PED/PEA-15 mediates TGF-beta1 effects in muscle. Muscles from transgenic mice overexpressing PED/PEA-15 featured a significant number of atrophic fibers, accompanied by increased light chain 3 (LC3)II to LC3I ratio and reduced PP2A/FoxO1 phosphorylation. Interestingly, these mice showed significantly impaired locomotor activity compared with their non-transgenic littermates. TGF-beta1 causes transcriptional upregulation of the autophagy-promoting gene PED/PEA-15, which in turn is capable to induce atrophic responses in skeletal muscle in vivo.

  12. Enzymes that hydrolyze adenine nucleotides in platelets and polymorphisms in the alpha2 gene of integrin alpha2beta1 in patients with von Willebrand disease.

    PubMed

    Santos, Karen Freitas; Battisti, Vanessa; Corrêa, Maísa de Carvalho; Mann, Thaís Rapachi; Pereira, Renata da Silva; Araújo, Maria do Carmo; Brülê, Alice Odete; Schetinger, Maria Rosa Chitolina; Morsch, Vera Maria

    2010-07-01

    Von Willebrand disease (VWD) is one of the most common inherited bleeding diseases caused by a qualitative or quantitative deficiency of the von Willebrand factor (FvW). FvW is a multimeric glycoprotein synthesized by megakaryocytes and endothelial cells and it is present in the subendothelial matrix, blood plasma, platelets, and endothelium. This glycoprotein plays an important role in thrombus formation by initiating platelet adhesion to sites of injury as well as platelet aggregation. The aim of this study was to evaluate the activities of enzymes that hydrolyze adenine nucleotides in platelets, ristocetin-induced platelet aggregation (RIPA), and polymorphisms of the alpha2 gene of alpha2beta1 integrin from VWD patients. Platelet nucleoside triphosphate diphosphohydrolase (NTPDase), 5'-nucleotidase, and ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) activities were verified in 14 VWD patients. For RIPA determination, a final concentration of 1.25 mg/ml of ristocetin was used. Polymorphisms of the alpha2 gene were analyzed through PCR. Platelet NTPDase and E-NPP were decreased in VWD patients. 5'-Nucleotidase activity was not statistically significant between controls and VWD patients. RIPA was significantly reduced, with an allelic frequency of 78.57% for 807C in VWD patients. Our results indicated reduced platelet NTPDase and E-NPP activities which might be related to the low platelet adhesiveness. The prevalence of the 807C allele might account for the variability in bleeding in VWD.

  13. Differential in vitro phenotype pattern, transforming growth factor-beta(1) activity and mRNA expression of transforming growth factor-beta(1) in Apert osteoblasts.

    PubMed

    Locci, P; Baroni, T; Pezzetti, F; Lilli, C; Marinucci, L; Martinese, D; Becchetti, E; Calvitti, M; Carinci, F

    1999-09-01

    The phenotype of Apert osteoblasts differs from that of normal osteoblasts in the accumulation of macromolecules in the extracellular matrix. Apert osteoblasts increase type I collagen, fibronectin and glycosaminoglycans secretion compared with normal osteoblasts. Because the extracellular matrix macromolecule accumulation is greatly modulated by transforming growth factor-beta(1), we examined the ability of normal and Apert osteoblasts to secrete transforming growth factor-beta(1) by CCL-64 assay and to produce transforming growth factor-beta(1 )by analysis of the mRNA expression of transforming growth factor-beta(1). Northern blot analysis revealed an increased amount of transforming growth factor-beta(1) mRNA expression in Apert osteoblasts compared with normal ones. Moreover, the level of the active transforming growth factor-beta(1) isoform was higher in Apert than in normal media. In pathologic cells, the increase in transforming growth factor-beta(1) gene expression was associated with a parallel increase in the factor secreted into the medium. The level of transforming growth factor-beta(1) was decreased by the addition of basic fibroblast growth factor. Transforming growth factor-beta(1) is controlled temporally and spatially during skeletal tissue development and produces complex stimulatory and inhibitory changes in osteoblast functions. We hypothesise that in vitro differences between normal and Apert osteoblasts may be correlated to different transforming growth factor-beta(1) cascade patterns, probably due to an altered balance between transforming growth factor-beta(1) and basic fibroblast growth factor.

  14. Apoptotic and anti-adhesion effect of ajoene, a garlic derived compound, on the murine melanoma B16F10 cells: possible role of caspase-3 and the alpha(4)beta(1) integrin.

    PubMed

    Ledezma, Eliades; Apitz-Castro, Rafael; Cardier, José

    2004-03-31

    In this study we evaluated the hypothesis that the antitumor activity of ajoene could be associated with its apoptosis-inducing effect, and with its ability to block the expression of the alpha(4)beta(1) integrin, in the murine melanoma B16F10 cells. Ajoene induced a significant reduction in B16F10 viability (IC(50)=62 microM), in a dose-dependent manner. Flow cytometric analysis showed that the cytotoxic effect of this compound was associated with caspase-3 activation. Ajoene at 25 microM altered the alpha(4)beta(1) integrin expression on B16F10, and induced a significant reduction in the adhesion of these cells to an endothelial cell monolayer.

  15. The Changing Integrin Expression and a Role for Integrin β8 in the Chondrogenic Differentiation of Mesenchymal Stem Cells

    PubMed Central

    LaPointe, Vanessa L. S.; Verpoorte, Amanda; Stevens, Molly M.

    2013-01-01

    Many cartilage tissue engineering approaches aim to differentiate human mesenchymal stem cells (hMSCs) into chondrocytes and develop cartilage in vitro by targeting cell-matrix interactions. We sought to better inform the design of cartilage tissue engineering scaffolds by understanding how integrin expression changes during chondrogenic differentiation. In three models of in vitro chondrogenesis, we studied the temporal change of cartilage phenotype markers and integrin subunits during the differentiation of hMSCs. We found that transcript expression of most subunits was conserved across the chondrogenesis models, but was significantly affected by the time-course of differentiation. In particular, ITGB8 was up-regulated and its importance in chondrogenesis was further established by a knockdown of integrin β8, which resulted in a non-hyaline cartilage phenotype, with no COL2A1 expression detected. In conclusion, we performed a systematic study of the temporal changes of integrin expression during chondrogenic differentiation in multiple chondrogenesis models, and revealed a role for integrin β8 in chondrogenesis. This work enhances our understanding of the changing adhesion requirements of hMSCs during chondrogenic differentiation and underlines the importance of integrins in establishing a cartilage phenotype. PMID:24312400

  16. Human macrophage differentiation involves an interaction between integrins and fibronectin

    SciTech Connect

    Laouar, A.; Chubb, C.B.H.; Collart, F.; Huberman, E.

    1996-11-15

    The authors have examined the role of the {beta}{sub 1} integrin family of adhesion receptors (VLA) and the extracellular matrix protein fibronectin (FN) in macrophage differentiation of (1) human HL-60 myeloid leukemia cells induced by phorbol 12-myristate 13-acetate (PMA) and (2) human peripheral blood monocytes induced by either PMA or macrophage-colony stimulating factor (M=CSF). Increased VLA and FN gene expression was observed as early as 4 h after PMA treatment of HL-60 cells and PMA- or M-CSF-treatment of monocytes, and it preceded the manifestation of macrophage markers. Treated HL-60 cells and monocytes also released and deposited FN on the surface of the tissue culture dishes. An HL-60 cell variant, HL-525, which is deficient in protein kinase C {beta} and resistant to PMA-induced differentiation, exhibited elevated levels of the VLA antigen but failed to express the FN gene. Incubation of HL-525 cells on dishes precoated with exogenous FN resulted in a macrophage differentiation. The macrophage phenotype induced in HL-60 cells, HL-525 cells, or monocytes was attenuated to various degrees by anti-VLA or anti-FN MAbs or by exogenous RGDS, a VLA-binding motif on FN. The authors suggest that macrophage differentiation is initiated by the activation of protein kinase C, which leads to the expression of the integrin, FN and related genes. The integrins mediate cell attachment and spreading on appropriate substrates by binding to deposited extracellular proteins such as FN. This attachment and spreading, in turn, leads to the expression of genes that code for the macrophage functions.

  17. The Effect of Conditional Inactivation of Beta 1 Integrins using Twist 2 Cre, Osterix Cre and Osteocalcin Cre Lines on Skeletal Phenotype

    PubMed Central

    Shekaran, Asha; Shoemaker, James T.; Kavanaugh, Taylor E.; Lin, Angela S.; LaPlaca, Michelle C.; Fan, Yuhong; Guldberg, Robert E.; García, Andrés J.

    2014-01-01

    Skeletal development and growth are complex processes regulated by multiple microenvironmental cues, including integrin-ECM interactions. The β1 sub-family of integrins is the largest integrin sub-family and constitutes the main integrin binding partners of collagen I, the major ECM component of bone. As complete β1 integrin integrin knockout results in embryonic lethality, studies of β1 integrin function in vivo rely on tissue-specific gene deletions. While multiple in vitro studies indicate that β1 integrins are crucial regulators of osteogenesis and mineralization, in vivo osteoblast-specific perturbations of β1 integrins have resulted in mild and sometimes contradictory skeletal phenotypes. To further investigate the role of β1 integrins on skeletal phenotype, we used the Twist2-Cre, Osterix-Cre and Osteocalcin-Cre lines to generate conditional β1 integrin deletions, where cre is expressed primarily in mesenchymal condensation, pre-osteoblast, and mature osteoblast lineage cells respectively within these lines. Mice with Twist2-specific β1 integrin disruption were smaller, had impaired skeletal development, especially in the craniofacial and vertebral tissues at E19.5, and did not survive beyond birth. Osterix-specific β1 integrin deficiency resulted in viable mice which were normal at birth but displayed early defects in calvarial ossification, incisor eruption and growth as well as femoral bone mineral density, structure, and mechanical properties. Although these defects persisted into adulthood, they became milder with age. Finally, a lack of β1 integrins in mature osteoblasts and osteocytes resulted in minor alterations to femur structure but had no effect on mineral density, biomechanics or fracture healing. Taken together, our data indicate that β1 integrin expression in early mesenchymal condensations play an important role in skeletal ossification, while β1 integrin-ECM interactions in pre-osteoblast, odontoblast- and hypertrophic

  18. The effect of conditional inactivation of beta 1 integrins using twist 2 Cre, Osterix Cre and osteocalcin Cre lines on skeletal phenotype.

    PubMed

    Shekaran, Asha; Shoemaker, James T; Kavanaugh, Taylor E; Lin, Angela S; LaPlaca, Michelle C; Fan, Yuhong; Guldberg, Robert E; García, Andrés J

    2014-11-01

    Skeletal development and growth are complex processes regulated by multiple microenvironmental cues, including integrin-ECM interactions. The β1 sub-family of integrins is the largest integrin sub-family and constitutes the main integrin binding partners of collagen I, the major ECM component of bone. As complete β1 integrin knockout results in embryonic lethality, studies of β1 integrin function in vivo rely on tissue-specific gene deletions. While multiple in vitro studies indicate that β1 integrins are crucial regulators of osteogenesis and mineralization, in vivo osteoblast-specific perturbations of β1 integrins have resulted in mild and sometimes contradictory skeletal phenotypes. To further investigate the role of β1 integrins on skeletal phenotype, we used the Twist2-Cre, Osterix-Cre and osteocalcin-Cre lines to generate conditional β1 integrin deletions, where Cre is expressed primarily in mesenchymal condensation, pre-osteoblast, and mature osteoblast lineage cells respectively within these lines. Mice with Twist2-specific β1 integrin disruption were smaller, had impaired skeletal development, especially in the craniofacial and vertebral tissues at E19.5, and did not survive beyond birth. Osterix-specific β1 integrin deficiency resulted in viable mice which were normal at birth but displayed early defects in calvarial ossification, incisor eruption and growth as well as femoral bone mineral density, structure, and mechanical properties. Although these defects persisted into adulthood, they became milder with age. Finally, a lack of β1 integrins in mature osteoblasts and osteocytes resulted in minor alterations to femur structure but had no effect on mineral density, biomechanics or fracture healing. Taken together, our data indicate that β1 integrin expression in early mesenchymal condensations play an important role in skeletal ossification, while β1 integrin-ECM interactions in pre-osteoblast, odontoblast- and hypertrophic chondryocyte

  19. Association of platelet glycoprotein receptor alpha2beta1 integrin and glycoprotein IIIa gene polymorphisms with diabetic retinopathy: evidence from 3007 subjects.

    PubMed

    Gong, Jian-Yang; Deng, Da-Tong; Sun, Ye-Huan

    2015-05-01

    Many epidemiological studies have evaluated associations of platelet glycoprotein receptor alpha2beta1 integrin (ITGA2) and glycoprotein IIIa (ITGB3) gene polymorphisms with diabetic retinopathy (DR), but the published data are inconclusive. The aim of the present study was to assess the associations by using meta-analysis. A comprehensive electronic search (PubMed, EMBASE, Elsevier Science Direct, CNKI and Wanfang) was carried out until 31 August 2013. Odds ratios (ORs) and its 95% confidence intervals (CIs) were used to assess the strength of the associations. Nine studies including 1678 cases and 1329 controls were included in the meta-analysis. Meta-analysis was performed for ITGA2 gene BgI II polymorphism (7 studies including 758 cases and 570 controls) and ITGB3 gene PlA1/A2 polymorphism (4 studies including 1047 cases and 861 controls). Significant associations were found for BgI II (+ versus -: OR = 1.42, 95% CI = 1.06-1.90, p = 0.02; +/- + +/+ versus -/-: OR = 1.46, 95% CI = 0.99-2.15, p = 0.06; +/+ versus -/- + +/-: OR = 1.90, 95% CI = 1.35-2.67, p = 0.0003) and PlA1/A2 (A2 versus A1: OR = 0.74, 95% CI = 0.52-1.07, p = 0.11; A1A2 + A2A2 versus A1A1: OR = 0.80, 95% CI = 0.64-0.99, p = 0.04; A2A2 versus A1A1 + A1A2: OR = 0.45, 95% CI = 0.27-0.75, p = 0.002) polymorphisms. This meta-analysis demonstrates that DR is associated with ITGA2 BgI II and ITGB3 PlA1/A2 polymorphisms.

  20. Differential Regulation of Human Thymosin Beta 15 Isoforms by Transforming Growth Factor Beta 1

    PubMed Central

    Banyard, Jacqueline; Barrows, Courtney; Zetter, Bruce R.

    2009-01-01

    We recently identified an additional isoform of human thymosin beta 15 (also known as NB-thymosin beta, gene name TMSB15A) transcribed from an independent gene, and designated TMSB15B. The purpose of this study was to investigate whether these isoforms were differentially expressed and functional. Our data show that the TMSB15A and TMSB15B isoforms have distinct expression patterns in different tumor cell lines and tissues. TMSB15A was expressed at higher levels in HCT116, DU145, LNCaP and LNCaP-LN3 cancer cells. In MCF-7, SKOV-3, HT1080 and PC-3MLN4 cells, TMSB15A and TMSB15B showed approximately equivalent levels of expression, while TMSB15B was the predominant isoform expressed in PC-3, MDA-MB-231, NCI-H322 and Caco-2 cancer cells. In normal human prostate and prostate cancer tissues, TMSB15A was the predominant isoform expressed. In contrast, normal colon and colon cancer tissue expressed predominantly TMSB15B. The two gene isoforms are also subject to different transcriptional regulation. Treatment of MCF-7 breast cancer cells with transforming growth factor beta 1 repressed TMSB15A expression but had no effect on TMSB15B. siRNA specific to the TMSB15B isoform suppressed cell migration of prostate cancer cells to epidermal growth factor, suggesting a functional role for this second isoform. In summary, our data reveal different expression patterns and regulation of a new thymosin beta 15 gene paralog. This may have important consequences in both tumor and neuronal cell motility. PMID:19296525

  1. Major Host Factors Involved in Epithelial Cell Invasion of Campylobacter jejuni: Role of Fibronectin, Integrin Beta1, FAK, Tiam-1, and DOCK180 in Activating Rho GTPase Rac1

    PubMed Central

    Boehm, Manja; Krause-Gruszczynska, Malgorzata; Rohde, Manfred; Tegtmeyer, Nicole; Takahashi, Seiichiro; Oyarzabal, Omar A.; Backert, Steffen

    2011-01-01

    Host cell entry by the food-borne pathogen Campylobacter jejuni has been reported as one of the primary reasons of tissue damage in infected humans, however, molecular invasion mechanisms and cellular factors involved in this process are widely unclear. Here we used knockout cell lines derived from fibronectin−/−, integrin beta1−/−, and focal adhesion kinase (FAK)−/− deficient mice and corresponding wild-type (WT) controls, to study C. jejuni-induced signaling cascades involved in the bacterial invasion process. Using high resolution scanning electron microscopy, GTPase pull-downs, G-LISA, and gentamicin protection assays we found that each of these host cell factors is indeed required for activation of the small Rho GTPase member Rac1 and maximal host cell invasion of this pathogen. Interestingly, membrane ruffling, tight engulfment of bacteria and invasion were only seen during infection of WT control cells, but not in fibronectin−/−, integrin beta1−/−, and FAK−/− knockout cell lines. We also demonstrate that C. jejuni activates FAK autophosphorylation activity at Y-397 and phosphorylation of Y-925, which is required for stimulating two downstream guanine exchange factors, DOCK180 and Tiam-1, which are upstream of Rac1. Small interfering (si) RNA studies further show that DOCK180 and Tiam-1 act cooperatively to trigger Rac1 activation and C. jejuni invasion. Moreover, mutagenesis data indicate that the bacterial fibronectin-binding protein CadF and the intact flagellum are involved in Rho GTPase activation and host cell invasion. Collectively, our results suggest that C. jejuni infection of host epithelial target cells hijacks a major fibronectin → integrin beta1 → FAK → DOCK180/Tiam-1 signaling cascade, which has a crucial role for Rac1 GTPase activity and bacterial entry into host target cells. PMID:22919583

  2. Neu differentiation factor upregulates epidermal migration and integrin expression in excisional wounds.

    PubMed

    Danilenko, D M; Ring, B D; Lu, J Z; Tarpley, J E; Chang, D; Liu, N; Wen, D; Pierce, G F

    1995-02-01

    Neu differentiation factor (NDF) is a 44-kD glycoprotein which was isolated from ras-transformed rat fibroblasts and indirectly induces tyrosine phosphorylation of the HER-2/neu receptor via binding to either the HER-3 or HER-4 receptor. NDF contains a receptor binding epidermal growth factor (EGF)-like domain and is a member of the EGF family. There are multiple different isoforms of NDF which arise by alternative splicing of a single gene. To date, in vivo biologic activities have not been demonstrated for any NDF isoform. Since NDF, HER-2/neu, and HER-3 are present in skin, and other EGF family members can influence wound keratinocytes in vivo, we investigated whether NDF would stimulate epidermal migration and proliferation in a rabbit ear model of excisional wound repair. In this model, recombinant human NDF-alpha 2 (rhNDF-alpha 2), applied once at the time of wounding, induced a highly significant increase in both epidermal migration and epidermal thickness at doses ranging from 4 to 40 micrograms/cm2. In contrast, rhNDF-alpha 1, rhNDF-beta 1, and rhNDF-beta 2 had no apparent biologic effects in this model. rhNDF-alpha 2 also induced increased neoepidermal expression of alpha 5 and alpha 6 integrins, two of the earliest integrins to appear during epidermal migration. In addition, rhNDF-alpha 2-treated wounds exhibited increased neoepidermal expression of cytokeratin 10 and filaggrin, both epidermal differentiation markers. NDF alpha isoforms were expressed in dermal fibroblasts of wounded and unwounded skin, while both HER-2/neu and HER-3 were expressed in unwounded epidermis and dermal adnexa. In wounds, HER-2/neu expression was markedly decreased in the wound neoepidermis while neoepidermal HER-3 expression was markedly upregulated. Taken together, these results suggest that endogenous NDF-alpha 2 may function as a paracrine mediator directing initial epidermal migration during cutaneous tissue repair.

  3. Early nuclear alterations and immunohistochemical expression of Ki-67, Erb-B2, vascular endothelial growth factor (VEGF), transforming growth factor (TGF-beta1) and integrine-linked kinase (ILK) two days after tamoxifen in breast carcinoma.

    PubMed

    Morena, A M L; Oshima, C T F; Gebrim, L H; Egami, M I; Silva, M R R; Segreto, R A; Giannotti Filho, O; Teixeira, V P C; Segreto, H R C

    2004-01-01

    The purpose of the present study was to evaluate breast carcinoma samples before and two days after treatment with tamoxifen in order to analyse early histopathological alterations--particularlynuclear alterations-- as well as immunohistochemical expression of Ki-67, Erb-B2, VEGF, TGF-beta1 and ILK proteins. Twenty one cases of invasive ductal and lobular breast carcinoma were studied. Patients were submitted to biopsy of the lesion and, after confirmation of the diagnosis, they received 20 mg of tamoxifen a day, beginning two days before surgery. The samples obtained during biopsy and after surgery were stained with HE for histopathological diagnosis. Estrogen receptor was positive in 18 cases and negative in 3. The immunohistochemical method was applied for the detection of Ki-67, Erb-B2, protein, vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-beta1) and integrin linked kinase (ILK). Two days after tamoxifen treatment, the following results were observed: 1) decrease in the cell volume, chomatine condensation, nucleoli less evident and clearly defined nuclear limits; 2) significant reduction in the expression of Erb-B2 protein and significant increase in the expression of TGF-beta1 protein; 3) expression of others proteins (Ki-67, VEGF and ILK) was not altered during the indicated time frame. Our results suggest that analyzing nuclear alterations and expression of Erb-B2 and TGF-beta1 proteins would be useful to assess the initial response to tamoxifen.

  4. Extracellular matrix proteins, integrin receptors (VLA-beta 1, VLA-alpha 2 and VLA-alpha 5) and growth fraction in atypical macroregenerative nodules of the liver: an immunocytochemical case study.

    PubMed

    Patriarca, C; Roncalli, M; Viale, G; Alfano, R M; Braidotti, P; Guddo, F; Coggi, G

    1994-08-01

    A case of cirrhotic liver harbouring three atypical macroregenerative nodules and an hepatocellular carcinoma was immunocytochemically investigated for the expression of VLA-beta 1, VLA-alpha 2 and VLA-alpha 5 integrins and for different extracellular matrix (ECM) components (collagen I, collagen IV, laminin, fibronectin and tenascin). In addition, the proliferative activity within the nodules was evaluated, using the MIB 1 monoclonal antibody (MAb). The cirrhotic liver disclosed a continuous staining pattern of the ECM proteins investigated, as well as a "sinusoidal" immunostaining of VLA-beta 1, VLA-alpha 2 and VLA-alpha 5. The macroregenerative nodules showed a discontinued immunoreactivity for ECM proteins while maintaining a VLA-beta 1 sinusoidal immunostaining, coupled with intercellular immunostaining. VLA-alpha 2 and VLA-alpha 5 expression was lacking. The growth fraction was low in both the above pathological conditions. The hepatocellular carcinoma was devoid of any ECM immunostaining. VLA-beta 1 immunoreactivity exhibited a honeycomb pattern of staining, whereas VLA alpha subunits were absent. MIB1 expression was high, being present in 30% of neoplastic nuclei. A possible relationship between atypical macroregenerative nodules and hepatocellular carcinoma is discussed.

  5. MMP-9 in B-cell chronic lymphocytic leukemia is up-regulated by alpha4beta1 integrin or CXCR4 engagement via distinct signaling pathways, localizes to podosomes, and is involved in cell invasion and migration.

    PubMed

    Redondo-Muñoz, Javier; Escobar-Díaz, Elizabeth; Samaniego, Rafael; Terol, María José; García-Marco, José A; García-Pardo, Angeles

    2006-11-01

    B-cell chronic lymphocytic leukemia (B-CLL) progression is determined by malignant cell extravasation and lymphoid tissue infiltration. We have studied the role and regulation of matrix metalloproteinase-9 (MMP-9) in B-CLL cell migration and invasion. Adhesion of B-CLL cells to the fibronectin fragment FN-H89, VCAM-1, or TNF-alpha-activated human umbilical vein endothelial cells (HUVECs) up-regulated MMP-9 production, measured by gelatin zymography. This effect was mediated by alpha4beta1 integrin and required PI3-K/Akt signaling. The chemokine CXCL12 also up-regulated MMP-9, independently of alpha4beta1 and involving ERK1/2 but not Akt activity. Accordingly, alpha4beta1 engagement activated the PI3-K/Akt/NF-kappaB pathway, while CXCL12/CXCR4 interaction activated ERK1/2/c-Fos signaling. Anti-MMP-9 antibodies, the MMP-9 inhibitor TIMP-1, or transfection with 3 different MMP-9 siRNAs significantly blocked migration through Matrigel or HUVECs. Cell-associated MMP-9 was mainly at the membrane and contained the proactive and mature forms. Moreover, B-CLL cells formed podosomes upon adhesion to FN-H89, VCAM-1, or fibronectin; MMP-9 localized to podosomes in a PI3-K-dependent manner and degraded a fibronectin/gelatin matrix. Our results are the first to show that MMP-9 is physiologically regulated by alpha4beta1 integrin and CXCL12 and plays a key role in cell invasion and transendothelial migration, thus contributing to B-CLL progression. MMP-9 could therefore constitute a target for treatment of this malignancy.

  6. Diet-induced muscle insulin resistance is associated with extracellular matrix remodeling and interaction with integrin alpha2beta1 in mice.

    PubMed

    Kang, Li; Ayala, Julio E; Lee-Young, Robert S; Zhang, Zhonghua; James, Freyja D; Neufer, P Darrell; Pozzi, Ambra; Zutter, Mary M; Wasserman, David H

    2011-02-01

    The hypothesis that high-fat (HF) feeding causes skeletal muscle extracellular matrix (ECM) remodeling in C57BL/6J mice and that this remodeling contributes to diet-induced muscle insulin resistance (IR) through the collagen receptor integrin α(2)β(1) was tested. The association between IR and ECM remodeling was studied in mice fed chow or HF diet. Specific genetic and pharmacological murine models were used to study effects of HF feeding on ECM in the absence of IR. The role of ECM-integrin interaction in IR was studied using hyperinsulinemic-euglycemic clamps on integrin α(2)β(1)-null (itga2(-/-)), integrin α(1)β(1)-null (itga1(-/-)), and wild-type littermate mice fed chow or HF. Integrin α(2)β(1) and integrin α(1)β(1) signaling pathways have opposing actions. HF-fed mice had IR and increased muscle collagen (Col) III and ColIV protein; the former was associated with increased transcript, whereas the latter was associated with reduced matrix metalloproteinase 9 activity. Rescue of muscle IR by genetic muscle-specific mitochondria-targeted catalase overexpression or by the phosphodiesterase 5a inhibitor, sildenafil, reversed HF feeding effects on ECM remodeling and increased muscle vascularity. Collagen remained elevated in HF-fed itga2(-/-) mice. Nevertheless, muscle insulin action and vascularity were increased. Muscle IR in HF-fed itga1(-/-) mice was unchanged. Insulin sensitivity in chow-fed itga1(-/-) and itga2(-/-) mice was not different from wild-type littermates. ECM collagen expansion is tightly associated with muscle IR. Studies with itga2(-/-) mice provide mechanistic insight for this association by showing that the link between muscle IR and increased collagen can be uncoupled by the absence of collagen-integrin α(2)β(1) interaction.

  7. Beta 1 integrin binding plays a role in the constant traction force generation in response to varying stiffness for cells grown on mature cardiac extracellular matrix.

    PubMed

    Gershlak, Joshua R; Black, Lauren D

    2015-01-15

    We have previously reported a unique response of traction force generation for cells grown on mature cardiac ECM, where traction force was constant over a range of stiffnesses. In this study we sought to further investigate the role of the complex mixture of ECM on this response and assess the potential mechanism behind it. Using traction force microscopy, we measured cellular traction forces and stresses for mesenchymal stem cells (MSCs) grown on polyacrylamide gels at a range of stiffnesses (9, 25, or 48 kPa) containing either adult rat heart ECM, different singular ECM proteins including collagen I, fibronectin, and laminin, or ECM mimics comprised of varying amounts of collagen I, fibronectin, and laminin. We also measured the expression of integrins on these different substrates as well as probed for β1 integrin binding. There was no significant change in traction force generation for cells grown on the adult ECM, as previously reported, whereas cells grown on singular ECM protein substrates had increased traction force generation with an increase in substrate stiffness. Cells grown on ECM mimics containing collagen I, fibronectin and laminin were found to be reminiscent of the traction forces generated by cells grown on native ECM. Integrin expression generally increased with increasing stiffness except for the β1 integrin, potentially implicating it as playing a role in the response to adult cardiac ECM. We inhibited binding through the β1 integrin on cells grown on the adult ECM and found that the inhibition of β1 binding led to a return to the typical response of increasing traction force generation with increasing stiffness. Our data demonstrates that cells grown on the mature cardiac ECM are able to circumvent typical stiffness related cellular behaviors, likely through β1 integrin binding to the complex composition. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Laminin-10 and Its Receptors in Breast Carcinoma: Cooperation of Alpha 6 Beta 4 and Alpha 3 Beta 1 Integrin Receptors in Breast Carcinoma Invasion

    DTIC Science & Technology

    2002-06-01

    integrin subunit. Surprisingly, TIP-2/ GIPC was found to interact with both oX6 isoforms, and the oc6B subunit was demonstrated to have a novel type of...serine at -2 position and an aliphatic residue at the C-terminal 0 position. Yeast two- hybrid screen suggested that TIP-2/ GIPC , a previously...TIP-2/ GIPC is sufficient for interaction with both c*6A and oX6B integrin subunits. In addition, the properties of the novel PDZ recognition sequence in

  9. Role of beta 1 and beta 2 integrins in the adhesion of human CD34hi stem cells to bone marrow stroma.

    PubMed Central

    Teixidó, J; Hemler, M E; Greenberger, J S; Anklesaria, P

    1992-01-01

    Hematopoietic stem cell interaction with elements of the underlying stroma is essential for sustained normal hematopoiesis. Here we have determined that adhesion receptors in the integrin family play a role in promoting adhesion of human hematopoietic stem cells to cultured human marrow stromal cells. Enriched CD34hi progenitor cells expressed VLA-4, VLA-5, and at least one or more beta 2 integrins. Homogeneous marrow stromal cell monolayers capable of supporting proliferation of cocultivated CD34hi cells expressed VCAM-1 and fibronectin (ligands for VLA-4 and VLA-5) as well as ICAM-1 (ligand for LFA-1 and Mac-1). Adhesion-blocking experiments indicated that VLA-4/VCAM-1, VLA-5/fibronectin, and beta 2-integrin/ICAM-1 pathways all are important for CD34hi cell attachment to stromal cells. Consistent with this suggestion, IL-1 stimulation of stromal cells caused both increased VCAM-1 and ICAM-1 expression and increased attachment by CD34hi bone marrow cells. In addition, CD34hi cells utilized VLA-4 to adhere to purified VCAM-1 and employed VLA-5 (and to a lesser extent VLA-4) to adhere to purified fibronectin. Together these results suggest that CD34hi stem cells may utilize multiple integrin-mediated adhesion pathways to localize within specialized microenvironmental niches created by marrow stromal cells. Images PMID:1379610

  10. Specific signals involved in the long-term maintenance of radiation-induced fibrogenic differentiation: a role for CCN2 and low concentration of TGF-beta1.

    PubMed

    Haydont, Valérie; Riser, Bruce L; Aigueperse, Jocelyne; Vozenin-Brotons, Marie-Catherine

    2008-06-01

    The fibrogenic differentiation of resident mesenchymal cells is a key parameter in the pathogenesis of radiation fibrosis and is triggered by the profibrotic growth factors transforming growth factor (TGF)-beta1 and CCN2. TGF-beta1 is considered the primary inducer of fibrogenic differentiation and is thought to control its long-term maintenance, whereas CCN2 is considered secondary effector of TGF-beta1. Yet, in long-term established fibrosis like that associated with delayed radiation enteropathy, in situ TGF-beta1 deposition is low, whereas CCN2 expression is high. To explore this apparent paradox, cell response to increasing doses of TGF-beta1 was investigated in cells modeling initiation and maintenance of fibrosis, i.e., normal and fibrosis-derived smooth muscle cells, respectively. Activation of cell-specific signaling pathways by low TGF-beta1 doses was demonstrated with a main activation of the Rho/ROCK pathway in fibrosis-derived cells, whereas the Smad pathway was mainly activated in normal cells. This leads to subsequent and cell-specific regulation of the CCN2 gene. These results suggested a specific profibrotic role of CCN2 in fibrosis-initiated cells. Furthermore, the modulation of CCN2 expression by itself and the combination of TGF-beta1 and CCN2 was investigated in fibrosis-derived cells. In fibrosis-initiated cells CCN2 triggered its autoinduction; furthermore, low concentration of TGF-beta1-potentiated CCN2 autoinduction. Our findings showed a differential requirement and action of TGF-beta1 in the fibrogenic response of normal vs. fibrosis-derived cells. This study defines a novel Rho/ROCK but Smad3-independent mode of TGF-beta signaling that may operate during the chronic stages of fibrosis and provides evidence of both specific and combinatorial roles of low TGF-beta1 dose and CCN2.

  11. Discovery, SAR, and Radiolabeling of Halogenated Benzimidazole Carboxamide Antagonists as Useful Tools for (alpha)4(beta)1 Integrin Expressed on T- and B-cell Lymphomas

    SciTech Connect

    Carpenter, R D; Natarajan, A; Lau, E Y; Andrei, M; Solano, D M; Lightstone, F C; DeNardo, S J; Lam, K S; Kurth, M J

    2010-02-08

    The cell surface receptor {alpha}{sub 4}{beta}{sub 1} integrin is an attractive yet poorly understood target for selective diagnosis and treatment of T- and B-cell lymphomas. This report focuses on the rapid microwave preparation of medicinally pertinent benzimidazole heterocycles, structure-activity relationships (SAR) of novel halobenzimidazole carboxamide antagonists 3-6, and preliminary biological evaluation of radioiodinated agents 7, 8, and 18. The I-125 derivative 18 had good tumor uptake (12 {+-} 1% ID/g at 24 h; 4.5 {+-} 1% ID/g at 48 h) and tumor:kidney ratio ({approx}4:1 at 24 h; 2.5:1 at 48 h) in xenograft murine models of B-cell lymphoma. Molecular homology models of {alpha}{sub 4}{beta}{sub 1} integrin have predicted that docked halobenzimidazole carboxamides have the halogen atom in a suitable orientation for halogen-hydrogen bonding. These high affinity ({approx} pM binding) halogenated ligands are attractive tools for medicinal and biological use; the fluoro and iodo derivatives are potential radiodiagnostic ({sup 18}F) or radiotherapeutic ({sup 131}I) agents, whereas the chloro and bromo analogues could provide structural insight into integrin-ligand interactions through photoaffinity cross-linking/mass spectroscopy experiments, as well as co-crystallization X-ray studies.

  12. Branching beta 1-6N-acetylglucosaminetransferases and polylactosamine expression in mouse F9 teratocarcinoma cells and differentiated counterparts.

    PubMed

    Heffernan, M; Lotan, R; Amos, B; Palcic, M; Takano, R; Dennis, J W

    1993-01-15

    beta-All-trans-retinoic acid (RA)-induced endodermal differentiation of mouse F9 teratocarcinoma cells is accompanied by changes in glycoprotein glycosylation, including expression of i antigen (i.e. polylactosamine) and leukophytohemagglutinin-reactive oligosaccharides (i.e. -GlcNAc beta 1-6Man alpha 1-6-branched N-linked). We have used the F9 teratocarcinoma cells as a model to study developmental regulation of glycosyltransferase activities which are responsible for the biosynthesis of beta 1-6GlcNAc-branched N- and O-linked oligosaccharides and polylactosamine. Growth of F9 cells in the presence of 10(-6) M RA for 4 days increased core 2 GlcNAc transferase and GlcNAc transferase V activities by 13- and 6-fold, respectively, whereas the activities of GlcNAc transferase I, beta 1-3GlcNAc transferase (i), beta 1-4Gal transferase, and beta 1-3Gal transferase increased 2-4-fold. Induction of glycosyltransferase activities by RA was dose-dependent and showed a biphasic response with approximately half of the increase observed 3 days after RA treatment and the remainder occurred by day 4. PYS-2, a parietal endoderm cell line, showed levels of glycosyltransferase activities similar to those of RA-treated F9 cells. Glycosyltransferase activities in the RA-resistant F9 cell line (RA-3-10) were low and showed only a small induction by RA. These observations suggest that differentiation of F9 cells is closely associated with induction of multiple glycosyltransferase activities, with most pronounced increases in GlcNAc transferase V and 2',5'-tetradenylate (core 2) GlcNAc transferase. The increase in GlcNAc transferase V was also reflected by the 4-6-fold increase in the binding of 125I-leukophytohemagglutinin to several cellular glycoproteins, which occurred after 3 days of RA treatment. The endo-beta-galactosidase-sensitive polylactosamine content of membrane glycoproteins and, in particular, the LAMP-1 glycoprotein was markedly increased after RA treatment of F9 cells

  13. Differential effects on mood of 12-15 (SMR) and 15-18 (beta1) Hz neurofeedback.

    PubMed

    Gruzelier, John H

    2014-07-01

    The common assumption in EEG-neurofeedback is one of functional specificity of the trained spectral bands, though it has been posited that only a nonspecific generalised learning process may be engaged. Earlier we reported differential effects on attention in healthy participants measured with continuous performance tests and the P300, following training of the sensory-motor rhythm band (SMR, 12-15 Hz) compared with the adjacent beta1 (15-18 hz) band. Here previously unreported results are presented with phenomenological data from an activation checklist in support of the putative calming effect of SMR neurofeedback. While within sessions both protocols induced tiredness, this was paralleled by an increase in calmness only following SMR training. The differential effect on mood was theoretically consistent and extends evidence of cognitive functional specificity with neurofeedback to affective processes.

  14. Transforming growth factor beta1 regulates melanocyte proliferation and differentiation in mouse neural crest cells via stem cell factor/KIT signaling.

    PubMed

    Kawakami, Tamihiro; Soma, Yoshinao; Kawa, Yoko; Ito, Masaru; Yamasaki, Emiko; Watabe, Hidenori; Hosaka, Eri; Yajima, Kenji; Ohsumi, Kayoko; Mizoguchi, Masako

    2002-03-01

    Stem cell factor is essential to the migration and differentiation of melanocytes during embryogenesis based on the observation that mutations in either the stem cell factor gene, or its ligand, KIT, result in defects in coat pigmentation in mice. Stem cell factor is also required for the survival of melanocyte precursors while they are migrating towards the skin. Transforming growth factor beta1 has been implicated in the regulation of both cellular proliferation and differentiation. NCC-melb4, an immortal cloned cell line, was cloned from a mouse neural crest cell. NCC-melb4 cells provide a model to study the specific stage of differentiation and proliferation of melanocytes. They also express KIT as a melanoblast marker. Using the NCC-melb4 cell line, we investigated the effect of transforming growth factor beta1 on the differentiation and proliferation of immature melanocyte precursors. Immunohistochemically, NCC-melb4 cells showed transforming growth factor beta1 expression. The anti-transforming growth factor beta1 antibody inhibited the cell growth, and downregulated the KIT protein and mRNA expression. To investigate further the activation of autocrine transforming growth factor beta1, NCC-melb4 cells were incubated in nonexogenous transforming growth factor beta1 culture medium. KIT protein decreased with anti-transforming growth factor beta1 antibody concentration in a concentration-dependent manner. We concluded that in NCC-melb4 cells, transforming growth factor beta1 promotes melanocyte precursor proliferation in autocrine and/or paracrine regulation. We further investigated the influence of transforming growth factor beta1 in vitro using a neural crest cell primary culture system from wild-type mice. Anti-transforming growth factor beta1 antibody decreased the number of KIT positive neural crest cell. In addition, the anti-transforming growth factor beta1 antibody supplied within the wild-type neural crest explants abolished the growth of the neural

  15. Tumor necrosis factor-α alters integrins and metalloprotease ADAM12 levels and signaling in differentiating myoblasts.

    PubMed

    Grzelkowska-Kowalczyk, K; Tokarska, J; Grabiec, K; Gajewska, M; Milewska, M; Błaszczyk, M

    2016-01-01

    The extracellular matrix (ECM) is important in the regulation of myogenesis. We hypothesized that tumor necrosis factor-α (TNF-α) modifies ECM during differentiation of mouse C2C12 myoblasts. Exogenous TNF-α (1 ng/ml) stimulated myoblast fusion on the 3rd day (by 160% vs control) but not on the 5th day of myogenesis. The level of integrin α5 was significantly augmented by TNF-α during 5 day-differentiation; however, integrin β1 was higher than control only on the 3rd day of cytokine treatment. Both the abundance of integrin α5 bound to actin and the level of integrin β1 complexed with integrin α5 increased in the presence of TNF-α, especially on the 3rd day of differentiation. Similarly, the stimulatory effects of TNF-α on integrin α3, metalloprotease ADAM12 and kinases related to integrins, FAK and ILK, were limited to the 3rd day of differentiation. We concluded that TNF-α-induced changes in ECM components in differentiating myogenic cells, i.e. i) increased expression of integrin α5, β1, α3, and metalloprotease ADAM12, ii) enhanced formation of α5β1 integrin receptors and interaction of integrin α5-cytoskeleton, and iii) increased expression of kinases associated with integrin signaling, FAK and ILK, were temporarily associated with the onset of myocyte fusion.

  16. Signals from the surface modulate differentiation of human pluripotent stem cells through glycosaminoglycans and integrins

    PubMed Central

    Wrighton, Paul J.; Klim, Joseph R.; Hernandez, Brandon A.; Koonce, Chad H.; Kamp, Timothy J.; Kiessling, Laura L.

    2014-01-01

    The fate decisions of human pluripotent stem (hPS) cells are governed by soluble and insoluble signals from the microenvironment. Many hPS cell differentiation protocols use Matrigel, a complex and undefined substrate that engages multiple adhesion and signaling receptors. Using defined surfaces programmed to engage specific cell-surface ligands (i.e., glycosaminoglycans and integrins), the contribution of specific matrix signals can be dissected. For ectoderm and motor neuron differentiation, peptide-modified surfaces that can engage both glycosaminoglycans and integrins are effective. In contrast, surfaces that interact selectively with glycosaminoglycans are superior to Matrigel in promoting hPS cell differentiation to definitive endoderm and mesoderm. The modular surfaces were used to elucidate the signaling pathways underlying these differences. Matrigel promotes integrin signaling, which in turn inhibits mesendoderm differentiation. The data indicate that integrin-activating surfaces stimulate Akt signaling via integrin-linked kinase (ILK), which is antagonistic to endoderm differentiation. The ability to attribute cellular responses to specific interactions between the cell and the substrate offers new opportunities for revealing and controlling the pathways governing cell fate. PMID:25422477

  17. Signals from the surface modulate differentiation of human pluripotent stem cells through glycosaminoglycans and integrins.

    PubMed

    Wrighton, Paul J; Klim, Joseph R; Hernandez, Brandon A; Koonce, Chad H; Kamp, Timothy J; Kiessling, Laura L

    2014-12-23

    The fate decisions of human pluripotent stem (hPS) cells are governed by soluble and insoluble signals from the microenvironment. Many hPS cell differentiation protocols use Matrigel, a complex and undefined substrate that engages multiple adhesion and signaling receptors. Using defined surfaces programmed to engage specific cell-surface ligands (i.e., glycosaminoglycans and integrins), the contribution of specific matrix signals can be dissected. For ectoderm and motor neuron differentiation, peptide-modified surfaces that can engage both glycosaminoglycans and integrins are effective. In contrast, surfaces that interact selectively with glycosaminoglycans are superior to Matrigel in promoting hPS cell differentiation to definitive endoderm and mesoderm. The modular surfaces were used to elucidate the signaling pathways underlying these differences. Matrigel promotes integrin signaling, which in turn inhibits mesendoderm differentiation. The data indicate that integrin-activating surfaces stimulate Akt signaling via integrin-linked kinase (ILK), which is antagonistic to endoderm differentiation. The ability to attribute cellular responses to specific interactions between the cell and the substrate offers new opportunities for revealing and controlling the pathways governing cell fate.

  18. Integrin function and regulation in development.

    PubMed

    Tarone, G; Hirsch, E; Brancaccio, M; De Acetis, M; Barberis, L; Balzac, F; Retta, S F; Botta, C; Altruda, F; Silengo, L; Retta, F

    2000-01-01

    Integrins are a large family of membrane receptors, consisting of alpha and beta subunits, that play a pivotal role in the interaction of cells with the extracellular matrix. Such interaction regulates the organization of cells in organs and tissues during development as well as cell differentiation and proliferation. We have shown that unfertilized oocytes express integrins that might be important during fertilization. We also analyzed nervous system and muscle tissue development showing that integrin expression is precisely regulated during organization of these tissues. The results indicate that two distinct integrin alpha subunits mediate the outgrowth of processes in nerve and glial cells. Alpha1 integrin, a laminin receptor, is up-regulated by nerve growth factor and other differentiation stimuli and is involved in neurite extension by nerve cells. In contrast, process extension by glial cells is likely to involve the alphaV integrin. Moreover, the latter integrin subunit is also transiently expressed in muscle of the embryo body where it localizes predominantly at developing myotendinous junctions. After birth this integrin disappears and is substituted by the alpha7 subunit. At the same time, important changes also occur in the expression of the associated beta subunit. In fact, the beta1A isoform which is expressed in fetal muscles, is substituted by beta1D. These isoforms are generated by alternative splicing and differ in only a few amino acid residues at the COOH terminus of the protein. This region of the molecule is exposed at the cytoplasmic face of the plasma membrane and is connected to the actin filaments. Our results show that beta1D, which is expressed only in striated muscle tissues, binds to both cytoskeletal and extracellular matrix proteins with an affinity higher than beta1A. Thus, beta1D provides a stronger link between the cytoskeleton and extracellular matrix necessary to support mechanical tension during muscle contraction. These

  19. Diminished expression of the alpha 5 beta 1 integrin (fibronectin receptor) by invasive clones of a human follicular thyroid cancer cell line.

    PubMed

    Demeure, M J; Doffek, K M; Rezaee, M; Goretzki, P E; Wilson, S D

    1994-01-01

    Altered adhesion plaques have been observed in transformed cell lines and are associated with enhanced metastatic potential. The prototypical adhesion plaque is formed by alpha 5 beta 1 fibronectin receptors (FnRs) interacting with the cellular actin network. We have found differences in the actin networks of noninvasive (FTC-133) and invasive (FTC-236, FTC-238) clones of a human follicular thyroid cancer cell line. Furthermore, thyroid-stimulating hormone (TSH) induces stress fibers in FTC-133. In order to investigate differences in adhesion plaques, expression of fibronectin (FN) and its receptor by these cells was analyzed. For these studies FTC-133, FTC-236, and FTC-238 were cultured in serum-depleted DME-H21 medium for 24 hours before the addition of TSH 30 mU/ml. No quantitative differences were noted in FN expression on Western blot in either the conditioned medium or cellular extracts. Western blots and immunohistochemical studies indicated that TSH induced secretion of FN only in FTC-133. Flow cytometry with an alpha 5 antibody demonstrated a 52% and 45% reduction (p < 0.01) in expression of FnR by FTC-236 and FTC-238, respectively, compared to FTC-133; this finding was supported by immunohistochemistry results. TSH treatment did not alter FnR expression. From these studies, we conclude that invasive clones of FTC decrease their expression of FnRs without changing their expression of FN. Furthermore, TSH treatment may promote FN secretion by FTC-133, although it does not seem to affect FnR or absolute FN expression. The diminished expression of FnR adhesion plaques may enhance metastatic potential in some follicular thyroid cancers.

  20. Differential gene expression in response to transforming growth factor-beta1 by fetal and postnatal dermal fibroblasts.

    PubMed

    Rolfe, Kerstin J; Irvine, Laurie M; Grobbelaar, Addie O; Linge, Claire

    2007-01-01

    The multipotent growth factor transforming growth factor (TGF)-beta1 is consistently linked with fibrosis and scarring. The perfect (scarless) healing of cutaneous wounds in early gestational age fetuses is proposed to be due to this tissue's predominance of the TGF-beta3 isoform over the profibrotic TGF-beta1 and 2. Nevertheless, TGF-beta1 is present during wound healing in the early fetus and recently we demonstrated that relevant intracellular signaling pathways are activated (albeit transiently) on TGF-beta1 stimulation. This study aimed to determine whether TGF-beta1 has different effects on gene transcription in human fetal (<14 weeks) vs. human postnatal dermal fibroblasts, using real-time polymerase chain reaction. The regulation pattern of a number of TGF-beta response genes differed dramatically between the two cell sources. The typical autocrine loop of TGF-beta1 autoinduction did not occur in fetal fibroblasts and genes that are normally up-regulated, connective tissue growth factor and collagen type I were actually down-regulated. Furthermore, other response genes responded in a delayed fashion (TGF-beta3) compared with that seen in the more developmentally mature postnatal fibroblasts. Finally, genes unaltered by TGF-beta stimulation in postnatal cells, TGF-beta2 and collagen III, were up-regulated in fetal cells. These developmentally related differences in fibroblast response to TGF-beta1 may influence wound-healing outcome, i.e., perfect regeneration or fibrosis.

  1. Differential effects of histone deacetylase inhibitors on phorbol ester- and TGF-beta1 induced murine tissue inhibitor of metalloproteinases-1 gene expression.

    PubMed

    Young, David A; Billingham, Olivia; Sampieri, Clara L; Edwards, Dylan R; Clark, Ian M

    2005-04-01

    Expression of the tissue inhibitor of metalloproteinases-1 (Timp-1) gene can be induced by either phorbol myristate acetate (PMA) or transforming growth factor beta1 (TGF-beta1), although the signalling pathways involved are not clearly defined. Canonically, histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA) or sodium butyrate (NaB) increase total cellular histone acetylation and activate expression of susceptible genes. Remarkably, PMA and TGF-beta1 stimulation of Timp-1 show a differential response to TSA or NaB. TSA or NaB potentiate PMA-induced Timp-1 expression but repress TGF-beta1-induced Timp-1 expression. The repression of TGF-beta1-induced Timp-1 by TSA was maximal at 5 ng.mL(-1), while for the superinduction of PMA-induced Timp-1 expression, the maximal dose is > 500 ng x mL(-1) TSA. A further HDACi, valproic acid, did not block TGF-beta1-induced Timp-1 expression, demonstrating that different HDACs impact on the induction of Timp-1. For either PMA or TGF-beta1 to induce Timp-1 expression, new protein synthesis is required, and the induction of AP-1 factors closely precedes that of Timp-1. The effects of the HDACi can be reiterated in transient transfection using Timp-1 promoter constructs. Mutation or deletion of the AP-1 motif (-59/-53) in the Timp-1 promoter diminishes PMA-induction of reporter constructs, however, the further addition of TSA still superinduces the reporter. In c-Jun-/- cells, PMA still stimulates Timp-1 expression, but TSA superinduction is lost. Transfection of a series of Timp-1 promoter constructs identified three regions through which TSA superinduces PMA-induced Timp-1 and we have demonstrated specific protein binding to two of these regions which contain either an avian erythroblastosis virus E26 (v-ets) oncogene homologue (Ets) or Sp1 binding motif.

  2. Cell density governs the ability of human bronchial epithelial cells to recognize serum and transforming growth factor beta-1 as squamous differentiation-inducing agents.

    PubMed Central

    Ke, Y.; Gerwin, B. I.; Ruskie, S. E.; Pfeifer, A. M.; Harris, C. C.; Lechner, J. F.

    1990-01-01

    Sparse (75 to 2000 cells/cm2) density cultures of normal human bronchial epithelial cells uniformly undergo terminal squamous differentiation when incubated in medium containing serum (fetal bovine serum [FBS]) or transforming growth factor beta-1 (TGF-beta 1). It was found that the cell density of the culture affects the probability that a cell will respond to these differentiation-inducing agents. Thus whereas irreversible inhibition of DNA synthesis occurs in sparse cell-density cultures within 24 hours after exposure, only a transient (less than 36 hours) depression in DNA synthesis was seen in high (more than 10,000 cells/cm2) density cultures. In addition, although phase microscopic image analysis revealed that virtually all of the cells displayed a squamous morphology within 1 hour after exposure to FBS or TGF-beta 1, observations made 48 to 72 hours later showed the presence of clusters of small prolate spheroid-shaped cells surrounded by many involucrin-positive squamous-appearing cells. Only the small cells were capable of DNA synthesis and cell division as determined by autoradiography and time-lapse photomicrographic images. These replicating cells immediately undergo squamous differentiation if they are subcultured and reinoculated at low cell density and incubated in medium supplemented with FBS or TGF-beta 1. Therefore the probability that a human bronchial epithelial cell will be refractive to FBS- or TGF-beta 1 induced terminal squamous differentiation is solely a function of the cell density of the culture. Images Figure 7 Figure 8 Figure 9 PMID:2221015

  3. Tissue engineering intrafusal fibers: dose- and time-dependent differentiation of nuclear bag fibers in a defined in vitro system using neuregulin 1-beta-1.

    PubMed

    Rumsey, John W; Das, Mainak; Kang, Jung-Fong; Wagner, Robert; Molnar, Peter; Hickman, James J

    2008-03-01

    While much is known about muscle spindle structure, innervation and function, relatively few factors have been identified that regulate intrafusal fiber differentiation and spindle development. Identification of these factors will be a crucial step in tissue engineering functional muscle systems. In this study, we investigated the role of the growth factor, neuregulin 1-beta-1 (Nrg 1-beta-1) EGF, for its ability to influence myotube fate specification in a defined culture system utilizing the non-biological substrate N-1[3-(trimethoxysilyl)propyl]-diethylenetriamine (DETA). Based on morphological and immunocytochemical criteria, Nrg 1-beta-1 treatment of developing myotubes increases the ratio of nuclear bag fibers to total myotubes from 0.019 to 0.100, approximately a five-fold increase. The myotube cultures were evaluated for expression of the intrafusal fiber-specific alpha cardiac-like myosin heavy chain and for the expression of the non-specific slow myosin heavy chain. Additionally, the expression of ErbB2 receptors on all myotubes was observed, while phosphorylated ErbB2 receptors were only observed in Nrg 1-beta-1-treated intrafusal fibers. After Nrg 1-beta-1 treatment, we were able to observe the expression of the intrafusal fiber-specific transcription factor Egr3 only in fibers exhibiting the nuclear bag phenotype. Finally, nuclear bag fibers were characterized electrophysiologically for the first time in vitro. This data shows conclusively, in a serum-free system, that Nrg 1-beta-1 is necessary to drive specification of forming myotubes to the nuclear bag phenotype.

  4. Interactions between integrin receptors and fibronectin are required for calvarial osteoblast differentiation in vitro

    NASA Technical Reports Server (NTRS)

    Moursi, A. M.; Globus, R. K.; Damsky, C. H.

    1997-01-01

    We previously showed that anti-fibronectin antibodies or soluble fibronectin fragments containing the central cell-binding domain inhibit formation of mineralized nodules by fetal calvarial osteoblasts in vitro. These findings suggest a critical role for fibronectin in osteoblast differentiation and morphogenesis. In this study we tested the hypothesis that fibronectin's effects on osteogenesis are mediated via direct interactions with integrin receptors for fibronectin on osteoblasts. Immunocytochemical analysis identified the integrin fibronectin receptor alpha5ss1 in fetal rat calvarial tissue and in cultured osteoblasts at all stages of differentiation. Three other integrins, alpha3ss1, alpha8ss1 and alphavss3, which can bind fibronectin, as well as other matrix components, were also identified in tissue and at all stages of cell culture. Immunoprecipitation data showed that alpha5ss1 levels are constant throughout osteoblast differentiation whereas levels of alpha3ss1 and alpha8ss1 decline in mature mineralized cultures. To determine whether integrin fibronectin receptors are required for osteoblast formation of mineralized nodules, we examined the extent of nodule formation in the presence and absence of function-perturbing anti-integrin antibodies. The antibodies were present continuously in cultures beginning at confluence (day 3), and nodule formation was measured at days 10 and 20. An anti-alpha5 integrin subunit antibody reduced nodule formation to less than 5% of control values at both time points. Inhibition of nodule formation was reversible and did not affect cell attachment and viability. Function-perturbing antibodies against alpha3ss1 and alpha8ss1 also reduced nodule formation, to less than 20% of control values. In contrast, function-perturbing antibodies to alphavss3 and alphavss5 did not affect nodule formation, indicating that the inhibitions noted were indeed specific. To determine the effect of antibody treatment on gene expression, steady

  5. Interactions between integrin receptors and fibronectin are required for calvarial osteoblast differentiation in vitro

    NASA Technical Reports Server (NTRS)

    Moursi, A. M.; Globus, R. K.; Damsky, C. H.

    1997-01-01

    We previously showed that anti-fibronectin antibodies or soluble fibronectin fragments containing the central cell-binding domain inhibit formation of mineralized nodules by fetal calvarial osteoblasts in vitro. These findings suggest a critical role for fibronectin in osteoblast differentiation and morphogenesis. In this study we tested the hypothesis that fibronectin's effects on osteogenesis are mediated via direct interactions with integrin receptors for fibronectin on osteoblasts. Immunocytochemical analysis identified the integrin fibronectin receptor alpha5ss1 in fetal rat calvarial tissue and in cultured osteoblasts at all stages of differentiation. Three other integrins, alpha3ss1, alpha8ss1 and alphavss3, which can bind fibronectin, as well as other matrix components, were also identified in tissue and at all stages of cell culture. Immunoprecipitation data showed that alpha5ss1 levels are constant throughout osteoblast differentiation whereas levels of alpha3ss1 and alpha8ss1 decline in mature mineralized cultures. To determine whether integrin fibronectin receptors are required for osteoblast formation of mineralized nodules, we examined the extent of nodule formation in the presence and absence of function-perturbing anti-integrin antibodies. The antibodies were present continuously in cultures beginning at confluence (day 3), and nodule formation was measured at days 10 and 20. An anti-alpha5 integrin subunit antibody reduced nodule formation to less than 5% of control values at both time points. Inhibition of nodule formation was reversible and did not affect cell attachment and viability. Function-perturbing antibodies against alpha3ss1 and alpha8ss1 also reduced nodule formation, to less than 20% of control values. In contrast, function-perturbing antibodies to alphavss3 and alphavss5 did not affect nodule formation, indicating that the inhibitions noted were indeed specific. To determine the effect of antibody treatment on gene expression, steady

  6. The Calcium-Sensing Receptor and Integrins in Cellular Differentiation and Migration

    PubMed Central

    Tharmalingam, Sujeenthar; Hampson, David R.

    2016-01-01

    The calcium-sensing receptor (CaSR) is a widely expressed homodimeric G-protein coupled receptor structurally related to the metabotropic glutamate receptors and GPRC6A. In addition to its well characterized role in maintaining calcium homeostasis and regulating parathyroid hormone release, evidence has accumulated linking the CaSR with cellular differentiation and migration, brain development, stem cell engraftment, wound healing, and tumor growth and metastasis. Elevated expression of the CaSR in aggressive metastatic tumors has been suggested as a potential novel prognostic marker for predicting metastasis, especially to bone tissue where extracellular calcium concentrations may be sufficiently high to activate the receptor. Recent evidence supports a model whereby CaSR-mediated activation of integrins promotes cellular migration. Integrins are single transmembrane spanning heterodimeric adhesion receptors that mediate cell migration by binding to extracellular matrix proteins. The CaSR has been shown to form signaling complexes with the integrins to facilitate both the movement and differentiation of cells, such as neurons during normal brain development and tumor cells under pathological circumstances. Thus, CaSR/integrin complexes may function as a universal cell migration or homing complex. Manipulation of this complex may be of potential interest for treating metastatic cancers, and for developmental disorders pertaining to aberrant neuronal migration. PMID:27303307

  7. Integrin-mediated interactions between human bone marrow stromal precursor cells and the extracellular matrix.

    PubMed

    Gronthos, S; Simmons, P J; Graves, S E; Robey, P G

    2001-02-01

    To date, the precise interactions between bone marrow stromal cells and the extracellular matrix that govern stromal cell development remain unclear. The integrin super-family of cell-surface adhesion molecules represents a major pathway used by virtually all cell types to interact with different extracellular matrix components. In this study, purified populations of stromal precursor cells were isolated from the STRO-1-positive fraction of normal human marrow, by fluoresence-activated cell sorting, and then assayed for their ability to initiate clonogenic growth in the presence of various integrin ligands. Bone marrow-derived stromal progenitors displayed differential growth to fibronectin, vitronectin, and laminin, over collagen types I and III, but showed a similar affinity for collagen type IV. The integrin heterodimers alpha1beta1, alpha2beta1, alpha5beta1, alpha6beta1, alpha(v)beta3, and alpha(v)beta5 were found to coexpress with the STRO-1 antigen on the cell surface of CFU-F, using dual-color analysis. Furthermore, only a proportion of stromal precursors expressed the integrin alpha4beta1, while no measurable levels of the integrin alpha3beta1 could be detected. Subsequent adhesion studies using functional blocking antibodies to different integrin alpha/beta heterodimers showed that stromal cell growth on collagen, laminin, and fibronectin was mediated by multiple beta1 integrins. In contrast, cloning efficiency in the presence of vitronectin was mediated in part by alpha(v)beta3. When human marrow stromal cells were cultured under osteoinductive conditions, their ability to form a mineralized matrix in vitro was significantly diminished in the presence of a functional blocking monoclonal antibody to the beta1 integrin subunit. The results of this study indicate that beta1 integrins appear to be the predominant adhesion receptor subfamily utilized by stromal precursor cells to adhere and proliferate utilizing matrix glycoproteins commonly found in the bone

  8. Integrin α6β1 Expressed in ESCs Instructs the Differentiation to Endothelial Cells.

    PubMed

    Toya, Sophie P; Wary, Kishore K; Mittal, Manish; Li, Fei; Toth, Peter T; Park, Changwon; Rehman, Jalees; Malik, Asrar B

    2015-06-01

    Adhesion of embryonic stem cells (ESCs) to the extracellular matrix may influence differentiation potential and cell fate decisions. Here, we investigated the inductive role of binding of integrin α6β1 expressed in mouse (m)ESCs to laminin-1 (LN1) in mediating the differentiation of ESCs to endothelial cells (ECs). We observed that α6β1 binding to LN1 was required for differentiation to ECs. α6β1 functioned by recruiting the adaptor tetraspanin protein CD151, which activated FAK and Akt signaling and mediated the EC lineage-specifying transcription factor Er71. In contrast, association of the ESC-expressed α3β1, another highly expressed LN1 binding integrin, with CD151, prevented α6β1-mediated differentiation. CD151 thus functioned as a bifurcation router to direct ESCs toward ECs when α6β1 associated with CD151, or prevented transition to ECs when α3β1 associated with CD151. These observations were recapitulated in mice in which α6 integrin or CD151 knockdown reduced the expression of Er71-regulated angiogenesis genes and development of blood vessels. Thus, interaction of α6β1 in ESCs with LN1 activates α6β1/CD151 signaling which programs ESCs toward the EC lineage fate. © 2015 AlphaMed Press.

  9. Keratinocytes display normal proliferation, survival and differentiation in conditional beta4-integrin knockout mice.

    PubMed

    Raymond, Karine; Kreft, Maaike; Janssen, Hans; Calafat, Jero; Sonnenberg, Arnoud

    2005-03-01

    The alpha6beta4 integrin is located at the basal surface of keratinocytes, in hemidesmosomal structures that mediate stable adhesion of epidermal cells to the underlying basement membrane component laminin-5. The absence of alpha6beta4 integrin causes junctional epidermolysis bullosa, a severe blistering disease of the skin leading to perinatal death, confirming its essential role in mediating strong keratinocyte adhesion. Several studies have suggested that alpha6beta4 integrin can also regulate signaling cascades that control cell proliferation, survival and migration through a mechanism independent of its adhesive function. We have generated a conditional knockout mouse strain, in which the gene encoding the beta4 integrin subunit (Itgb4) was inactivated only in small stretches of the skin. These mice were viable and permitted an accurate analysis of the consequences of the loss of beta4 on various biological processes by comparing beta4-positive and -negative parts of the skin in the same animal. Despite the complete loss of hemidesmosomes in regions lacking alpha6beta4 integrin, the distribution of a range of adhesion receptors and basement membrane proteins was unaltered. Moreover, loss of alpha6beta4 did not affect squamous differentiation, proliferation or survival, except for areas in which keratinocytes had detached from the basement membrane. These in vivo observations were confirmed in vitro by using immortalized keratinocytes - derived from beta4-subunit conditional knockout mice - from which the gene encoding beta4 had been deleted by Cre-mediated recombination. Consistent with the established role of alpha6beta4 in adhesion strengthening, its loss from cells was found to increase their motility. Our findings clearly demonstrate that, after birth, epidermal differentiation, proliferation and survival all proceed normally in the absence of alpha6beta4, provided that cell adhesion is not compromised.

  10. Integrin associated proteins differentially regulate neutrophil polarity and directed migration in 2D and 3D.

    PubMed

    Yamahashi, Yukie; Cavnar, Peter J; Hind, Laurel E; Berthier, Erwin; Bennin, David A; Beebe, David; Huttenlocher, Anna

    2015-10-01

    Directed neutrophil migration in blood vessels and tissues is critical for proper immune function; however, the mechanisms that regulate three-dimensional neutrophil chemotaxis remain unclear. It has been shown that integrins are dispensable for interstitial three-dimensional (3D) leukocyte migration; however, the role of integrin regulatory proteins during directed neutrophil migration is not known. Using a novel microfluidic gradient generator amenable to 2D and 3D analysis, we found that the integrin regulatory proteins Kindlin-3, RIAM, and talin-1 differentially regulate neutrophil polarization and directed migration to gradients of chemoattractant in 2D versus 3D. Both talin-1-deficient and RIAM-deficient neutrophil-like cells had impaired adhesion, polarization, and migration on 2D surfaces whereas in 3D the cells polarized but had impaired 3D chemotactic velocity. Kindlin-3 deficient cells were able to polarize and migrate on 2D surfaces but had impaired directionality. In a 3D environment, Kindlin-3 deficient cells displayed efficient chemotaxis. These findings demonstrate that the role of integrin regulatory proteins in cell polarity and directed migration can be different in 2D and 3D.

  11. Differential effect of purified spruce chitinases and beta-1,3-glucanases on the activity of elicitors from ectomycorrhizal fungi.

    PubMed

    Salzer, P; Hübner, B; Sirrenberg, A; Hager, A

    1997-07-01

    Two chitinases (EC 3.2.1.14) and two beta-1,3-glucanases (EC 3.2.1.39) were purified from the culture medium of spruce (Picea abines [L.] Karst.) cells to study their role in modifying elicitors, cell walls, growth, and hyphal morphology of ectomycorrhizal fungi. The 36-kD class I chitinase (isoelectric point [pl] 8.0) and the 28-kD chitinase (pl 8.7) decreased the activity of elicitor preparations from Hebeloma crustuliniforme (Bull. ex Fries.) Quél., Amanita muscaria (L.) Pers., and Suillus variegatus (Sw.: Fr.) O.K., as demonstrated by using the elicitor-induced extracellular alkalinization in spruce cells as a test system. In addition, chitinases released monomeric products from the walls of these ectomycorrhizal fungi. The beta-1,3-glucanases (35 kD, pl 3.7 and 3.9), in contrast, had little influence on the activity of the fungal elicitors and released only from walls of A. muscaria some polymeric products. Furthermore, chitinases alone and in combination with beta-1,3-glucanases had no effect on the growth and morphology of the hyphae. Thus, it is suggested that apoplastic chitinases in the root cortex destroy elicitors from the ectomycorrhizal fungi without damaging the fungus. By this mechanism the host plant could attenuate the elicitor signal and adjust its own defense reactions to a level allowing symbiotic interaction.

  12. Tac-beta1 inhibits FAK activation and Src signaling.

    PubMed

    Berrier, Allison L; Jones, Christopher W; LaFlamme, Susan E

    2008-03-28

    The binding of integrins to extracellular matrix triggers signals that promote cell spreading. We previously demonstrated that expression of the integrin beta1 cytoplasmic domain in the context of a chimeric transmembrane receptor with the Tac subunit of the interleukin-2 receptor (Tac-beta1) inhibits cell spreading. To study the mechanism whereby Tac-beta1 inhibits cell spreading, we examined the effect of Tac-beta1 on early signaling events following integrin engagement namely FAK and Src signaling. We infected primary fibroblasts with adenoviruses expressing Tac or Tac-beta1 and found that Tac-beta1 prevented FAK activation by inhibiting the phosphorylation of FAK at Tyr-397. In contrast, Src activation was maintained, as phosphorylation of Src at Tyr-419 and Tyr-530 were not responsive to expression of Tac-beta1. Importantly, adhesion-induced tyrosine phosphorylation of the Src substrates p130Cas and paxillin was inhibited, indicating that Src signaling was blocked by Tac-beta1. These Src-dependent signaling events were found to require FAK signaling. Our results suggest that Tac-beta1 inhibits cell spreading, at least in part, by preventing the phosphorylation of FAK at Tyr-397 and the assembly of signaling complexes necessary for phosphorylation of p130Cas and other downstream effectors.

  13. Differential integrin expression regulates cell sensing of the matrix nanoscale geometry.

    PubMed

    Di Cio, Stefania; Bøggild, Thea M L; Connelly, John; Sutherland, Duncan S; Gautrot, Julien E

    2017-03-01

    The nanoscale geometry and topography of the extra-cellular matrix (ECM) is an important parameter controlling cell adhesion and phenotype. Similarly, integrin expression and the geometrical maturation of adhesions they regulate have been correlated with important changes in cell spreading and phenotype. However, how integrin expression controls the nanoscale sensing of the ECM geometry is not clearly understood. Here we develop a new nanopatterning technique, electrospun nanofiber lithography (ENL), which allows the production of a quasi-2D fibrous nanopattern with controlled dimensions (250-1000nm) and densities. ENL relies on electrospun fibres to act as a mask for the controlled growth of protein-resistant polymer brushes. SEM, AFM and immunofluorescence imaging were used to characterise the resulting patterns and the adsorption of the extra-cellular matrix protein fibronectin to the patterned fibres. The control of adhesion formation was studied, as well as the remodelling and deposition of novel matrix. Cell spreading was found to be regulated by the size of fibres, similarly to previous observations made on circular nanopatterns. However, cell shape and polarity were more significantly affected. These changes correlated with important cytoskeleton reorganisation, with a gradual decrease in stress fibre formation as the pattern dimensions decrease. Finally, the differential expression of αvβ3 and α5β1 integrins in engineered cell lines was found to be an important mediator of cell sensing of the nanoscale geometry of the ECM.

  14. Differentiation of Human Skeletal Muscle Stem Cells into Odontoblasts Is Dependent on Induction of α1 Integrin Expression*

    PubMed Central

    Ozeki, Nobuaki; Mogi, Makio; Yamaguchi, Hideyuki; Hiyama, Taiki; Kawai, Rie; Hase, Naoko; Nakata, Kazuhiko; Nakamura, Hiroshi; Kramer, Randall H.

    2014-01-01

    Skeletal muscle stem cells represent an abundant source of autologous cells with potential for regenerative medicine that can be directed to differentiate into multiple lineages including osteoblasts and adipocytes. In the current study, we found that α7 integrin-positive human skeletal muscle stem cells (α7+hSMSCs) could differentiate into the odontoblast lineage under specific inductive conditions in response to bone morphogenetic protein-4 (BMP-4). Cell aggregates of FACS-harvested α7+hSMSCs were treated in suspension with retinoic acid followed by culture on a gelatin scaffold in the presence of BMP-4. Following this protocol, α7+hSMSCs were induced to down-regulate myogenic genes (MYOD and α7 integrin) and up-regulate odontogenic markers including dentin sialophosphoprotein, matrix metalloproteinase-20 (enamelysin), dentin sialoprotein, and alkaline phosphatase but not osteoblastic genes (osteopontin and osteocalcin). Following retinoic acid and gelatin scaffold/BMP-4 treatment, there was a coordinated switch in the integrin expression profile that paralleled odontoblastic differentiation where α1β1 integrin was strongly up-regulated with the attenuation of muscle-specific α7β1 integrin expression. Interestingly, using siRNA knockdown strategies revealed that the differentiation-related expression of the α1 integrin receptor positively regulates the expression of the odontoblastic markers dentin sialophosphoprotein and matrix metalloproteinase-20. These results strongly suggest that the differentiation of α7+hSMSCs along the odontogenic lineage is dependent on the concurrent expression of α1 integrin. PMID:24692545

  15. Geometric guidance of integrin mediated traction stress during stem cell differentiation.

    PubMed

    Lee, Junmin; Abdeen, Amr A; Tang, Xin; Saif, Taher A; Kilian, Kristopher A

    2015-11-01

    Cells sense and transduce the chemical and mechanical properties of their microenvironment through cell surface integrin receptors. Traction stress exerted by cells on the extracellular matrix mediates focal adhesion stabilization and regulation of the cytoskeleton for directing biological activity. Understanding how stem cells integrate biomaterials properties through focal adhesions during differentiation is important for the design of soft materials for regenerative medicine. In this paper we use micropatterned hydrogels containing fluorescent beads to explore force transmission through integrins from single mesenchymal stem cells (MSCs) during differentiation. When cultured on polyacrylamide gels, MSCs will express markers associated with osteogenesis and myogenesis in a stiffness dependent manner. The shape of single cells and the composition of tethered matrix protein both influence the magnitude of traction stress applied and the resultant differentiation outcome. We show how geometry guides the spatial positioning of focal adhesions to maximize interaction with the matrix, and uncover a relationship between αvβ3, α5β1 and mechanochemical regulation of osteogenesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Integrin α4 impacts on differential adhesion of preadipocytes and stem cells on synthetic polymers.

    PubMed

    Hoss, Mareike; Apel, Christian; Dhanasingh, Anandhan; Suschek, Christoph V; Hemmrich, Karsten; Salber, Jochen; Zenke, Martin; Neuss, Sabine

    2013-04-01

    Stem cells represent an ideal cell source for tissue engineering and regenerative medicine, because they can be readily isolated, expanded, differentiated and transplanted. For stem cell-based therapies, biomaterials are required to allow for a spatial distribution of the stem cells within a defined area in the body. In our recent studies, we analysed the interaction of a large panel of stem cell types with an array of biomaterials and demonstrated that a rational prediction of stem cell behaviour on a specific biomaterial is so far not possible. Interestingly, even ontogenetically related stem cell types, such as mesenchymal stem cells (MSCs), preadipocytes and dental pulp stem cells (DPSCs), exhibit distinct adhesion properties on the very same biomaterial surface. Therefore, we investigated integrin and extracellular matrix (ECM) protein expression of stem cells to relate gene expression to adhesion behaviour. MSCs, preadipocytes and DPSCs were cultured on selected synthetic polymers, such as Texin, a thermoplastic polyurethane, poly(dimethyl siloxane) (PDMS), poly-d,l-lactic acid (PDLLA) and l-lactic acid-trimehylene carbonate (Resomer® LT706). Integrins and ECM proteins were analysed by RT-PCR, real-time PCR and immunohistochemistry. Analysis of several adhesion molecules yielded that only one molecule, integrin α4, might play a significant role in differential adhesion on polymers for preadipocytes compared to DPSCs and MSCs. Thus, our studies on the molecular interactions of stem cells and polymers are expected to lead to a more profound understanding of the stem cell-biomaterial interactions to eventually allow for a rational biomaterial design. Copyright © 2012 John Wiley & Sons, Ltd.

  17. Effect of transforming growth factor-beta1 on embryonic and posthatch muscle growth and development in normal and low score normal chicken.

    PubMed

    Li, X; Velleman, S G

    2009-02-01

    During skeletal muscle development, transforming growth factor-beta1 (TGF-beta1) is a potent inhibitor of muscle cell proliferation and differentiation. The TGF-beta1 signal is carried by Smad proteins into the cell nucleus, inhibiting the expression of key myogenic regulatory factors including MyoD and myogenin. However, the molecular mechanism by which TGF-beta1 inhibits muscle cell proliferation and differentiation has not been well documented in vivo. The present study investigated the effect of TGF-beta1 on in vivo skeletal muscle growth and development. A chicken line, Low Score Normal (LSN) with reduced muscling and upregulated TGF-beta1 expression, was used and compared to a normal chicken line. The injection of TGF-beta1 at embryonic day (ED) 3 significantly reduced the pectoralis major (p. major) muscle weight in the normal birds at 1 wk posthatch, whereas no significant difference was observed in the LSN birds. The difference between normal and LSN birds in response to TGF-beta1 is likely due to different levels of endogenous TGF-beta1 where the LSN birds have increased TGF-beta1 expression in their p. major muscle at both 17 ED and 6 wk posthatch. Smad3 expression was reduced by TGF-beta1 from 10 ED to 1 wk posthatch in normal p. major muscle. Unlike Smad3, Smad7 expression was not significantly affected by TGF-beta1 until posthatch in both normal and LSN p. major muscle. Expression of MyoD was reduced 35% by TGF-beta1 during embryonic development in normal p. major muscle, whereas LSN p. major muscle showed a delayed decrease at 1 d posthatch in MyoD expression in response to the TGF-beta1 treatment. Myogenin expression was reduced 29% by TGF-beta1 after hatch in normal p. major muscle. In LSN p. major muscle, TGF-beta1 treatment significantly decreased myogenin expression by 43% at 1 d posthatch and 32% at 1 wk posthatch. These data suggested that TGF-beta1 reduced p. major muscle growth by inhibiting MyoD and myogenin expression during both embryonic

  18. Integrin-linked kinase is involved in matrix-induced hepatocyte differentiation

    SciTech Connect

    Gkretsi, Vasiliki; Bowen, William C.; Yang, Yu; Wu, Chuanyue; Michalopoulos, George K. . E-mail: michalopoulosgk@UPMC.edu

    2007-02-16

    Hepatocytes have restricted proliferative capacity in culture and when cultured without matrix, lose the hepatocyte-specific gene expression and characteristic cellular micro-architecture. Overlay of matrix-preparations on de-differentiated hepatocytes restores differentiation. Integrin-linked kinase (ILK) is a cell-matrix-adhesion protein crucial in fundamental processes such as differentiation and survival. In this study, we investigated the role of ILK, and its binding partners PINCH, {alpha}-parvin, and Mig-2 in matrix-induced hepatocyte differentiation. We report here that ILK is present in the liver and localizes at cell-matrix adhesions of cultured hepatocytes. We also show that ILK, PINCH, {alpha}-parvin, and Mig-2 expression level is dramatically reduced in the re-differentiated hepatocytes. Interestingly, hepatocytes lacking ILK undergo matrix-induced differentiation but their differentiation is incomplete, as judged by monitoring cell morphology and production of albumin. Our results show that ILK and cell-matrix adhesion proteins play an important role in the process of matrix-induced hepatocyte differentiation.

  19. PLC-beta 1 regulates the expression of miR-210 during mithramycin-mediated erythroid differentiation in K562 cells

    PubMed Central

    Fiume, Roberta; Blalock, William; Matteucci, Alessandro; Ramazzotti, Giulia; McCubrey, James A.; Cocco, Lucio; Faenza, Irene

    2014-01-01

    PLC-beta 1 (PLCβ1) inhibits in human K562 cells erythroid differentiation induced by mithramycin (MTH) by targeting miR-210 expression. Inhibition of miR-210 affects the erythroid differentiation pathway and it occurs to a greater extent in MTH-treated cells. Overexpression of PLCβ1 suppresses the differentiation of K562 elicited by MTH as demonstrated by the absence of γ-globin expression. Inhibition of PLCβ1 expression is capable to promote the differentiation process leading to a recovery of γ-globin gene even in the absence of MTH. Our experimental evidences suggest that PLCβ1 signaling regulates erythropoiesis through miR-210. Indeed overexpression of PLCβ1 leads to a decrease of miR-210 expression after MTH treatment. Moreover miR-210 is up-regulated when PLCβ1 expression is down-regulated. When we silenced PKCα by RNAi technique, we found a decrease in miR-210 and γ-globin expression levels, which led to a severe slowdown of cell differentiation in K562 cells and these effects were the same encountered in cells overexpressing PLCβ1. Therefore we suggest a novel role for PLCβ1 in regulating miR-210 and our data hint at the fact that, in human K562 erythroleukemia cells, the modulation of PLCβ1 expression is able to exert an impairment of normal erythropoiesis as assessed by γ-globin expression. PMID:24962066

  20. α2 Integrin, extracellular matrix metalloproteinase inducer, and matrix metalloproteinase-3 act sequentially to induce differentiation of mouse embryonic stem cells into odontoblast-like cells

    SciTech Connect

    Ozeki, Nobuaki; Kawai, Rie; Hase, Naoko; Hiyama, Taiki; Yamaguchi, Hideyuki; Kondo, Ayami; Nakata, Kazuhiko; Mogi, Makio

    2015-02-01

    We previously reported that interleukin 1β acts via matrix metalloproteinase (MMP)-3 to regulate cell proliferation and suppress apoptosis in α2 integrin-positive odontoblast-like cells differentiated from mouse embryonic stem (ES) cells. Here we characterize the signal cascade underpinning odontoblastic differentiation in mouse ES cells. The expression of α2 integrin, extracellular matrix metalloproteinase inducer (Emmprin), and MMP-3 mRNA and protein were all potently increased during odontoblastic differentiation. Small interfering RNA (siRNA) disruption of the expression of these effectors potently suppressed the expression of the odontoblastic biomarkers dentin sialophosphoprotein, dentin matrix protein-1 and alkaline phosphatase, and blocked odontoblast calcification. Our siRNA, western blot and blocking antibody analyses revealed a unique sequential cascade involving α2 integrin, Emmprin and MMP-3 that drives ES cell differentiation into odontoblasts. This cascade requires the interaction between α2 integrin and Emmprin and is potentiated by exogenous MMP-3. Finally, although odontoblast-like cells potently express α2, α6, αV, β1, and β3, integrins, we confirmed that β1 integrin acts as the trigger for ES cell differentiation, apparently in complex with α2 integrin. These results demonstrate a unique and unanticipated role for an α2 integrin-, Emmprin-, and MMP-3-mediated signaling cascade in driving mouse ES cell differentiation into odontoblast-like cells. - Highlights: • Odontoblast differentiation requires activation of α2 integrin, Emmprin and MMP-3. • α2 integrin, Emmprin and MMP-3 form a sequential signaling cascade. • β1 integrin acts a specific trigger for odontoblast differentiation. • The role of these effectors is highly novel and unanticipated.

  1. Lysyl oxidase like 4, a novel target gene of TGF-{beta}1 signaling, can negatively regulate TGF-{beta}1-induced cell motility in PLC/PRF/5 hepatoma cells

    SciTech Connect

    Kim, Dong Joon; Lee, Dong Chul; Yang, Suk-Jin; Lee, Jung Ju; Bae, Eun Mi; Kim, Dong Min; Min, Sang Hyun; Kim, Soo Jung; Kang, Dong Chul; Sang, Byung Chan; Myung, Pyung Keun; Park, Kyung Chan Yeom, Young Il

    2008-09-05

    Transforming growth factor-{beta}1 (TGF-{beta}1) is a multi-functional cytokine involved in the regulation of cell proliferation, differentiation and extracellular matrix formation. In search for novel genes mediating the TGF-{beta}1 function at downstream signaling, we performed a cDNA microarray analysis and identified 60 genes whose expression is regulated by TGF-{beta}1 in the liver cancer cell line PLC/PRF/5. Among them, we report here lysyl oxidase like 4 (LOXL4) as a novel target of TGF-{beta}1 signaling, and provide experimental evidence for its expression regulation and function. LOXL4 was found to be the only member of LOX family whose expression is induced by TGF-{beta}1 in hepatoma cells. Deletion mapping of the LOXL4 promoter indicated that the TGF-{beta}1 regulation of LOXL4 expression is mediated through the binding of AP1 transcription factor to a conserved region of the promoter. This was confirmed by the chromatin immunoprecipitation assay that captured c-Fos-bound chromatin from TGF-{beta}1-treated cells. Forced expression of LOXL4 in PLC/PRF/5 cells resulted in inhibition of cell motility through Matrigel in the presence of TGF-{beta}1 treatment. In parallel, LOXL4 suppressed the expression of laminins and {alpha}3 integrin and the activity of MMP2. These results suggest that LOXL4 may function as a negative feedback regulator of TGF-{beta}1 in cell invasion by inhibiting the metabolism of extracellular matrix (ECM) components.

  2. Differential regulation of monocyte cytokine release by αV and β(2) integrins that bind CD23.

    PubMed

    Edkins, Adrienne L; Borland, Gillian; Acharya, Mridu; Cogdell, Richard J; Ozanne, Bradford W; Cushley, William

    2012-06-01

    The human soluble CD23 (sCD23) protein displays highly pleiotropic cytokine-like activity. Monocytic cells express the sCD23-binding integrins αVβ(3), αVβ(5), αMβ(2) and αXβ(2), but it is unclear which of these four integrins most acutely regulates sCD23-driven cytokine release. The hypothesis that ligation of different sCD23-binding integrins promoted release of distinct subsets of cytokines was tested. Lipopolysaccharide (LPS) and sCD23 promoted release of distinct groups of cytokines from the THP-1 model cell line. The sCD23-driven cytokine release signature was characterized by elevated amounts of RANTES (CCL5) and a striking increase in interleukin-8 (IL-8; CXCL8) secretion, but little release of macrophage inflammatory protein 1β (MIP-1β; CCL4). Antibodies to αVβ(3) or αXβ(2) both promoted IL-8 release, consistent with the sCD23-driven pattern, but both also evoked strong MIP-1β secretion; simultaneous ligation of these two integrins further increased cytokine secretion but did not alter the pattern of cytokine output. In both model cell lines and primary tissue, integrin-mediated cytokine release was more pronounced in immature monocyte cells than in mature cells. The capacity of anti-integrin monoclonal antibodies to elicit a cytokine release response is epitope-dependent and also reflects the differentiation state of the cell. Although a pattern of cytokine release identical to that provoked by sCD23 could not be elicited with any individual anti-integrin monoclonal antibody, αXβ(2) and αVβ(3) appear to regulate IL-8 release, a hallmark feature of sCD23-driven cytokine secretion, more acutely than αMβ(2) or αVβ(5).

  3. Electro-magnetic fields in the home environment (color TV, computer monitor, microwave oven, cellular phone, etc) as potential contributing factors for the induction of oncogen C-fos Ab1, oncogen C-fos Ab2, integrin alpha 5 beta 1 and development of cancer, as well as effects of microwave on amino acid composition of food and living human brain.

    PubMed

    Omura, Y; Losco, M

    1993-01-01

    The effects, on normal human subjects, of 3 minutes exposure to electro-magnetic fields (EMFs) emitted from: A) personal computers, B) color television sets, or C) microwave-ovens, or cellular phones were compared by placing the same large sheet of aluminum foil with a square hole or rectangular band-shaped hole at the chest level (or at the side of the head with the cellular phone), with or without grounding the aluminum foil, using the Bi-Digital O-Ring Test Dysfunction Localization and Molecular Identification Methods with cancer related substances (i.e., Oncogen C-fos Ab2 and mercury in the cell nucleus, Integrin alpha 5 beta 1 in the cell & nuclear membranes, and disappearance of Acetylcholine) as reference control substances. All the above sources of the EMFs not only induced the following various transitional abnormalities on the EMF entry area, but also induced similar abnormalities at the EMF exit area on the back (where the abnormality was found in the same shape as exposed EMF entry area, and the effect lasted for a shorter time than the entry point of the EMF): A) Exposure of the body at about 50 cm from the monitor of some of the typical personal computers resulted in: A1) decrease in Acetylcholine; A2) appearance of circulatory disturbance with the appearance of Thromboxane B2; A3) short-lasting appearance of Oncogen C-fos Ab2; A4) short-lasting appearance of Oncogen C-fos Ab1, though it lasted longer than C-fos Ab2; A5) no appearance of Integrin alpha 5 beta 1. B) part of the chest was exposed at a distance between 1 meter and up to 3 meters from a color television sized anywhere from 13'' to 21'', resulting in: B1) decrease in Acetylcholine; B2) appearance of circulatory disturbance with the appearance of Thromboxane B2; B3) short-lasting appearance of Oncogen C-fos Ab2; B4) short-lasting appearance of Oncogen C-fos Ab1, though it lasted longer than C-fos Ab2; B5) very short-lasting appearance of Integrin alpha 5 beta 1. C) When body was exposed, at

  4. Specific β-containing Integrins Exert Differential Control on Proliferation and Two-dimensional Collective Cell Migration in Mammary Epithelial Cells*

    PubMed Central

    Jeanes, Alexa I.; Wang, Pengbo; Moreno-Layseca, Paulina; Paul, Nikki; Cheung, Julia; Tsang, Ricky; Akhtar, Nasreen; Foster, Fiona M.; Brennan, Keith; Streuli, Charles H.

    2012-01-01

    Understanding how cell cycle is regulated in normal mammary epithelia is essential for deciphering defects of breast cancer and therefore for developing new therapies. Signals provided by both the extracellular matrix and growth factors are essential for epithelial cell proliferation. However, the mechanisms by which adhesion controls cell cycle in normal epithelia are poorly established. In this study, we describe the consequences of removing the β1-integrin gene from primary cultures of mammary epithelial cells in situ, using CreER. Upon β1-integrin gene deletion, the cells were unable to progress efficiently through S-phase, but were still able to undergo collective two-dimensional migration. These responses are explained by the presence of β3-integrin in β1-integrin-null cells, indicating that integrins containing different β-subunits exert differential control on mammary epithelial proliferation and migration. β1-Integrin deletion did not inhibit growth factor signaling to Erk or prevent the recruitment of core adhesome components to focal adhesions. Instead the S-phase arrest resulted from defective Rac activation and Erk translocation to the nucleus. Rac inhibition prevented Erk translocation and blocked proliferation. Activated Rac1 rescued the proliferation defect in β1-integrin-depleted cells, indicating that this GTPase is essential in propagating proliferative β1-integrin signals. These results show that β1-integrins promote cell cycle in mammary epithelial cells, whereas β3-integrins are involved in migration. PMID:22511753

  5. Integrin α1β1 is involved in the differentiation into myofibroblasts in adult reactive tissues in vivo

    PubMed Central

    Rodriguez, Alejandro; Karen, Jakob; Gardner, Humphrey; Gerdin, Bengt; Rubin, Kristofer; Sundberg, Christian

    2009-01-01

    Connective tissue cell activation is of importance during reactive conditions such as solid tumour growth, wound healing and pannus formation in rheumatoid arthritis. Here, we have compared connective tissue cells of mesenchymal origin in human tissues from these conditions and their normal counterparts using a panel of cell-type-specific markers. In particular, we investigated variations of integrin expression among connective tissue cell phenotypes. Connective tissue cell populations were defined based on their association with the microvasculature and their expression of activation markers. The phenotype of these cells varied according to the type of pathological connective tissue examined. Our morphological data from human tissues suggested that the α1β1 integrin, a collagen/laminin receptor, is involved in the differentiation of precursor cells into myofibroblasts. To mechanistically investigate this hypothesis, we employed experimental models for carcinoma growth and wound healing utilizing α1 integrin-deficient mice. The data confirmed that the α1β1 integrin is of importance not only for the differentiation of mesenchymal cells into myofibroblasts but also for the neovascularization and connective tissue organization and emphasize the importance of myofibroblasts in the pathophysiology of tissue repair, inflammation and tumour growth. PMID:19397781

  6. Autocrine transforming growth factor-{beta}1 activation mediated by integrin {alpha}V{beta}3 regulates transcriptional expression of laminin-332 in Madin-Darby canine kidney epithelial cells.

    PubMed

    Moyano, Jose V; Greciano, Patricia G; Buschmann, Mary M; Koch, Manuel; Matlin, Karl S

    2010-11-01

    Laminin (LM)-332 is an extracellular matrix protein that plays a structural role in normal tissues and is also important in facilitating recovery of epithelia from injury. We have shown that expression of LM-332 is up-regulated during renal epithelial regeneration after ischemic injury, but the molecular signals that control expression are unknown. Here, we demonstrate that in Madin-Darby canine kidney (MDCK) epithelial cells LM-332 expression occurs only in subconfluent cultures and is turned-off after a polarized epithelium has formed. Addition of active transforming growth factor (TGF)-β1 to confluent MDCK monolayers is sufficient to induce transcription of the LM α3 gene and LM-332 protein expression via the TGF-β type I receptor (TβR-I) and the Smad2-Smad4 complex. Significantly, we show that expression of LM-332 in MDCK cells is an autocrine response to endogenous TGF-β1 secretion and activation mediated by integrin αVβ3 because neutralizing antibodies block LM-332 production in subconfluent cells. In confluent cells, latent TGF-β1 is secreted apically, whereas TβR-I and integrin αVβ3 are localized basolaterally. Disruption of the epithelial barrier by mechanical injury activates TGF-β1, leading to LM-332 expression. Together, our data suggest a novel mechanism for triggering the production of LM-332 after epithelial injury.

  7. Regulation of α5 and αV Integrin Expression by GDF-5 and BMP-7 in Chondrocyte Differentiation and Osteoarthritis

    PubMed Central

    Garciadiego-Cázares, David; Aguirre-Sánchez, Hilda I.; Abarca-Buis, René F.; Kouri, Juan B.; Velasquillo, Cristina; Ibarra, Clemente

    2015-01-01

    The Integrin β1 family is the major receptors of the Extracellular matrix (ECM), and the synthesis and degradation balance of ECM is seriously disrupted during Osteoarthritis (OA). In this scenario, integrins modify their pattern expression and regulate chondrocyte differen-tiation in the articular cartilage. Members of the Transforming growth factor beta (Tgf-β) Su-perfamily, such as Growth differentiation factor 5 (Gdf-5) and Bone morphogenetic protein 7 (Bmp-7), play a key role in joint formation and could regulate the integrin expression during chondrocyte differentiation and osteoarthritis progression in an experimental OA rat model. Decrease of α5 integrin expression in articular cartilage was related with chondrocyte dedif-ferentiation during OA progression, while increase of α1, α2, and α3 integrin expression was related with fibrous areas in articular cartilage during OA. Hypertrophic chondrocytes expressedαV integrin and was increased in the articular cartilage of rats with OA. Integrin expression during chondrocyte differentiation was also analyzed in a micromass culture system of mouse embryo mesenchymal cells, micromass cultures was treated with Gdf-5 or Bmp-7 for 4 and 6 days, respectively. Gdf-5 induced the expression of theα5 sub-unit, while Bmp-7 induced the expression of the αV sub-unit. This suggests a switch in signaling for prehypertrophic chondrocyte differentiation towards hypertrophy, where Gdf-5 could maintain the articular chondrocyte phenotype and Bmp-7 would induce hypertrophy. Decrease of Ihh expression during late stages of OA in rat model suggest that the ossification in OA rat knees and endochondral ossification could be activated by Bmp-7 and αV integrin in absence of Ihh. Thus, chondrocyte phenotype in articular cartilage is similar to prehypetrophic chondrocyte in growth plate, and is preserved due to the presence of Indian hedgehog (Ihh), Gdf-5 and α5 integrin to maintain articular cartilage and prevent hy

  8. Regulation of α5 and αV Integrin Expression by GDF-5 and BMP-7 in Chondrocyte Differentiation and Osteoarthritis.

    PubMed

    Garciadiego-Cázares, David; Aguirre-Sánchez, Hilda I; Abarca-Buis, René F; Kouri, Juan B; Velasquillo, Cristina; Ibarra, Clemente

    2015-01-01

    The Integrin β1 family is the major receptors of the Extracellular matrix (ECM), and the synthesis and degradation balance of ECM is seriously disrupted during Osteoarthritis (OA). In this scenario, integrins modify their pattern expression and regulate chondrocyte differentiation in the articular cartilage. Members of the Transforming growth factor beta (Tgf-β) Superfamily, such as Growth differentiation factor 5 (Gdf-5) and Bone morphogenetic protein 7 (Bmp-7), play a key role in joint formation and could regulate the integrin expression during chondrocyte differentiation and osteoarthritis progression in an experimental OA rat model. Decrease of α5 integrin expression in articular cartilage was related with chondrocyte dedifferentiation during OA progression, while increase of α1, α2, and α3 integrin expression was related with fibrous areas in articular cartilage during OA. Hypertrophic chondrocytes expressed αV integrin and was increased in the articular cartilage of rats with OA. Integrin expression during chondrocyte differentiation was also analyzed in a micromass culture system of mouse embryo mesenchymal cells, micromass cultures was treated with Gdf-5 or Bmp-7 for 4 and 6 days, respectively. Gdf-5 induced the expression of the α5 sub-unit, while Bmp-7 induced the expression of the αV sub-unit. This suggests a switch in signaling for prehypertrophic chondrocyte differentiation towards hypertrophy, where Gdf-5 could maintain the articular chondrocyte phenotype and Bmp-7 would induce hypertrophy. Decrease of Ihh expression during late stages of OA in rat model suggest that the ossification in OA rat knees and endochondral ossification could be activated by Bmp-7 and αV integrin in absence of Ihh. Thus, chondrocyte phenotype in articular cartilage is similar to prehypetrophic chondrocyte in growth plate, and is preserved due to the presence of Indian hedgehog (Ihh), Gdf-5 and α5 integrin to maintain articular cartilage and prevent hypertrophy.

  9. Magnetically Tuning Tether Mobility of Integrin Ligand Regulates Adhesion, Spreading, and Differentiation of Stem Cells.

    PubMed

    Wong, Dexter S H; Li, Jinming; Yan, Xiaohui; Wang, Ben; Li, Rui; Zhang, Li; Bian, Liming

    2017-03-08

    Cells sense and respond to the surrounding microenvironment through binding of membranous integrin to ligands such as the Arg-Gly-Asp (RGD) peptide. Previous studies show that the RGD tether properties on substrate influence cell adhesion and spreading, but few studies have reported strategies to control the tether mobility of RGD on substrate via a physical and noncontact approach. Herein, we demonstrate a novel strategy to tune the tether mobility of RGD on substrate via magnetic force. We conjugate a monolayer of RGD-bearing magnetic nanoparticles (MNPs) on a glass substrate via the flexible and coiled poly(ethylene glycol) linker of large molecular weight (PEG, average MW: 2000), and this increases the RGD tether mobility, which can be significantly reduced by applying magnetic attraction on MNPs. Our data show that high RGD tether mobility delays the early adhesion and spreading of human mesenchymal stem cells (hMSCs), leading to compromised osteogenic differentiation at later stage. In contrast, hMSCs cultured on substrate with restricted RGD tether mobility, achieved either via a shorter PEG linker (MW: 200) or magnetic force, show significantly better adhesion, spreading, and osteogenic differentiation. The control utilizing RGD-bearing nonmagnetic nanoparticles shows no such enhancing effect of magnetic field on cellular events, further supporting our conjecture of magnetic tuning of RGD tether mobility. We hypothesize that high tether mobility of RGD entails additional time and effort by the cells to fully develop traction force and mechanical feedback, thereby delaying the maturation of FAs and activation of subsequent mechanotransduction signaling. Our staining results of vinculin, a critical component of FAs, and Yes-associated protein (YAP), an important mechanosensitive transcriptional factor, support our hypothesis. We believe that our work not only sheds light on the impact of dynamic presentation of cell adhesive ligands on cellular behaviors

  10. Fibrillar Type I Collagen Enhances the Differentiation and Proliferation of Myofibroblasts by Lowering α2β1 Integrin Expression in Cardiac Fibrosis

    PubMed Central

    Hong, Jian; Chu, Ming; Qian, Lijun; Wang, Junhong; Guo, Yan

    2017-01-01

    Many studies have shown that α2β1 integrin plays an important role in the development of cardiac fibrosis. However, the mechanism of how α2β1 integrin regulates the differentiation and proliferation of myofibroblasts in cardiac fibrosis through fibrillar collagen (FC) remains uncertain. We established that FC mimicked the 3-dimensional extracellular matrix (ECM) of fibroblasts from post-myocardial infarction (MI) patients in vivo. This allowed us to explore the differentiation and proliferation of cardiac fibroblasts on FC. Here, we report that low expression of α2β1 integrin increased protein kinase B (AKT) activation and α-smooth muscle actin (α-SMA) expression. This occurred due to the instability of phosphatase and tensin homolog (PTEN) in myofibroblasts on FC. We also demonstrated that FC reduced protein phosphatase type 2A (PP2A) activity of myofibroblasts, which was coincident with low α2β1 integrin expression and activation of AKT, but not mitogen-activated protein kinase (ERK). In addition, knock-down of both β1 integrin and PP2A in fibroblasts promoted differentiation and proliferation via AKT activation and increased α-SMA expression. In summary, our study demonstrated that low α2β1 integrin expression regulated its downstream targets PTEN and AKT via crosstalk with PP2A, a critical cell signaling pathway that permits aberrant differentiation and proliferation of myofibroblasts on FC. PMID:28251149

  11. The role of integrin αv in proliferation and differentiation of human dental pulp cell response to calcium silicate cement.

    PubMed

    Hung, Chi-Jr; Hsu, Hsin-I; Lin, Chi-Chang; Huang, Tsui-Hsien; Wu, Buor-Chang; Kao, Chia-Tze; Shie, Ming-You

    2014-11-01

    It has been proved that integrin αv activity is related to cell proliferation, differentiation, migration, and organ development. However, the biological functions of integrin αv in human dental pulp cells (hDPCs) cultured on silicate-based materials have not been explored. The aim of this study was to investigate the role of integrin αv in the proliferation and odontogenic differentiation of hDPCs cultured with the effect of calcium silicate (CS) cement and β-tricalcium phosphate (TCP) cement. In this study, hDPCs were cultured on CS and TCP materials, and we evaluated fibronectin (FN) secretion and integrin αv expression during the cell attachment stage. After small interfering RNA transfection targeting integrin αv, the proliferation and odontogenesis differentiation behavior of hDPCs were analyzed. The results indicate that CS releases Si ion-increased FN secretion and adsorption, which promote cell attachment more effectively than TCP. The CS cement facilitates FN and αv subintegrin expression. However, the FN adsorption and integrin expression of TCP are similar to that observed in the control dish. Integrin αv small interfering RNA inhibited odontogenic differentiation of hDPCs with the decreased formation of mineralized nodules on CS. It also down-regulated the protein expression of multiple markers of odontogenesis and the expression of dentin sialophosphoprotein protein. These results establish composition-dependent differences in integrin binding and its effectiveness as a mechanism regulating cellular responses to biomaterial surface. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  12. Interferon Beta-1b Injection

    MedlinePlus

    Interferon beta-1b injection is used to reduce episodes of symptoms in patients with relapsing-remitting (course ... and problems with vision, speech, and bladder control). Interferon beta-1b is in a class of medications ...

  13. Biomimetic integrin-specific surfaces to direct osteoblastic function and tissue healing

    NASA Astrophysics Data System (ADS)

    Petrie, Timothy Andrew

    Current orthopedic implant technologies used suffer from slow rates of osseointegration, short lifetime, and lack of mechanical integrity as a result of poorly controlled cell-surface interactions. Recent biologically-inspired surface strategies (biomimetic) have focused on mimicking the biofunctionality of the extracellular matrix (ECM) by using short, adhesive oligopeptides, such as arginine-glycine-aspartic acid (RGD) present in numerous ECM components. However, these strategies have yielded mixed results in vivo and marginal bone healing responses. The central goal of this dissertation project was to engineer bioactive surfaces that specifically target integrin receptors important for osteogenic functions in order to improve bone tissue repair. In order to create integrin-specific interfaces, integrin-specific ligands reconstituting the fibronectin (FN) secondary/tertiary structure were first engineered and functionalized on material surfaces using several robust presentation schemes. We demonstrated that FN-mimetic-functionalized surfaces that directed alpha 5beta1 binding enhanced osteoblast and stromal cell integrin binding and adhesion, osteogenic signaling, and osteoblastic differentiation compared to various other RGD-based ligand-functionalized surfaces. Next, we investigated the effect of integrin-specific biointerfaces to modulate bone healing in a rat tibia implant bone model. We demonstrated, using a robust polymer brush system, that bioactive coatings on titanium implants that conferred high alpha5beta1 integrin specificity in vitro enhanced bone formation and implant integration in vivo. Moreover, we showed that integrin specificity can be engineered using different immobilization schemes, including clinically-relevant ligand dip-coating, and promote the same robust in vivo effect. Furthermore, we investigate the synergistic roles of integrin specificity and ligand clustering on cell response by engineering biointerfaces presenting trimeric and

  14. Localization of αvβ3-like integrin in cultivated larval cells of the mussel Mytilus trossulus during neuronal and muscle differentiation.

    PubMed

    Odintsova, Nelly A; Maiorova, Maria A

    2012-08-01

    Using immunofluorescence phenotyping, the expression of αvβ3-like integrin was examined during neuronal and muscle differentiation in cell cultures derived from trochophore larvae of the mussel Mytilus trossulus. We have demonstrated that some mussel cells grown on fibronectin in vitro express the extracellular matrix (ECM) αvβ3 integrin-like receptor. At the same time, the distribution of αvβ3-like integrin is not ubiquitous, i.e. it depends on the cell type and the time of cultivation. Using immunohistochemical staining, we have found that only in some cells this integrin is co-localized with molluscan neuronal markers, neurotransmitters serotonin (5-HT) or Phe-Met-Arg-Phe-NH(2) neuropeptide (FMRFamide), and also with filament actin but not with paramyosin. Although we have previously shown that an integrin-dependent mechanism is involved in cell adhesion and differentiation of muscle cells of Mytilus, in this study, αvβ3-like integrin has not been found to participate in fibronectin adhesion of muscle cells but may be a linking agent between the ECM and the neuron-like cells.

  15. The Platelet Integrin αIIbβ3 Differentially Interacts with Fibrin Versus Fibrinogen.

    PubMed

    Litvinov, Rustem I; Farrell, David H; Weisel, John W; Bennett, Joel S

    2016-04-08

    Fibrinogen binding to the integrin αIIbβ3 mediates platelet aggregation and spreading on fibrinogen-coated surfaces. However,in vivoαIIbβ3 activation and fibrinogen conversion to fibrin occur simultaneously, although the relative contributions of fibrinogenversusfibrin to αIIbβ3-mediated platelet functions are unknown. Here, we compared the interaction of αIIbβ3 with fibrin and fibrinogen to explore their differential effects. A microscopic bead coated with fibrinogen or monomeric fibrin produced by treating the immobilized fibrinogen with thrombin was captured by a laser beam and repeatedly brought into contact with surface-attached purified αIIbβ3. When αIIbβ3-ligand complexes were detected, the rupture forces were measured and displayed as force histograms. Monomeric fibrin displayed a higher probability of interacting with αIIbβ3 and a greater binding strength. αIIbβ3-fibrin interactions were also less sensitive to inhibition by abciximab and eptifibatide. Both fibrinogen- and fibrin-αIIbβ3 interactions were partially inhibited by RGD peptides, suggesting the existence of common RGD-containing binding motifs. This assumption was supported using the fibrin variants αD97E or αD574E with mutated RGD motifs. Fibrin made from a fibrinogen γ'/γ' variant lacking the γC αIIbβ3-binding motif was more reactive with αIIbβ3 than the parent fibrinogen. These results demonstrate that fibrin is more reactive with αIIbβ3 than fibrinogen. Fibrin is also less sensitive to αIIbβ3 inhibitors, suggesting that fibrin and fibrinogen have distinct binding requirements. In particular, the maintenance of αIIbβ3 binding activity in the absence of the γC-dodecapeptide and the α-chain RGD sequences suggests that the αIIbβ3-binding sites in fibrin are not confined to its known γ-chain and RGD motifs.

  16. Laminin regulates the osteogenic differentiation of dental follicle cells via integrin-α2/-β1 and the activation of the FAK/ERK signaling pathway.

    PubMed

    Viale-Bouroncle, Sandra; Gosau, Martin; Morsczeck, Christian

    2014-07-01

    Dental follicle cells (DFCs) are ideal for studies concerning the differentiation of dental precursor cells into alveolar osteoblasts and cementoblasts. Previous investigations have suggested that the extracellular matrix (ECM) protein laminin and the ECM receptor integrin-α2/-β1 play regulatory roles during the osteogenic differentiation of DFCs. Our present data indicate that laminin impairs alkaline phosphatase (ALP) activity following osteogenic induction while inducing integrin-α2/-β1 expression, osteogenic differentiation marker elevation, and DFC biomineralization. Integrin-α2/-β1 facilitates the laminin-dependent expression of osteogenic differentiation markers and the laminin-dependent inhibition of ALP activity. Moreover, these laminin-dependent effects on the osteogenic differentiation of DFCs can be reversed by the inhibition of the FAK/ERK signaling pathway. Thus, laminin regulates the inhibition of early osteogenic differentiation markers and the induction of late osteogenic differentiation markers via integrin-α2/-β1 and the activation of the FAK/ERK signaling pathway.

  17. α4-integrins control viral meningoencephalitis through differential recruitment of T helper cell subsets

    PubMed Central

    2014-01-01

    Introduction Natalizumab blocks α4-integrins and is a prototypic agent for a series of anti-inflammatory drugs that impair trafficking of immune cells into the CNS. However, modulation of the access of immune cells to the CNS is associated with impaired immune surveillance and detrimental viral infections of the CNS. Here, we explored the potency of cellular immune responses within the CNS to protect against viral encephalitis in mice with T cell conditional disruption of VLA-4 integrin (α4β1) expression. Results While VLA-4 expression in virus specific Th1 cells is non-redundant for their ability to access the CNS, α4-integrin deficient Th17 cells enter the CNS compartment and generate an inflammatory milieu upon intrathecal vaccinia virus (VV) infection. However, in contrast to Th1 cells that can adopt direct cytotoxic properties, Th17 cells fail to clear the virus due to insufficient Eomes induced perforin-1 expression. Conclusion The quality of the intrathecal cellular antiviral response under conditions of impaired VLA-4 function jeopardizes host protection. Our functional in vivo data extend our mechanistic understanding of anti-viral immunity in the CNS and help to estimate the risk potential of upcoming therapeutic agents that target the trafficking of immune cells into distinct anatomical compartments. PMID:24606807

  18. Mimicking bone extracellular matrix: integrin-binding peptidomimetics enhance osteoblast-like cells adhesion, proliferation, and differentiation on titanium.

    PubMed

    Fraioli, Roberta; Rechenmacher, Florian; Neubauer, Stefanie; Manero, José M; Gil, Javier; Kessler, Horst; Mas-Moruno, Carlos

    2015-04-01

    Interaction between the surface of implants and biological tissues is a key aspect of biomaterials research. Apart from fulfilling the non-toxicity and structural requirements, synthetic materials are asked to direct cell response, offering engineered cues that provide specific instructions to cells. This work explores the functionalization of titanium with integrin-binding peptidomimetics as a novel and powerful strategy to improve the adhesion, proliferation and differentiation of osteoblast-like cells to implant materials. Such biomimetic strategy aims at targeting integrins αvβ3 and α5β1, which are highly expressed on osteoblasts and are essential for many fundamental functions in bone tissue development. The successful grafting of the bioactive molecules on titanium is proven by contact angle measurements, X-ray photoelectron spectroscopy and fluorescent labeling. Early attachment and spreading of cells are statistically enhanced by both peptidomimetics compared to unmodified titanium, reaching values of cell adhesion comparable to those obtained with full-length extracellular matrix proteins. Moreover, an increase in alkaline phosphatase activity, and statistically higher cell proliferation and mineralization are observed on surfaces coated with the peptidomimetics. This study shows an unprecedented biological activity for low-molecular-weight ligands on titanium, and gives striking evidence of the potential of these molecules to foster bone regeneration on implant materials.

  19. The C-terminal region of laminin beta chains modulates the integrin binding affinities of laminins.

    PubMed

    Taniguchi, Yukimasa; Ido, Hiroyuki; Sanzen, Noriko; Hayashi, Maria; Sato-Nishiuchi, Ryoko; Futaki, Sugiko; Sekiguchi, Kiyotoshi

    2009-03-20

    Laminins are major cell-adhesive proteins in basement membranes that are capable of binding to integrins. Laminins consist of three chains (alpha, beta, and gamma), in which three laminin globular modules in the alpha chain and the Glu residue in the C-terminal tail of the gamma chain have been shown to be prerequisites for binding to integrins. However, it remains unknown whether any part of the beta chain is involved in laminin-integrin interactions. We compared the binding affinities of pairs of laminin isoforms containing the beta1 or beta2 chain toward a panel of laminin-binding integrins, and we found that beta2 chain-containing laminins (beta2-laminins) bound more avidly to alpha3beta1 and alpha7X2beta1 integrins than beta1 chain-containing laminins (beta1-laminins), whereas alpha6beta1, alpha6beta4, and alpha7X1beta1 integrins did not show any preference toward beta2-laminins. Because alpha3beta1 contains the "X2-type" variable region in the alpha3 subunit and alpha6beta1 and alpha6beta4 contain the "X1-type" region in the alpha6 subunit, we hypothesized that only integrins containing the X2-type region were capable of discriminating between beta1-laminins and beta2-laminins. In support of this possibility, a putative X2-type variant of alpha6beta1 was produced and found to bind preferentially to beta2-laminins. Production of a series of swap mutants between the beta1 and beta2 chains revealed that the C-terminal 20 amino acids in the coiled-coil domain were responsible for the enhanced integrin binding by beta2-laminins. Taken together, the results provide evidence that the C-terminal region of beta chains is involved in laminin recognition by integrins and modulates the binding affinities of laminins toward X2-type integrins.

  20. Integrins in point contacts mediate cell spreading: factors that regulate integrin accumulation in point contacts vs. focal contacts

    PubMed Central

    1993-01-01

    We have studied the function and distribution of the alpha 1 beta 1, alpha 5 beta 1 and alpha 6 beta 1 heterodimers on type-1 astrocytes with antibodies specific for integrin subunits (alpha 1, alpha 5, alpha 6, and beta 1). The alpha 1 beta 1 heterodimer mediates adhesion to laminin and collagen, the alpha 5 beta 1 to fibronectin in an RGD- dependent manner. The alpha 5 beta 1 integrin is found in focal contacts in long-term cultures of well-spread astrocytes colocalizing with vinculin and the termini of actin stress fibers. alpha 1 beta 1 heterodimers can occasionally be found as small aggregates within focal contacts but they do not accumulate there. Instead, alpha 1 beta 1 integrins are found in punctate deposits called point contacts which are distributed over the upper and the lower cell surfaces whether laminin, collagen, fibronectin or polylysine is used as a substratum. Unlike focal contacts, point contacts contain clathrin but rarely codistribute with actin or vinculin. Two observations indicate that these point contacts are functional. First, mAb 3A3, directed against the rat alpha 1 subunit, inhibits the attachment of astrocytes to laminin and collagen. Second, during the spreading of astrocytes, a band of point contacts forms around the cell perimeter at a time when no focal contacts are visible. While alpha 1 beta 1 integrins are found only in point contacts in astrocytes, the alpha 6 beta 1 integrin, another laminin receptor, is localized within focal contacts. Moreover, alpha 1 beta 1 heterodimers accumulate in focal contacts in fibroblasts. Thus, the alpha subunit contributes, independent of its ligand, to functional integrin heterodimer accumulation in focal contacts or in point contacts. This accumulation varies among different cell types with apparently identical heterodimers as well as with the motile state (spreading vs. flattened) of the same cells. PMID:8416993

  1. Differential expression of homing receptors and vascular addressins in tonsils and draining lymph nodes: Effect of Brucella infection in sheep.

    PubMed

    Suraud, Vanessa; Olivier, Michel; Bodier, Christelle C; Guilloteau, Laurence A

    2007-02-15

    The differential expression of homing receptors (HR) and complementary vascular addressins was studied in T and B lymphocytes from ovine tonsils and draining lymph nodes (LN) in uninfected and Brucella melitensis-infected sheep. In uninfected sheep, CD4+CD25+ T cells expressed proportionally more L-selectin and beta1 integrin than beta7 integrin in pharyngeal and palatine tonsils and in parotid LN (PLN), retropharyngeal LN (RLN) and the peripheral prescapular LN (PSLN). In contrast, memory CD4+CD45RA- T cells expressed an equivalent proportion of the three HR in PLN and PSLN, whereas beta1 and beta7 integrins were proportionally more expressed than L-selectin in pharyngeal tonsil. beta7 integrin was proportionally more expressed than beta1 integrin or L-selectin in palatine tonsils, RLN and the mucosal mesenteric LN (MLN). beta1 integrin was proportionally more expressed in IgG+ and IgA+ cells than beta7 integrin and L-selectin in tonsils, PLN and RLN. The main endothelial addressin expressed on venules in both pharyngeal and palatine tonsils, the PLN and RLN, as well as in the PSLN, was the peripheral PNAd, while in the MLN it was MAdCAM-1. Conjunctival infection by Brucella resulted in an increase of CD4+CD25+ and CD4+CD45RA- T cell subsets, which was associated to modifications of HR expression. CD4+CD45RA- T cells expressed proportionally more beta1 and beta7 integrins than L-selectin in regional PLN and RLN, but also in PSLN. The infection induced an increase of IgG+ and IgA+ cell percentages expressing beta1 integrin in all LN, and also beta7 integrin in the RLN. PNAd continued to be expressed on venules of tonsils and draining LN after Brucella infection, and MAdCAM-1 was also weakly expressed on RLN venules. These results suggest that lymphocyte trafficking through tonsils and draining LN could involve L-selectin/PNAd interactions, as well as beta1 or beta7 integrin, possibly in interaction with VCAM-1 or MAdCAM-1. The homing of antigen-specific lymphocytes

  2. Integrin expression is altered after acute and chronic cocaine.

    PubMed

    Wiggins, Armina T; Pacchioni, Alejandra M; Kalivas, Peter W

    2009-02-06

    Cocaine addiction is associated with an increase in actin cycling and alterations in dendritic spines in the nucleus accumbens. Both actin polymerization and spine morphology are regulated in part by beta-(beta) integrins. Mice were administered acute or daily injections of cocaine or saline for 7 days. After 3 weeks of withdrawal, the level of beta-integrins in the postsynaptic density enriched subfraction from nucleus accumbens tissue was quantified by immunoblotting at 0, 30 or 120min following an a cocaine challenge injection. After chronic treatment and withdrawal the basal level of beta1-integrin was increased while beta3-integrin was unaltered. However, following a cocaine challenge in chronic cocaine, but not saline-treated animals, beta3-integrin was transiently up-regulated while beta1-integrin was transiently downregulated. These data demonstrate a bidirectional regulation of beta-integrins by chronic cocaine treatment that may contribute to cocaine-induced changes in actin cycling and dendrite morphology.

  3. VLA-4 integrin concentrates at the peripheral supramolecular activation complex of the immune synapse and drives T helper 1 responses.

    PubMed

    Mittelbrunn, María; Molina, Ana; Escribese, María M; Yáñez-Mó, María; Escudero, Ester; Ursa, Angeles; Tejedor, Reyes; Mampaso, Francisco; Sánchez-Madrid, Francisco

    2004-07-27

    The integrin alpha 4 beta 1 (VLA-4) not only mediates the adhesion and transendothelial migration of leukocytes, but also provides costimulatory signals that contribute to the activation of T lymphocytes. However, the behavior of alpha 4 beta 1 during the formation of the immune synapse is currently unknown. Here, we show that alpha 4 beta 1 is recruited to both human and murine antigen-dependent immune synapses, when the antigen-presenting cell is a B lymphocyte or a dendritic cell, colocalizing with LFA-1 at the peripheral supramolecular activation complex. However, when conjugates are formed in the presence of anti-alpha 4 antibodies, VLA-4 colocalizes with the CD3-zeta chain at the center of the synapse. In addition, antibody engagement of alpha 4 integrin promotes polarization toward a T helper 1 (Th1) response in human in vitro models of CD4(+) T cell differentiation and naïve T cell priming by dendritic cells. The in vivo administration of anti-alpha 4 integrin antibodies also induces an immune deviation to Th1 response that dampens a Th2-driven autoimmune nephritis in Brown Norway rats. These data reveal a regulatory role of alpha 4 integrins on T lymphocyte-antigen presenting cell cognate immune interactions.

  4. Differential expression of the pro-inflammatory cytokines IL-1beta-1, TNFalpha-1 and IL-8 in vaccinated pink (Oncorhynchus gorbuscha) and chum (Oncorhynchus keta) salmon juveniles.

    PubMed

    Fast, M D; Johnson, S C; Jones, S R M

    2007-04-01

    Laboratory-reared pink and chum salmon juveniles (approximately 2g) received an intraperitoneal injection with a commercial, unadjuvanted Aeromonas salmonicida bacterin or sterile saline. Relative to elongation factor-1A, expression levels of genes encoding the proinflammatory cytokines interleukin-1beta-1 (IL-1beta), tumour necrosis factor-alpha-1 (TNFalpha) and interleukin-8 (IL-8) in pools of kidney and liver were examined 6- and 24-h after injection. Expression of IL-1beta was significantly elevated in pink and chum salmon by 6-h, and declined in pink salmon but not in chum salmon by 24-h. Similarly, expression of TNFalpha was significantly elevated in both species at 6h and only in chum salmon after 24-h. Expression of IL-8 was significantly elevated in both species at 6- and 24-h after injection. Expression of the three proinflammatory cytokine genes differed between salmon species both in the timing and magnitude of their expression. The significance of these differences with respect to immune function in these fish requires further research.

  5. Integrin alpha(3)-subunit expression modulates alveolar epithelial cell monolayer formation.

    PubMed

    Lubman, R L; Zhang, X L; Zheng, J; Ocampo, L; Lopez, M Z; Veeraraghavan, S; Zabski, S M; Danto, S I; Borok, Z

    2000-07-01

    We investigated expression of the alpha(3)-integrin subunit by rat alveolar epithelial cells (AECs) grown in primary culture as well as the effects of monoclonal antibodies with blocking activity against the alpha(3)-integrin subunit on AEC monolayer formation. alpha(3)-Integrin subunit mRNA and protein were detectable in AECs on day 1 and increased with time in culture. alpha(3)- and beta(1)-integrin subunits coprecipitated in immunoprecipitation experiments with alpha(3)- and beta(1)-subunit-specific antibodies, consistent with their association as the alpha(3)beta(1)-integrin receptor at the cell membrane. Treatment with blocking anti-alpha(3) monoclonal antibody from day 0 delayed development of transepithelial resistance, reduced transepithelial resistance through day 5 compared with that in untreated AECs, and resulted in large subconfluent patches in monolayers viewed by scanning electron microscopy on day 3. These data indicate that alpha(3)- and beta(1)-integrin subunits are expressed in AEC monolayers where they form the heterodimeric alpha(3)beta(1)-integrin receptor at the cell membrane. Blockade of the alpha(3)-integrin subunit inhibits formation of confluent AEC monolayers. We conclude that the alpha(3)-integrin subunit modulates formation of AEC monolayers by virtue of the key role of the alpha(3)beta(1)-integrin receptor in AEC adhesion.

  6. β8 Integrin Binds Rho GDP Dissociation Inhibitor-1 and Activates Rac1 to Inhibit Mesangial Cell Myofibroblast Differentiation*

    PubMed Central

    Lakhe-Reddy, Sujata; Khan, Shenaz; Konieczkowski, Martha; Jarad, George; Wu, Karen L.; Reichardt, Louis F.; Takai, Yoshimi; Bruggeman, Leslie A.; Wang, Bingcheng; Sedor, John R.; Schelling, Jeffrey R.

    2009-01-01

    αvβ8 integrin expression is restricted primarily to kidney, brain, and placenta. Targeted αv or β8 deletion is embryonic lethal due to defective placenta and brain angiogenesis, precluding investigation of kidney αvβ8 function. We find that kidney β8 is localized to glomerular mesangial cells, and expression is decreased in mouse models of glomerulosclerosis, suggesting that β8 regulates normal mesangial cell differentiation. To interrogate β8 signaling pathways, yeast two-hybrid and co-precipitation studies demonstrated β8 interaction with Rho guanine nucleotide dissociation inhibitor-1 (GDI). Selective β8 stimulation enhanced β8-GDI interaction as well as Rac1 (but not RhoA) activation and lamellipodia formation. Mesangial cells from itgb8−/− mice backcrossed to a genetic background that permitted survival, or gdi−/− mice, which develop glomerulosclerosis, demonstrated RhoA (but not Rac1) activity and α-smooth muscle actin assembly, which characterizes mesangial cell myofibroblast transformation in renal disease. To determine whether Rac1 directly modulates RhoA-associated myofibroblast differentiation, mesangial cells were transduced with inhibitory Rac peptide fused to human immunodeficiency virus-Tat, resulting in enhanced α-smooth muscle actin organization. We conclude that the β8 cytosolic tail in mesangial cells organizes a signaling complex that culminates in Rac1 activation to mediate wild-type differentiation, whereas decreased β8 activation shifts mesangial cells toward a RhoA-dependent myofibroblast phenotype. PMID:16690620

  7. The role of integrin-α5 in the proliferation and odontogenic differentiation of human dental pulp stem cells.

    PubMed

    Cui, Li; Xu, Shuaimei; Ma, Dandan; Gao, Jie; Liu, Ying; Yue, Jing; Wu, Buling

    2014-02-01

    It has been reported that integrin-α5 (ITGA5) activity is related to cell proliferation, differentiation, migration, and organ development. However, the involvement of ITGA5 in the biological functions of human dental pulp stem cells (hDPSCs) has not been explored. The aim of this study was to investigate the role of ITGA5 in the proliferation and odontogenic differentiation of hDPSCs. We knocked down ITGA5 in hDPSCs using lentivirus-mediated ITGA5 short hairpin RNA (shRNA). Changes in the proliferation in hDPSCs infected with lentiviruses expressing ITGA5-specific shRNA or negative control shRNA were examined using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and 5-ethynyl-2'-deoxyuridine labeling. Both ITGA5 knockdown cells and shMock cells were cultured in mineralization medium for 3 weeks, and the differentiation of cells was detected with alizarin red S staining. The expression of odontogenic differentiation-related molecular markers was assessed using real-time polymerase chain reaction and Western blot assays. The knockdown of ITGA5 decreased the proliferation capacity of hDPSCs. ITGA5 shRNA promoted odontogenic differentiation of hDPSCs with the enhanced formation of mineralized nodules. It also up-regulated the messenger RNA expression of multiple markers of odontogenesis and the expression of dentin sialophosphoprotein protein. These findings suggest that ITGA5 plays an important role in maintaining hDPSCs in a proliferative state. The inhibition of ITGA5 signaling promotes the odontogenic differentiation of hDPSCs. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  8. Differential time-series expression of immune-related genes of Pacific white shrimp Litopenaeus vannamei in response to dietary inclusion of beta-1,3-glucan.

    PubMed

    Wang, Yu-Chi; Chang, Poh-Shing; Chen, Houng-Yung

    2008-01-01

    Time-series changes in transcript abundance of nine genes encoding important immune proteins in haemocytes or hepatopancreas of Pacific white shrimp Litopenaeus vannamei fed daily in a 1-week feeding trial diets containing three levels (0%, 0.2% or 1%) of beta-1,3-glucan from Schizophyllum commune were quantified by real-time PCR. As a whole, the immune modulation elicited by beta-glucan is bimodal, one swift reaction of up- or down-regulation occurred within 24h and a delayed regulation was commenced as late as 3-7days. Haemocyanin, crustin, prophenoloxidase (proPO) and transglutaminase (TGase) did not respond to the glucan treatment. While penaeidin 3 (Litvan PEN3) was swiftly down-regulated (0-24h), lysozyme and cytosolic manganese superoxide dismutase (cMnSOD) were swiftly up-regulated (0-24h). In contrast, the two pattern recognition proteins (PRPs), beta-glucan binding protein-high density lipoprotein (BGBP-HDL) and lipopolysaccharide/beta-glucan binding protein (LGBP), showed a delayed up-regulation. Their expressions were not maximized until as late as 72h or 7days, respectively, which coincide with the initiation of reported immune enhancement (6-24days) of PO and SOD activity, phagocytosis and superoxide anion production in penaeid shrimp receiving glucan-containing diet. These immune responses could be the downstream effects of the two PRP gene up-regulation that predispose the shrimp to a state of high immune responsiveness. Increased dosage of beta-glucan from 2 to 10gkg(-1) diet did not affect the expressions of the genes, indicating the sufficiency of beta-glucan supplementation at 2gkg(-1) diet.

  9. Effects of transcutaneous electrical stimulation (1 pulse/sec) through custom-made disposable surface electrodes covering Omura's ST36 area of both legs on normal cell telomeres, oncogen C-fosAb2, integrin alpha5beta1, chlamydia trachomatis, etc. in breast cancer & alzheimer patients.

    PubMed

    Omura, Yoshiaki; Chen, Yemeng; Lermand, Olivia; Jones, Marilyn; Duvvi, Harsha; Shimotsuura, Yasuhiro

    2010-01-01

    Our previous study indicated that when extremely reduced normal cell (NC) telomeres in various cancer patients are increased over 500 ng BDORT units, abnormally high cancer cell telomeres and cancer-related markers such as Oncogen C-fosAb2 (Onco.)& Integrin alpha5beta1 (Integ.), & 8-OH-dG as well as bacterial & viral infections, mercury, asbestos, chromium, & beta-amyloid (1-42) markedly reduced due to improved circulation & excretion of these substances in urine. Since 1995, we have been using press-needle stimulation of Omura's ST36 with 200x press-release procedure 4x a day, with significant improvements in various cancer patients. In this study, Transcutaneous Electrical Stimulation (TES) at 60 pulses/min, which is close to patient's heart rate, was given between Omura's ST36 of both legs of the breast cancer & Alzheimer's patients. After about 10 minutes of TES, NC telomeres increased from 1 yg (= 10-24 g) to 500-525 ng; Integ. reduced from 85-75 ng to 0.5 ng & Chlamydia trachomatis (CT) reduced from 4500-3500 ng to 0.5 ng. An additional 10 minutes TES increased NC telomeres to 800-875 ng, while Integ. reduced to 0.5 yg & CT became less than 0.1 yg. After a total 30 minutes of TES, NC telomeres increased to 1000-1200ng BDORT units, with decreases in Integ. and Onco. to less than 0.1 yg. CT reduced to < 0.1 yg. About 24 hours later, NC telomeres were still 300 ng & both Integ. and Onco. were 2.5 ng. CT was approximately 20 ng. In Alzheimer patient, abnormally high beta-Amyloid (1-42) of 7-12 ng markedly reduced to within normal value of less than 1.5 ng by 20-30 min TES. Stimulation beyond 30 minutes gradually reduced NC telomeres. TES pulse rate of 4 pulses/sec for the same patient initially increased NC telomere up to 750-950 ng BDORT units within 20 minutes, but when stimulation continued more than 20 min, NC telomeres rapidly reduced to -150 ng in less than 10 min of TES with reduced beneficial effects.

  10. The integrins.

    PubMed

    Takada, Yoshikazu; Ye, Xiaojing; Simon, Scott

    2007-01-01

    The integrins are a superfamily of cell adhesion receptors that bind to extracellular matrix ligands, cell-surface ligands, and soluble ligands. They are transmembrane alphabeta heterodimers and at least 18 alpha and eight beta subunits are known in humans, generating 24 heterodimers. Members of this family have been found in mammals, chicken and zebrafish, as well as lower eukaryotes, including sponges, the nematode Caenorhabditis elegans (two alpha and one beta subunits, generating two integrins) and the fruitfly Drosophila melanogaster (five alpha and one beta, generating five integrins). The alpha and beta subunits have distinct domain structures, with extracellular domains from each subunit contributing to the ligand-binding site of the heterodimer. The sequence arginine-glycine-aspartic acid (RGD) was identified as a general integrin-binding motif, but individual integrins are also specific for particular protein ligands. Immunologically important integrin ligands are the intercellular adhesion molecules (ICAMs), immunoglobulin superfamily members present on inflamed endothelium and antigen-presenting cells. On ligand binding, integrins transduce signals into the cell interior; they can also receive intracellular signals that regulate their ligand-binding affinity. Here we provide a brief overview that concentrates mostly on the organization, structure and function of mammalian integrins, which have been more extensively studied than integrins in other organisms.

  11. Altered vascular endothelium integrin expression in psoriasis.

    PubMed Central

    Creamer, D.; Allen, M.; Sousa, A.; Poston, R.; Barker, J.

    1995-01-01

    Considerable evidence indicates that microvascular changes observed in psoriasis are a result of vascular proliferation. A critical step in the sequence of events leading to neovascularization involves interactions between endothelial cells and extracellular matrix proteins mediated in part by the integrin family of adhesion molecules. A number of endothelial integrins have been shown to participate in neovascularization, including members of the beta 1, beta 3, and beta 4 subfamilies. To investigate the role of these integrins in psoriasis, specimens of lesional and nonlesional skin were taken from 10 patients with active, untreated plaque disease. Vascular endothelium was labeled with monoclonal antibodies specific for alpha 2, alpha 5, alpha 6, beta 1, av beta 3, and beta 4 integrins. The use of image analysis permitted quantification of immunoperoxidase staining and comparison of endothelial labeling in lesional and nonlesional skin. There was a significant increase in endothelial staining of av beta 3 integrin in lesional compared with nonlesional skin, both in superficial and deep vasculature. In contrast, there was a significant decrease in endothelial beta 4 staining in lesional compared with nonlesional superficial dermal vessels, alpha 2, alpha 5, alpha 6, and beta 1 staining showed no significant difference between the two groups. These results demonstrate an important role of av beta 3 and beta 4 integrins in the microvascular changes of psoriatic lesions. Images Figure 1 Figure 3 PMID:7495291

  12. Immunolocalization of integrins and fibronectin in tubal pregnancy.

    PubMed

    Inan, Sevinc; Giray, Gulsen; Vatansever, H Seda; Ozbilgin, Kemal; Kuscu, N Kemal; Sayhan, Sevil

    2004-01-01

    Integrins are a large family of cell adhesion molecules that serve as receptors involved in cell-to-cell and cell-to-matrix interactions during implantation. We studied immunohistochemical staining of integrins (alpha 3, alpha V, beta 1, and alpha 2 beta 1) and fibronectin in ectopic tubal pregnancy. Thirty fallopian tube samples with ectopic pregnancies and five normal tubal segments were obtained during ligation operations; the latter specimens served as controls in the study. Formalin-fixed paraffin-embedded tissue sections were stained with hematoxylin-eosin or primary antibodies against alpha 3, beta 1, alpha V, and alpha 2 beta 1 integrins and fibronectin, using the avidin-biotin-peroxidase method. A semi-quantitative grading system was used to compare staining intensities. In the control samples, immunostaining of all integrins was found in a single layer of tall columnar epithelial cells, the lamina propria (Lp) and the muscular layer. Fibronectin staining was detected in the Lp and the muscular layer. Staining intensities of alpha 3 and beta 1 integrins and fibronectin were increased in the normal part of fallopian tubes with ectopic pregnancies. Staining of beta 1 integrin was more intense than staining of alpha 3 and fibronectin, whereas there was no difference in alpha V and alpha 2 beta 1 integrin expression between normal tubal tissue in the ectopic pregnancy group and control tubal tissue. In the tubal pregnancy group at the site of implantation, staining intensity of alpha 3 and beta 1 integrins and fibronectin was strong in decidual cells, supporting tissue and placental villi, whereas alpha V and alpha 2 beta 1 staining was mild. We concluded that integrins, especially beta 1 and alpha 3, and fibronectin may play a role in progression of tubal implantation. Although the role of integrins has not yet been clearly defined, these molecules may function as markers of normal and abnormal states of receptivity. We like to suggest that integrins and

  13. Transforming growth factor-beta1 to the bone.

    PubMed

    Janssens, Katrien; ten Dijke, Peter; Janssens, Sophie; Van Hul, Wim

    2005-10-01

    TGF-beta1 is a ubiquitous growth factor that is implicated in the control of proliferation, migration, differentiation, and survival of many different cell types. It influences such diverse processes as embryogenesis, angiogenesis, inflammation, and wound healing. In skeletal tissue, TGF-beta1 plays a major role in development and maintenance, affecting both cartilage and bone metabolism, the latter being the subject of this review. Because it affects both cells of the osteoblast and osteoclast lineage, TGF-beta1 is one of the most important factors in the bone environment, helping to retain the balance between the dynamic processes of bone resorption and bone formation. Many seemingly contradictory reports have been published on the exact functioning of TGF-beta1 in the bone milieu. This review provides an overall picture of the bone-specific actions of TGF-beta1 and reconciles experimental discrepancies that have been reported for this multifunctional cytokine.

  14. Differential Influence of Components Resulting from Atmospheric-Pressure Plasma on Integrin Expression of Human HaCaT Keratinocytes

    PubMed Central

    Haertel, Beate; Straßenburg, Susanne; Wende, Kristian; von Woedtke, Thomas

    2013-01-01

    Adequate chronic wound healing is a major problem in medicine. A new solution might be non-thermal atmospheric-pressure plasma effectively inactivating microorganisms and influencing cells in wound healing. Plasma components as, for example, radicals can affect cells differently. HaCaT keratinocytes were treated with Dielectric Barrier Discharge plasma (DBD/air, DBD/argon), ozone or hydrogen peroxide to find the components responsible for changes in integrin expression, intracellular ROS formation or apoptosis induction. Dependent on plasma treatment time reduction of recovered cells was observed with no increase of apoptotic cells, but breakdown of mitochondrial membrane potential. DBD/air plasma increased integrins and intracellular ROS. DBD/argon caused minor changes. About 100 ppm ozone did not influence integrins. Hydrogen peroxide caused similar effects compared to DBD/air plasma. In conclusion, effects depended on working gas and exposure time to plasma. Short treatment cycles did neither change integrins nor induce apoptosis or ROS. Longer treatments changed integrins as important for influencing wound healing. Plasma effects on integrins are rather attributed to induction of other ROS than to generation of ozone. Changes of integrins by plasma may provide new solutions of improving wound healing, however, conditions are needed which allow initiating the relevant influence on integrins without being cytotoxic to cells. PMID:23936843

  15. Differential influence of components resulting from atmospheric-pressure plasma on integrin expression of human HaCaT keratinocytes.

    PubMed

    Haertel, Beate; Straßenburg, Susanne; Oehmigen, Katrin; Wende, Kristian; von Woedtke, Thomas; Lindequist, Ulrike

    2013-01-01

    Adequate chronic wound healing is a major problem in medicine. A new solution might be non-thermal atmospheric-pressure plasma effectively inactivating microorganisms and influencing cells in wound healing. Plasma components as, for example, radicals can affect cells differently. HaCaT keratinocytes were treated with Dielectric Barrier Discharge plasma (DBD/air, DBD/argon), ozone or hydrogen peroxide to find the components responsible for changes in integrin expression, intracellular ROS formation or apoptosis induction. Dependent on plasma treatment time reduction of recovered cells was observed with no increase of apoptotic cells, but breakdown of mitochondrial membrane potential. DBD/air plasma increased integrins and intracellular ROS. DBD/argon caused minor changes. About 100 ppm ozone did not influence integrins. Hydrogen peroxide caused similar effects compared to DBD/air plasma. In conclusion, effects depended on working gas and exposure time to plasma. Short treatment cycles did neither change integrins nor induce apoptosis or ROS. Longer treatments changed integrins as important for influencing wound healing. Plasma effects on integrins are rather attributed to induction of other ROS than to generation of ozone. Changes of integrins by plasma may provide new solutions of improving wound healing, however, conditions are needed which allow initiating the relevant influence on integrins without being cytotoxic to cells.

  16. Silencing of VAMP3 inhibits cell migration and integrin-mediated adhesion

    SciTech Connect

    Luftman, Kevin; Hasan, Nazarul; Day, Paul; Hardee, Deborah; Hu Chuan

    2009-02-27

    Integrins are transmembrane receptors for cell adhesion to the extracellular matrix. In cell migration, integrins are endocytosed from the plasma membrane or the cell surface, transported in vesicles and exocytosed actively at the cell front. In the present study, we examined the roles of VAMP3, a SNARE protein that mediates exocytosis, in cell migration and integrin trafficking. Small interfering RNA (siRNA)-induced silencing of VAMP3 inhibited chemotactic cell migration by more than 60% without affecting cell proliferation. VAMP3 silencing reduced the levels of {beta}1 integrin at the cell surface but had no effect on total cellular {beta}1 integrin, indicating that VAMP3 is required for trafficking of {beta}1 integrin to the plasma membrane. Furthermore, VAMP3 silencing diminished cell adhesion to laminin but not to fibronectin or collagen. Taken together, these data suggest that VAMP3-dependent integrin trafficking is crucial in cell migration and cell adhesion to laminin.

  17. Slug regulates integrin expression and cell proliferation in human epidermal keratinocytes.

    PubMed

    Turner, Frances E; Broad, Simon; Khanim, Farhat L; Jeanes, Alexa; Talma, Sonia; Hughes, Sharon; Tselepis, Chris; Hotchin, Neil A

    2006-07-28

    The human epidermis is a self-renewing epithelial tissue composed of several layers of keratinocytes. Within the epidermis there exists a complex array of cell adhesion structures, and many of the cellular events within the epidermis (differentiation, proliferation, and migration) require that these adhesion structures be remodeled. The link between cell adhesion, proliferation, and differentiation within the epidermis is well established, and in particular, there is strong evidence to link the process of terminal differentiation to integrin adhesion molecule expression and function. In this paper, we have analyzed the role of a transcriptional repressor called Slug in the regulation of adhesion molecule expression and function in epidermal keratinocytes. We report that activation of Slug, which is expressed predominantly in the basal layer of the epidermis, results in down-regulation of a number of cell adhesion molecules, including E-cadherin, and several integrins, including alpha3, beta1, and beta4. We demonstrate that Slug binds to the alpha3 promoter and that repression of alpha3 transcription by Slug is dependent on an E-box sequence within the promoter. This reduction in integrin expression is reflected in decreased cell adhesion to fibronectin and laminin-5. Despite the reduction in integrin expression and function, we do not observe any increase in differentiation. We do, however, find that activation of Slug results in a significant reduction in keratinocyte proliferation.

  18. The integrin alpha 6 beta 4 is a laminin receptor

    PubMed Central

    1992-01-01

    In this study, the putative laminin receptor function of the alpha 6 beta 4 integrin was assessed. For this purpose, we used a human cell line, referred to as clone A, that was derived from a highly invasive, colon adenocarcinoma. This cell line, which expresses the alpha 6 beta 4 integrin, adheres to the E8 and not to the P1 fragment of laminin. The adhesion of clone A cells to laminin is extremely rapid with half- maximal adhesion observed at 5 min after plating. Adhesion to laminin is blocked by GoH3, and alpha 6 specific antibody (60% inhibition), as well as by A9, a beta 4 specific antibody (30% inhibition). Most importantly, we demonstrate that alpha 6 beta 4 binds specifically to laminin-Sepharose columns in the presence of either Mg2+ or Mn2+ and it is eluted from these columns with EDTA but not with NaCl. The alpha 6 beta 4 integrin does not bind to collagen-Sepharose, but the alpha 2 beta 1 integrin does bind. Clone A cells do not express alpha 6 beta 1 as evidenced by the following observations: (a) no beta 1 integrin is detected in beta 1 immunoblots of GoH3 immunoprecipitates; and (b) no alpha 6 beta 1 integrin is seen in GoH3 immunoprecipitates of clone A extracts that had been immunodepleted of all beta 4 containing integrin using the A9 antibody. These data establish that laminin is a ligand for the alpha 6 beta 4 integrin and that this integrin can function as a laminin receptor independently of alpha 6 beta 1. PMID:1533398

  19. Effects of a bioactive scaffold containing a sustained TGF-beta1-releasing nanoparticle system on the migration and differentiation of stem cells from the apical papilla

    NASA Astrophysics Data System (ADS)

    Bellamy, Craig D.

    The study aimed to develop and characterize a novel chitosan-based scaffold (CMCS) containing TGF-β1-releasing chitosan nanoparticles (TGF-β1-CSnp) to enhance migration and differentiation of SCAP. Part I concerned synthesis and characterization of the scaffold (CMCS) and TGF-β1-CSnp. Part II examined the effect of sustained TGF-β1 release from scaffold containing TGF-β1-CSnp on odontogenic differentiation of SCAP. The scaffold demonstrated properties conducive to cellular activities. Incorporation of TGF-β1 in CSnp allowed sustained release of TGF-β1 facilitating delivery of a critical concentration of TGF-β1 at the opportune time. SCAP showed greater viability, migration and biomineralization in the presence of TGF-β1-CSnp than in the presence of Free TGF-β1. SCAP cultured in TGF-β1-CSnp + scaffold showed significantly higher dentin matrix protein (DMP)-1 and dentin sialophosphoprotein (DSPP) signals compared to Free TGF-β1 + scaffold or CSnp + scaffold. These experiments highlighted the potential of a CMCS based scaffold with growth factor releasing nanoparticles to promote migration and differentiation of SCAP.

  20. Differential β3 and β1 Integrin Expression in Bone Marrow and Cortical Bone of Estrogen Deficient Rats.

    PubMed

    Voisin, Muriel; McNamara, Laoise M

    2015-09-01

    Integrin-based (β3 ) attachments to the extracellular matrix (ECM) on osteocyte cell processes have recently been proposed to play an important role in facilitating osteocyte mechanosensation. However, it is not yet known whether integrin expression is altered in the mechanoregulatory osteocytes during osteoporosis. The objective of this study was to test the hypothesis that the expression of integrin-based mechanosensory complexes (β1 and β3 integrins) is altered as a direct response to estrogen deficiency, in an estrogen deficient animal model of osteoporosis. Four weeks post-operatively, immunohistochemistry was used to detect for β1 and β3 integrin subunits in bone tissue and marrow of ovariectomized (OVX; N = 4) and SHAM (N = 4) operated animals. A tartrate resistant acid phosphatase (TRAP) control stain was performed to quantify the presence of osteoclasts in the bone marrow and bone surfaces. Image analysis was performed to quantify expression patterns in different biological compartments, that is, bone marrow, endosteum, and cortical bone. Our results showed that β1 integrins were ubiquitously expressed throughout the bone and marrow, for both OVX and SHAM groups. β3 integrin subunit expression was lower in bone cells from osteoporotic animals compared to controls, whereas β3 expression in marrow cells did not differ significantly between groups. At the endosteum no difference was observed in β3 integrin subunit expression. As expected, the number of osteoclasts was higher in the OVX group validating an imbalance in bone remodeling. We propose that a reduction in β3 integrin expression in osteocytes might impair mechanosensation by bone cells during estrogen deficiency.

  1. Role of vitronectin and fibronectin receptors in oral mucosal and dermal myofibroblast differentiation.

    PubMed

    Lygoe, Kate A; Wall, Ivan; Stephens, Philip; Lewis, Mark P

    2007-11-01

    The activation of fibroblasts into myofibroblasts is a crucial event in healing that is linked to remodelling and scar formation, therefore we determined whether regulation of myofibroblast differentiation via integrins might affect wound healing responses in populations of patient-matched HOFs (human oral fibroblasts) compared with HDFs (human dermal fibroblasts). Both the HOF and HDF cell types underwent TGF-beta1 (transforming growth factor-beta1)-induced myofibroblastic differentiation [upregulation of the expression of alpha-sma (alpha-smooth muscle actin)], although analysis of unstimulated cells indicated that HOFs contained higher basal levels of alpha-sma than HDFs (P<0.05). Functional blocking antibodies against the integrin subunits alpha 5 (fibronectin) or alpha v (vitronectin) were used to determine whether the effects of TGF-beta1 were regulated via integrin signalling pathways. alpha-sma expression in both HOFs and HDFs was down-regulated by antibodies against both alpha 5 and alpha v. Functionally, TGF-beta1 inhibited cell migration in an in vitro wound model and increased the contraction of collagen gels. Greater contraction was evident for HOFs compared with HDFs, both with and without stimulation by TGF-beta1 (P<0.05). When TGF-beta1-stimulated cells were incubated with blocking antibodies against alpha 5 and alpha v, gel contraction was decreased to that of non-stimulated cells; however, blocking alpha v or alpha 5 could not restore cellular migration in both HOFs and HDFs. Despite intrinsic differences in their basal state, the cellular events associated with TGF-beta1-induced myofibroblastic differentiation are common to both HOFs and HDFs, and appear to require differential integrin usage; up-regulation of alpha-sma expression and increases in collagen gel contraction are vitronectin- and fibronectin-receptor-dependent processes, whereas wound re-population is not.

  2. The involvement of integrin β1 signaling in the migration and myofibroblastic differentiation of skin fibroblasts on anisotropic collagen-containing nanofibers.

    PubMed

    Huang, Chengyang; Fu, Xiaoling; Liu, Jie; Qi, Yanmei; Li, Shaohua; Wang, Hongjun

    2012-02-01

    Utilization of nanofibrous matrices for skin wound repair holds great promise due to their morphological and dimensional similarity to native extracellular matrix (ECM). It becomes highly desired to understand how various nanofibrous matrices regulate skin cell behaviors and intracellular signaling pathways, important to tuning the functionality of tissue-engineered skin grafts and affecting the wound healing process. In this study, the phenotypic expressions of normal human dermal fibroblasts (NHDFs) on collagen-containing nanofibrous matrices with either isotropic (i.e., fibers collected randomly with no alignment) or anisotropic (i.e., fibers collected with alignment) fiber organizations were studied by immunostaining, migration assay and molecular analyses. Results showed that both nanofibrous matrices supported the attachment and growth of NHDFs similarly, while showing different cell morphology with distinct variation in focal adhesion formation and distribution. Anisotropic nanofibers significantly triggered the integrin β1 signaling pathway in NHDFs as evidenced by an increase of active integrin β1 (130 kD mature form) and phosphorylation of focal adhesion kinase (FAK) at Tyr-397. Anisotropic matrices also promoted the migration of NHDFs along the fibers, while neutralization of the integrin β1 activity abolished this promotion. Moreover, the fibroblast-to-myofibroblast differentiation was greatly enhanced for the NHDFs cultured on anisotropic nanofibrous matrices over a period of 48 h. Inhibition of cellular integrin β1 activity by neutralizing antibody eliminated this enhancement. These findings suggest the important role of integrin β1 signaling pathway in regulating the nanofiber-induced fibroblast phenotypic alteration and providing insightful understanding of the possible application of collagen-containing nanofibrous matrices for skin regeneration.

  3. Differential distribution and functional impact of BK channel beta1 subunits across mesenteric, coronary, and different cerebral arteries of the rat.

    PubMed

    Kuntamallappanavar, Guruprasad; Bisen, Shivantika; Bukiya, Anna N; Dopico, Alex M

    2017-02-01

    Large conductance, Ca(2+)i- and voltage-gated K(+) (BK) channels regulate myogenic tone and, thus, arterial diameter. In smooth muscle (SM), BK channels include channel-forming α and auxiliary β1 subunits. BK β1 increases the channel's Ca(2+) sensitivity, allowing BK channels to negatively feedback on depolarization-induced Ca(2+) entry, oppose SM contraction and favor vasodilation. Thus, endothelial-independent vasodilation can be evoked though targeting of SM BK β1 by endogenous ligands, including lithocholate (LCA). Here, we investigated the expression of BK β1 across arteries of the cerebral and peripheral circulations, and the contribution of such expression to channel function and BK β1-mediated vasodilation. Data demonstrate that endothelium-independent, BK β1-mediated vasodilation by LCA is larger in coronary (CA) and basilar (BA) arteries than in anterior cerebral (ACA), middle cerebral (MCA), posterior cerebral (PCA), and mesenteric (MA) arteries, all arterial segments having a similar diameter. Thus, differential dilation occurs in extracranial arteries which are subjected to similar vascular pressure (CA vs. MA) and in arteries that irrigate different brain regions (BA vs. ACA, MCA, and PCA). SM BK channels from BA and CA displayed increased basal activity and LCA responses, indicating increased BK β1 functional presence. Indeed, in the absence of detectable changes in BK α, BA and CA myocytes showed an increased location of BK β1 in the plasmalemma/subplasmalemma. Moreover, these myocytes distinctly showed increased BK β1 messenger RNA (mRNA) levels. Supporting a major role of enhanced BK β1 transcripts in artery dilation, LCA-induced dilation of MCA transfected with BK β1 complementary DNA (cDNA) was as high as LCA-induced dilation of untransfected BA or CA.

  4. Quantitative evaluation of endothelial progenitors and cardiac valve endothelial cells: proliferation and differentiation on poly-glycolic acid/poly-4-hydroxybutyrate scaffold in response to vascular endothelial growth factor and transforming growth factor beta1.

    PubMed

    Dvorin, Evan L; Wylie-Sears, Jill; Kaushal, Sunjay; Martin, David P; Bischoff, Joyce

    2003-06-01

    Three-dimensional scaffolds made of bioabsorbable polymeric constituents are currently being tested for use in tissue engineering of various tissues. A composite scaffold of poly-glycolic acid (PGA) non-woven mesh dip-coated in a 1% solution of poly-4-hydroxybutyrate (P4HB) was shown to be suitable as a scaffold for creation of tissue-engineered trileaflet pulmonic valve replacements in sheep [Hoerstrup, S.P., et al., Circulation 102(Suppl. 3), III44, 2000]. However, little is known about how cells seeded on PGA/P4HB respond in vitro to soluble factors supplied in the culture medium. To optimize tissue development in vitro, before implantation, we set out to develop quantitative biochemical assays to measure how cells seeded on PGA/P4HB respond to growth and differentiation factors. Herein we show that ovine aortic valvular endothelial cells and circulating endothelial progenitor cells (EPCs) seeded onto PGA/P4HB proliferate in response to vascular endothelial growth factor and transdifferentiate to a mesenchymal phenotype in response to transforming growth factor beta(1). Transdifferentiation from an endothelial to mesenchymal phenotype is a critical step during embryonic development of cardiac valves. Our results demonstrate that valvular endothelial cells and EPCs isolated from peripheral blood can recapitulate critical developmental steps on PGA/P4HB. These results demonstrate that PGA/P4HB provides a conducive environment for cellular proliferation, differentiation, and tissue development.

  5. Interferon Beta-1a Intramuscular Injection

    MedlinePlus

    Interferon beta-1a intramuscular injection is used to reduce the number of episodes of symptoms and slow ... and problems with vision, speech, and bladder control). Interferon beta-1a is in a class of medications ...

  6. Interferon Beta-1a Subcutaneous Injection

    MedlinePlus

    Interferon beta-1a subcutaneous injection is used to reduce episodes of symptoms and slow the development of ... and problems with vision, speech, and bladder control). Interferon beta-1a is in a class of medications ...

  7. Integrin signaling in inflammatory and neuropathic pain in the rat.

    PubMed

    Dina, Olayinka A; Parada, Carlos A; Yeh, Jenny; Chen, Xiaojie; McCarter, Gordon C; Levine, Jon D

    2004-02-01

    Many painful conditions are associated with alterations in the extracellular matrix (ECM) of affected tissues. While several integrins, the receptors for ECM proteins, are present on sensory neurons that mediate pain, the possible role of these cell adhesion molecules in inflammatory or neuropathic pain has not been explored. We found that the intradermal injection of peptide fragments of domains of laminin and fibronectin important for adhesive signaling selectively inhibited the hyperalgesia caused by prostaglandin E2 (PGE2) and epinephrine (EPI), respectively. The block of EPI hyperalgesia was mimicked by other peptides containing the RGD integrin-binding sequence. Monoclonal antibodies (mAbs) against the alpha1 or alpha3 integrin subunits, which participate in laminin binding, selectively blocked PGE2 hyperalgesia, while a mAb against the alpha5 subunit, which participates in fibronectin binding, blocked only EPI-induced hyperalgesia. A mAb against the beta1 integrin subunit, common to receptors for both laminin and fibronectin, inhibited hyperalgesia caused by both agents, as did the knockdown of beta1 integrin expression by intrathecal injection of antisense oligodeoxynucleotides. The laminin peptide, but not the fibronectin peptides, also reversibly abolished the longer lasting inflammatory hyperalgesia induced by carrageenan. Finally, the neuropathic hyperalgesia caused by systemic administration of the cancer chemotherapy agent taxol was reversibly inhibited by antisense knockdown of beta1 integrin. These results strongly implicate specific integrins in the maintenance of inflammatory and neuropathic hyperalgesia.

  8. Integrins as architects of cell behavior.

    PubMed

    Streuli, Charles H

    2016-10-01

    Integrins are cell surface receptors that bind cells to their physical external environment, linking the extracellular matrix to cell function. They are essential in the biology of all animals. In the late 1980s, we discovered that integrins are required for the ability of breast epithelia to do what they are programmed to do, which is to differentiate and make milk. Since then, integrins have been shown to control most other aspects of phenotype: to stay alive, to divide, and to move about. Integrins also provide part of the mechanism that allows cells to form tissues. Here I discuss how we discovered that integrins control mammary gland differentiation and explore the role of integrins as central architects of other aspects of cell behavior.

  9. Integrins as architects of cell behavior

    PubMed Central

    Streuli, Charles H.

    2016-01-01

    Integrins are cell surface receptors that bind cells to their physical external environment, linking the extracellular matrix to cell function. They are essential in the biology of all animals. In the late 1980s, we discovered that integrins are required for the ability of breast epithelia to do what they are programmed to do, which is to differentiate and make milk. Since then, integrins have been shown to control most other aspects of phenotype: to stay alive, to divide, and to move about. Integrins also provide part of the mechanism that allows cells to form tissues. Here I discuss how we discovered that integrins control mammary gland differentiation and explore the role of integrins as central architects of other aspects of cell behavior. PMID:27687254

  10. Integrin-α FG-GAP repeat-containing protein 2 is critical for normal B cell differentiation and controls disease development in a lupus model.

    PubMed

    Al-Shami, Amin; Crisostomo, Jeannette; Wilkins, Carrie; Xu, Nianhua; Humphries, Juliane; Chang, Wei C; Anderson, Stephen J; Oravecz, Tamas

    2013-10-01

    The phenylalanyl-glycyl-glycyl-alanyl-prolyl (FG-GAP) domain plays an important role in protein-protein interactions, including interaction of integrins with their ligands. Integrin-α FG-GAP repeat-containing protein 2 (Itfg2) is a highly conserved protein in vertebrates that carries two FG-GAP domains, but its role in mammalian physiology is unknown. In this article, we show that Itfg2 is an intracellular protein and it plays a critical role in B cell differentiation and development of autoimmunity. Itfg2-deficient mice displayed a phenotype consistent with retention of B cells in the spleen and had a lower concentration of IgG in the blood when compared with wild-type littermates. Itfg2-deficient splenocytes also showed a defect in cell migration in vitro. After immunization with a thymus-dependent Ag, the absence of Itfg2 caused a shift in B cell maturation from the germinal centers to the extrafollicular regions of the spleen and blocked deposition of Ag-specific plasma cells in the bone marrow. In support of hematopoietic cell intrinsic activity of Itfg2, bone marrow transplantation of Itfg2-deficient cells was sufficient to impair germinal center development in wild-type mice. Furthermore, Itfg2 deficiency exacerbated development of autoimmune disease in MRL/lpr lupus-prone mice. These results identify Itfg2 as a novel contributor to B cell differentiation and a negative regulator of the autoimmune response during lupus.

  11. Laminins 411 and 421 differentially promote tumor cell migration via α6β1 integrin and MCAM (CD146).

    PubMed

    Ishikawa, Taichi; Wondimu, Zenebech; Oikawa, Yuko; Gentilcore, Giusy; Kiessling, Rolf; Egyhazi Brage, Suzanne; Hansson, Johan; Patarroyo, Manuel

    2014-09-01

    α4-laminins, such as laminins 411 and 421, are mesenchymal laminins expressed by blood and lymphatic vessels and some tumor cells. Laminin-411 promotes migration of leukocytes and endothelial cells, but the effect of this laminin and laminin-421 on tumor cells is poorly understood. In the present study, we demonstrate that laminin-411 and, to a greater extent, laminin-421 significantly promote migration of tumor cells originated from melanomas, gliomas and different carcinomas via α6β1 integrin. In solid-phase binding assays, both laminins similarly bound α6β1 integrin but only laminin-421, among several laminin isoforms, readily bound MCAM (CD146), a cell-surface adhesion molecule strongly associated with tumor progression. Accordingly, a function-blocking mAb to MCAM inhibited tumor cell migration on laminin-421 but not on laminins 411 or 521. In tumor tissues, melanoma cells co-expressed MCAM, laminin α4, β1, β2 and γ1 chains, and integrin α6 and β1 chains. The present data highlight the novel role of α4-laminins in tumor cell migration and identify laminin-421 as a primary ligand for MCAM and a putative mediator of tumor invasion and metastasis.

  12. Characterization of integrin receptors in normal and neoplastic human brain.

    PubMed Central

    Paulus, W.; Baur, I.; Schuppan, D.; Roggendorf, W.

    1993-01-01

    We studied the immunohistochemical expression of integrin alpha and beta chains in the normal and neoplastic human brain. Normal astrocytes expressed alpha 2, alpha 3, alpha 6, beta 1, and beta 4 chains in some areas facing major interstitial tissues, but they were consistently negative for the other integrins examined (alpha 4, alpha 5, alpha V, alpha L, alpha M, alpha X, beta 2, beta 3). Neoplastic astrocytes in vivo and in vitro showed increased expression of alpha 3 and beta 1, and some also of alpha 5, alpha V, beta 3, and beta 4. Neoexpression of alpha 4 and reduced levels of beta 4 were detected in glioblastoma vascular proliferations compared with normal endothelial cells. Oligodendroglioma, ependymoma, choroid plexus papilloma, pituitary adenoma, and meningioma cells showed the same integrin pattern as their normal counterparts. Adhesion assays using the astrocytoma cell lines U-138 MG and U-373 MG revealed strong attachment to collagen types I to VI and undulin, which was inhibited by antibodies to beta 1, but not by those to alpha 2, alpha 3, alpha 6, and alpha V. We conclude that astrocytomas show increased levels or neoexpression of various integrins and strong attachment to various extracellular matrix components, which appears to be almost exclusively mediated by beta 1-integrins. Images Figure 1 PMID:8317546

  13. Immunoexpression of integrins in ameloblastoma, adenomatoid odontogenic tumor, and human tooth germs.

    PubMed

    de Souza Andrade, Emanuel Sávio; Miguel, Márcia Cristina da Costa; de Almeida Freitas, Roseana; Pereira Pinto, Leão; Batista de Souza, Lélia

    2008-07-01

    The expression of integrins alpha2beta1, alpha3beta1, and alpha5beta1 in 30 ameloblastomas (20 solid and 10 unicystic tumors), 12 adenomatoid odontogenic tumors (AOTs), and 5 human tooth germs in different stages of odontogenesis was analyzed. The distribution, location, pattern, and intensity of immunohistochemical expression were evaluated. Intensity was analyzed using scores (0 = absence, 1 = weak staining, and 2 = strong staining). No difference in the immunoexpression of the integrins was observed between solid and unicystic ameloblastomas. When these two ameloblastoma types were pooled into a single group, the following significant differences were found: immunoexpression of integrin alpha2beta1 was stronger in ameloblastomas than in AOTs and tooth germs, and the expression of integrin alpha5beta1 was stronger in ameloblastomas than in AOTs. The lack of detection of integrin alpha3beta1 in tooth germs and its detection in the odontogenic tumors studied suggest that this integrin might be used as a marker of neoplastic transformation in odontogenic tissues.

  14. Dendritic Cells from Rheumatoid Arthritis Patient Peripheral Blood Induce Th17 Cell Differentiation via miR-363/Integrin αv/TGF-β Axis.

    PubMed

    Pan, F; Xiang, H; Yan, J; Hong, L; Zhang, L; Liu, Y; Feng, X; Cai, C

    2017-06-01

    Dendritic cells (DCs) are critical regulators of immune responses. This study was to observe the effect of DCs from peripheral blood on the differentiation of Th17 in patients with rheumatoid arthritis (RA). Peripheral blood samples were collected from 30 patients with RA and 20 healthy controls, respectively. Flow cytometry results showed that in contrast to Treg cells, the proportion of Th17 cells in T cells and the Th17/Treg ratio were both increased in patients with RA. The RT-PCR results showed that Foxp3、ROR γt and miR-363 expression in PBMC of patients with RA were reduced, but the ITGAV expression was increased, which was negatively related to miR-363 expression. IL-17, TGF-β and IL-6 levels detected by ELISA were increased in peripheral blood serum of patients with RA. Moreover, we noted that the CD11C(+) αν(+) /CD11C(+) DCs ratio was obvious increased in patients with RA and has positive correlation to the Th17/Treg ratio. In cocultured system, Th17 cell differentiation was significantly inhibited in the presence of ITGF-β suggesting that Th17 cell differentiation was controlled by active TGF-β (aTGF-β). After DCs transfecting with miR-363 mimics and cocultured with T cells, Th17 cell number, IL-17 level and ROR-γt expression were significantly reduced in the presence of latent TGF-β (ITGF-β). In addition, the integrin αv protein expression was both reduced in the presence of aTGF-β or ITGF-β. These data demonstrated that DCs induced Th17 cell differentiation through miR-363/Integrin αv/TGF-β pathway in patients with RA. © 2017 The Foundation for the Scandinavian Journal of Immunology.

  15. Regulation of integrin-mediated adhesions

    PubMed Central

    Iwamoto, Daniel V.; Calderwood, David A.

    2015-01-01

    Integrins are heterodimeric transmembrane adhesion receptors that couple the actin cytoskeleton to the extracellular environment and bidirectionally relay signals across the cell membrane. These processes are critical for cell attachment, migration, differentiation, and survival, and therefore play essential roles in metazoan development, physiology, and pathology. Integrin-mediated adhesions are regulated by diverse factors, including the conformation-specific affinities of integrin receptors for their extracellular ligands, the clustering of integrins and their intracellular binding partners into discrete adhesive structures, mechanical forces exerted on the adhesion, and the intracellular trafficking of integrins themselves. Recent advances shed light onto how the interaction of specific intracellular proteins with the short cytoplasmic tails of integrins controls each of these activities. PMID:26189062

  16. Differential regulation of phosphoinositide metabolism by alphaVbeta3 and alphaVbeta5 integrins upon smooth muscle cell migration.

    PubMed

    Paulhe, F; Racaud-Sultan, C; Ragab, A; Albiges-Rizo, C; Chap, H; Iberg, N; Morand, O; Perret, B

    2001-11-09

    Smooth muscle cell migration is a key step of atherosclerosis and angiogenesis. We demonstrate that alpha(V)beta(3) and alpha(V)beta(5) integrins synergistically regulate smooth muscle cell migration onto vitronectin. Using an original haptotactic cell migration assay, we measured a strong stimulation of phosphoinositide metabolism in migrating vascular smooth muscle cells. Phosphatidic acid production and phosphoinositide 3-kinase IA activation were triggered only upon alpha(V)beta(3) engagement. Blockade of alpha(V)beta(3) engagement or phospholipase C activity resulted in a strong inhibition of smooth muscle cell spreading on vitronectin. By contrast, blockade of alpha(V)beta(5) reinforced elongation and polarization of cell shape. Moreover, Pyk2-associated tyrosine kinase and phosphoinositide 4-kinase activities measured in Pyk2 immunoprecipitates were stimulated upon cell migration. Blockade of either alpha(V)beta(3) or alpha(V)beta(5) function, as well as inhibition of phospholipase C activity, decreased both Pyk2-associated activities. We demonstrated that the Pyk2-associated phosphoinositide 4-kinase corresponded to the beta isoform. Our data point to the metabolism of phosphoinositides as a regulatory pathway for the differential roles played by alpha(V)beta(3) and alpha(V)beta(5) upon cell migration and identify the Pyk2-associated phosphoinositide 4-kinase beta as a common target for both integrins.

  17. Alpha3-integrins are required for hippocampal long-term potentiation and working memory.

    PubMed

    Chan, Chi-Shing; Levenson, Jonathan M; Mukhopadhyay, Partha S; Zong, Lin; Bradley, Allan; Sweatt, J David; Davis, Ronald L

    2007-09-01

    Integrins comprise a large family of heterodimeric, transmembrane cell adhesion receptors that mediate diverse neuronal functions in the developing and adult CNS. Recent pharmacological and genetic studies have suggested that beta1-integrins are critical in synaptic plasticity and memory formation. To further define the role of integrins in these processes, we generated a postnatal forebrain and excitatory neuron-specific knockout of alpha3-integrin, one of several binding partners for beta1 subunit. At hippocampal Schaffer collateral-CA1 synapses, deletion of alpha3-integrin resulted in impaired long-term potentiation (LTP). Basal synaptic transmission and paired-pulse facilitation were normal in the absence of alpha3-integrin. Behavioral studies demonstrated that the mutant mice were selectively defective in a hippocampus-dependent, nonmatch-to-place working memory task, but were normal in other hippocampus-dependent spatial tasks. The impairment in LTP and working memory is similar to that observed in beta1-integrin conditional knockout mice, suggesting that alpha3-integrin is the functional binding partner for beta1 for these processes in the forebrain.

  18. Integrin Cytoplasmic Tail Interactions

    PubMed Central

    2015-01-01

    Integrins are heterodimeric cell surface adhesion receptors essential for multicellular life. They connect cells to the extracellular environment and transduce chemical and mechanical signals to and from the cell. Intracellular proteins that bind the integrin cytoplasmic tail regulate integrin engagement of extracellular ligands as well as integrin localization and trafficking. Cytoplasmic integrin-binding proteins also function downstream of integrins, mediating links to the cytoskeleton and to signaling cascades that impact cell motility, growth, and survival. Here, we review key integrin-interacting proteins and their roles in regulating integrin activity, localization, and signaling. PMID:24467163

  19. Semaphorin 7A plays a critical role in TGF-beta1-induced pulmonary fibrosis.

    PubMed

    Kang, Hye-Ryun; Lee, Chun Geun; Homer, Robert J; Elias, Jack A

    2007-05-14

    Semaphorin (SEMA) 7A regulates neuronal and immune function. In these studies, we tested the hypothesis that SEMA 7A is also a critical regulator of tissue remodeling. These studies demonstrate that SEMA 7A and its receptors, plexin C1 and beta1 integrins, are stimulated by transforming growth factor (TGF)-beta(1) in the murine lung. They also demonstrate that SEMA 7A plays a critical role in TGF-beta(1)-induced fibrosis, myofibroblast hyperplasia, alveolar remodeling, and apoptosis. TGF-beta(1) stimulated SEMA 7A via a largely Smad 3-independent mechanism and stimulated SEMA 7A receptors, matrix proteins, CCN proteins, fibroblast growth factor 2, interleukin 13 receptor components, proteases, antiprotease, and apoptosis regulators via Smad 2/3-independent and SEMA 7A-dependent mechanisms. SEMA 7A also played an important role in the pathogenesis of bleomycin-induced pulmonary fibrosis. TGF-beta(1) and bleomycin also activated phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB)/AKT via SEMA 7A-dependent mechanisms, and PKB/AKT inhibition diminished TGF-beta(1)-induced fibrosis. These observations demonstrate that SEMA 7A and its receptors are induced by TGF-beta(1) and that SEMA 7A plays a central role in a PI3K/PKB/AKT-dependent pathway that contributes to TGF-beta(1)-induced fibrosis and remodeling. They also demonstrate that the effects of SEMA 7A are not specific for transgenic TGF-beta(1), highlighting the importance of these findings for other fibrotic stimuli.

  20. Integrins in epithelial cell polarity: using antibodies to analyze adhesive function and morphogenesis.

    PubMed

    Matlin, Karl S; Haus, Brian; Zuk, Anna

    2003-07-01

    Epithelial cells polarize in response to cell-substratum and cell-cell adhesive interactions. Contacts between cells and proteins of the extracellular matrix are mediated by integrin receptors. Of the 24 recognized integrin heterodimers, epithelial cells typically express four or more distinct integrins, with the exact complement dependent on the tissue of origin. Investigation of the roles of integrins in epithelial cell polarization has depended on the use of function-blocking antibodies both to determine ligand specificity of individual integrins and to disrupt and redirect normal morphogenesis. In this article we describe techniques for employing function-blocking anti-integrin antibodies in adhesion assays of the polarized Madin-Darby canine kidney (MDCK) cell line and to demonstrate the involvement of beta1 integrins in collagen-induced tubulocyst formation. These techniques can be easily expanded to other antibodies and epithelial cell lines to characterize specific functions of individual integrins in epithelial morphogenesis.

  1. Beta 1-adrenoreceptors regulate resting metabolic rate.

    PubMed

    Lamont, L S; Romito, R A; Finkelhor, R S; Kalhan, S C

    1997-06-01

    This was a randomized, cross-over experiment designed to determine which beta-adrenergic receptors, beta 1, beta 2, or both, regulate metabolic rate in humans. All subjects (3 women, 4 men) were administered a 7-d therapeutic dose of a selective beta 1-antagonist (atenolol 50 mg BID), a combined beta 1, beta 2-antagonist (propranolol 80 mg BID), and a placebo control (BID). Indirect calorimetry was determined before and after 1 h of submaximal exercise. Exercise was performed at 50% of the trial specific VO2peak because maximal exercise was significantly decreased in the presence of the nonselective beta 1, beta 2-antagonist (VO2peak placebo: 44.90 +/- 4.40 mL.kg-1.min-1 vs beta 1, beta 2-antagonism: 39.20 +/- 3.00 mL.kg-1.min-1; P < 0.05). Both the beta 1 and the combined beta 1, beta 2-adrenoreceptor antagonists reduced resting oxygen consumption to a similar extent (0.247 +/- 0.007 L.min-1 placebo, vs 0.218 +/- 0.007 L.min-1 beta 1-antagonism, vs 0.226 +/- 0.007 L.min-1 beta 1, beta 2-antagonism; P < 0.05). However, the 30-min and 60-min excess post-exercise oxygen consumption (mean EPOC) remained unchanged. It is concluded that the beta 1-receptors are regulating the effects of the sympathetic nervous system on resting but not exercise recovery metabolic rate. These metabolic side effects may suggest that changes need to be made in the nutritional requirements of patients using beta-adrenergic antagonists.

  2. Cell-cell adhesion via the ECM: integrin genetics in fly and worm.

    PubMed

    Brown, N H

    2000-07-01

    Integrins are essential for the development of the two genetically tractable invertebrate model organisms, the nematode worm Caenorhabditis elegans and the fruit fly Drosophila melanogaster. Just two integrins are present in C. elegans: one putative RGD binding integrin alphapat-2betapat-3, corresponding to Drosophila alphaPS2betaPS and vertebrate alpha5beta1, alphaVbeta1 and alpha8beta1, and one putative laminin binding integrin alphaina-1betapat-3, corresponding to Drosophila alphaPS1betaPS and vertebrate alpha3beta1, alpha6beta1 and alpha7beta1. In this review, the function of this minimal set of integrins during the development of these two invertebrates is compared. Despite the differences in bodyplan and developmental strategy, integrin adhesion to the extracellular matrix is required for similar processes: the formation of the link that translates muscle contraction into movement of the exoskeleton, cell migration, and morphogenetic interactions between epithelia. Other integrin functions, such as regulation of gene expression, have not yet been experimentally demonstrated in both organisms. Additional proteins have been characterised in each organism that are essential for integrin function, including extracellular matrix ligands and intracellular interacting proteins, but so far different proteins have been found in the two organisms. This in part represents the fact that the characterisation of the full set of interacting proteins is not complete in either system. However, in other cases different proteins appear to be used for similar functions in the two animals. The continued use of genetic approaches to identify proteins required for integrin function in these two model organisms should lead to the identification of the minimal set of conserved components that form integrin adhesive structures.

  3. Expression of GPI-80, a beta2-integrin-associated glycosylphosphatidylinositol-anchored protein, requires neutrophil differentiation with dimethyl sulfoxide in HL-60 cells.

    PubMed

    Takeda, Yuji; Fu, Junfen; Suzuki, Kichiya; Sendo, Dai; Nitto, Takeaki; Sendo, Fujiro; Araki, Yoshihiko

    2003-06-10

    GPI-80 is a member of the amidohydrolase family that has been proposed as a potential regulator of beta2-integrin-dependent leukocyte adhesion. GPI-80 is expressed mainly in human neutrophils. Our previous studies suggested that GPI-80 expression might be associated with myeloid differentiation. To verify this, we examined whether GPI-80 is expressed on the human promyelocytic leukemia cell line HL-60 following treatment with differentiation inducers. GPI-80 expression was induced in cells treated with dimethyl sulfoxide (DMSO) to stimulate differentiation down the neutrophil pathway. On the other hand, all-trans-retinoic acid (ATRA), another neutrophil-inducing reagent, induced no clear GPI-80 expression. Potent monocyte-inducing reagents such as 1alpha,25-dihydroxyvitamin D(3) or phorbol 12-myristate 13-acetate also had no significant effect on the protein expression. GPI-80-positive cells were found in the well-differentiated CD11b-positive and transferrin-receptor-negative cell population. Granulocyte colony-stimulating factor, which augments neutrophil differentiation of HL-60 cells, up-regulated GPI-80 expression in the presence of DMSO. Granulocyte/macrophage colony-stimulating factor, which is known to suppress the neutrophil maturation of cells, inhibited expression. Adhesion of DMSO-induced cells was regulated by anti-GPI-80 monoclonal antibody, similar to the regulation observed in neutrophils. These results suggest that use of DMSO to induce neutrophil differentiation provides suitable conditions for GPI-80 expression, and that this culture system may be a helpful model for further study of the regulation of GPI-80 expression during myeloid differentiation.

  4. Alpha1 and Alpha2 Integrins Mediate Invasive Activity of Mouse Mammary Carcinoma Cells through Regulation of Stromelysin-1 Expression

    SciTech Connect

    Lochter, Andre; Navre, Marc; Werb, Zena; Bissell, Mina J

    1998-06-29

    Tumor cell invasion relies on cell migration and extracellular matrix proteolysis. We investigated the contribution of different integrins to the invasive activity of mouse mammary carcinoma cells. Antibodies against integrin subunits {alpha}6 and {beta}1, but not against {alpha}1 and {alpha}2, inhibited cell locomotion on a reconstituted basement membrane in two-dimensional cell migration assays, whereas antibodies against {beta}1, but not against a6 or {alpha}2, interfered with cell adhesion to basement membrane constituents. Blocking antibodies against {alpha}1 integrins impaired only cell adhesion to type IV collagen. Antibodies against {alpha}1, {alpha}2, {alpha}6, and {beta}1, but not {alpha}5, integrin subunits reduced invasion of a reconstituted basement membrane. Integrins {alpha}1 and {alpha}2, which contributed only marginally to motility and adhesion, regulated proteinase production. Antibodies against {alpha}1 and {alpha}2, but not {alpha}6 and {beta}1, integrin subunits inhibited both transcription and protein expression of the matrix metalloproteinase stromelysin-1. Inhibition of tumor cell invasion by antibodies against {alpha}1 and {alpha}2 was reversed by addition of recombinant stromelysin-1. In contrast, stromelysin-1 could not rescue invasion inhibited by anti-{alpha}6 antibodies. Our data indicate that {alpha}1 and {alpha}2 integrins confer invasive behavior by regulating stromelysin-1 expression, whereas {alpha}6 integrins regulate cell motility. These results provide new insights into the specific functions of integrins during tumor cell invasion.

  5. Method of increasing radiation sensitivity by inhibition of beta one integrin

    DOEpatents

    Park, Catherine; Bissell, Mina J.

    2009-11-17

    A method for increasing or monitoring apoptosis in tumor cells by the co-administration of ionizing radiation and an anti-integrin antibody. Increasing apoptosis reduces tumor growth in vivo and in a cell culture model. The antibody is directed against the beta-1 integrin subunit and is inhibitory of beta-1 integrin signaling. Other molecules having an inhibitory effect on beta-1 integrin, either in signaling or in binding to its cognate extracellular receptors may also be used. The present method is particularly of interest in treatment of tumor cells associated with breast cancer, wherein radiation is currently used alone. The present method further contemplates a monoclonal antibody suitable for human administration that may further comprise a radioisotope attached thereto.

  6. Primary afferent second messenger cascades interact with specific integrin subunits in producing inflammatory hyperalgesia.

    PubMed

    Dina, Olayinka A; Hucho, Tim; Yeh, Jenny; Malik-Hall, Misbah; Reichling, David B; Levine, Jon D

    2005-05-01

    We recently reported that hyperalgesia induced by the inflammatory mediator prostaglandin E(2) (PGE(2)) requires intact alpha1, alpha3 and beta1 integrin subunit function, whereas epinephrine-induced hyperalgesia depends on alpha5 and beta1. PGE(2)-induced hyperalgesia is mediated by protein kinase A (PKA), while epinephrine-induced hyperalgesia is mediated by a combination of PKA, protein kinase Cepsilon (PKCepsilon) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK). We hypothesized that inflammatory mediator-induced hyperalgesia involves specific interactions between different subsets of integrin subunits and particular second messenger species. In the present study, function-blocking anti-integrin antibodies and antisense oligodeoxynucleotides were used to elucidate these interactions in rat. Hyperalgesia produced by an activator of adenylate cyclase (forskolin) depended on alpha1, alpha3 and beta1 integrins. However, hyperalgesia induced by activation of the cascade at a point farther downstream (by cAMP analog or PKA catalytic subunit) was independent of any integrins tested. In contrast, hyperalgesia induced by a specific PKCepsilon agonist depended only on alpha5 and beta1 integrins. Hyperalgesia induced by agonism of MAPK/ERK depended on all four integrin subunits tested (alpha1, alpha3, alpha5 and beta1). Finally, disruption of lipid rafts antagonized hyperalgesia induced by PGE(2) and by forskolin, but not that induced by epinephrine. Furthermore, alpha1 integrin, but not alpha5, was present in detergent-resistant membrane fractions (which retain lipid raft components). These observations suggest that integrins play a critical role in inflammatory pain by interacting with components of second messenger cascades that mediate inflammatory hyperalgesia, and that such interaction with the PGE(2)-activated pathway may be organized by lipid rafts.

  7. Expression, topography, and function of integrin receptors are severely altered in keratinocytes from involved and uninvolved psoriatic skin.

    PubMed

    Pellegrini, G; De Luca, M; Orecchia, G; Balzac, F; Cremona, O; Savoia, P; Cancedda, R; Marchisio, P C

    1992-06-01

    Psoriasis is a hyperproliferative cutaneous disease of unknown etiology and etiopathogenesis. Alteration of keratinocyte adhesiveness to basal lamina has been proposed as the initial disturbance leading to poorly controlled proliferation. Keratinocyte adhesion to basal lamina and lateral interactions among basal epidermal cells are mediated, besides other molecules, by integrin receptors that are segregated to discrete membrane domains. In this paper, the expression and function of integrins in psoriatic keratinocytes were examined, both in vivo and in vitro. We found that: (a) in psoriatic keratinocytes the integrin heterodimers alpha 2 beta 1, alpha 3 beta 1, and alpha 6 beta 4 have lost their polarized distribution on the plasma membrane; (b) the role of these integrins in mediating keratinocyte adhesion in vitro is altered; (c) psoriatic keratinocytes form focal contacts containing both beta 1 and beta 4 integrins. In normal adult keratinocytes the alpha 5 beta 1 fibronectin receptor is poorly expressed and diffusely distributed on the basal keratinocyte plasma membrane and is not organized in defined adhesive structures. In contrast, psoriatic keratinocytes show a clear fibronectin receptor staining in vivo, and organize alpha 5 beta 1 in typical focal contacts in vitro without any obvious increase of its expression and synthesis. These multiple alterations of integrins are also present in uninvolved keratinocytes from psoriatic patients, suggesting a key role for altered integrin-mediated adhesion in the pathogenesis of this disease.

  8. Expression, topography, and function of integrin receptors are severely altered in keratinocytes from involved and uninvolved psoriatic skin.

    PubMed Central

    Pellegrini, G; De Luca, M; Orecchia, G; Balzac, F; Cremona, O; Savoia, P; Cancedda, R; Marchisio, P C

    1992-01-01

    Psoriasis is a hyperproliferative cutaneous disease of unknown etiology and etiopathogenesis. Alteration of keratinocyte adhesiveness to basal lamina has been proposed as the initial disturbance leading to poorly controlled proliferation. Keratinocyte adhesion to basal lamina and lateral interactions among basal epidermal cells are mediated, besides other molecules, by integrin receptors that are segregated to discrete membrane domains. In this paper, the expression and function of integrins in psoriatic keratinocytes were examined, both in vivo and in vitro. We found that: (a) in psoriatic keratinocytes the integrin heterodimers alpha 2 beta 1, alpha 3 beta 1, and alpha 6 beta 4 have lost their polarized distribution on the plasma membrane; (b) the role of these integrins in mediating keratinocyte adhesion in vitro is altered; (c) psoriatic keratinocytes form focal contacts containing both beta 1 and beta 4 integrins. In normal adult keratinocytes the alpha 5 beta 1 fibronectin receptor is poorly expressed and diffusely distributed on the basal keratinocyte plasma membrane and is not organized in defined adhesive structures. In contrast, psoriatic keratinocytes show a clear fibronectin receptor staining in vivo, and organize alpha 5 beta 1 in typical focal contacts in vitro without any obvious increase of its expression and synthesis. These multiple alterations of integrins are also present in uninvolved keratinocytes from psoriatic patients, suggesting a key role for altered integrin-mediated adhesion in the pathogenesis of this disease. Images PMID:1534817

  9. Investigation of the effect of mutations of rat albumin on the binding affinity to the alpha(4)beta(1) integrin antagonist, 4-[1-[3-chloro-4-[N'-(2-methylphenyl)ureido]phenylacetyl]-(4S)-fluoro-(2S)-pyrrolidine-2-yl]methoxybenzoic acid (D01-4582), using recombinant rat albumins.

    PubMed

    Ito, Takashi; Takahashi, Masayuki; Okazaki, Osamu; Sugiyama, Yuichi

    2010-08-02

    The authors reported previously rat strain differences in plasma protein binding to alpha(4)beta(1) antagonist D01-4582, resulting in a great strain difference in its pharmacokinetics (19-fold differences in the AUC). The previous study suggested that amino acid changes of V238L and/or T293I in albumin reduced the binding affinity. In order to elucidate the relative significance of these mutations, an expression system was developed to obtain recombinant rat albumins (rRSA) using Pichia pastoris, followed by a binding analysis of four rRSAs by the ultracentrifugation method. The equilibrium dissociation constant (K(d)) of wild-type rRSA was 210 nM, while K(d) of rRSA that carried both V238L and T293I mutations was 974 nM. K(d) of artificial rRSA that carried only V238L was 426 nM, and K(d) of artificial rRSA that carried only T293I was 191 nM. These results suggested that V238L would be more important in the alteration of K(d). However, since none of the single mutations were sufficient to explain the reduction of affinity, the possibility was also suggested that T293I interacted cooperatively to reduce the binding affinity of rat albumin to D01-4582. Further investigation is required to elucidate the mechanism of the possible cooperative interaction.

  10. New osmoregulated beta(1-3),beta(1-6) glucosyltransferase(s) in Azospirillum brasilense.

    PubMed Central

    Altabe, S G; Iñón de Iannino, N; de Mendoza, D; Ugalde, R A

    1994-01-01

    A linear beta(1-3),beta(1-6) glucan was detected in the periplasm of Azospirillum brasilense cells growing in a medium of low osmotic strength. This glucan was produced in vitro by purified bacterial inner membranes with UDP-glucose as the sugar donor in the presence of Mg2+. Growth in a high-osmotic-strength medium strongly reduced the amount of this glucan accumulated in the periplasmic space, and the inhibition was associated with a reduction in the enzymatic activity of the beta(1-3),beta(1-6) glucosyltransferase(s). Images PMID:8051002

  11. Characterization of Laminin Binding Integrin Internalization in Prostate Cancer Cells.

    PubMed

    Das, Lipsa; Anderson, Todd A; Gard, Jaime M C; Sroka, Isis C; Strautman, Stephanie R; Nagle, Raymond B; Morrissey, Colm; Knudsen, Beatrice S; Cress, Anne E

    2017-05-01

    Laminin binding integrins α6 (CD49f) and α3 (CD49c) are persistently but differentially expressed in prostate cancer (PCa). Integrin internalization is an important determinant of their cell surface expression and function. Using flow cytometry, and first order kinetic modeling, we quantitated the intrinsic internalization rates of integrin subunits in a single cycle of internalization. In PCa cell line DU145, α6 integrin internalized with a rate constant (kactual ) of 3.25 min(-1) , threefold faster than α3 integrin (1.0 min(-1) ), 1.5-fold faster than the vitronectin binding αv integrin (CD51) (2.2 min(-1) ), and significantly slower than the unrelated transferrin receptor (CD71) (15 min(-1) ). Silencing of α3 integrin protein expression in DU145, PC3, and PC3B1 cells resulted in up to a 1.71-fold increase in kactual for α6 integrin. The internalized α6 integrin was targeted to early endosomes but not to lamp1 vesicles. Depletion of α3 integrin expression resulted in redistribution of α6β4 integrin to an observed cell-cell staining pattern that is consistent with a suprabasal distribution observed in epidermis and early PIN lesions in PCa. Depletion of α3 integrin increased cell migration by 1.8-fold, which was dependent on α6β1 integrin. Silencing of α6 integrin expression however, had no significant effect on the kactual of α3 integrin or its distribution in early endosomes. These results indicate that α3 and α6 integrins have significantly different internalization kinetics and that coordination exists between them for internalization. J. Cell. Biochem. 118: 1038-1049, 2017. © 2016 Wiley Periodicals, Inc.

  12. Depressed adrenomedullin in the embryonic transforming growth factor-beta1 null mouse becomes elevated postnatally.

    PubMed

    Bodegas, Elena; Martínez, Alfredo; Ozbun, Laurent L; Garayoa, Mercedes; Letterio, John J; Montuenga, Luis M; Jakowlew, Sonia B

    2004-02-01

    Transforming growth factor-beta (TGF-beta) and adrenomedullin are multifunctional regulatory proteins which are expressed in developing embryonic and adult tissues. Because of their colocalization, TGF-beta1 and adrenomedullin may be able to coordinately act to influence development and differentiation. In order to learn more about the biology of adrenomedullin in the absence of the effects of TGF-beta1 in vivo, we examined adrenomedullin in the TGF-beta1 null mouse. A generally lower amount of adrenomedullin was detected by immunohistochemical staining analysis in multiple tissues from embryonic TGF-beta1 null mice compared to wildtype animals, including the heart, lung, brain, liver, and kidney, among others. In contrast, immunohistochemical staining for adrenomedullin was more intense in tissues of the postnatal TGF-beta1 null mouse compared to the wildtype mouse. These observations were confirmed by quantitative real time RT-PCR for adrenomedullin in both embryos and postnatal animals, as well as in cultured mouse embryo fibroblasts from TGF-beta1 null and wildtype mice. In addition, when cultured mouse embryo fibroblasts were treated with a neutralizing monoclonal antibody against TGF-beta1, the levels of adrenomedullin expression were statistically reduced compared to untreated cells. Our data show that expression of adrenomedullin is reduced in tissues of the developing embryonic TGF-beta1 null mouse compared to the wildtype mouse, but increases during postnatal development in TGF-beta1 null mice. The elevated expression of adrenomedullin which occurs postnatally in the TGF-beta1 null mouse may be a cause or a consequence of the multifocal wasting syndrome which is characteristic of postnatal TGF-beta1 null mice.

  13. Transforming growth factor beta 1: an autocrine regulator of adrenocortical steroidogenesis.

    PubMed

    Feige, J J; Cochet, C; Savona, C; Shi, D L; Keramidas, M; Defaye, G; Chambaz, E M

    1991-01-01

    Transforming growth factor beta 1 (TGF beta 1) is a member of a large family of structurally related regulatory polypeptides which comprises both functionally similar (TGF beta 1, TGF beta 2, TGF beta 3, TGF beta 4 and TGF beta 5) and functionally distinct proteins. In the past few years, TGF beta 1 has emerged as a multifunctional protein. One of its remarkable properties is its capacity to negatively modulate the differentiated, steroidogenic adrenocortical functions. We present here a review of the results from our recent work related to the effects of TGF beta 1 on bovine adrenocortical cell (zona fasciculata-reticularis) functions. We identified the steroid 17 alpha-hydroxylase (P-450 17 alpha) biosynthetic enzyme and the angiotensin II receptor as major targets whose expression are negatively regulated by TGF beta 1 in these cells. We characterized TGF beta 1 receptors at the surface of adrenocortical cells (mainly type I and type III receptors) and observed that their number is increased under ACTH treatment. Furthermore, we could detect the presence of immunoreactive TGF beta 1 in the bovine adrenal cortex whereas it was undetectable in the adrenal medulla and in the capsule. We also observed that adrenocortical cells secrete TGF beta 1 under a latent form together with large amounts of alpha 2-macroglobulin, a protease inhibitor known to be implied in the latency of TGF beta in serum. Taken together, these observations led us to a working hypothesis, proposing TGF beta 1 as an autocrine and/or paracrine regulator of adrenocortical steroidogenic functions. This concept points out the physiological activation of the latent TGF beta 1 complex as the important limiting step controlling its action in the adrenal cortex.

  14. Epiligrin, a component of epithelial basement membranes, is an adhesive ligand for alpha 3 beta 1 positive T lymphocytes

    PubMed Central

    1993-01-01

    The cutaneous T cell lymphomas (CTCL), typified by mycosis fungoides, and several chronic T cell mediated dermatoses are characterized by the migration of T lymphocytes into the epidermis (epidermotropism). Alternatively, other types of cutaneous inflammation (malignant cutaneous B cell lymphoma, CBCL, or lymphocytoma cutis, non-malignant T or B cell type) do not show evidence of epidermotropism. This suggests that certain T lymphocyte subpopulations are able to interact with and penetrate the epidermal basement membrane. We show here that T lymphocytes derived from patients with CTCL (HUT 78 or HUT 102 cells), adhere to the detergent-insoluble extracellular matrix prepared from cultured basal keratinocytes (HFK ECM). HUT cell adhesion to HFK ECM was inhibitable with monoclonal antibodies (mAbs) directed to the alpha 3 (P1B5) or beta 1 (P4C10) integrin receptors, and could be up- regulated by an activating anti-beta 1 mAb (P4G11). An inhibitory mAb, P3H9-2, raised against keratinocytes identified epiligrin as the ligand for alpha 3 beta 1 positive T cells in HFK ECM. Interestingly, two lymphocyte populations could be clearly distinguished relative to expression of alpha 3 beta 1 by flow cytometry analysis. Lymphokine activated killer cells, alloreactive cytotoxic T cells and T cells derived from patients with CTCL expressed high levels of alpha 3 beta 1 (alpha 3 beta 1high). Non-adherent peripheral blood mononuclear cells, acute T or B lymphocytic leukemias, or non-cutaneous T or B lymphocyte cell lines expressed low levels of alpha 3 beta 1 (alpha 3 beta 1low). Resting PBL or alpha 3 beta 1low T or B cell lines did not adhere to HFK ECM or purified epiligrin. However, adhesion to epiligrin could be up-regulated by mAbs which activate the beta 1 subunit indicating that alpha 3 beta 1 activity is a function of expression and affinity. In skin derived from patients with graft-vs.-host (GVH) disease, experimentally induced delayed hypersensitivity reactions, and CTCL

  15. Improved cartilage regeneration utilizing mesenchymal stem cells in TGF-beta1 gene-activated scaffolds.

    PubMed

    Diao, Huajia; Wang, Jinliang; Shen, Chao; Xia, Suhua; Guo, Ting; Dong, Lei; Zhang, Chenyu; Chen, Jiangning; Zhao, Jianning; Zhang, Junfeng

    2009-09-01

    Recently, bone marrow-derived mesenchymal stem cells (MSCs) have been paid more attention for cartilage regeneration. This study evaluated the potential of using MSCs seeded in plasmid transforming growth factor beta1 (pTGF-beta1)-activated three-dimensional chitosan/gelatin scaffolds for improving cartilage repair in vivo. Significant cell proliferation and transforming growth factor beta1 protein expression were observed in vitro in pTGFbeta1-activated scaffolds. Transforming growth factor beta1-activated scaffolds showed high collagen type II and aggrecan expression and low collagen type I expression during in vitro cultivation. MSC-based pTGF-beta1-activated scaffolds also exhibited cartilage histology with high secretion of collagen type II in vitro under the stimulation of pTGF-beta1. In rabbits with full-thickness cartilage defects, the implantation of MSC-based pTGF-beta1-activated scaffolds not only significantly promoted chondrogenic differentiation of MSCs and hyalin-like cartilage matrix synthesis, but also remarkably improved the overall repair of rabbit cartilage defects and exhibited favorable tissue integrity at 10 weeks postsurgery. These results suggest that MSC-based localized pTGF-beta1-activated scaffolds have potential applications for in vivo cartilage repair.

  16. Spatiotemporal segregation of endothelial cell integrin and nonintegrin extracellular matrix-binding proteins during adhesion events

    PubMed Central

    1990-01-01

    Bovine aortic endothelial cell (BAEC) attachments to laminin, fibronectin, and fibrinogen are inhibited by soluble arginine-glycine- aspartate (RGD)-containing peptides, and YGRGDSP activity is responsive to titration of either soluble peptide or matrix protein. To assess the presence of RGD-dependent receptors, immunoprecipitation and immunoblotting studies were conducted and demonstrated integrin beta 1, beta 3, and associated alpha subunits as well as a beta 1 precursor. Immunofluorescence of BAECs plated on laminin, fibronectin, and fibrinogen reveals different matrix-binding specificities of each of these integrin subclasses. By 1 h after plating, organization of beta 1 integrin into fibrillar streaks is influenced by laminin and fibronectin, whereas beta 3 integrin punctate organization is influenced by fibrinogen and the integrin spatial distribution changes with time in culture. In contrast, the nonintegrin laminin-binding protein LB69 only organizes after cell-substrate contact is well established several hours after plating. Migration of BAECs is also mediated by both integrin and nonintegrin matrix-binding proteins. Specifically, BAEC migration on laminin is remarkably sensitive to RGD peptide inhibition, and, in its presence, beta 1 integrin organization dissipates and reorganizes into perinuclear vesicles. However, RGD peptides do not alter LB69 linear organization during migration. Similarly, agents that block LB69--e.g., antibodies to LB69 as well as YIGSR-NH2 peptide--do not inhibit attachment of nonmotile BAECs to laminin. However, both anti-LB69 and YIGSR-NH2 inhibit late adhesive events such as spreading. Accordingly, we propose that integrin and nonintegrin extracellular matrix-binding protein organizations in BAECs are both temporally and spatially segregated during attachment processes. High affinity nonintegrin interaction with matrix may create necessary stable contacts for longterm attachment, while lower affinity integrins may be important

  17. Integrin Targeted Therapeutics

    PubMed Central

    Millard, Melissa; Odde, Srinivas; Neamati, Nouri

    2011-01-01

    Integrins are heterodimeric, transmembrane receptors that function as mechanosensors, adhesion molecules and signal transduction platforms in a multitude of biological processes. As such, integrins are central to the etiology and pathology of many disease states. Therefore, pharmacological inhibition of integrins is of great interest for the treatment and prevention of disease. In the last two decades several integrin-targeted drugs have made their way into clinical use, many others are in clinical trials and still more are showing promise as they advance through preclinical development. Herein, this review examines and evaluates the various drugs and compounds targeting integrins and the disease states in which they are implicated. PMID:21547158

  18. Keratins Stabilize Hemidesmosomes through Regulation of β4-Integrin Turnover.

    PubMed

    Seltmann, Kristin; Cheng, Fang; Wiche, Gerhard; Eriksson, John E; Magin, Thomas M

    2015-06-01

    Epidermal integrity and wound healing depend on remodeling of cell-matrix contacts including hemidesmosomes. Mutations in β4-integrin and plectin lead to severe epidermolysis bullosa (EB). Whether mutations in keratins K5 or K14, which cause EB simplex, also compromise cell-matrix adhesion through altering hemidesmosomal components is not well investigated. In particular, the dependence of β4-integrin endocytosis and turnover on keratins remains incompletely understood. Here, we show that the absence of keratins causes loss of plectin-β4-integrin interaction and elevated β4-integrin phosphorylation at Ser1354 and Ser1362. This triggered a caveolin-dependent endocytosis of β4-integrin but not of other integrins through Rab5 and Rab11 compartments in keratinocytes. Expressing a phospho-deficient β4-integrin mutant reduces β4-integrin endocytosis and rescues plectin localization in keratin-free cells. β4-integrin phosphorylation in the absence of keratins resulted from elevated Erk1/2 activity downstream of increased EGFR and PKCα signaling. Further, increased Erk1/2 phosphorylation and altered plectin localization occur in keratin-deficient mouse epidermis in vivo. Strikingly, expression of the K14-R125P EBS mutant also resulted in plectin mislocalization and elevated β4-integrin turnover, suggesting disease relevance. Our data underscore a major role of keratins in controlling β4-integrin endocytosis involving a plectin-Erk1/2-dependent mechanism relevant for epidermal differentiation and pathogenesis.

  19. Differential dephosphorylation of the Protein Kinase C-zeta (PKCζ) in an integrin αIIbβ3-dependent manner in platelets

    PubMed Central

    Mayanglambam, Azad; Bhavanasi, Dheeraj; Vijayan, K. Vinod; Kunapuli, Satya P.

    2011-01-01

    Protein kinase C-zeta (PKCζ), an atypical isoform of the PKC family of protein serine/threonine kinases, is expressed in human platelets. However, the mechanisms of its activation and the regulation of its activity in platelets are not known. We have found that under basal resting conditions, PKCζ has a high phosphorylation status at the activation loop threonine 410 (T410) and the turn motif (autophosphorylation site) threonine 560 (T560), both of which have been shown to be important for its catalytic activity. After stimulation with agonist under stirring conditions, the T410 residue was dephosphorylated in a time- and concentration-dependent manner, while the T560 phosphorylation remained unaffected. The T410 dephosphorylation could be significantly prevented by blocking the binding of fibrinogen to integrin αIIbβ3 with an antagonist, SC-57101; or by okadaic acid used at concentrations that inhibits protein serine/threonine phosphatases PP1 and PP2A in vitro. The dephosphorylation of T410 residue on PKCζ was also observed in PP1cγ null murine platelets after agonist stimulation, suggesting that other isoforms of PP1c or another phosphatase could be responsible for this dephosphorylation event. We conclude that human platelets express PKCζ, and it may be constitutively phosphorylated at the activation loop threonine 410 and the turn motif threonine 560 under basal resting conditions, which are differentially dephosphorylated by outside-in signaling. This differential dephosphorylation of PKCζ might be an important regulatory mechanism for platelet functional responses. PMID:21645497

  20. The role of phosphorylation in activation of the alpha 6A beta 1 laminin receptor.

    PubMed

    Hogervorst, F; Kuikman, I; Noteboom, E; Sonnenberg, A

    1993-09-05

    The phorbol ester phorbol 12-myristate 13-acetate (PMA) induces phosphorylation of serine residues in the cytoplasmic domain of the alpha 6A integrin subunit, as well as activation of the alpha 6A beta 1 laminin receptor. We examined whether phosphorylation correlates with the induction of high affinity binding of laminin by the alpha 6A beta 1 receptor. Two potential phosphorylation sites for protein kinase C, serine 1041 and serine 1048, are present in the cytoplasmic domain of the alpha 6A subunit. We introduced point mutations into the alpha 6A cDNA, replacing either one or both of the serine residues with alanine. Wild-type and mutant alpha 6A cDNAs were transfected into K562 cells. All alpha 6A subunit mutants were expressed at levels similar to those of wild-type alpha 6A and formed heterodimers with endogenous beta 1. Analysis of the phosphorylation state of wild-type and mutant alpha 6A subunits in resting K562 cells and after treatment with PMA showed that serine 1041, but not serine 1048, is the target residue of PMA-induced phosphorylation. Cells expressing alpha 6A mutant subunits or wild-type alpha 6A transfectants all bound laminin in the presence, but not in the absence of PMA; however, the extent of binding differed. Cells transfected with alpha 6A containing the serine to alanine mutation showed a 2-3-fold higher binding to laminin than cells transfected with alpha 6A containing serine 1041. The results indicate that phosphorylation of the alpha 6A cytoplasmic domain is not required for the induction of high affinity of the alpha 6A beta 1 receptor by PMA, and suggest that, in contrast, it may reduce the affinity of this integrin for ligand.

  1. Immunolocalization of integrin-like proteins in Arabidopsis and Chara

    NASA Technical Reports Server (NTRS)

    Katembe, W. J.; Swatzell, L. J.; Makaroff, C. A.; Kiss, J. Z.

    1997-01-01

    Integrins are a large family of integral plasma membrane proteins that link the extracellular matrix to the cytoskeleton in animal cells. As a first step in determining if integrin-like proteins are involved in gravitropic signal transduction pathways, we have used a polyclonal antibody against the chicken beta1 integrin subunit in western blot analyses and immunofluorescence microscopy to gain information on the size and location of these proteins in plants. Several different polypeptides are recognized by the anti-integrin antibody in roots and shoots of Arabidopsis and in the internodal cells and rhizoids of Chara. These cross-reactive polypeptides are associated with cellular membranes, a feature which is consistent with the known location of integrins in animal systems. In immunofluorescence studies of Arabidopsis roots, a strong signal was obtained from labeling integrin-like proteins in root cap cells, and there was little or no immunolabel in other regions of the root tip. While the antibody stained throughout Chara rhizoids, the highest density of immunolabel was at the tip. Thus, in both Arabidopsis roots and Chara rhizoids, the sites of gravity perception/transduction appear to be enriched in integrin-like molecules.

  2. Immunolocalization of integrin-like proteins in Arabidopsis and Chara

    NASA Technical Reports Server (NTRS)

    Katembe, W. J.; Swatzell, L. J.; Makaroff, C. A.; Kiss, J. Z.

    1997-01-01

    Integrins are a large family of integral plasma membrane proteins that link the extracellular matrix to the cytoskeleton in animal cells. As a first step in determining if integrin-like proteins are involved in gravitropic signal transduction pathways, we have used a polyclonal antibody against the chicken beta1 integrin subunit in western blot analyses and immunofluorescence microscopy to gain information on the size and location of these proteins in plants. Several different polypeptides are recognized by the anti-integrin antibody in roots and shoots of Arabidopsis and in the internodal cells and rhizoids of Chara. These cross-reactive polypeptides are associated with cellular membranes, a feature which is consistent with the known location of integrins in animal systems. In immunofluorescence studies of Arabidopsis roots, a strong signal was obtained from labeling integrin-like proteins in root cap cells, and there was little or no immunolabel in other regions of the root tip. While the antibody stained throughout Chara rhizoids, the highest density of immunolabel was at the tip. Thus, in both Arabidopsis roots and Chara rhizoids, the sites of gravity perception/transduction appear to be enriched in integrin-like molecules.

  3. Crystal structure of a heparin- and integrin-binding segment of human fibronectin.

    PubMed Central

    Sharma, A; Askari, J A; Humphries, M J; Jones, E Y; Stuart, D I

    1999-01-01

    The crystal structure of human fibronectin (FN) type III repeats 12-14 reveals the primary heparin-binding site, a clump of positively charged residues in FN13, and a putative minor site approximately 60 A away in FN14. The IDAPS motif implicated in integrin alpha4beta1 binding is at the FN13-14 junction, rendering the critical Asp184 inaccessible to integrin. Asp184 clamps the BC loop of FN14, whose sequence (PRARI) is reminiscent of the synergy sequence (PHSRN) of FN9. Mutagenesis studies prompted by this observation reveal that both arginines of the PRARI sequence are important for alpha4beta1 binding to FN12-14. The PRARI motif may represent a new class of integrin-binding sites. The spatial organization of the binding sites suggests that heparin and integrin may bind in concert. PMID:10075919

  4. The serine/threonine kinase Ndr2 controls integrin trafficking and integrin-dependent neurite growth.

    PubMed

    Rehberg, Kati; Kliche, Stefanie; Madencioglu, Deniz A; Thiere, Marlen; Müller, Bettina; Meineke, Bernhard Manuel; Freund, Christian; Budinger, Eike; Stork, Oliver

    2014-04-09

    Integrins have been implicated in various processes of nervous system development, including proliferation, migration, and differentiation of neuronal cells. In this study, we show that the serine/threonine kinase Ndr2 controls integrin-dependent dendritic and axonal growth in mouse hippocampal neurons. We further demonstrate that Ndr2 is able to induce phosphorylation at the activity- and trafficking-relevant site Thr(788/789) of β1-integrin to stimulate the PKC- and CaMKII-dependent activation of β1-integrins, as well as their exocytosis. Accordingly, Ndr2 associates with integrin-positive early and recycling endosomes in primary hippocampal neurons and the surface expression of activated β1-integrins is reduced on dendrites of Ndr2-deficient neurons. The role of Ndr2 in dendritic differentiation is also evident in vivo, because Ndr2-null mutant mice show arbor-specific alterations of dendritic complexity in the hippocampus. This indicates a role of Ndr2 in the fine regulation of dendritic growth; in fact, treatment of primary neurons with Semaphorin 3A rescues Ndr2 knock-down-induced dendritic growth deficits but fails to enhance growth beyond control level. Correspondingly, Ndr2-null mutant mice show a Semaphorin 3A(-/-)-like phenotype of premature dendritic branching in the hippocampus. The results of this study show that Ndr2-mediated integrin trafficking and activation are crucial for neurite growth and guidance signals during neuronal development.

  5. Integrin αvβ6 Critically Regulates Hepatic Progenitor Cell Function and Promotes Ductular Reaction, Fibrosis, and Tumorigenesis

    PubMed Central

    Peng, Zhen-Wei; Ikenaga, Naoki; Liu, Susan B.; Sverdlov, Deanna Y.; Vaid, Kahini A.; Dixit, Richa; Weinreb, Paul H.; Violette, Shelia; Sheppard, Dean; Schuppan, Detlef; Popov, Yury

    2017-01-01

    Integrin αvβ6 is rapidly up-regulated on cells of epithelial lineage during tissue injury, where one of its primary functions is activation of latent transforming growth factor beta 1 (TGFβ1). In human liver cirrhosis, αvβ6 is overexpressed by cells comprising the ductular reaction, and its inhibition suppresses experimental biliary fibrosis in rodents. Here, we show that αvβ6 is expressed on the actively proliferating subset of hepatic progenitor cells and is required for their progenitor function in vivo and in vitro through integrin αvβ6-dependent TGFβ1 activation. Freshly isolated αvβ6+ liver cells demonstrate clonogenic potential and differentiate into cholangiocytes and functional hepatocytes in vitro, whereas colony formation by epithelial cell adhesion molecule-positive progenitor cells is blocked by αvβ6-neutralizing antibody and in integrin beta 6-deficient cells. Inhibition of progenitors by anti-αvβ6 antibody is recapitulated by TGFβ1 neutralization and rescued by addition of bioactive TGFβ1. Genetic disruption or selective targeting of αvβ6 with 3G9 antibody potently inhibits progenitor cell responses in mouse models of chronic biliary injury and protects from liver fibrosis and tumorigenesis, two conditions clinically associated with exacerbated ductular reaction. Conclusion These results suggest that αvβ6 is a promising target for chronic fibrotic liver diseases and associated cancers. PMID:26448099

  6. Integrin signaling in atherosclerosis.

    PubMed

    Finney, Alexandra C; Stokes, Karen Y; Pattillo, Christopher B; Orr, A Wayne

    2017-06-01

    Atherosclerosis, a chronic lipid-driven inflammatory disease affecting large arteries, represents the primary cause of cardiovascular disease in the world. The local remodeling of the vessel intima during atherosclerosis involves the modulation of vascular cell phenotype, alteration of cell migration and proliferation, and propagation of local extracellular matrix remodeling. All of these responses represent targets of the integrin family of cell adhesion receptors. As such, alterations in integrin signaling affect multiple aspects of atherosclerosis, from the earliest induction of inflammation to the development of advanced fibrotic plaques. Integrin signaling has been shown to regulate endothelial phenotype, facilitate leukocyte homing, affect leukocyte function, and drive smooth muscle fibroproliferative remodeling. In addition, integrin signaling in platelets contributes to the thrombotic complications that typically drive the clinical manifestation of cardiovascular disease. In this review, we examine the current literature on integrin regulation of atherosclerotic plaque development and the suitability of integrins as potential therapeutic targets to limit cardiovascular disease and its complications.

  7. Nanoparticle-formulated siRNA targeting integrins inhibits hepatocellular carcinoma progression in mice

    PubMed Central

    Bogorad, Roman L; Yin, Hao; Zeigerer, Anja; Nonaka, Hidenori; Ruda, Vera; Zerial, Marino; Anderson, Daniel G; Koteliansky, Victor

    2014-01-01

    Integrins play an important role during development, regulating cell differentiation, proliferation and survival. Here we show that knockdown of integrin subunits slows down the progression of hepatocellular carcinoma (HCC). Using nanoparticulate delivery of short interfering RNAs targeting β1 and αv integrin subunits we downregulate all integrin receptors in hepatocytes. Short-term integrin knockdown (two weeks) does not cause apparent structural or functional perturbations of normal liver tissue. Alterations in liver morphology accumulate upon sustained integrin downregulation (seven weeks). The integrin knockdown leads to significant retardation of HCC progression, reducing proliferation and increasing tumour cell death. This tumour retardation is accompanied by reduced activation of MET oncogene as well as expression of its mature form on the cell surface. Our data suggest that transformed proliferating cells from HCC are more sensitive to knockdown of integrins than normal quiescent hepatocytes, highlighting the potential of siRNA-mediated inhibition of integrins as an anti-cancer therapeutic approach. PMID:24844798

  8. Hepatic progenitor cell resistance to TGF-{beta}1's proliferative and apoptotic effects

    SciTech Connect

    Clark, J. Brian; Rice, Lisa; Sadiq, Tim; Brittain, Evan; Song, Lujun; Wang Jian; Gerber, David A. . E-mail: David_Gerber@med.unc.edu

    2005-04-01

    The success of hepatocellular therapies using stem or progenitor cell populations is dependent upon multiple factors including the donor cell, microenvironment, and etiology of the liver injury. The following experiments investigated the impact of TGF-{beta}1 on a previously described population of hepatic progenitor cells (HPC). The majority of the hepatic progenitor cells were resistant to endogenously produced TGF-{beta}1's proapoptotic and anti-proliferative effects unlike more well-differentiated cellular populations (e.g., mature hepatocytes). Surprisingly, in vitro TGF-{beta}1 supplementation significantly inhibited de novo hepatic progenitor cell colony formation possibly via an indirect mechanism(s). Therefore despite the HPC's direct resistance to supplemental TGF-{beta}1, this cytokine's inhibitory effect on colony formation could have a potential negative impact on the use of these cells as a therapy for patients with liver disease.

  9. The MIG-2/integrin interaction strengthens cell-matrix adhesion and modulates cell motility.

    PubMed

    Shi, Xiaohua; Ma, Yan-Qing; Tu, Yizeng; Chen, Ka; Wu, Shan; Fukuda, Koichi; Qin, Jun; Plow, Edward F; Wu, Chuanyue

    2007-07-13

    Integrin-mediated cell-matrix adhesion plays an important role in control of cell behavior. We report here that MIG-2, a widely expressed focal adhesion protein, interacts with beta1 and beta3 integrin cytoplasmic domains. Integrin binding is mediated by a single site within the MIG-2 FERM domain. Functionally, the MIG-2/integrin interaction recruits MIG-2 to focal adhesions. Furthermore, using alphaIIbbeta3 integrin-expressing Chinese hamster ovary cells, a well described model system for integrin activation, we show that MIG-2 promotes integrin activation and enhances cell-extracellular matrix adhesion. Although MIG-2 is expressed in many cell types, it is deficient in certain colon cancer cells. Expression of MIG-2, but not of an integrin binding-defective MIG-2 mutant, in MIG-2-null colon cancer cells strengthened cell-matrix adhesion, promoted focal adhesion formation, and reduced cell motility. These results suggest that the MIG-2/integrin interaction is an important element in the cellular control of integrin-mediated cell-matrix adhesion and that loss of this interaction likely contributes to high motility of colon cancer cells.

  10. JAM-A promotes neutrophil chemotaxis by controlling integrin internalization and recycling.

    PubMed

    Cera, Maria Rosaria; Fabbri, Monica; Molendini, Cinzia; Corada, Monica; Orsenigo, Fabrizio; Rehberg, Markus; Reichel, Christoph A; Krombach, Fritz; Pardi, Ruggero; Dejana, Elisabetta

    2009-01-15

    The membrane-associated adhesion molecule JAM-A is required for neutrophil infiltration in inflammatory or ischemic tissues. JAM-A expressed in both endothelial cells and neutrophils has such a role, but the mechanism of action remains elusive. Here we show that JAM-A has a cell-autonomous role in neutrophil chemotaxis both in vivo and in vitro, which is independent of the interaction of neutrophils with endothelial cells. On activated neutrophils, JAM-A concentrates in a polarized fashion at the leading edge and uropod. Surprisingly, a significant amount of this protein is internalized in intracellular endosomal-like vesicles where it codistributes with integrin beta1. Clustering of beta1 integrin leads to JAM-A co-clustering, whereas clustering of JAM-A does not induce integrin association. Neutrophils derived from JAM-A-null mice are unable to correctly internalize beta1 integrins upon chemotactic stimuli and this causes impaired uropod retraction and cell motility. Consistently, inhibition of integrin internalization upon treatment with BAPTA-AM induces a comparable phenotype. These data indicate that JAM-A is required for the correct internalization and recycling of integrins during cell migration and might explain why, in its absence, the directional migration of neutrophils towards an inflammatory stimulus is markedly impaired.

  11. Targeting ILK and {beta}4 integrin abrogates the invasive potential of ovarian cancer

    SciTech Connect

    Choi, Yoon Pyo; Kim, Baek Gil; Gao, Ming-Qing; Kang, Suki; Cho, Nam Hoon

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer The potential of targeting ILK and integrins for highly aggressive ovarian cancer. Black-Right-Pointing-Pointer Unanticipated synergistic effect for the combination of ILK/{beta}4 integrin. Black-Right-Pointing-Pointer Combination of ILK/{beta}4 integrin effectively inhibited the PI3K/Akt/Rac1 cascade. Black-Right-Pointing-Pointer Targeting of {beta}4 integrin/ILK had potent inhibitory effects in ovarian cancer. -- Abstract: Integrins and integrin-linked kinase (ILK) are essential to cancerous invasion because they mediate physical interactions with the extracellular matrix, and regulate oncogenic signaling pathways. The purpose of our study is to determine whether deletion of {beta}1 and {beta}4 integrin and ILK, alone or in combination, has antitumoral effects in ovarian cancer. Expression of {beta}1 and {beta}4 integrin and ILK was analyzed by immunohistochemistry in 196 ovarian cancer tissue samples. We assessed the effects of depleting these molecules with shRNAs in ovarian cancer cells by Western blot, conventional RT-PCR, cell proliferation, migration, invasion, and in vitro Rac1 activity assays, and in vivo xenograft formation assays. Overexpression of {beta}4 integrin and ILK in human ovarian cancer specimens was found to correlate with tumor aggressiveness. Depletion of these targets efficiently suppresses ovarian cancer cell proliferation, migration, and invasion in vitro and xenograft tumor formation in vivo. We also demonstrated that single depletion of ILK or combination depletion of {beta}4 integrin/ILK inhibits phosphorylation of downstream signaling targets, p-Ser 473 Akt and p-Thr202/Tyr204 Erk1/2, and activation of Rac1, as well as reduce expression of MMP-2 and MMP-9 and increase expression of caspase-3 in vitro. In conclusion, targeting {beta}4 integrin combined with ILK can instigate the latent tumorigenic potential and abrogate the invasive potential in ovarian cancer.

  12. Selective down-regulation of the alpha6-integrin subunit in melanocytes by UVB light.

    PubMed

    Krengel, Sven; Stark, Imke; Geuchen, Christian; Knoppe, Bettina; Scheel, Gabriele; Schlenke, Peter; Gebert, Andreas; Wünsch, Lutz; Brinckmann, Jürgen; Tronnier, Michael

    2005-06-01

    In vivo, melanocytes bind to laminin (LM) molecules of the basement membrane (BM) via the integrins alpha3beta1 and alpha6beta1, and they adhere to neighbouring keratinocytes via E-cadherin. Only few studies have addressed the impact of ultraviolet (UV) light on the interaction of melanocytes with their microenvironment. In this report, we examined the influence of UVB irradiation on the expression of the most important melanocyte-adhesion molecules (E-, N-cadherin, alpha2-, alpha3-, alpha5-, alpha6-, alphaV-, beta1-, beta3-integrins and ICAM-1) in vitro by flow cytometry. We were able to demonstrate that the alpha6-integrin subunit is selectively and reversibly down-regulated by UVB in a dwzm 150ose-dependent manner. In comparison, keratinocytes lacked UVB-inducible alterations in the expression of alpha6-integrin. In the presence of LM-1, the UVB-induced down-regulation of alpha6-integrin in melanocytes was significantly reduced. Moreover, LM-1 increased the resistance of melanocytes to UVB-induced cell death, as measured by annexinV-binding analysis. This effect was reversed by preincubation with an alpha6-integrin-blocking antibody. By immunofluorescence, we could demonstrate that UVB leads to a dose-dependent internalization of alpha6-integrin, providing an obvious explanation for the down-regulation on the outer cell surface observed by flow cytometry. We suggest that adhesion to LM-1 through alpha6-integrin represents a protective mechanism for melanocytes to withstand UVB damage. Through alpha6-integrin internalization, sunburns might alter the interaction between melanocytes and the BM, resulting in apoptosis induced by loss of anchorage (anoikis). Repeated sunburns may then lead to the selection of a population of melanocytes which are capable of anchorage-independent survival, culminating in solar nevogenesis and melanoma development.

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

    SciTech Connect

    Samandari, Elika; Visarius, Theresa; Zingg, Jean-Marc; Azzi, Angelo . E-mail: angelo.azzi@tufts.edu

    2006-04-21

    The effect of vitamin E on proliferation, integrin expression, adhesion, and migration in human glioma cells has been studied. {gamma}-tocopherol at 50 {mu}M concentration exerted more inhibitory effect than {alpha}-tocopherol at the same concentration on glioma cell proliferation. Integrin {alpha}5 and {beta}1 protein levels were increased upon both {alpha}- and {gamma}-tocopherol treatments. In parallel, an increase in the {alpha}5{beta}1 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 {alpha}5 and {beta}1 expression. Cell adhesion is also negatively affected by tocopherols, despite a small increase in the surface appearance of the {alpha}5{beta}1 heterodimer. Cell migration is stimulated by {gamma}-tocopherol. It is concluded that {alpha}5 and {beta}1 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.

  14. TGF-beta1 release from biodegradable polymer microparticles: its effects on marrow stromal osteoblast function

    NASA Technical Reports Server (NTRS)

    Lu, L.; Yaszemski, M. J.; Mikos, A. G.; McIntire, L. V. (Principal Investigator)

    2001-01-01

    BACKGROUND: Controlled release of transforming growth factor-beta1 (TGF-beta1) to a bone defect may be beneficial for the induction of a bone regeneration cascade. The objectives of this work were to assess the feasibility of using biodegradable polymer microparticles as carriers for controlled TGF-beta1 delivery and the effects of released TGF-beta1 on the proliferation and differentiation of marrow stromal cells in vitro. METHODS: Recombinant human TGF-beta1 was incorporated into microparticles of blends of poly(DL-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG). Fluorescein isothiocynate-labeled bovine serum albumin (FITC-BSA) was co-encapsulated as a porogen. The effects of PEG content (0, 1, or 5% by weight [wt%]) and buffer pH (3, 5, or 7.4) on the protein release kinetics and the degradation of PLGA were determined in vitro for as long as 28 days. Rat marrow stromal cells were seeded on a biodegradable poly(propylene fumarate) (PPF) substrate. The dose response and biological activity of released TGF-beta1 was determined after 3 days in culture. The effects of TGF-beta1 released from PLGA/PEG microparticles on marrow stromal cell proliferation and osteoblastic differentiation were assessed during a 21-day period. RESULTS: TGF-beta1 was encapsulated along with FITC-BSA into PLGA/PEG blend microparticles and released in a multiphasic fashion including an initial burst for as long as 28 days in vitro. Increasing the initial PEG content resulted in a decreased cumulative mass of released proteins. Aggregation of FITC-BSA occurred at lower buffer pH, which led to decreased release rates of both proteins. The degradation of PLGA was increased at higher PEG content and significantly accelerated at acidic pH conditions. Rat marrow stromal cells cultured on PPF substrates showed a dose response to TGF-beta1 released from the microparticles similar to that of added TGF-beta1, indicating that the activity of TGF-beta1 was retained during microparticle

  15. Integrins and metastasis

    PubMed Central

    Ganguly, Kirat Kumar; Pal, Sekhar; Moulik, Shuvojit; Chatterjee, Amitava

    2013-01-01

    Metastasis is a combination of biological events that makes the difference between cancer and other diseases. Metastasis requires flow of erroneous but precisely coordinated basic cellular activities like cell migration–invasion, cell survival–apoptosis, cell proliferation, etc. All of these processes require efficient regulation of cell attachment and detachment, which recruit integrin receptors in this flow of events. World literatures show several aspects of interrelation of integrins and metastasis. Integrin molecules are being used as prime target to battle metastasis. In this review we are collating the observations showing importance of integrin biology in regulation of metastasis and the strategies where integrin receptors are being used as targets to regulate metastasis. PMID:23563505

  16. Molecular requirements for induction of CTGF expression by TGF-beta1 in primary osteoblasts.

    PubMed

    Arnott, J A; Zhang, X; Sanjay, A; Owen, T A; Smock, S L; Rehman, S; DeLong, W G; Safadi, F F; Popoff, S N

    2008-05-01

    Connective tissue growth factor (CTGF/CCN2) is a cysteine rich, extracellular matrix protein that acts as an anabolic growth factor to regulate osteoblast differentiation and function. In osteoblasts, CTGF is induced by TGF-beta1 where it acts as a downstream mediator of TGF-beta1 induced matrix production. The molecular mechanisms that control CTGF induction by TGF-beta1 in osteoblasts are not known. To assess the role of individual Smads in mediating the induction of CTGF by TGF-beta1, we used specific Smad siRNAs to block Smad expression. These studies demonstrated that Smads 3 and 4, but not Smad 2, are required for TGF-beta1 induced CTGF promoter activity and expression in osteoblasts. Since the activation of MAPKs (Erk, Jnk and p38) by TGF-beta1 is cell type specific, we were interested in determining the role of individual MAPKs in TGF-beta1 induction of CTGF promoter activity and expression. Using dominant negative (DN) mutants for Erk, Jnk and p38, we demonstrated that the expression of DN-Erk caused a significant inhibition of TGF-beta1 induced CTGF promoter activity. In contrast, the expression of DN-p38 or DN-Jnk failed to inhibit activation of CTGF promoter activity. To confirm the vital role of Erk, we used the Erk inhibitor (PD98059) to block its activation, demonstrating that it prevented TGF-beta1 activation of the CTGF promoter and up-regulation of CTGF expression in osteoblasts. Since Src can also act as a downstream signaling effector for TGF-beta in some cell types, we determined its role in TGF-beta1 induction of CTGF in osteoblasts. Treatment of osteoblasts with a Src family kinase inhibitor, PP2, or the expression of two independent kinase-dead Src mutant constructs caused significant inhibition of TGF-beta1 induced CTGF promoter activity and expression. Additionally, blocking Src activation prevented Erk activation by TGF-beta1 demonstrating a role for Src as an upstream mediator of Erk in regulating CTGF expression in osteoblasts. To

  17. Differential Role of β1C and β1A Integrin Cytoplasmic Variants in Modulating Focal Adhesion Kinase, Protein Kinase B/AKT, and Ras/Mitogen-activated Protein Kinase Pathways

    PubMed Central

    Fornaro, Mara; Steger, Craig A.; Bennett, Anton M.; Wu, J. Julie; Languino, Lucia R.

    2000-01-01

    The integrin cytoplasmic domain modulates cell proliferation, adhesion, migration, and intracellular signaling. The β1 integrin subunits, β1C and β1A, that contain variant cytoplasmic domains differentially affect cell proliferation; β1C inhibits proliferation, whereas β1A promotes it. We investigated the ability of β1C and β1A to modulate integrin-mediated signaling events that affect cell proliferation and survival in Chinese hamster ovary stable cell lines expressing either human β1C or human β1A. The different cytodomains of either β1C or β1A did not affect either association with the endogenous α2, αV, and α5 subunits or cell adhesion to fibronectin or TS2/16, a mAb to human β1. Upon engagement of endogenous and exogenous integrins by fibronectin, cells expressing β1C showed significantly inhibited extracellular signal–regulated kinase (ERK) 2 activation compared with β1A stable cell lines. In contrast, focal adhesion kinase phosphorylation and Protein Kinase B/AKT activity were not affected. Selective engagement of the exogenously expressed β1C by TS2/16 led to stimulation of Protein Kinase B/AKT phosphorylation but not of ERK2 activation; in contrast, β1A engagement induced activation of both proteins. We show that Ras activation was strongly reduced in β1C stable cell lines in response to fibronectin adhesion and that expression of constitutively active Ras, Ras 61 (L), rescued β1C-mediated down-regulation of ERK2 activation. Inhibition of cell proliferation in β1C stable cell lines was attributable to an inhibitory effect of β1C on the Ras/MAP kinase pathway because expression of activated MAPK kinase rescued β1C antiproliferative effect. These findings show that the β1C variant, by means of a unique signaling mechanism, selectively inhibits the MAP kinase pathway by preventing Ras activation without affecting either survival signals stimulated by integrins or cellular interactions with the extracellular matrix. These findings

  18. Influence of Snail on Integrin Beta 1 Expression/Activity in Breast Carcinoma

    DTIC Science & Technology

    2008-09-01

    Lipscomb E, Wendt MA, Eickholt BJ, and Mercurio AM. 2003. Semaphorin 3A is an endogenous suppressor of breast carcinoma chemotaxis/invasion...human semaphorin - 3A /Fc, recombinant human IgG1 Fc and neuropilin-1-neutralizing antibody (MAB566) were obtained from R and D Systems, Inc...expressing cells (Fig. 3a ). Importantly, SEMA3A shRNA-expressing cells exhibited a significantly reduced ability to bind to collagen I as compared to

  19. Role of ALK5/Smad2/3 and MEK1/ERK Signaling in Transforming Growth Factor Beta 1-modulated Growth, Collagen Turnover, and Differentiation of Stem Cells from Apical Papilla of Human Tooth.

    PubMed

    Chang, Hsiao-Hua; Chang, Mei-Chi; Wu, I-Hua; Huang, Guay-Fen; Huang, Wei-Ling; Wang, Yin-Lin; Lee, Sheng-Yang; Yeh, Chien-Yang; Guo, Ming-Kuang; Chan, Chiu-Po; Hsien, Hsiang-Chi; Jeng, Jiiang-Huei

    2015-08-01

    Transforming growth factor β1 (TGF-β1) plays an important role in cell proliferation, matrix formation, and odontogenesis. This study investigated the effects of TGF-β1 on stem cells from apical papilla (SCAPs) and its signaling by MEK/ERK and Smad2. SCAPs were exposed to TGF-β1 with/without pretreatment and coincubation by SB431542 (an ALK5/Smad 2/3 inhibitor) or U0126 (a MEK/ERK inhibitor). Cell growth was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay or direct counting of viable cells. Collagen content was determined by using the Sircol collagen assay (Biocolor Ltd, Newtownabbey, Northern Ireland). Cell differentiation was evaluated by measuring alkaline phosphatase (ALP) activity. Smad2 and ERK1/2 phosphorylation was analyzed by Western blotting or PathScan phospho-enzyme-linked immunosorbent assay (Cell Signaling Technology Inc, Danvers, MA). TGF-β1 stimulated the growth and collagen content of cultured SCAPs. TGF-β1 stimulated ERK1/2 and Smad2 phosphorylation within 60 minutes of exposure. Pretreatment by U0126 and SB431542 effectively prevented the TGF-β1-induced cell growth and collagen content in SCAPs. TGF-β1 stimulated ALP activity at lower concentrations (0.1-1 ng/mL) but down-regulated ALP at higher concentrations (>5 ng/mL). U0126 prevented 0.5 ng/mL TGF-β1-induced ALP activity but showed little effect on 10 ng/mL TGF-β1-induced decline of ALP in SCAPs. Interestingly, SB431542 attenuated both the stimulatory and inhibitory effects on ALP by TGF-β1. TGF-β1 may affect the proliferation, collagen turnover, and differentiation of SCAPs via differential activation of ALK5/Smad2 and MEK/ERK signaling. These results highlight the future use of TGF-β1 and SCAP for engineering of pulpal regeneration and apexogenesis. Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  20. A skin-depth analysis of integrins: role of the integrin network in health and disease.

    PubMed

    Hegde, Samarth; Raghavan, Srikala

    2013-12-01

    In the skin epidermis, adhesion to the underlying basement membrane is mediated through trans-membrane integrin receptors. In addition to a structural role, integrins can signal in a bi-directional manner though the membrane and thus play a crucial role in cell adhesion, migration, proliferation, and differentiation. In this review we will discuss the role of integrins and their network of partner proteins in normal skin development, and how dysregulation influences disease states such as skin blistering disorders and cancers. We also discuss major integrin-specific therapeutic advances that have been made over the past few years in treating these skin disorders as well as targeting angiogenesis, neo-vasculature, and tumorigenesis.

  1. Integrins in periodontal disease.

    PubMed

    Larjava, Hannu; Koivisto, Leeni; Heino, Jyrki; Häkkinen, Lari

    2014-07-15

    Cell surface integrin receptors mediate cell adhesion, migration and cellular signaling in all nucleated cells. They are activated by binding to extracellular ligands or by intracellular proteins, such as kindlins that engage with their cytoplasmic tails. Cells in the periodontal tissues express several integrins with overlapping ligand-binding capabilities. A distinct phenotype in the periodontium has only been described for knockouts or mutations of three integrin subunits, α11, β6 and β2. Integrin α11β1 appears to have some regulatory function in the periodontal ligament of continuously erupting incisors in mice. Integrin αvβ6 is expressed in the junctional epithelium (JE) of the gingiva. Animals deficient in this receptor develop classical signs of periodontal disease, including inflammation, apical migration of the JE and bone loss, suggesting that it plays a role in the regulation of periodontal inflmmation, likely through activation of transforming growth factor-β1. Lack of integrin activation in the JE is also associated with periodontitis. Patients with kindlin-1 mutations have severe early-onset periodontal disease. Finally, patients with mutations in the leukocyte-specific β2 integrin subunit have severe periodontal problems due to lack of transiting neutrophils in the periodontal tissues.

  2. Mechanotransduction through Integrins

    NASA Technical Reports Server (NTRS)

    Ingber, Donald

    2004-01-01

    The goal of this project was to characterize the molecular mechanism by which cells recognize and respond to physical forces in their local environment. The project was based on the working hypothesis that cells sense mechanical stresses through cell surface integrin receptors and through their interconnections with the underlying cytoskeleton. Work completed and published in past funding period had provided direct support for this hypothesis. In particular, we demonstrated that application of mechanical stresses to activated integrin receptors (but not inactive integrins or other control transmembrane receptors) resulted in stress-dependent activation of the CAMP signaling pathway leading to gene transcription. We also showed that this form of mechanotransduction requires activation of heterotrimeric G proteins. In this grant, our specific aims included: 1) to characterize the signal processing capabilities of different integrins and other cell surface receptors, 2) to identify heterotrimeric G proteins that mediate CAMP signaling by stresses applied to integrins, 3) to identify molecules that mediate transmembrane mechanochemical coupling between integrins and G proteins, and 4) to use genome-wide gene expression profiling techniques to identify other genes and signaling pathways that are activated by mechanical forces transmitted over specific cell surface receptors. Elucidation of the mechanism by which cells sense mechanical stresses through integrins and translate them into a biochemical response should help us to understand the molecular basis of the cellular response to gravity as well as many other forms of mechanosensation and tissue regulation.

  3. Differentiation between isomeric triantennary N-linked glycans by negative ion tandem mass spectrometry and confirmation of glycans containing galactose attached to the bisecting (beta1-4-GlcNAc) residue in N-glycans from IgG.

    PubMed

    Harvey, David J; Crispin, Max; Scanlan, Chris; Singer, Bernhard B; Lucka, Lothar; Chang, Veronica T; Radcliffe, Catherine M; Thobhani, Smita; Yuen, Chun-Ting; Rudd, Pauline M

    2008-04-01

    Negative ion tandem mass spectrometry (MS/MS) spectra of three isomeric triantennary N-linked glycans provided clear differentiation between the isomers and confirmed the occurrence of an isomer that was substituted with galactose on a bisecting GlcNAc (1 --> 4-substituted on the core mannose) residue recently reported by Takegawa et al. from N-glycans released from human immunoglobulin G (IgG). We extend this analysis of human serum IgG to reveal an analogue of the fucosylated triantennary glycan reported by Takegawa et al. together with a third compound that lacked both the sialic acid and the fucose residues. In addition, we demonstrate the biosynthesis of bisected hybrid-type glycans with the galactose modification, with and without core fucose, on the stem cell marker glycoprotein, 19A, expressed in a partially ricin-resistant human embryonic kidney cell line. It would appear, therefore, that this modification of N-linked glycans containing a galactosylated bisecting GlcNAc residue may be more common than originally thought. Negative ion MS/MS analysis of glycans is likely to prove an invaluable tool in the analysis and monitoring of therapeutic glycoproteins. (c) 2008 John Wiley & Sons, Ltd.

  4. Effect of shear stress on migration and integrin expression in macaque trophoblast cells.

    PubMed

    Soghomonians, Arlen; Barakat, Abdul I; Thirkill, Twanda L; Blankenship, Thomas N; Douglas, Gordon C

    2002-05-08

    During fetal development, trophoblast cells enter endometrial capillaries, migrate within the uterine vasculature, and eventually reside within spiral arteries of the uterus. This invasive activity is accompanied by upregulation of trophoblast beta1 integrin expression. Fluid mechanical shear stress regulates migration and expression of adhesion molecules in vascular endothelial cells, but nothing is known about the effects of shear stress on trophoblast cells. We tested the hypothesis that shear stress regulates the motility and beta1 integrin expression of trophoblast cells. Early gestation macaque trophoblast cells were cultured in 1 x 1-mm square cross-section capillary tubes within which the flow field was determined using three-dimensional computational fluid dynamic simulations. Trophoblast cells in the capillary tubes were exposed to a steady shear stress of 7.5, 15, or 30 dyn/cm2 for up to 24 h. In the absence of flow, trophoblast cells were highly dynamic with constant nondirectional positional shifts but with no net cell migration. Exposure of the cells to shear stress within 24-72 h of cell plating significantly increased the level of this activity and led to net cell migration in the direction of flow. Shear stress also increased the expression and altered the topography of beta1 integrin. These results suggest that shear stress regulates trophoblast motility and beta1 integrin expression in vitro.

  5. Integrins and Integrin-Associated Proteins in the Cardiac Myocyte

    PubMed Central

    Ross, Robert S.

    2014-01-01

    Integrins are heterodimeric, transmembrane receptors that are expressed in all cells, including those in the heart. They participate in multiple critical cellular processes including adhesion, extracellular matrix organization, signaling, survival, and proliferation. Particularly relevant for a contracting muscle cell, integrins are mechanotransducers, translating mechanical to biochemical information. While it is likely that cardiovascular clinicians and scientists have highest recognition of integrins in the cardiovascular system from drugs used to inhibit platelet aggregation, the focus of this article will be on the role of integrins specifically in the cardiac myocyte. Following a general introduction to integrin biology, the manuscript will discuss important work on integrin signaling, mechanotransduction, and lessons learned about integrin function from a range of model organisms. Then we will detail work on integrin-related proteins in the myocyte, how integrins may interact with ion channels and mediate viral uptake into cells, and also play a role in stem cell biology. Finally, we will discuss directions for future study. PMID:24481847

  6. Breast Cancer Cells in Three-dimensional Culture Display an Enhanced Radioresponse after Coordinate Targeting of Integrin ?5?1 and Fibronectin

    SciTech Connect

    Nam, Jin-Min; Onodera, Yasuhito; Bissell, Mina J; Park, Catherine C

    2010-04-07

    Tactics to selectively enhance cancer radioresponse are of great interest. Cancer cells actively elaborate and remodel their extracellular matrix (ECM) to aid in survival and progression. Previous work has shown that {beta}1-integrin inhibitory antibodies can enhance the growth-inhibitory and apoptotic responses of human breast cancer cell lines to ionizing radiation, either when cells are cultured in three-dimensional laminin-rich ECM (3D lrECM) or grown as xenografts in mice. Here, we show that a specific {alpha} heterodimer of {beta}1-integrin preferentially mediates a prosurvival signal in human breast cancer cells that can be specifically targeted for therapy. 3D lrECM culture conditions were used to compare {alpha}-integrin heterodimer expression in malignant and nonmalignant cell lines. Under these conditions, we found that expression of {alpha}5{beta}1-integrin was upregulated in malignant cells compared with nonmalignant breast cells. Similarly, we found that normal and oncofetal splice variants of fibronectin, the primary ECM ligand of {alpha}5{beta}1-integrin, were also strikingly upregulated in malignant cell lines compared with nonmalignant acini. Cell treatment with a peptide that disrupts the interactions of {alpha}5{beta}1-integrin with fibronectin promoted apoptosis in malignant cells and further heightened the apoptotic effects of radiation. In support of these results, an analysis of gene expression array data from breast cancer patients revealed an association of high levels of {alpha}5-integrin expression with decreased survival. Our findings offer preclinical validation of fibronectin and {alpha}5{beta}1-integrin as targets for breast cancer therapy.

  7. Covisualization by computational optical-sectioning microscopy of integrin and associated proteins at the cell membrane of living onion protoplasts

    NASA Technical Reports Server (NTRS)

    Gens, J. S.; Reuzeau, C.; Doolittle, K. W.; McNally, J. G.; Pickard, B. G.; Evans, M. L. (Principal Investigator)

    1996-01-01

    Using higher-resolution wide-field computational optical-sectioning fluorescence microscopy, the distribution of antigens recognized by antibodies against animal beta 1 integrin, fibronectin, and vitronectin has been visualized at the outer surface of enzymatically protoplasted onion epidermis cells and in depectinated cell wall fragments. On the protoplast all three antigens are colocalized in an array of small spots, as seen in raw images, in Gaussian filtered images, and in images restored by two different algorithms. Fibronectin and vitronectin but not beta 1 integrin antigenicities colocalize as puncta in comparably prepared and processed images of the wall fragments. Several control visualizations suggest considerable specifity of antibody recognition. Affinity purification of onion cell extract with the same anti-integrin used for visualization has yielded protein that separates in SDS-PAGE into two bands of about 105-110 and 115-125 kDa. These bands are again recognized by the visualization antibody, which was raised against the extracellular domain of chicken beta 1 integrin, and are also recognized by an antibody against the intracellular domain of chicken beta 1 integrin. Because beta 1 integrin is a key protein in numerous animal adhesion sites, it appears that the punctate distribution of this protein in the cell membranes of onion epidermis represents the adhesion sites long known to occur in cells of this tissue. Because vitronectin and fibronection are matrix proteins that bind to integrin in animals, the punctate occurrence of antigenically similar proteins both in the wall (matrix) and on enzymatically prepared protoplasts reinforces the concept that onion cells have adhesion sites with some similarity to certain kinds of adhesion sites in animals.

  8. Covisualization by computational optical-sectioning microscopy of integrin and associated proteins at the cell membrane of living onion protoplasts

    NASA Technical Reports Server (NTRS)

    Gens, J. S.; Reuzeau, C.; Doolittle, K. W.; McNally, J. G.; Pickard, B. G.; Evans, M. L. (Principal Investigator)

    1996-01-01

    Using higher-resolution wide-field computational optical-sectioning fluorescence microscopy, the distribution of antigens recognized by antibodies against animal beta 1 integrin, fibronectin, and vitronectin has been visualized at the outer surface of enzymatically protoplasted onion epidermis cells and in depectinated cell wall fragments. On the protoplast all three antigens are colocalized in an array of small spots, as seen in raw images, in Gaussian filtered images, and in images restored by two different algorithms. Fibronectin and vitronectin but not beta 1 integrin antigenicities colocalize as puncta in comparably prepared and processed images of the wall fragments. Several control visualizations suggest considerable specifity of antibody recognition. Affinity purification of onion cell extract with the same anti-integrin used for visualization has yielded protein that separates in SDS-PAGE into two bands of about 105-110 and 115-125 kDa. These bands are again recognized by the visualization antibody, which was raised against the extracellular domain of chicken beta 1 integrin, and are also recognized by an antibody against the intracellular domain of chicken beta 1 integrin. Because beta 1 integrin is a key protein in numerous animal adhesion sites, it appears that the punctate distribution of this protein in the cell membranes of onion epidermis represents the adhesion sites long known to occur in cells of this tissue. Because vitronectin and fibronection are matrix proteins that bind to integrin in animals, the punctate occurrence of antigenically similar proteins both in the wall (matrix) and on enzymatically prepared protoplasts reinforces the concept that onion cells have adhesion sites with some similarity to certain kinds of adhesion sites in animals.

  9. β1 integrin

    PubMed Central

    Howe, Grant A.; Addison, Christina L.

    2012-01-01

    Historically, a hallmark of tumorigenesis was the ability to grow in an anchorage-independent manner. Hence, tumors were thought to proliferate and survive independently of integrin attachment to the substratum. However, recent data suggest that integrins regulate not only tumor cell proliferation, survival and migration, but may also influence their response to anti-cancer agents. Interestingly, these influences are largely masked by growth of tumor cells in the standard, yet artificial, environment of 2D cell culture, but are readily apparent under 3D in vitro culture conditions and in tumor growth in vivo. We, and others, have recently demonstrated that the β1 integrin subunit controls the growth and invasion of prostate tumor cells in 3D culture conditions. Recently, the importance of integrins has also been demonstrated using tissue specific conditional knockout strategies in transgenic mouse tumor models, where they control primary tumor growth and dictate the site of metastatic spread. Furthermore, integrin-extracellular matrix interactions may modulate the response of tumors to standard chemotherapy agents or radiation. Taken together, these results highlight the important role of integrins in regulating tumor growth and metastasis; however, point out that the evaluation of their contribution to these processes requires appropriate contextual modeling. PMID:22568952

  10. Inhibition of spermidine synthase gene expression by transforming growth factor-beta 1 in hepatoma cells.

    PubMed Central

    Nishikawa, Y; Kar, S; Wiest, L; Pegg, A E; Carr, B I

    1997-01-01

    We screened genes responsive to transforming growth factor-beta (TGF-beta 1) protein in a human hepatoma cell line (Hep3B) using a PCR-mediated differential display technique, in order to investigate the mechanisms involved in TGF-beta-induced growth suppression. We found a gene that was down-regulated by TGF-beta 1 to be completely identical in an approx. 620 bp segment to the gene for the enzyme spermidine synthase, which mediates the conversion of putrescine into spermidine. Both spermidine synthase mRNA expression and its enzyme activity were decreased after TGF-beta 1 treatment of Hep3B cells. The inhibition of spermidine synthase gene expression by TGF-beta 1 protein was also observed in other hepatoma cell lines. The expression of genes for other biosynthetic enzymes in polyamine metabolism (ornithine decarboxylase and S-adenosylmethionine decarboxylase) was also inhibited to the same extent as for spermidine synthase, while the gene expression of spermidine/spermine N1-acetyltransferase, a catabolic enzyme, was relatively resistant to TGF-beta 1. Spermine levels in Hep3B cells were decreased by TGF-beta 1 treatment, although the levels of spermidine and putrescine were unchanged, probably due to compensation by remaining spermidine/spermine N1-acetyltransferase activity. Exogenously added spermidine or spermine, but not putrescine, partially antagonized the growth-inhibitor effects of TGF-beta 1 on Hep3B cells. Our data suggest that down-regulation of gene expression of the enzymes involved in polyamine metabolism, including spermidine synthase, may be associated with the mechanism of TGF-beta-induced growth suppression. PMID:9020892

  11. Transforming growth factor beta1 and aldosterone

    PubMed Central

    Matsuki, Kota; Hathaway, Catherine K.; Chang, Albert S.; Smithies, Oliver; Kakoki, Masao

    2016-01-01

    Purpose of review It is well established that blocking renin-angiotensin II-aldosterone system (RAAS) is effective for the treatment of cardiovascular and renal complications in hypertension and diabetes mellitus. Although the induction of transforming growth factor beta1 (TGFbeta1) by components of RAAS mediates the hypertrophic and fibrogenic changes in cardiovascular-renal complications, it is still controversial as to whether TGFbeta1 can be a target to prevent such complications. Here we review recent findings on the role of TGFbeta1 in fluid homeostasis, focusing on the relationship with aldosterone. Recent findings TGFbeta1 suppresses adrenal production of aldosterone and renal tubular sodium reabsorption. We have generated mice with TGFbeta1 mRNA expression graded in five steps from 10% to 300% normal, and found that blood pressure and plasma volume are negatively regulated by TGFbeta1. Notably, the 10 % hypomorph exhibits primary aldosteronism and sodium and water retention due to markedly impaired urinary excretion of water and electrolytes. Summary These results identify TGFbeta signaling as an important counterregulatory system against aldosterone. Understanding the molecular mechanisms for the suppressive effects of TGFbeta1 on adrenocortical and renal function may further our understanding of primary aldosteronism as well as assist in the development of novel therapeutic strategies for hypertension. PMID:25587902

  12. Exercise promotes alpha7 integrin gene transcription and protection of skeletal muscle.

    PubMed

    Boppart, Marni D; Volker, Sonja E; Alexander, Nicole; Burkin, Dean J; Kaufman, Stephen J

    2008-11-01

    The alpha7beta1 integrin is increased in skeletal muscle in response to injury-producing exercise, and transgenic overexpression of this integrin in mice protects against exercise-induced muscle damage. The present study investigates whether the increase in the alpha7beta1 integrin observed in wild-type mice in response to exercise is due to transcriptional regulation and examines whether mobilization of the integrin at the myotendinous junction (MTJ) is a key determinant in its protection against damage. A single bout of downhill running exercise selectively increased transcription of the alpha7 integrin gene in 5-wk-old wild-type mice 3 h postexercise, and an increased alpha7 chain was detected in muscle sarcolemma adjacent to tendinous tissue immediately following exercise. The alpha7B, but not alpha7A isoform, was found concentrated and colocalized with tenascin-C in muscle fibers lining the MTJ. To further validate the importance of the integrin in the protection against muscle damage following exercise, muscle injury was quantified in alpha7(-/-) mice. Muscle damage was extensive in alpha7(-/-) mice in response to both a single and repeated bouts of exercise and was largely restricted to areas of high MTJ concentration and high mechanical force near the Achilles tendon. These results suggest that exercise-induced muscle injury selectively increases transcription of the alpha7 integrin gene and promotes a rapid change in the alpha7beta integrin at the MTJ. These combined molecular and cellular alterations are likely responsible for integrin-mediated attenuation of exercise-induced muscle damage.

  13. GIPC interacts with the beta1-adrenergic receptor and regulates beta1-adrenergic receptor-mediated ERK activation.

    PubMed

    Hu, Liaoyuan A; Chen, Wei; Martin, Negin P; Whalen, Erin J; Premont, Richard T; Lefkowitz, Robert J

    2003-07-11

    Beta1-adrenergic receptors, expressed at high levels in the human heart, have a carboxyl-terminal ESKV motif that can directly interact with PDZ domain-containing proteins. Using the beta1-adrenergic receptor carboxyl terminus as bait, we identified the novel beta1-adrenergic receptor-binding partner GIPC in a yeast two-hybrid screen of a human heart cDNA library. Here we demonstrate that the PDZ domain-containing protein, GIPC, co-immunoprecipitates with the beta1-adrenergic receptor in COS-7 cells. Essential for this interaction is the Ser residue of the beta1-adrenergic receptor carboxyl-terminal ESKV motif. Our data also demonstrate that beta1-adrenergic receptor stimulation activates the mitogen-activated protein kinase, ERK1/2. beta1-adrenergic receptor-mediated ERK1/2 activation was inhibited by pertussis toxin, implicating Gi, and was substantially decreased by the expression of GIPC. Expression of GIPC had no observable effect on beta1-adrenergic receptor sequestration or receptor-mediated cAMP accumulation. This GIPC effect was specific for the beta1-adrenergic receptor and was dependent on an intact PDZ binding motif. These data suggest that GIPC can regulate beta1-adrenergic receptor-stimulated, Gi-mediated, ERK activation while having no effect on receptor internalization or Gs-mediated cAMP signaling.

  14. Integrin-like proteins are localized to plasma membrane fractions, not plastids, in Arabidopsis

    NASA Technical Reports Server (NTRS)

    Swatzell, L. J.; Edelmann, R. E.; Makaroff, C. A.; Kiss, J. Z.

    1999-01-01

    Integrins are a large family of integral membrane proteins that function in signal transduction in animal systems. These proteins are conserved in vertebrates, invertebrates, and fungi. Evidence from previous research suggests that integrin-like proteins may be present in plants as well, and that these proteins may function in signal transduction during gravitropism. In past studies, researchers have used monoclonal and polyclonal antibodies to localize beta 1 integrin-like proteins in plants. However, there is a disparity between data collected from these studies, especially since molecular weights obtained from these investigations range from 55-120 kDa for integrin-like proteins. To date, a complete investigation which employs all three basic immunolabeling procedures, immunoblotting, immunofluorescence microscopy, and immunogold labeling, in addition to extensive fractionation and exhaustive controls, has been lacking. In this paper, we demonstrate that use of a polyclonal antibody against the cytoplasmic domain of avian beta 1-integrin can produce potential artifacts in immunolocalization studies. However, these problems can be eliminated through use of starchless mutants or proper specimen preparation prior to electrophoresis. We also show that this antibody, when applied within the described parameters and with careful controls, identifies a large (100 kDa) integrin-like protein that is localized to plasma membrane fractions in Arabidopsis.

  15. Integrin-like proteins are localized to plasma membrane fractions, not plastids, in Arabidopsis

    NASA Technical Reports Server (NTRS)

    Swatzell, L. J.; Edelmann, R. E.; Makaroff, C. A.; Kiss, J. Z.

    1999-01-01

    Integrins are a large family of integral membrane proteins that function in signal transduction in animal systems. These proteins are conserved in vertebrates, invertebrates, and fungi. Evidence from previous research suggests that integrin-like proteins may be present in plants as well, and that these proteins may function in signal transduction during gravitropism. In past studies, researchers have used monoclonal and polyclonal antibodies to localize beta 1 integrin-like proteins in plants. However, there is a disparity between data collected from these studies, especially since molecular weights obtained from these investigations range from 55-120 kDa for integrin-like proteins. To date, a complete investigation which employs all three basic immunolabeling procedures, immunoblotting, immunofluorescence microscopy, and immunogold labeling, in addition to extensive fractionation and exhaustive controls, has been lacking. In this paper, we demonstrate that use of a polyclonal antibody against the cytoplasmic domain of avian beta 1-integrin can produce potential artifacts in immunolocalization studies. However, these problems can be eliminated through use of starchless mutants or proper specimen preparation prior to electrophoresis. We also show that this antibody, when applied within the described parameters and with careful controls, identifies a large (100 kDa) integrin-like protein that is localized to plasma membrane fractions in Arabidopsis.

  16. Cell adhesion and integrin expression are modulated by oxidative stress in EA.hy 926 cells.

    PubMed

    Lamari, Foudil; Braut-Boucher, Francoise; Pongnimitprasert, Nushjira; Bernard, Maguy; Foglietti, Marie-Jose; Derappe, Christian; Aubery, Michele

    2007-07-01

    The effects of oxidative stress on integrin-mediated cell adhesion to the extracellular matrix (ECM) and related apoptosis were investigated using the EA.hy926 endothelial cells treated (or not) with two oxidants: the hypoxanthine/xanthine oxidase system (HX/XO) or the tert-butyl hydroperoxide (t-BHP) which both increased cell apoptosis. Cell adhesion onto vitronectin (Vn) and fibronectin (Fn) was increased at low concentrations of HX/XO (up to 5 mU/ml) or t-BHP (up to 125 microM) and prevented ROS-induced apoptosis. Flow cytometry analysis of integrin expression showed that the expression of integrin alphav and alpha5 subunits was, respectively, increased and decreased. Cell adhesion inhibition experiments using function-blocking monoclonal antibodies against integrin subunits indicated that alphavbeta1 and alphavbeta3 integrins were involved in adhesion of cells to Vn, and alphavbeta3 integrin played a major role in oxidant-treated cells. For adhesion to Fn, alpha5beta1 and alphavbeta1 integrins were required for oxidant-treated cells. Taken together, the results suggest that reactive oxygen species (ROS) produced either by HX/XO or t-BHP could affect expression and/or activation of specific integrins in the interaction of EA.hy926 cells with ECM.

  17. Relative roles of TGF-beta1 and Wnt in the systemic regulation and aging of satellite cell responses.

    PubMed

    Carlson, Morgan E; Conboy, Michael J; Hsu, Michael; Barchas, Laurel; Jeong, Jaemin; Agrawal, Anshu; Mikels, Amanda J; Agrawal, Smita; Schaffer, David V; Conboy, Irina M

    2009-12-01

    Muscle stem (satellite) cells are relatively resistant to cell-autonomous aging. Instead, their endogenous signaling profile and regenerative capacity is strongly influenced by the aged P-Smad3, differentiated niche, and by the aged circulation. With respect to muscle fibers, we previously established that a shift from active Notch to excessive transforming growth factor-beta (TGF-beta) induces CDK inhibitors in satellite cells, thereby interfering with productive myogenic responses. In contrast, the systemic inhibitor of muscle repair, elevated in old sera, was suggested to be Wnt. Here, we examined the age-dependent myogenic activity of sera TGF-beta1, and its potential cross-talk with systemic Wnt. We found that sera TGF-beta1 becomes elevated within aged humans and mice, while systemic Wnt remained undetectable in these species. Wnt also failed to inhibit satellite cell myogenicity, while TGF-beta1 suppressed regenerative potential in a biphasic fashion. Intriguingly, young levels of TGF-beta1 were inhibitory and young sera suppressed myogenesis if TGF-beta1 was activated. Our data suggest that platelet-derived sera TGF-beta1 levels, or endocrine TGF-beta1 levels, do not explain the age-dependent inhibition of muscle regeneration by this cytokine. In vivo, TGF-beta neutralizing antibody, or a soluble decoy, failed to reduce systemic TGF-beta1 and rescue myogenesis in old mice. However, muscle regeneration was improved by the systemic delivery of a TGF-beta receptor kinase inhibitor, which attenuated TGF-beta signaling in skeletal muscle. Summarily, these findings argue against the endocrine path of a TGF-beta1-dependent block on muscle regeneration, identify physiological modalities of age-imposed changes in TGF-beta1, and introduce new therapeutic strategies for the broad restoration of aged organ repair.

  18. Effect of transforming growth factor-beta1 on decorin expression and muscle morphology during chicken embryonic and posthatch growth and development.

    PubMed

    Li, X; Velleman, S G

    2009-02-01

    During skeletal muscle development, transforming growth factor-beta1 (TGF-beta1) is a potent inhibitor of muscle cell proliferation and differentiation, as well as a regulator of extracellular matrix (ECM) production. Decorin, a member of the small leucine-rich ECM proteoglycans, binds to TGF-beta1 and modulates TGF-beta1-dependent cell growth stimulation or inhibition. The expression of decorin can be regulated by TGF-beta1 during muscle proliferation and differentiation. How TGF-beta1 affects decorin and muscle growth, however, has not been well documented in vivo. The present study investigated the effect of TGF-beta1 on decorin expression and intracellular connective tissue development during skeletal muscle growth. Exogenous TGF-beta1 significantly decreased the number of myofibers in a given area at both 1 d and 6 wk posthatch. The TGF-beta1-treated muscle had a significant decrease in decorin mRNA expression at embryonic day (ED) 10, whereas protein amounts decreased at 17 ED and 1 d posthatch compared to the control muscle. Decorin was localized in both the endomysium and perimysium in the control pectoralis major muscle. Transforming growth factor-beta1 reduced decorin in both the endomysium and perimysium from 17 ED to 6 wk posthatch. Compared to the control muscle, the perimysium space in the pectoralis major muscle was dramatically decreased by TGF-beta1 during embryonic development through posthatch growth. Because decorin regulates collagen fibrillogenesis, a major component of the ECM, the reduction of decorin by TGF-beta1 treatment may cause the irregular formation of collagen fibrils, leading to the decrease in endomysium and perimysium space. The results from the current study suggest that the effect of TGF-beta1 on decorin expression and localization was likely associated with altered development of the perimysium and the regulation of muscle fiber development.

  19. Alteration of pulmonary artery integrin levels in chronic hypoxia and monocrotaline-induced pulmonary hypertension.

    PubMed

    Umesh, Anita; Paudel, Omkar; Cao, Yuan-Ning; Myers, Allen C; Sham, James S K

    2011-01-01

    Pulmonary hypertension is associated with vascular remodeling and increased extracellular matrix (ECM) deposition. While the contribution of ECM in vascular remodeling is well documented, the roles played by their receptors, integrins, in pulmonary hypertension have received little attention. Here we characterized the changes of integrin expression in endothelium-denuded pulmonary arteries (PAs) and aorta of chronic hypoxia as well as monocrotaline-treated rats. Immunoblot showed increased α(1)-, α(8)- and α(v)-integrins, and decreased α(5)-integrin levels in PAs of both models. β(1)- and β(3)-integrins were reduced in PAs of chronic hypoxia and monocrotaline-treated rats, respectively. Integrin expression in aorta was minimally affected. Differential expression of α(1)- and α(5)-integrins induced by chronic hypoxia was further examined. Immunostaining showed that they were expressed on the surface of PA smooth muscle cells (PASMCs), and their distribution was unaltered by chronic hypoxia. Phosphorylation of focal adhesion kinase was augmented in PAs of chronic hypoxia rats, and in chronic hypoxia PASMCs cultured on the α(1)-ligand collagen IV. Moreover, α(1)-integrin binding hexapeptide GRGDTP elicited an enhanced Ca(2+) response, whereas the response to α(5)-integrin binding peptide GRGDNP was reduced in CH-PASMCs. Integrins in PASMCs are differentially regulated in pulmonary hypertension, and the dynamic integrin-ECM interactions may contribute to the vascular remodeling accompanying disease progression. Copyright © 2011 S. Karger AG, Basel.

  20. Alteration of Pulmonary Artery Integrin Levels in Chronic Hypoxia and Monocrotaline-Induced Pulmonary Hypertension

    PubMed Central

    Umesh, Anita; Paudel, Omkar; Cao, Yuan-Ning; Myers, Allen C.; Sham, James S.K.

    2011-01-01

    Background Pulmonary hypertension is associated with vascular remodeling and increased extracellular matrix (ECM) deposition. While the contribution of ECM in vascular remodeling is well documented, the roles played by their receptors, integrins, in pulmonary hypertension have received little attention. Here we characterized the changes of integrin expression in endothelium-denuded pulmonary arteries (PAs) and aorta of chronic hypoxia as well as monocrotaline-treated rats. Methods and Results Immunoblot showed increased α1-, α8- and αv-integrins, and decreased α5-integrin levels in PAs of both models. β1- and β3-integrins were reduced in PAs of chronic hypoxia and monocrotaline-treated rats, respectively. Integrin expression in aorta was minimally affected. Differential expression of α1- and α5-integrins induced by chronic hypoxia was further examined. Immunostaining showed that they were expressed on the surface of PA smooth muscle cells (PASMCs), and their distribution was unaltered by chronic hypoxia. Phosphorylation of focal adhesion kinase was augmented in PAs of chronic hypoxia rats, and in chronic hypoxia PASMCs cultured on the α1-ligand collagen IV. Moreover, α1-integrin binding hexapeptide GRGDTP elicited an enhanced Ca2+ response, whereas the response to α5-integrin binding peptide GRGDNP was reduced in CH-PASMCs. Conclusion Integrins in PASMCs are differentially regulated in pulmonary hypertension, and the dynamic integrin-ECM interactions may contribute to the vascular remodeling accompanying disease progression. PMID:21829038

  1. Cloning and characterization of the human phosphoinositide-specific phospholipase C-beta 1 (PLC beta 1).

    PubMed

    Caricasole, A; Sala, C; Roncarati, R; Formenti, E; Terstappen, G C

    2000-12-15

    Phospholipase C-beta (PLC beta) catalyses the generation of inositol 1,4,5-trisphosphate (IP(3)) and diacylglycerol (DAG) from phosphatidylinositol 4,5-bisphosphate (IP(2)), a key step in the intracellular transduction of a large number of extracellular signals, including neurotransmitters and hormones modulating diverse developmental and functional aspects of the mammalian central nervous system. Four mammalian isozymes are known (PLC beta 1-4), which differ in their function and expression patterns in vivo. We have characterized the human PLC beta 1 genomic locus (PLC beta 1), cloned two distinct PLC beta 1 cDNAs (PLC beta 1a and b) and analysed their respective expression patterns in a comprehensive panel of human tissues using quantitative TaqMan technology. The two cDNAs derive from transcripts generated through alternative splicing at their 3' end, and are predicted to encode for PLC beta 1 isoforms differing at their carboxy-terminus. The human PLC beta 1 isoforms are co-expressed in the same tissues with a distinctly CNS-specific profile of expression. Quantitative differences in PLC beta 1 isoform expression levels are observed in some tissues. Transient expression of epitope-tagged versions of the two isoforms followed by immunofluorescence revealed localization of the proteins to the cytoplasm and the inner side of the cell membrane. Finally, we characterized the structure of the PLC beta 1 locus and confirmed its mapping to human chromosome 20.

  2. Integrin endosomal signalling suppresses anoikis

    PubMed Central

    Alanko, Jonna; Mai, Anja; Jacquemet, Guillaume; Schauer, Kristine; Kaukonen, Riina; Saari, Markku; Goud, Bruno; Ivaska, Johanna

    2016-01-01

    Integrin containing focal adhesions (FAs) transmit extracellular signals across the plasma membrane to modulate cell adhesion, signalling and survival. Although integrins are known to undergo continuous endo/exocytic traffic, potential impact of endocytic traffic on integrin-induced signals is unknown. Here, we demonstrate that integrin signalling is not restricted to cell-ECM adhesions and identify an endosomal signalling platform that supports integrin signalling away from the plasma membrane. We show that active focal adhesion kinase (FAK), an established marker of integrin-ECM downstream signalling, localises with active integrins on endosomes. Integrin endocytosis positively regulates adhesion-induced FAK activation, which is early endosome antigen-1 (EEA1) and small GTPase Rab21 dependent. FAK binds directly to purified endosomes and becomes activated on them, suggesting a role for endocytosis in enhancing distinct integrin downstream signalling events. Finally, endosomal integrin signalling contributes to cancer-related processes such as anoikis resistance, anchorage-independence and metastasis. Integrins are heterodimeric cell surface adhesion receptors functioning as integrators of the extra-cellular matrix (ECM) driven cues, the cellular cytoskeleton and the cellular signalling apparatus 1.Upon adhesion, integrins trigger the formation of plasma-membrane proximal large mechanosensing and signal-transmitting protein clusters depicted as “adhesomes” 2, 3. In addition, integrins undergo constant endocytic traffic to facilitate focal adhesion turnover, cell migration, invasion and cytokinesis 4. For other receptor systems it is well established that endocytic membrane traffic regulates bioavailability of cell-surface molecules and therefore the intensity and/or specificity of receptor-initiated signals 5, 6. Although active integrins and their ligands have been detected in endosomes 7–9 and increased integrin recycling to the plasma membrane contributes

  3. Pulmonary administration of integrin-nanoparticles regenerates collapsed alveoli.

    PubMed

    Horiguchi, Michiko; Kojima, Hisako; Sakai, Hitomi; Kubo, Hiroshi; Yamashita, Chikamasa

    2014-08-10

    Chronic obstructive pulmonary disease (COPD) is an intractable pulmonary disease, causes widespread and irreversible alveoli collapse. In search of a treatment target molecule, which is able to regenerate collapsed alveoli, we sought to identify a factor that induces differentiation in human alveolar epithelial stem cells using all-trans retinoic acid (ATRA), whose alveolar repair capacity has been reported in animal experiments. When human alveolar epithelial stem cells were exposed to ATRA at a concentration of 10μM for over seven days, approximately 20% of the cells differentiated into each of the type-I and type-II alveolar epithelial cells that constitute the alveoli. In a microarray analysis, integrin-α1 and integrin-β3 showed the largest variation in the ATRA-treated group compared with the controls. Furthermore, the effect of the induction of differentiation in human alveolar epithelial stem cells using ATRA was suppressed by approximately one-fourth by siRNA treatments with integrin α1 and integrin β3. These results suggested that integrin α1 and β3 are factors responsible for the induction of differentiation in human alveolar epithelial stem cells. We accordingly investigated whether integrin nanoparticles also had a regenerative effect in vivo. Elastase-induced COPD model mouse was produced, and the alveolar repair effect of pulmonary administration using nanoparticles of integrin protein was evaluated by X-ray CT scanning. Improvement in the CT value in comparison with an untreated group indicated that there was an alveolar repair effect. In this study, it was shown that the differentiation-inducing effect on human alveolar epithelial stem cells by ATRA was induced by increased expression of integrin, and that the induced integrin enhanced phosphorylation signaling of AKT, resulting in inducing differentiations. Furthermore, the study demonstrated that lung administration of nanoparticles with increased solubility and stability of integrin

  4. Epithelial-mesenchymal interactions and lung branching morphogenesis. Role of polyamines and transforming growth factor beta1.

    PubMed

    Stabellini, G; Locci, P; Calvitti, M; Evangelisti, R; Marinucci, L; Bodo, M; Caruso, A; Canaider, S; Carinci, P

    2001-01-01

    Lung branching morphogenesis is a result of epithelial-mesenchymal interactions, which are in turn dependent on extracellular matrix composition and cytokine regulation. Polyamines have recently been demonstrated as able to modify chick embryo skin differentiation. In this work we have examined the effects of putrescine and spermidine during chick embryo lung morphogenesis in organotypic cultures by morphological, histochemical and biochemical examination. To verify the role of polyamines, we used specific inhibitors, such as bis-cyclohexylammonium sulphate and alfa-difluoromethylornithine, and transforming growth factor beta1, an ornithine decarboxylase and polyamine stimulator. Our data show that lung morphogenesis is significantly altered following the induced mesenchymal glycosaminoglycan changes. The increase of mesenchymal glycosaminoglycans is correlated with a stimulation of lung development in the presence of polyamines, and with its inhibition when transforming growth factor beta1 is added to the culture medium. The morphometric data show a uniform increase of both the mesenchyme and epithelial branching with spermidine and putrescine stimulus, whereas the mesenchymal substance alone is significantly increased in apical-median lung sections with transforming growth factor beta1 and transforming growth factor beta1 + spermidine lung cultures. Transforming growth factor beta1 and transforming growth factor beta1 + spermidine confirm the blocking of epithelial branching formations and fibroblast activation, and show that polyamines are unable to prevent the blocking of epithelial cells due to the inhibitory effect of transforming growth factor beta1.

  5. RhoC is essential for TGF-{beta}1-induced invasive capacity of rat ascites hepatoma cells

    SciTech Connect

    Mukai, M.; Endo, H.; Iwasaki, T.; Tatsuta, M.; Togawa, A.; Nakamura, H.; Inoue, M. . E-mail: inoue-ma2@mc.pref.osaka.jp

    2006-07-21

    Transforming growth factor-{beta}1 (TGF-{beta}1) is a multifunctional growth factor that plays a role in cell proliferation, differentiation, extracellular matrix production, apoptosis, and cell motility. We show here that TGF-{beta}1 increased the invasiveness of MM1 cells, which are a highly invasive clone of rat ascites hepatoma cells. Both mRNA and protein levels of RhoC but not RhoA in TGF-{beta}1-treated MM1 cells increased. In parallel with this increase in expression, RhoC activity was induced by TGF-{beta}1 treatment. When RhoC was overexpressed in MM1 cells, the invasive capacity increased. The RhoC-overexpressing cells formed more nodules than did mock cells when injected into rat peritoneum. Furthermore, when RhoC expression was reduced by transfection with shRNA/RhoC, the invasiveness of MM1 cells decreased with concomitant suppression of RhoC expression. Thus, the induced expression of RhoC by TGF-{beta}1 in MM1 cells plays a critical role in TGF-{beta}1-induced cell migration.

  6. Integrin β1 regulates leiomyoma cytoskeletal integrity and growth

    PubMed Central

    Malik, Minnie; Segars, James; Catherino, William H.

    2014-01-01

    Uterine leiomyomas are characterized by an excessive extracellular matrix, increased mechanical stress, and increased active RhoA. Previously, we observed that mechanical signaling was attenuated in leiomyoma, but the mechanisms responsible remain unclear. Integrins, especially integrin β1, are transmembrane adhesion receptors that couple extracellular matrix stresses to the intracellular cytoskeleton to influence cell proliferation and differentiation. Here we characterized integrin and laminin to signaling in leiomyoma cells. We observed a 2.25 ± 0.32 fold increased expression of integrin β1 in leiomyoma cells, compared to myometrial cells. Antibody-mediated inhibition of integrin β1 led to significant growth inhibition in leiomyoma cells and a loss of cytoskeletal integrity. Specifically, polymerization of actin filaments and formation of focal adhesions were reduced by inhibition of integrin p1. Inhibition of integrin β1 in leiomyoma cells led to 0.81 ± 0.02 fold decrease in active RhoA, and resembled levels found in serum-starved cells. Likewise, inhibition of integrin β1 was accompanied by a decrease in phospho-ERK. Compared to myometrial cells, leiomyoma cells demonstrated increased expression of integrin α6 subunit to laminin receptor (1.91 ± 0.11 fold), and increased expression of laminin 5α (1.52±0.02), laminin 5β (3.06±0.92), and laminin 5γ (1.66 ± 0.06). Of note, leiomyoma cells grown on laminin matrix appear to realign themselves. Taken together, the findings reveal that the attenuated mechanical signaling in leiomyoma cells is accompanied by an increased expression and a dependence on integrin β1 signaling in leiomyoma cells, compared to myometrial cells. PMID:23023061

  7. Paxillin binding to the alpha 4 integrin subunit stimulates LFA-1 (integrin alpha L beta 2)-dependent T cell migration by augmenting the activation of focal adhesion kinase/proline-rich tyrosine kinase-2.

    PubMed

    Rose, David M; Liu, Shouchun; Woodside, Darren G; Han, Jaewon; Schlaepfer, David D; Ginsberg, Mark H

    2003-06-15

    Engagement of very late Ag-4 (integrin alpha(4)beta(1)) by ligands such as VCAM-1 markedly stimulates leukocyte migration mediated by LFA-1 (integrin alpha(L)beta(2)). This form of integrin trans-regulation in T cells requires the binding of paxillin to the alpha(4) integrin cytoplasmic domain. This conclusion is based on the abolition of trans-regulation in Jurkat T cells by an alpha(4) mutation (alpha(4)(Y991A)) that disrupts paxillin binding. Furthermore, cellular expression of an alpha(4)-binding fragment of paxillin that blocks the alpha(4)-paxillin interaction, selectively blocked VCAM-1 stimulation of alpha(L)beta(2)-dependent cell migration. The alpha(4)-paxillin association mediates trans-regulation by enhancing the activation of tyrosine kinases, focal adhesion kinase (FAK) and/or proline-rich tyrosine kinase-2 (Pyk2), based on two lines of evidence. First, disruption of the paxillin-binding site in the alpha(4) tail resulted in much less alpha(4)beta(1)-mediated phosphorylation of Pyk2 and FAK. Second, transfection with cDNAs encoding C-terminal fragments of Pyk2 and FAK, which block the function of the intact kinases, blocked alpha(4)beta(1) stimulation of alpha(L)beta(2)-dependent migration. These results define a proximal protein-protein interaction of an integrin cytoplasmic domain required for trans-regulation between integrins, and establish that augmented activation of Pyk2 and/or FAK is an immediate signaling event required for the trans-regulation of integrin alpha(L)beta(2) by alpha(4)beta(1).

  8. Integrating with integrins

    NASA Technical Reports Server (NTRS)

    Schwartz, M. A.; Ingber, D. E.

    1994-01-01

    Our central claim is that signaling by integrins provides a mechanism by which signals generated in response to adhesion, soluble hormones, and mechanical forces can interact. Such interactions permit cells to integrate these different classes of external stimuli and hence to orchestrate an efficient response. This integrating function of integrins is likely to be essential for much of development and physiology, as well as complex pathologies such as cancer. Understanding in detail how these signals are transduced and processed is likely to be an important area of research in the near future.

  9. PKCalpha-induced drug resistance in pancreatic cancer cells is associated with transforming growth factor-beta1.

    PubMed

    Chen, Ying; Yu, Guanzhen; Yu, Danghui; Zhu, Minghua

    2010-08-05

    Drug resistance remains a great challenge in the treatment of pancreatic cancer. The goal of this study was to determine whether TGF-beta1 is associated with drug resistance in pancreatic cancer. Pancreatic cancer BxPC3 cells were stably transfected with TGF-beta1 cDNA. Cellular morphology and cell cycle were determined and the suppressive subtracted hybridization (SSH) assay was performed to identify differentially expressed genes induced by TGF-beta1. Western blotting and immunohistochemistry were used to detect expression of TGF-beta1-related genes in the cells and tissue samples. After that, the cells were further treated with an anti-cancer drug (e.g., cisplatin) after pre-incubated with the recombinant TGF-beta1 plus PKCalpha inhibitor Gö6976. TGF-beta1 type II receptor, TbetaRII was also knocked down using TbetaRII siRNA to assess the effects of these drugs in the cells. Cell viability was assessed by MTT assay. Overexpression of TGF-beta1 leads to a markedly increased invasion potential but a reduced growth rate in BxPC3 cells. Recombinant TGF-beta1 protein increases expression of PKCalpha in BxPC3 cells, a result that we confirmed by SSH. Moreover, TGF-beta1 reduced the sensitivity of BxPC3 cells to cisplatin treatment, and this was mediated by upregulation of PKCalpha. However, blockage of PKCalpha with Gö6976 and TbetaRII with siRNA reversed the resistance of BxPC3 cells to gemcitabine, even in the presence of TGF-beta1. Immunohistochemical data show that pancreatic cancers overexpress TGF-beta1 and P-gp relative to normal tissues. In addition, TGF-beta1 expression is associated with P-gp and membranous PKCalpha expression in pancreatic cancer. TGF-beta1-induced drug resistance in pancreatic cancer cells was associated with PKCalpha expression. The PKCalpha inhibitor Gö6976 could be a promising agent to sensitize pancreatic cancer cells to chemotherapy.

  10. Critical role of integrin-linked kinase in granule cell precursor proliferation and cerebellar development.

    PubMed

    Mills, Julia; Niewmierzycka, Agnieszka; Oloumi, Arusha; Rico, Beatriz; St-Arnaud, Rene; Mackenzie, Ian R; Mawji, Nasrin M; Wilson, Jason; Reichardt, Louis F; Dedhar, Shoukat

    2006-01-18

    Integrin-linked kinase (ILK) is a serine/threonine protein kinase that plays an important role in integrin signaling and cell proliferation. We used Cre recombinase (Cre)-loxP technology to study CNS restricted knock-out of the ilk gene by either Nestin-driven or gfap-driven Cre-mediated recombination. Developmental changes in ilk-excised brain regions are similar to those observed in mice lacking the integrin beta1 subunit in the CNS, including defective laminin deposition, abnormal glial morphology, and alterations in granule cell migration. Decreases in 6-bromodeoxyuridine (BrdU) pulse labeling and proliferating cell nuclear antigen expression in the external granule cell layer of the cerebellum demonstrated that proliferation is disrupted in granule cells lacking ILK. Previous studies have shown that laminin-sonic hedgehog (Shh)-induced granule cell precursor (GCP) proliferation is dependent on beta1 integrins, several of which bind laminin and interact with ILK through the beta1 cytoplasmic domain. Both ex vivo deletion of ilk and a small molecule inhibitor of ILK kinase activity decreased laminin-Shh-induced BrdU labeling in cultured GCPs. Together, these results implicate ILK as a critical effector in a signaling pathway necessary for granule cell proliferation and cerebellar development.

  11. Integrin-linked kinase stabilizes myotendinous junctions and protects muscle from stress-induced damage.

    PubMed

    Wang, Hao-Ven; Chang, Ling-Wei; Brixius, Klara; Wickström, Sara A; Montanez, Eloi; Thievessen, Ingo; Schwander, Martin; Müller, Ulrich; Bloch, Wilhelm; Mayer, Ulrike; Fässler, Reinhard

    2008-03-10

    Skeletal muscle expresses high levels of integrin-linked kinase (ILK), predominantly at myotendinous junctions (MTJs) and costameres. ILK binds the cytoplasmic domain of beta1 integrin and mediates phosphorylation of protein kinase B (PKB)/Akt, which in turn plays a central role during skeletal muscle regeneration. We show that mice with a skeletal muscle-restricted deletion of ILK develop a mild progressive muscular dystrophy mainly restricted to the MTJs with detachment of basement membranes and accumulation of extracellular matrix. Endurance exercise training enhances the defects at MTJs, leads to disturbed subsarcolemmal myofiber architecture, and abrogates phosphorylation of Ser473 as well as phosphorylation of Thr308 of PKB/Akt. The reduction in PKB/Akt activation is accompanied by an impaired insulin-like growth factor 1 receptor (IGF-1R) activation. Coimmunoprecipitation experiments reveal that the beta1 integrin subunit is associated with the IGF-1R in muscle cells. Our data identify the beta1 integrin-ILK complex as an important component of IGF-1R/insulin receptor substrate signaling to PKB/Akt during mechanical stress in skeletal muscle.

  12. Sheep (Ovis aries) integrins alphavbeta1 and alphavbeta6 related to foot-and-mouth disease virus infection: molecular cloning, sequence analysis and comparison with homologues.

    PubMed

    Du, Junzheng; Chang, Huiyun; Gao, Shandian; Cong, Guozheng; Shao, Junjun; Lin, Tong; Liu, Zaixin; Liu, Xiangtao; Cai, Xuepeng

    2009-10-01

    Four members of the alphav integrin family of cellular receptors, alphavbeta1, alphavbeta3, alphavbeta6, and alphavbeta8, have been identified as receptors for foot-and-mouth disease virus (FMDV) in vitro, and integrins are believed to be the receptors used to target epithelial cells in the infected animals. To analyse roles of the alphav integrins from a susceptible species as viral receptors, we have cloned sheep alphav, beta1, and beta6 integrin cDNAs and compared them to those of other species. The coding sequences for sheep integrin alphav, beta1, and beta6 were found to be 3147, 2397, and 2364 nuclotides in length, encoding 1048, 798, and 787 amino acids, respectively. The sheep alphav, beta1, and beta6 subunits share many structural features including ligand binding domain and cysteine-rich region with homologues of other species. Phylogenetic trees and similarity analyses showed the close relationship of integrin genes among sheep, pigs, cattle and Bactrian camels that are susceptible to FMDV infection, which were distinct from the order Rodentia, Primates, Perissodactyla, Carnivora, Galliformes. We postulate that host tropism of FMDV may be related to divergence in integrin subunits among different species.

  13. The tumor microenvironment: possible role of integrins and the extracellular matrix in tumor biological behavior of intratubular germ cell neoplasia and testicular seminomas.

    PubMed Central

    Timmer, A.; Oosterhuis, J. W.; Schraffordt Koops, H.; Sleijfer, D. T.; Szabo, B. G.; Timens, W.

    1994-01-01

    In the present study, we examined the distribution of integrin subunits and extracellular matrix proteins in normal testis, intratubular germ cell neoplasia (ITGCN), and primary and metastatic seminomas. Compared to normal testis in ITGCN, Sertoli cells showed increased expression of alpha 3, alpha 6, and beta 1 integrin subunits. Malignant intratubular germ cells stained for alpha 3, alpha 6, and beta 1 integrin subunits. Progression of ITGCN to invasive seminoma was associated with loss of alpha 3 integrin subunit expression by tumor cells. Consequent to this loss, it can be speculated that the strong expression on ITGCN may be related to the noninvasive character of the lesion as is also known from other noninvasive tumors. All tumors showed a strong expression of alpha 6 and beta 1 integrin subunits. The alpha 5 integrin subunit was weakly expressed in primary seminomas in all stages. No differences were observed in integrin expression between primary and metastatic tumors. The distribution of extracellular matrix proteins was heterogeneous and revealed clear architectural differences between seminomas that may reflect different stages of tumor stroma formation. To our knowledge, the results presented in this study provide the first information on the possible role of tumor-extracellular matrix interactions in the biological behavior of ITGCN and testicular seminomas. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8178927

  14. Role of transforming growth factor-[beta]1 in inhibiting endothelial cell proliferation in experimental alcoholic liver disease.

    PubMed Central

    Nanji, A. A.; Tahan, S. R.; Golding, M.; Khwaja, S.; Rahemtulla, A.; Lalani, E. N.

    1996-01-01

    We used the intragastric feeding rat model for alcoholic liver disease to investigate the relationship between transforming growth factor (TGF)-beta 1 and inhibition of endothelial cell proliferation. Twelve groups of male Wistar rats (four to five rats per group) were fed ethanol or dextrose with either corn oil or saturated fat for 1-, 2-, and 4-week periods. All control animals were pair fed the same diets as ethanol-fed rats except that ethanol was isocalorically replaced by dextrose. In the ethanol-fed groups, nonparenchymal cells were isolated and TGF-beta 1 was measured in the nonparenchymal cell supernatant. Liver pathology and endothelial cell proliferation with an antibody to proliferating cell nuclear antigen were studied in all groups. Plasma TGF-beta 1 was measured in all rats. Pathological changes (fatty liver, necrosis, and inflammation) were observed only in the corn oil/ethanol-fed rats at 4 weeks. Significantly higher levels of TGF-beta 1 were seen in both plasma and nonparenchymal cell supernatant in rats fed corn oil and ethanol; plasma levels of TGF-beta 1 were not significantly different between the dextrose-fed controls and saturated fat/ethanol-fed rats. A significant inverse correlation (r = -0.89, P < 0.01) was seen between plasma TGF-beta 1 and the number of endothelial cells arrested at G1/S. Immunohistochemistry revealed the presence of TGF-beta 1 staining in interstitial macrophages only in rats fed corn oil and ethanol. The present study provides evidence for a role for TGF-beta 1 in inhibiting endothelial cell proliferation in experimental alcoholic liver disease. Arrest of endothelial cells may lead to their differentiation and/or to produce mediators that could stimulate other cells such as Ito cells. Sustained TGF-beta 1 may also lead to Ito cell production of extracellular matrix. Images Figure 1 Figure 2 Figure 4 Figure 5 Figure 6 Figure 7 PMID:8774130

  15. Reorganization of endothelial cord-like structures on basement membrane complex (Matrigel): involvement of transforming growth factor beta 1.

    PubMed

    Kuzuya, M; Kinsella, J L

    1994-11-01

    The formation of capillary-like network structures by cultured vascular endothelial cells on reconstituted basement membrane matrix, Matrigel, models endothelial cell differentiation, the final step of angiogenesis (Kubota et al., 1988; Grant et al., 1989). When endothelial cells derived from bovine aorta and brain capillaries were plated on Matrigel, DNA synthesis was suppressed and a network of capillary-like structures rapidly formed in 8-12 h. With time, the network broke down, resulting in dense cellular cords radiating from multiple cellular clusters in 16-24 h. Finally, multicellular aggregates of cells were formed as the network underwent further retraction. Network regression was prevented when either dithiothreitol (DTT) or anti-TGF-beta 1 antibodies were added during the assay. The addition of exogenous TGF-beta 1 promoted the regression of endothelial cells into the clusters. This response to TGF-beta 1 was blocked by potent serine threonine protein kinase inhibitors, H-7 and HA100. TGF-beta 1 was released from polymerized Matrigel by incubation with Dulbecco's modified eagle's medium (DMEM) in the absence of cells. The Matrigel-conditioned DMEM inhibited endothelial DNA synthesis even in the presence of anti-TGF-beta 1 antibodies. These results suggest that TGF-beta 1 and possibly other soluble factors from Matrigel may be important for differentiation and remodeling of endothelial cells in a capillary network with possible implications for wound healing and development.

  16. Nuclear phosphoinositide specific phospholipase C (PI-PLC)-beta 1: a central intermediary in nuclear lipid-dependent signal transduction.

    PubMed

    Martelli, A M; Fiume, R; Faenza, I; Tabellini, G; Evangelista, C; Bortul, R; Follo, M Y; Falà, F; Cocco, L

    2005-10-01

    Several studies have demonstrated the existence of an autonomous intranuclear phospho-inositide cycle that involves the activation of nuclear PI-PLC and the generation of diacylglycerol (DG) within the nucleus. Although several distinct isozymes of PI-PLC have been detected in the nucleus, the isoform that has been most consistently highlighted as being nuclear is PI-PLC-beta1. Nuclear PI-PLC-beta1 has been linked with either cell proliferation or differentiation. Remarkably, the activation mechanism of nuclear PI-PLC-beta1 has been shown to be different from its plasma membrane counterpart, being dependent on phosphorylation effected by p44/42 mitogen activated protein (MAP) kinase. In this review, we report the most up-dated findings about nuclear PI-PLC-beta1, such as the localization in nuclear speckles, the activity changes during the cell cycle phases, and the possible involvement in the progression of myelodisplastic syndrome to acute myeloid leukemia.

  17. Integrin dynamics on the tail region of migrating fibroblasts.

    PubMed

    Palecek, S P; Schmidt, C E; Lauffenburger, D A; Horwitz, A F

    1996-05-01

    Cell migration is a complex process that can be considered as a repeated cycle of lamellipod extension and attachment, cytoskeletal contraction, and tail detachment. While lamellipodial and cytoskeletal phenomena are currently the focus of considerable research on cell migration, under many conditions locomotion appears to be rate-limited by events at the cell rear, especially release of cell/substratum adhesions. To study the mechanism of tail detachment, we have developed a novel experimental system that permits observation of integrin dynamics on the ventral surface of migrating fibroblasts. Photoactivatable caged fluorescein is coupled to a non-adhesion-perturbing anti-avian-beta 1 integrin subunit antibody, which labels integrins on chicken fibroblasts migrating on a laminin-coated glass coverslip. Ultraviolet light is focused through a pinhole to photoactivate the caged fluorophore in a 10-micron-diameter spot at the rear of a polarized cell. The fate of integrins initially present in this spot is monitored using a cooled CCD camera to follow the movement of fluorescent intensity as a function of time over a 2 to 3 hour period. We find that a substantial fraction of the integrins is left behind on the substratum as the cell detaches and locomotes, while another fraction collects into vesicles which are transported along the cell body as the cell migrates. As aggregates rip from the cell membrane, the integrin-cytoskeletal bonds are preferentially fractured resulting in 81 +/- 15% of the integrin remaining attached to the substratum. We additionally find that adhesions sometimes disperse into integrins which can form new adhesions at other locations in the cell. Adhesions along the cell edge can release from the substrate and translocate with the cell. They either disperse in the cell membrane, rip from the cell membrane and remain attached to the substratum, or form a new aggregate. These observations indicate that the behavior of integrins at the cell rear

  18. Reduced growth and integrin expression of prostate cells cultured with lycopene, vitamin E and fish oil in vitro.

    PubMed

    Bureyko, T; Hurdle, H; Metcalfe, J B; Clandinin, M T; Mazurak, Vera C

    2009-04-01

    Integrins are transmembrane proteins that facilitate the interaction of cells with the extracellular environment. They have also been implicated in cancer progression. The effects of nutrients thought to be involved in the prevention of prostate cancer on integrin expression have not been determined. Prostate cancer cell lines representing a range of malignancy from normal (RWPE-1) to highly invasive phenotypes (22Rv1 < LNCaP < PC-3) were cultured with or without lycopene (10 nM), vitamin E (5 microm) or fish oil (100 microm) for 48 h. Growth and integrin (alpha2beta1, alphavbeta3 and alphavbeta5) expression were assessed using Trypan Blue exclusion and monoclonal antibodies combined with flow cytometry. Vitamin E enhanced (P < 0.001) whereas fish oil reduced the growth of all the cell lines tested (P < 0.001). Lycopene had no effect on growth. All the malignant cell lines exhibited lower expression of alpha2beta1 with the addition of lycopene to culture media. Supplemental fish oil reduced alpha2beta1 in most invasive cell lines (LNCaP and PC-3). Each nutrient at physiological levels reduced integrins alphavbeta3 and alphavbeta5 in most invasive cell lines (PC-3). The results suggest that integrins may represent an additional target of bioactive nutrients and that the effects of nutrients may be dependent on the type of cell line used.

  19. Antisense targeting of TGF-{beta}1 augments BMP-induced upregulation of osteopontin, type I collagen and Cbfa1 in human Saos-2 cells

    SciTech Connect

    Shen, Zhong-Jian . E-mail: zshen2@wisc.edu; Kook Kim, Sang; Youn Jun, Do; Park, Wan; Ho Kim, Young; Malter, James S.; Jo Moon, Byung . E-mail: bjmoon@mail.knu.ac.kr

    2007-04-15

    Despite commonalities in signal transduction in osteoblasts from different species, the role of TGF-{beta}1 on bone formation remains elusive. In particular, the role of autocrine TGF-{beta}1 on human osteoblasts is largely unknown. Here we show the effect of TGF-{beta}1 knock-down on the proliferation and differentiation of osteoblasts induced by BMP2. Treatment with antisense TGF-{beta}1 moderately increased the rate of cell proliferation, which was completely reversed by the exogenous addition of TGF-{beta}1. Notably, TGF-{beta}1 blockade significantly enhanced BMP2-induced upregulation of mRNAs encoding osteopontin, type I collagen and Cbfa1, which was suppressed by exogenous TGF-{beta}1. Moreover, TGF-{beta}1 knock-down increased BMP2-induced phosphorylation of Smad1/5 as well as their nuclear import, which paralleled a reduction of inhibitory Smad6. These data suggest autocrine TGF-{beta}1 antagonizes BMP signaling through modulation of inducible Smad6 and the activity of BMP specific Smad1/5.

  20. Integrin-β1 regulates chondrocyte proliferation and apoptosis through the upregulation of GIT1 expression.

    PubMed

    Zhang, Long-Qiang; Zhao, Guang-Zong; Xu, Xiao-Yan; Fang, Jun; Chen, Jing-Ming; Li, Ji-Wen; Gao, Xue-Jian; Hao, Li-Juan; Chen, Yun-Zhen

    2015-04-01

    Chondrocytes play a critical role in the repair process of osteoarthritis, which is also known as degenerative arthritis. Integrins, as the key family of cell surface receptors, are responsible for the regulation of chondrocyte proliferation, differentiation, survival and apoptosis through the recruitment and activation of downstream adaptor proteins. Moreover, G-protein-coupled receptor kinase interacting protein-1 (GIT1) exerts its effects on cell proliferation and migration through interaction with various cytokines. It has been previously suggested that GIT1 acts as a vital protein downstream of the integrin-mediated pathway. In the present study, we investigated the effects of integrin-β1 on cell proliferation and apoptosis, as well as the underlying mechanisms in chondrocytes in vitro. Following transfection with a vector expressing integrin-β1, our results revealed that the overexpression of integrin-β1 enhanced GIT1 expression, whereas the knockdown of integrin-β1 by siRNA suppressed GIT1 expression. However, no significant effect was observed on integrin-β1 expression following the enforced overexpression of GIT1, which suggests that GIT1 is localized downstream of integrin-β1. In other words, integrin-β1 regulates the expression of GIT1. Furthermore, this study demonstrated that integrin-β1 and GIT1 increased the expression levels of aggrecan and type II collagen, thus promoting chondrocyte proliferation; however, they inhibited chondrocyte apoptosis. Taken together, our data demonstrate that integrin-β1 plays a vital role in chondrocyte proliferation, differentiation and apoptosis. GIT1 exerts effects similar to those of integrin-β1 and is a downstream target of integrin-β1.

  1. Conformational mAb as a tool for integrin ligand discovery.

    PubMed

    Njus, Ben H; Chigaev, Alexandre; Waller, Anna; Wlodek, Danuta; Ostopovici-Halip, Liliana; Ursu, Oleg; Wang, Wei; Oprea, Tudor I; Bologa, Cristian G; Sklar, Larry A

    2009-10-01

    alpha(4)beta(1)-Integrin (very late antigen-4 (VLA-4)) mediates cell adhesion to cell surface ligands (VCAM-1). Binding of VLA-4 to VCAM-1 initiates rolling and firm adhesion of leukocytes to vascular endothelium followed by the extravasation into the tissue. VLA-4-dependent adhesion plays a key role in controlling leukocyte adhesive events. Small molecules that bind to the integrin ligand-binding site and block its interaction with natural ligands represent promising candidates for treatment of several diseases. Following a flow cytometric screen for small molecule discovery, we took advantage of a conformationally sensitive anti-beta(1)-integrin antibody (HUTS-21) and a small LDV-containing ligand (LDV-FITC) with known affinity to study binding affinities of several known and recently discovered integrin ligands. We found that binding of the LDV-containing small molecule induced exposure of HUTS-21 epitope and that the EC(50) for antibody binding was equal to previously reported K(d) for fluorescent LDV (LDV-FITC). Thus, binding of HUTS-21 can be used to report ligand-binding site occupancy. We studied binding of two known integrin ligands (YLDV and TR14035), as well as of two novel compounds. EC(50) values for HUTS-21 binding showed good correlation with K(i)s determined in the competition assay with LDV-FITC for all ligands. A docking model suggests a common mode of binding for the small molecule VLA-4 ligands. This novel approach described here can be used to determine ligand-binding affinities for unlabeled integrin ligands, and can be adapted to a high-throughput screening format for identification of unknown integrin ligands.

  2. Suramin inhibits growth and transforming growth factor-beta 1 (TGF-beta 1) binding in osteosarcoma cell lines.

    PubMed

    Kloen, P; Jennings, C L; Gebhardt, M C; Springfield, D S; Mankin, H J

    1994-01-01

    Autocrine production of growth factors has been shown to be involved in the multistep process of tumorigenesis. The ability of suramin, a polyanionic anti-parasitic drug, to block growth factor-induced cell proliferation makes it a potential antineoplastic drug. We studied the effects of suramin on seven osteosarcoma cell lines. Using clinically achievable concentrations of suramin (50-400 micrograms/ml), we found a time- and dose-dependent inhibition of [3H]thymidine incorporation. We also showed that suramin is able, dose-dependently, to prevent binding of transforming growth factor (TGF)-beta 1 to its receptors. DNA synthesis inhibition by suramin was attenuated by TGF-beta 1 in some cell lines. Two cell lines that were inhibited by TGF-beta 1 were affected similarly by suramin as cell lines that were stimulated by TGF-beta 1. In conclusion, in five out of seven osteosarcoma cell lines, we showed a correlation between inhibition of growth factor-stimulated mitogenesis and binding of TGF-beta 1 to its receptor. Similar effects in TGF-beta 1-inhibited osteosarcoma cell lines suggest involvement of other mechanisms and/or growth factors. However, suramin proves to be a potent inhibitor of osteosarcoma cell proliferation in vitro.

  3. Lumican induces human corneal epithelial cell migration and integrin expression via ERK 1/2 signaling

    SciTech Connect

    Seomun, Young; Joo, Choun-Ki

    2008-07-18

    Lumican is a major proteoglycans of the human cornea. Lumican knock-out mice have been shown to lose corneal transparency and to display delayed wound healing. The purpose of this study was to define the role of lumican in corneal epithelial cell migration. Over-expression of lumican in human corneal epithelial (HCE-T) cells increased both cell migration and proliferation, and increased levels of integrins {alpha}2 and {beta}1. ERK 1/2 was also activated in lumican over-expressed cells. When we treated HCE-T cells with the ERK-specific inhibitor U0126, cell migration and the expression of integrin {beta}1 were completely blocked. These data provide evidence that lumican stimulates cell migration in the corneal epithelium by activating ERK 1/2, and point to a novel signaling pathway implicated in corneal epithelial cell migration.

  4. Tumor suppressor KAI1 affects integrin {alpha}v{beta}3-mediated ovarian cancer cell adhesion, motility, and proliferation

    SciTech Connect

    Ruseva, Zlatna; Geiger, Pamina Xenia Charlotte; Hutzler, Peter; Kotzsch, Matthias; Luber, Birgit; Schmitt, Manfred; Gross, Eva; Reuning, Ute

    2009-06-10

    The tetraspanin KAI1 had been described as a metastasis suppressor in many different cancer types, a function for which associations of KAI1 with adhesion and signaling receptors of the integrin superfamily likely play a role. In ovarian cancer, integrin {alpha}v{beta}3 correlates with tumor progression and its elevation in vitro provoked enhanced cell adhesion accompan