Science.gov

Sample records for beta1 integrins differentially

  1. RhoA-dependent switch between alpha2beta1 and alpha3beta1 integrins is induced by laminin-5 during early stage of HT-29 cell differentiation.

    PubMed

    Gout, S P; Jacquier-Sarlin, M R; Rouard-Talbot, L; Rousselle, P; Block, M R

    2001-10-01

    Integrin-mediated interactions between the basement membrane and epithelial cells control the differentiation of epithelia. We characterized the modulation of adhesive behaviors to basement membrane proteins and of integrin function in the human colon adenocarcinoma HT-29 cell line, which differentiates into enterocytes after the substitution of galactose for glucose in the medium. We demonstrate an increased capability of these cells to adhere to collagen type IV during the early stage of differentiation. This effect occurs without any changes in integrin cell surface expression but rather results from an alpha2beta1/alpha3beta1 integrin switch, alpha3beta1 integrin becoming the major collagen receptor. The increase in laminin-5 secretion and deposit on the matrix is a key factor in the mechanism regulating cell adhesion, because it is responsible for the activation of alpha3beta1 integrin. Furthermore, down-regulation of RhoA GTPase activity occurs during HT-29 cell differentiation and correlates with the activation of the integrin alpha3beta1. Indeed, C3 transferase, a RhoA GTPase inhibitor, induces a similar alpha2beta1/alpha3beta1 switch in undifferentiated HT-29 cells. These results indicate that the decrease in RhoA activation is the biochemical mechanism underlying this integrin switch observed during cell differentiation. The physiological relevance of such modulation of integrin activity in the functioning of the crypt-villus axis is discussed. PMID:11598208

  2. Integrins alpha(6A)beta 1 and alpha(6B)beta 1 promote different stages of chondrogenic cell differentiation.

    PubMed

    Segat, Daniela; Comai, Riccardo; Di Marco, Eddi; Strangio, Antonella; Cancedda, Ranieri; Franzi, Adriano T; Tacchetti, Carlo

    2002-08-30

    The differentiation of chondrocytes and of several other cell types is associated with a switch from the alpha(6B) to the alpha(6A) isoform of the laminin alpha(6)beta(1) integrin receptor. To define whether this event plays a functional role in cell differentiation, we used an in vitro model system that allows chick chondrogenic cells to remain undifferentiated when cultured in monolayer and to differentiate into chondrocytes when grown in suspension culture. We report that: (i) upon over-expression of the human alpha(6B), adherent chondrogenic cells differentiate to stage I chondrocytes (i.e. increased type II collagen, reduced type I collagen, fibronectin, alpha(5)beta(1) and growth rate, loss of fibroblast morphology); (ii) the expression of type II collagen requires the activation of p38 MAP kinase; (iii) the over-expression of alpha(6A) induces an incomplete differentiation to stage I chondrocytes, whereas no differentiation was observed in alpha(5) and mock-transfected control cells; (iv) a prevalence of the alpha(6A) subunit is necessary to stabilize the differentiated phenotype when cells are transferred to suspension culture. Altogether, these results indicate a functional role for the alpha(6B) to alpha(6A) switch in chondrocyte differentiation; the former promotes chondrocyte differentiation, and the latter is necessary in stabilizing the differentiated phenotype. PMID:12077132

  3. Role of alpha 5 beta 1 integrin in TGF-beta 1-costimulated CD8+ T cell growth and apoptosis.

    PubMed

    Rich, S; Van Nood, N; Lee, H M

    1996-10-01

    TGF-beta 1 regulates cell growth, differentiation, and adhesion and is a potent immunosuppressant, in part through its well-recognized growth-inhibitory effects. However, certain T cell subsets, particularly of naive phenotype, can instead be costimulated to proliferate by TGF-beta 1. We have previously demonstrated that naive murine CD8+ T cells, TCR activated by platebound anti-CD3 Ab or SEB superantigen, are growth stimulated by TGF-beta 1, acquire a memory phenotype, express elevated IL-10 and TGF-beta 1, and cause T cell growth inhibition as effector CD8+ T cells. TGF-beta 1 causes growth among certain nonlymphoid cells in part by inducing or mimicking integrin activation. The present studies thus addressed mediation of TGF-beta 1-dependent growth and survival of anti-CD3-triggered CD8+ T cells via beta 1 integrins. TGF-beta 1 reduced anti-CD3-activated alpha 4 beta 1 integrin expression and constitutive adhesion to fibronectin, while initial alpha 5 beta 1 expression was heightened and adhesive function sustained. Fibronectin-based RGD peptides that bind alpha 5 beta 1 integrins and alpha 5 or beta 1 integrin chain-specific Abs blocked TGF-beta 1-dependent proliferation, while connecting segment-1 peptide that binds alpha 4 beta 1 integrin and alpha 4 chain-specific Abs had no effect. Cross-linked alpha 5- but not alpha 4-specific Ab mimicked TGF-beta 1 function by costimulating CD8+ T cell growth. TGF-beta 1 also caused RGD peptide-sensitive CD8+ T cell aggregation. Additionally, TGF-beta 1-costimulated proliferation correlated with TGF-beta 1 protection of CD8+ T cells from anti-CD3-induced apoptosis. RGD peptides and alpha 5 integrin-specific Ab abolished TGF-beta 1 prevention of activation-induced apoptosis. Therefore, TGF-beta 1 costimulates CD8+ T cell growth via activation of the alpha 5 beta 1 integrin and/or its ligand and supports sustained growth at least in part by alpha 5 beta 1-mediated protection from activation-induced apoptosis. PMID:8816397

  4. 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.

  5. 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.

  6. Cross-talk between integrins {alpha}1{beta}1 and {alpha}2{beta}1 in renal epithelial cells

    SciTech Connect

    Abair, Tristin D.; Sundaramoorthy, Munirathinam; Chen, Dong; Heino, Jyrki; Ivaska, Johanna; Hudson, Billy G.; Sanders, Charles R.; Pozzi, Ambra; Zent, Roy

    2008-11-15

    The collagen-binding integrins {alpha}1{beta}1 and {alpha}2{beta}1 have profoundly different functions, yet they are often co-expressed in epithelial cells. When both integrins are expressed in the same cell, it has been suggested that {alpha}1{beta}1 negatively regulates integrin {alpha}2{beta}1-dependent functions. In this study we utilized murine ureteric bud (UB) epithelial cells, which express no functionally detectable levels of endogenous integrins {alpha}1{beta}1 and {alpha}2{beta}1, to determine the mechanism whereby this regulation occurs. We demonstrate that UB cells expressing integrin {alpha}2{beta}1, but not {alpha}1{beta}1 adhere, migrate and proliferate on collagen I as well as form cellular cords in 3D collagen I gels. Substitution of the transmembrane domain of the integrin {alpha}2 subunit with that of {alpha}1 results in decreased cell adhesion, migration and cord formation. In contrast, substitution of the integrin {alpha}2 cytoplasmic tail with that of {alpha}1, decreases cell migration and cord formation, but increases proliferation. When integrin {alpha}1 and {alpha}2 subunits are co-expressed in UB cells, the {alpha}1 subunit negatively regulates integrin {alpha}2{beta}1-dependent cord formation, adhesion and migration and this inhibition requires expression of both {alpha}1 and {alpha}2 tails. Thus, we provide evidence that the transmembrane and cytoplasmic domains of the {alpha}2 integrin subunit, as well as the {alpha}1 integrin subunit, regulate integrin {alpha}2{beta}1 cell function.

  7. Integrins alpha1beta1 and alpha2beta1 are receptors for the rotavirus enterotoxin.

    PubMed

    Seo, Neung-Seon; Zeng, Carl Q-Y; Hyser, Joseph M; Utama, Budi; Crawford, Sue E; Kim, Kate J; Höök, Magnus; Estes, Mary K

    2008-07-01

    Rotavirus NSP4 is a viral enterotoxin capable of causing diarrhea in neonatal mice. This process is initiated by the binding of extracellular NSP4 to target molecule(s) on the cell surface that triggers a signaling cascade leading to diarrhea. We now report that the integrins alpha1beta1 and alpha2beta1 are receptors for NSP4. NSP4 specifically binds to the alpha1 and alpha2 I domains with apparent K(d) = 1-2.7 muM. Binding is mediated by the I domain metal ion-dependent adhesion site motif, requires Mg(2+) or Mn(2+), is abolished with EDTA, and an NSP4 point mutant, E(120)A, fails to bind alpha2 integrin I domain. NSP4 has two distinct integrin interaction domains. NSP4 amino acids 114-130 are essential for binding to the I domain, and NSP4 peptide 114-135 blocks binding of the natural ligand, collagen I, to integrin alpha2. NSP4 amino acids 131-140 are not associated with the initial binding to the I domain, but elicit signaling that leads to the spreading of attached C2C12-alpha2 cells, mouse myoblast cells stably expressing the human alpha2 integrin. NSP4 colocalizes with integrin alpha2 on the basolateral surface of rotavirus-infected polarized intestinal epithelial (Caco-2) cells as well as surrounding noninfected cells. NSP4 mutants that fail to bind or signal through integrin alpha2 were attenuated in diarrhea induction in neonatal mice. These results indicate that NSP4 interaction with integrin alpha1 and alpha2 is an important component of enterotoxin function and rotavirus pathogenesis, further distinguishing this viral virulence factor from other microbial enterotoxins. PMID:18587047

  8. 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.

  9. Modulation of the transforming growth factor-beta1-induced Smad phosphorylation by the extracellular matrix receptor beta1-integrin.

    PubMed

    Hamajima, Hiroshi; Ozaki, Iwata; Zhang, Hao; Iwane, Shinji; Kawaguchi, Yasunori; Eguchi, Yuichiro; Matsuhashi, Sachiko; Mizuta, Toshihiko; Matsuzaki, Koichi; Fujimoto, Kazuma

    2009-12-01

    Integrins, heterodimeric receptors for the extracellular matrix, are known to modulate transforming growth factor-beta1 (TGF-beta1)-mediated cell behavior. However, the interplay between beta1-integrin and Smad signaling, regulated by TGF-beta1, is not clearly understood. This study focuses on the alterations of the regulatory Smads (R-Smads) by TGF-beta1 in beta1-integrin-transfected HepG2 cells. The phosphorylation at the C-terminal site of R-Smads by TGF-beta1 was impaired in the beta1-integrin-transfected cells. However, the R-Smads were constitutively phosphorylated at the linker region in a MAP kinase-dependent manner. Furthermore, the expression of a mutant Smad3, that lacks the phosphorylation sites in the linker region, restored the TGF-beta1-induced Smad transcriptional activity. These results suggest that beta1-integrin impairs the TGF-beta1-mediated signals through the altered phosphorylation of the R-Smads. PMID:19885568

  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. beta1,4-N-Acetylglucosaminyltransferase III potentiates beta1 integrin-mediated neuritogenesis induced by serum deprivation in Neuro2a cells.

    PubMed

    Shigeta, Masaki; Shibukawa, Yukinao; Ihara, Hideyuki; Miyoshi, Eiji; Taniguchi, Naoyuki; Gu, Jianguo

    2006-06-01

    Aspects of the biological significance of the bisecting N-acetylglucosamine (GlcNAc) structure on N-glycans introduced by beta1,4-N-acetylglucosaminyltransferase III (GnT-III) in Neuro2a cell differentiation are demonstrated. The overexpression of GnT-III in the cells led to the induction of axon-like processes with numerous neurites and swellings, in which beta1 integrin was localized, under conditions of serum starvation. This enhancement in neuritogenesis was suppressed by either the addition of a bisecting GlcNAc-containing N-glycan or erythroagglutinating phytohemagglutinin (E(4)-PHA), which preferentially recognizes the bisecting GlcNAc. GnT-III-promoted neuritogenesis was also significantly perturbed by treatment with a functional blocking anti-beta1 integrin antibody. In fact, beta1 integrin was found to be one of the target proteins of GnT-III, as confirmed by a pull-down assay with E(4)-PHA. These data suggest that N-glycans with a bisecting GlcNAc on target molecules, such as beta1 integrin, play important roles in the regulation of neuritogenesis. PMID:16531477

  12. Molecular basis of the recognition of nephronectin by integrin alpha8beta1.

    PubMed

    Sato, Yuya; Uemura, Toshihiko; Morimitsu, Keisuke; Sato-Nishiuchi, Ryoko; Manabe, Ri-Ichiroh; Takagi, Junichi; Yamada, Masashi; Sekiguchi, Kiyotoshi

    2009-05-22

    Integrin alpha8beta1 interacts with a variety of Arg-Gly-Asp (RGD)-containing ligands in the extracellular matrix. Here, we examined the binding activities of alpha8beta1 integrin toward a panel of RGD-containing ligands. Integrin alpha8beta1 bound specifically to nephronectin with an apparent dissociation constant of 0.28 +/- 0.01 nm, but showed only marginal affinities for fibronectin and other RGD-containing ligands. The high-affinity binding to alpha8beta1 integrin was fully reproduced with a recombinant nephronectin fragment derived from the RGD-containing central "linker" segment. A series of deletion mutants of the recombinant fragment identified the LFEIFEIER sequence on the C-terminal side of the RGD motif as an auxiliary site required for high-affinity binding to alpha8beta1 integrin. Alanine scanning mutagenesis within the LFEIFEIER sequence defined the EIE sequence as a critical motif ensuring the high-affinity integrin-ligand interaction. Although a synthetic LFEIFEIER peptide failed to inhibit the binding of alpha8beta1 integrin to nephronectin, a longer peptide containing both the RGD motif and the LFEIFEIER sequence was strongly inhibitory, and was approximately 2,000-fold more potent than a peptide containing only the RGD motif. Furthermore, trans-complementation assays using recombinant fragments containing either the RGD motif or LFEIFEIER sequence revealed a clear synergism in the binding to alpha8beta1 integrin. Taken together, these results indicate that the specific high-affinity binding of nephronectin to alpha8beta1 integrin is achieved by bipartite interaction of the integrin with the RGD motif and LFEIFEIER sequence, with the latter serving as a synergy site that greatly potentiates the RGD-driven integrin-ligand interaction but has only marginal activity to secure the interaction by itself. PMID:19342381

  13. Fibronectin receptor functions in embryonic cells deficient in alpha 5 beta 1 integrin can be replaced by alpha V integrins.

    PubMed Central

    Yang, J T; Hynes, R O

    1996-01-01

    alpha 5 beta 1 integrin mediates cell adhesion to extracellular matrix by interacting with fibronectin (FN). Mouse lines carrying null mutations in genes encoding either the alpha 5 integrin subunit or FN have been generated previously. Both mutations are embryonic lethal with overlapping defects, but the defects of alpha 5-null embryos are less severe. Primary embryonic cells lacking alpha 5 beta 1 are able to adhere to FN, form focal contacts, migrate on FN, and assemble FN matrix. These results suggest the involvement of (an)other FN receptors(s). In this study, we examined functions of alpha 4 beta 1 and alpha V integrins in embryonic cells lacking alpha 5 beta 1. Our analysis of cells lacking both alpha 4 beta 1 and alpha 5 beta 1 showed that alpha 4 beta 1 is also not required for these FN-dependent functions. Using alpha V-specific blocking reagents, we showed that alpha V integrins are required for alpha 5-null cells, but not wild-type cells, to adhere and spread on FN. Our data also showed that, although the expression levels of alpha V integrins on the wild-type and alpha 5-null cells are similar, there is an increase in recruitment of alpha V integrins into focal contacts in alpha 5-null cells plated on FN, indicating that alpha V integrins can compensate functionally for the loss of alpha 5 beta 1 in focal contacts of alpha 5-null cells. Finally, our data suggested possible roles for alpha V integrins in replacing the role of alpha 5 beta 1 in FN matrix assembly in vitro and in FN-dependent embryonic functions in vivo. Images PMID:8930896

  14. 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

  15. alpha 11beta 1 integrin recognizes the GFOGER sequence in interstitial collagens.

    PubMed

    Zhang, Wan-Ming; Kapyla, Jarmo; Puranen, J Santeri; Knight, C Graham; Tiger, Carl-Fredrik; Pentikainen, Olli T; Johnson, Mark S; Farndale, Richard W; Heino, Jyrki; Gullberg, Donald

    2003-02-28

    The integrins alpha(1)beta(1), alpha(2)beta(1), alpha(10)beta(1), and alpha(11)beta(1) are referred to as a collagen receptor subgroup of the integrin family. Recently, both alpha(1)beta(1) and alpha(2)beta(1) integrins have been shown to recognize triple-helical GFOGER (where single letter amino acid nomenclature is used, O = hydroxyproline) or GFOGER-like motifs found in collagens, despite their distinct binding specificity for various collagen subtypes. In the present study we have investigated the mechanism whereby the latest member in the integrin family, alpha(11)beta(1), recognizes collagens using C2C12 cells transfected with alpha(11) cDNA and the bacterially expressed recombinant alpha(11) I domain. The ligand binding properties of alpha(11)beta(1) were compared with those of alpha(2)beta(1). Mg(2+)-dependent alpha(11)beta(1) binding to type I collagen required micromolar Ca(2+) but was inhibited by 1 mm Ca(2+), whereas alpha(2)beta(1)-mediated binding was refractory to millimolar concentrations of Ca(2+). The bacterially expressed recombinant alpha(11) I domain preference for fibrillar collagens over collagens IV and VI was the same as the alpha(2) I domain. Despite the difference in Ca(2+) sensitivity, alpha(11)beta(1)-expressing cells and the alpha(11) I domain bound to helical GFOGER sequences in a manner similar to alpha(2)beta(1)-expressing cells and the alpha(2) I domain. Modeling of the alpha I domain-collagen peptide complexes could partially explain the observed preference of different I domains for certain GFOGER sequence variations. In summary, our data indicate that the GFOGER sequence in fibrillar collagens is a common recognition motif used by alpha(1)beta(1), alpha(2)beta(1), and also alpha(11)beta(1) integrins. Although alpha(10) and alpha(11) chains show the highest sequence identity, alpha(2) and alpha(11) are more similar with regard to collagen specificity. Future studies will reveal whether alpha(2)beta(1) and alpha(11)beta(1

  16. Beta1 integrin expression by podocytes is required to maintain glomerular structural integrity.

    PubMed

    Pozzi, Ambra; Jarad, George; Moeckel, Gilbert W; Coffa, Sergio; Zhang, Xi; Gewin, Leslie; Eremina, Vera; Hudson, Billy G; Borza, Dorin-Bogdan; Harris, Raymond C; Holzman, Lawrence B; Phillips, Carrie L; Fassler, Reinhard; Quaggin, Susan E; Miner, Jeffrey H; Zent, Roy

    2008-04-15

    Integrins are transmembrane heteromeric receptors that mediate interactions between cells and extracellular matrix (ECM). beta1, the most abundantly expressed integrin subunit, binds at least 12 alpha subunits. beta1 containing integrins are highly expressed in the glomerulus of the kidney; however their role in glomerular morphogenesis and maintenance of glomerular filtration barrier integrity is poorly understood. To study these questions we selectively deleted beta1 integrin in the podocyte by crossing beta1(flox/flox) mice with podocyte specific podocin-cre mice (pod-Cre), which express cre at the time of glomerular capillary formation. We demonstrate that podocyte abnormalities are visualized during glomerulogenesis of the pod-Cre;beta1(flox/flox) mice and proteinuria is present at birth, despite a grossly normal glomerular basement membrane. Following the advent of glomerular filtration there is progressive podocyte loss and the mice develop capillary loop and mesangium degeneration with little evidence of glomerulosclerosis. By 3 weeks of age the mice develop severe end stage renal failure characterized by both tubulointerstitial and glomerular pathology. Thus, expression of beta1 containing integrins by the podocyte is critical for maintaining the structural integrity of the glomerulus. PMID:18328474

  17. 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.

  18. Signaling through urokinase and urokinase receptor in lung cancer cells requires interactions with beta1 integrins.

    PubMed

    Tang, Chi-Hui; Hill, Marla L; Brumwell, Alexis N; Chapman, Harold A; Wei, Ying

    2008-11-15

    The urokinase receptor (uPAR) is upregulated upon tumor cell invasion and correlates with poor lung cancer survival. Although a cis-interaction with integrins has been ascribed to uPAR, whether this interaction alone is critical to urokinase (uPA)- and uPAR-dependent signaling and tumor promotion is unclear. Here we report the functional consequences of point mutations of uPAR (H249A-D262A) that eliminate beta1 integrin interactions but maintain uPA binding, vitronectin attachment and association with alphaV integrins, caveolin and epidermal growth factor receptor. Disruption of uPAR interactions with beta1 integrins recapitulated previously reported findings with beta1-integrin-derived peptides that attenuated matrix-dependent ERK activation, MMP expression and in vitro migration by human lung adenocarcinoma cell lines. The uPAR mutant cells acquired enhanced capacity to adhere to vitronectin via uPAR-alphaVbeta5-integrin, rather than through the uPAR-alpha3beta1-integrin complex and they were unable to initiate uPA signaling to activate ERK, Akt or Stat1. In an orthotopic lung cancer model, uPAR mutant cells exhibited reduced tumor size compared with cells expressing wild-type uPAR. Taken together, the results indicate that uPAR-beta1-integrin interactions are essential to signals induced by integrin matrix ligands or uPA that support lung cancer cell invasion in vitro and progression in vivo. PMID:18940913

  19. 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.

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

    PubMed

    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

  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. Mouse egg integrin alpha 6 beta 1 functions as a sperm receptor.

    PubMed

    Almeida, E A; Huovila, A P; Sutherland, A E; Stephens, L E; Calarco, P G; Shaw, L M; Mercurio, A M; Sonnenberg, A; Primakoff, P; Myles, D G; White, J M

    1995-06-30

    Binding between sperm and egg plasma membranes is an essential step in fertilization. Whereas fertilin, a mammalian sperm surface protein, is involved in this crucial interaction, sperm receptors on the egg plasma membrane have not been identified. Because fertilin contains a predicted integrin ligand domain, we investigated the expression and function of integrin subunits in unfertilized mouse eggs. Polymerase chain reactions detected mRNAs for alpha 5, alpha 6, alpha v, beta 1, beta 3, and beta 5. Immunofluorescence revealed alpha 6 beta 1 and alpha v beta 3 on the plasma membrane. GoH3, a function-blocking anti-alpha 6 monoclonal antibody, abolished sperm binding, but a nonfunction-blocking anti-alpha 6 monoclonal antibody, a function-blocking anti-alpha v beta 3 polyclonal antibody, and an RGD peptide had no effect. Somatic cells bound sperm avidly, but only if they expressed alpha 6 beta 1. A peptide analog of the fertilin integrin ligand domain inhibited sperm binding to eggs and alpha 6 beta 1+ cells and diminished GoH3 staining of eggs. Our results indicate a novel role for the integrin alpha 6 beta 1 as a cell-cell adhesion receptor that mediates sperm-egg binding. PMID:7600577

  3. Integrin alpha3beta1, a novel receptor for alpha3(IV) noncollagenous domain and a trans-dominant Inhibitor for integrin alphavbeta3.

    PubMed

    Borza, Corina M; Pozzi, Ambra; Borza, Dorin-Bogdan; Pedchenko, Vadim; Hellmark, Thomas; Hudson, Billy G; Zent, Roy

    2006-07-28

    Exogenous soluble human alpha3 noncollagenous (NC1) domain of collagen IV inhibits angiogenesis and tumor growth. These biological functions are attributed to the binding of alpha3NC1 to integrin alphavbeta3. However, in some tumor cells that express integrin alphavbeta3, the alpha3NC1 domain does not inhibit proliferation, suggesting that integrin alphavbeta3 expression is not sufficient to mediate the anti-tumorigenic activity of this domain. Therefore, in the present study, we searched for novel binding receptors for the soluble alpha3NC1 domain in cells lacking alphavbeta3 integrin. In these cells, soluble alpha3NC1 bound integrin alpha3beta1; however, unlike alphavbeta3, alpha3beta1 integrin did not mediate cell adhesion to immobilized alpha3NC1 domain. Interestingly, in cells lacking integrin alpha3beta1, adhesion to the alpha3NC1 domain was enhanced due to activation of integrin alphavbeta3. These findings indicate that integrin alpha3beta1 is a receptor for the alpha3NC1 domain and transdominantly inhibits integrin alphavbeta3 activation. Thus integrin alpha3beta1, in conjunction with integrin alphavbeta3, modulates cellular responses to the alpha3NC1 domain, which may be pivotal in the mechanism underpinning its anti-angiogenic and anti-tumorigenic activities. PMID:16731529

  4. 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.

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

    PubMed Central

    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

    2016-01-01

    Abstract 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.

  6. 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

  7. Widespread histologic distribution of the alpha 2 beta 1 integrin cell-surface collagen receptor.

    PubMed Central

    Zutter, M. M.; Santoro, S. A.

    1990-01-01

    The alpha 2 beta 1 integrin (platelet membrane glycoprotein Ia-IIa, VLA-2, ECMR-II) functions as a cell surface receptor for collagen. The authors have determined the histologic distribution of the alpha 2 beta 1 receptor in normal tissues by immunohistochemical technique. The studies revealed that the alpha 2 beta 1 receptor was expressed on fibroblasts, endothelial cells, and epithelial cells from multiple sites including skin, tonsil, breast, sweat gland, gastrointestinal tract, lung, bladder, cervix, and prostate. Follicular dendritic cells of the lymph node, tonsil, and spleen and dendritic cells of the thymus also expressed the alpha 2 beta 1 receptor. The receptor also was present on Schwann cells of ganglia and on neuroglia. Greatly enhanced expression of the receptor in regions of proliferating epithelium suggests that enhanced expression of alpha 2 beta 1 is associated with orderly, regulated cell proliferation. The circumferential staining pattern of the alpha 2 beta 1 integrin within many epithelia is virtually identical to that observed for other adhesive receptors, such as the cadherins, which have been implicated in cell-cell adhesion. Images Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 16 Figure 17 PMID:2164774

  8. Halogenated benzimidazole carboxamides target integrin alpha4 beta1 on T and B cell lymphomas3456

    PubMed Central

    Carpenter, Richard D.; Natarajan, Arutselvan; Lau, Edmond Y.; Andrei, Mirela; Solano, Danielle M.; Lightstone, Felice C.; DeNardo, Sally J.; Lam, Kit S.; Kurth, Mark J.

    2011-01-01

    Integrin alpha-4 beta-1 (alpha4 beta1, alpha(4)beta(1), α4β1) is an attractive but poorly understood target for selective diagnosis and treatment of T and B cell lymphomas. This report focuses on the rapid microwave preparation, structure-activity relationships and biological evaluation of medicinally pertinent benzimidazole heterocycles as integrin α4β1 antagonists. We documented tumor uptake of derivatives labelled with I-125 in xenograft murine models of B-cell lymphoma. Molecular homology models of integrin α4β1 predicted that docked halobenzimidazole carboxamides have the halogen atom in a suitable orientation for halogen-hydrogen bonding. The high affinity halogenated ligands identified offer attractive tools for medicinal and biological use, including fluoro and iodo derivatives with potential radiodiagnostic (18F) or radiotherapeutic (131I) applications, or chloro and bromo analogues that could provide structural insights into integrin-ligand interactions through photoaffinity, cross-linking/mass spectroscopy, and X-ray crystallographic studies. PMID:20530664

  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. 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

  11. A preliminary optical and electron microscopic study of the beta(1) integrin distribution pattern of human osteosarcoma-derived cells.

    PubMed

    Banai, Kiarash; Brady, Ken; McDonald, Fraser

    2004-07-01

    Immunogold labelling was used to study the organisation of the beta(1) integrins on osteosarcoma-derived osteoblasts (Saos-2 and MG-63). Monolayers of cells were prepared in multiwell culture plates on both uncovered and collagen-covered coverslips, and beta(1) integrins were primarily labelled using mouse monoclonal antibodies to beta(1) integrins. Indirect immunofluorescence labels using an anti-mouse fluorescein-conjugated goat antibody showed an even distribution of the beta(1) integrins on the cell membranes of all cell types used. A concentration of 2 microg/ml of the primary antibodies and a 1:100 dilution of the secondary antibodies were determined as the optimal concentration for labelling to use with indirect localisation of the primary antibodies gold conjugated to goat anti-mouse antibodies and viewed under an electron microscope. Ten nanometre gold particles were used for transmission electron microscopy (TEM) and 40 nm gold particles for scanning electron microscopy. TEM showed that beta(1) integrins were mainly clustered on the cell membrane processes with less labelling on the cell membranes themselves. The distribution of beta(1) integrins on osteosarcoma cells supports the concept that integrins may function by forming focal adhesions at the site of the cytoplasmic membrane processes. PMID:15241608

  12. Prolyl hydroxylation of collagen type I is required for efficient binding to integrin alpha 1 beta 1 and platelet glycoprotein VI but not to alpha 2 beta 1.

    PubMed

    Perret, Stéephanie; Eble, Johannes A; Siljander, Pia R-M; Merle, Christine; Farndale, Richard W; Theisen, Manfred; Ruggiero, Florence

    2003-08-01

    Collagen is a potent adhesive substrate for cells, an event essentially mediated by the integrins alpha 1 beta 1 and alpha 2 beta 1. Collagen fibrils also bind to the integrin alpha 2 beta 1 and the platelet receptor glycoprotein VI to activate and aggregate platelets. The distinct triple helical recognition motifs for these receptors, GXOGER and (GPO)n, respectively, all contain hydroxyproline. Using unhydroxylated collagen I produced in transgenic plants, we investigated the role of hydroxyproline in the receptor-binding properties of collagen. We show that alpha 2 beta 1 but not alpha 1 beta 1 mediates cell adhesion to unhydroxylated collagen. Soluble recombinant alpha 1 beta 1 binding to unhydroxylated collagen is considerably reduced compared with bovine collagens, but binding can be restored by prolyl hydroxylation of recombinant collagen. We also show that platelets use alpha 2 beta 1 to adhere to the unhydroxylated recombinant molecules, but the adhesion is weaker than on fully hydroxylated collagen, and the unhydroxylated collagen fibrils fail to aggregate platelets. Prolyl hydroxylation is thus required for binding of collagen to platelet glycoprotein VI and to cells by alpha 1 beta 1. These observations give new insights into the molecular basis of collagen-receptor interactions and offer new selective applications for the recombinant unhydroxylated collagen I. PMID:12771137

  13. 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

  14. Integrin alpha5beta1 function is regulated by XGIPC/kermit2 mediated endocytosis during Xenopus laevis gastrulation.

    PubMed

    Spicer, Erin; Suckert, Catherine; Al-Attar, Hyder; Marsden, Mungo

    2010-01-01

    During Xenopus gastrulation alpha5beta1 integrin function is modulated in a temporally and spatially restricted manner, however, the regulatory mechanisms behind this regulation remain uncharacterized. Here we report that XGIPC/kermit2 binds to the cytoplasmic domain of the alpha5 subunit and regulates the activity of alpha5beta1 integrin. The interaction of kermit2 with alpha5beta1 is essential for fibronectin (FN) matrix assembly during the early stages of gastrulation. We further demonstrate that kermit2 regulates alpha5beta1 integrin endocytosis downstream of activin signaling. Inhibition of kermit2 function impairs cell migration but not adhesion to FN substrates indicating that integrin recycling is essential for mesoderm cell migration. Furthermore, we find that the alpha5beta1 integrin is colocalized with kermit2 and Rab 21 in embryonic and XTC cells. These data support a model where region specific mesoderm induction acts through kermit2 to regulate the temporally and spatially restricted changes in adhesive properties of the alpha5beta1 integrin through receptor endocytosis. PMID:20498857

  15. Loss of integrin alpha1beta1 ameliorates Kras-induced lung cancer.

    PubMed

    Macias-Perez, Ines; Borza, Corina; Chen, Xiwu; Yan, Xuexian; Ibanez, Raquel; Mernaugh, Glenda; Matrisian, Lynn M; Zent, Roy; Pozzi, Ambra

    2008-08-01

    The collagen IV binding receptor integrin alpha1beta1 has been shown to regulate lung cancer due to its proangiogenic properties; however, it is unclear whether this receptor also plays a direct role in promoting primary lung tumors. To investigate this possibility, integrin alpha1-null mice were crossed with KrasLA2 mice that carry an oncogenic mutation of the Kras gene (G12D) and develop spontaneous primary tumors with features of non-small cell lung cancer. We provide evidence that KrasLA2/alpha1-null mice have a decreased incidence of primary lung tumors and longer survival compared with KrasLA2/alpha1 wild-type controls. Tumors from KrasLA2/alpha1-null mice were also smaller, less vascularized, and exhibited reduced cell proliferation and increased apoptosis, as determined by proliferating cell nuclear antigen and terminal deoxynucleotidyl-transferase-mediated dUTP nick-end staining, respectively. Moreover, tumors from the KrasLA2/alpha1-null mice showed diminished extracellular signal-regulated kinase (ERK) but enhanced p38 mitogen-activated protein kinase activation. Primary lung tumor epithelial cells isolated from KrasLA2/alpha1-null mice showed a significant decrease in anchorage-independent colony formation, collagen-mediated cell proliferation, ERK activation, and, most importantly, tumorigenicity when injected into nude mice compared with KrasLA2/alpha1 wild-type tumor cells. These results indicate that loss of the integrin alpha1 subunit decreases the incidence and growth of lung epithelial tumors initiated by oncogenic Kras, suggesting that both Kras and integrin alpha1beta1 cooperate to drive the growth of non-small cell lung cancer in vivo. PMID:18676835

  16. Role of the von Hippel-Lindau tumor suppressor gene in the formation of beta1-integrin fibrillar adhesions.

    PubMed

    Esteban-Barragán, Miguel A; Avila, Pilar; Alvarez-Tejado, Miguel; Gutiérrez, M Dolores; García-Pardo, Angeles; Sánchez-Madrid, Francisco; Landázuri, Manuel O

    2002-05-15

    The von Hippel-Lindau tumor suppressor gene (VHL) is absent or inactivated in the VHLcancer syndrome and in most sporadic renal cancers. VHL is requiredfor the assembly of a proper extracellular fibronectin matrix, although the exact mechanism remains unknown. In this report, we demonstrate that 786-O renal cancer cells are unable to organize an adequate matrix even in the presence of an excess of exogenous fibronectin. Because the formation of integrin fibrillar adhesions plays a pivotal role in the organization of extracellular fibronectin, we next examined the expression and subcellular distribution of integrins in VHL- cells and their wild-type VHL stably transfected counterparts. The levels of beta1 and alphav integrins were increased in VHL- cells when compared with VHL+ transfectants. Early after plating, both VHL+ and VHL- cells were capable of assembling classic "patch-like" alphav focal contacts. As the culture advanced and cells became confluent, alphav integrins partly relocated to the intercellular junctions in VHL+ transfectants, which then developed large beta1 fibrillar-type adhesions and anchored firmly to the substrate. In contrast, confluent VHL- cells were unable to assemble beta1 fibrillar adhesions, and alphav focal contacts remained unchanged at all stages of the culture. Exogenous activation of beta1 integrins with either divalent cations or activating antibodies partly restored the capability of VHL- cells to assemble beta1 fibrillar adhesions and fibronectin fibers. Finally, pulse-chase studies of metabolically labeled 786-O cells revealed that the maturation of the common beta1-integrin chain was delayed in VHL- cells when compared with VHL+ cells. Our results show that VHL is an important regulator of integrins and is essential for the formation of beta1 fibrillar adhesions. These findings help to explain the abnormal extracellular matrix organization and increased motility of VHL- renal cancer cells. PMID:12019174

  17. Polymorphonuclear leucocyte migration through human dermal fibroblast monolayers is dependent on both beta 2-integrin (CD11/CD18) and beta 1-integrin (CD29) mechanisms.

    PubMed Central

    Gao, J X; Issekutz, A C

    1995-01-01

    Accumulation of leucocytes in inflammation involves their migration through vascular endothelium and then in the connective tissue. We investigated human polymorphonuclear leucocyte (PMNL) migration through a biological barrier of human dermal fibroblasts grown on microporous filters, as a model of PMNL migration in the connective tissue. PMNL did not migrate through a fibroblast monolayer unless a chemotactic factor, e.g. C5a, interleukin-8 (IL-8) or zymosan-activated plasma (ZAP; C5adesArg), was added. This migration was partially inhibited (35-70%, depending on the stimulus) by treatment of PMNL with monoclonal antibody (mAb) to CD18 (beta 2-integrins). Most of the CD18-independent migration was inhibited by mAb to beta 1-integrins (CD29). Inhibition by mAb to beta 1 was observed when the PMNL, but not the fibroblasts, were treated with mAb. The role of beta 1-integrins in PMNL transfibroblast migration was detectable only when the function of the CD11-CD18 complex was blocked, because mAb to beta 1-integrin alone had no significant effect on PMNL migration. Migration induced by C5a was more CD18-independent compared to IL-8 or C5adesArg. The CD18-independent migration was also inhibited by mAb to the beta 1-integrin subunits alpha 5 (of very late antigens-5; VLA-5) and alpha 6 (of VLA-6). Treatment of the fibroblasts (4 hr) with tumour necrosis factor-alpha (TNF-alpha) or IL-1 alpha enhanced C5a-induced PMNL transfibroblast migration and increased the proportion of migration utilizing the CD11-CD18 mechanism. However, TNF-alpha treatment had no effect on the degree of beta 1-integrin-dependent migration. These findings suggest that in response to the chemotactic factors C5a, IL-8 and C5adesArg, PMNL migration in the connective tissue is mediated by both CD11-CD18 (beta 2) and beta 1-integrins on the PMNL. The VLA-5 and VLA-6 members of beta 1-integrins are involved in this process. This is in contrast to PMNL migration across endothelium in this system, which

  18. Focal adhesion kinase (FAK) phosphorylation is not required for genistein-induced FAK-beta-1-integrin complex formation.

    PubMed

    Liu, Y; Kyle, E; Lieberman, R; Crowell, J; Kellof, G; Bergan, R C

    2000-01-01

    It has previously been shown that changes in the activity of focal adhesion kinase (FAK), and its binding to beta-1-integrin, accompany genistein-induced adhesion of prostate cells. Consumption of genistein world wide is associated with a lower incidence of metastatic prostate cancer. Early human clinical trials of genistein are under way to evaluate genistein's potential causal role in this regard. Though an important cell adhesion-associated signaling molecule, FAK's role in regulating prostate cell adhesion was not clear. Elucidation of this process would provide important information relating to both biology and potential clinical endpoints. It was hypothesized that FAK activation and complex formation are temporally related in prostate cells, and can thus be separated. Significant activation of FAK was demonstrated when cells adhered to fibronectin, as compared to poly-L-lysine, thus demonstrating that beta-1-integrin plays a significant role in activating FAK. Neither FAK activation, nor FAK-integrin complex formation, required beta-1-integrin ligand. However, disruption of the cellular cytoskeleton by cytochalasin D prevented FAK activation, but did not block genistein-induced complex formation. In the face of a disrupted cytoskeleton, signaling through FAK could not be restored through either integrin cross linking, or re-establishment of tensile forces via attachment to solid matrix. These studies demonstrate that FAK-beta-1-integrin complex formation does not require FAK activation, suggesting that it is an early event in prostate cell adhesion. An intact cytoskeleton is necessary for FAK activation. The functional importance of beta-1-integrin in prostate cells is demonstrated. Current findings support plans to test genistein in prostate cancer. PMID:11315093

  19. Osteoblast mineralization requires beta1 integrin/ICAP-1-dependent fibronectin deposition.

    PubMed

    Brunner, Molly; Millon-Frémillon, Angélique; Chevalier, Genevieve; Nakchbandi, Inaam A; Mosher, Deane; Block, Marc R; Albigès-Rizo, Corinne; Bouvard, Daniel

    2011-07-25

    The morphogenetic and differentiation events required for bone formation are orchestrated by diffusible and insoluble factors that are localized within the extracellular matrix. In mice, the deletion of ICAP-1, a modulator of β1 integrin activation, leads to severe defects in osteoblast proliferation, differentiation, and mineralization and to a delay in bone formation. Deposition of fibronectin and maturation of fibrillar adhesions, adhesive structures that accompany fibronectin deposition, are impaired upon ICAP-1 loss, as are type I collagen deposition and mineralization. Expression of β1 integrin with a mutated binding site for ICAP-1 recapitulates the ICAP-1-null phenotype. Follow-up experiments demonstrated that ICAP-1 negatively regulates kindlin-2 recruitment onto the β1 integrin cytoplasmic domain, whereas an excess of kindlin-2 binding has a deleterious effect on fibrillar adhesion formation. These results suggest that ICAP-1 works in concert with kindlin-2 to control the dynamics of β1 integrin-containing fibrillar adhesions and, thereby, regulates fibronectin deposition and osteoblast mineralization. PMID:21768292

  20. 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

  1. Cell-cell contact between marrow stromal cells and myeloma cells via VCAM-1 and alpha(4)beta(1)-integrin enhances production of osteoclast-stimulating activity.

    PubMed

    Michigami, T; Shimizu, N; Williams, P J; Niewolna, M; Dallas, S L; Mundy, G R; Yoneda, T

    2000-09-01

    Myeloma is a unique hematologic malignancy that exclusively homes in the bone marrow and induces massive osteoclastic bone destruction presumably by producing cytokines that promote the differentiation of the hematopoietic progenitors to osteoclasts (osteoclastogenesis). It is recognized that neighboring bone marrow stromal cells influence the expression of the malignant phenotype in myeloma cells. This study examined the role of the interactions between myeloma cells and neighboring stromal cells in the production of osteoclastogenic factors to elucidate the mechanism underlying extensive osteoclastic bone destruction. A murine myeloma cell line 5TGM1, which causes severe osteolysis, expresses alpha(4)beta(1)-integrin and tightly adheres to the mouse marrow stromal cell line ST2, which expresses the vascular cell adhesion molecule-1 (VCAM-1), a ligand for alpha(4)beta(1)-integrin. Co-cultures of 5TGM1 with primary bone marrow cells generated tartrate-resistant acid phosphatase-positive multinucleated bone-resorbing osteoclasts. Co-cultures of 5TGM1 with ST2 showed increased production of bone-resorbing activity and neutralizing antibodies against VCAM-1 or alpha(4)beta(1)-integrin inhibited this. The 5TGM1 cells contacting recombinant VCAM-1 produced increased osteoclastogenic and bone-resorbing activity. The activity was not blocked by the neutralizing antibody to known osteoclastogenic cytokines including interleukin (IL)-1, IL-6, tumor necrosis factor, or parathyroid hormone-related peptide. These data suggest that myeloma cells are responsible for producing osteoclastogenic activity and that establishment of direct contact with marrow stromal cells via alpha(4)beta(1)-integrin/VCAM-1 increases the production of this activity by myeloma cells. They also suggest that the presence of stromal cells may provide a microenvironment that allows exclusive colonization of myeloma cells in the bone marrow. (Blood. 2000;96:1953-1960) PMID:10961900

  2. 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.

  3. Use of synthetic peptides to locate novel integrin alpha2beta1-binding motifs in human collagen III.

    PubMed

    Raynal, Nicolas; Hamaia, Samir W; Siljander, Pia R-M; Maddox, Ben; Peachey, Anthony R; Fernandez, Rafael; Foley, Loraine J; Slatter, David A; Jarvis, Gavin E; Farndale, Richard W

    2006-02-17

    A set of 57 synthetic peptides encompassing the entire triplehelical domain of human collagen III was used to locate binding sites for the collagen-binding integrin alpha(2)beta(1). The capacity of the peptides to support Mg(2+)-dependent binding of several integrin preparations was examined. Wild-type integrins (recombinant alpha(2) I-domain, alpha(2)beta(1) purified from platelet membranes, and recombinant soluble alpha(2)beta(1) expressed as an alpha(2)-Fos/beta(1)-Jun heterodimer) bound well to only three peptides, two containing GXX'GER motifs (GROGER and GMOGER, where O is hydroxyproline) and one containing two adjacent GXX'GEN motifs (GLKGEN and GLOGEN). Two mutant alpha(2) I-domains were tested: the inactive T221A mutant, which recognized no peptides, and the constitutively active E318W mutant, which bound a larger subset of peptides. Adhesion of activated human platelets to GER-containing peptides was greater than that of resting platelets, and HT1080 cells bound well to more of the peptides compared with platelets. Binding of cells and recombinant proteins was abolished by anti-alpha(2) monoclonal antibody 6F1 and by chelation of Mg(2+). We describe two novel high affinity integrin-binding motifs in human collagen III (GROGER and GLOGEN) and a third motif (GLKGEN) that displays intermediate activity. Each motif was verified using shorter synthetic peptides. PMID:16326707

  4. 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.

  5. The integrin alpha 6 beta 1 promotes the survival of metastatic human breast carcinoma cells in mice.

    PubMed Central

    Wewer, U. M.; Shaw, L. M.; Albrechtsen, R.; Mercurio, A. M.

    1997-01-01

    The role of the integrin alpha 6 beta 1 in breast carcinoma progression was studied by targeted elimination of this integrin in MDA-MB-435 cells, a human breast carcinoma cell line that is highly metastatic in athymic mice. The strategy used is based on the finding that expression of a cytoplasmic domain deletion mutant of the beta 4-integrin subunit (beta 4-delta CYT) in MDA-MB-435 cells eliminates formation of the alpha 6 beta 1 heterodimer. MDA-MB-435 cells that lacked alpha 6 beta 1 expression (beta 4-delta CYT transfectants) formed tumors in athymic mice that were suppressed in their growth and that exhibited a significant increase in apoptosis in comparison to the control tumors. Unlike the control MDA-MB-435 cells, the beta 4-delta CYT transfectants were unable to establish metastatic foci in the lungs. Also, the control transfectants grew substantially better than the beta 4-delta CYT transfectants in the liver after intrahepatic injection because of extensive apoptosis in the beta 4-delta CYT transfectants. These data suggest that a major function of the alpha 6 beta 1 integrin in breast carcinoma is to facilitate tumorigenesis and promote tumor cell survival in distant organs. Images Figure 1 Figure 2 Figure 3 PMID:9358743

  6. CD31 promotes beta1 integrin-dependent engulfment of apoptotic Jurkat T lymphocytes opsonized for phagocytosis by fibronectin.

    PubMed

    Vernon-Wilson, Elizabeth F; Auradé, Frédéric; Brown, Simon B

    2006-06-01

    Phagocyte integrins, by binding "bridging" molecules, mediate the ingestion of late apoptotic cells and apoptotic bodies by mechanisms that remain obscure. We recently reported that human monocyte-derived macrophages capture viable and apoptotic human leukocytes through homophilic interactions involving CD31 and that CD31 then promotes the engulfment of apoptotic cells or the detachment of viable cells. We now report that CD31 homophilic interactions between phagocyte and target cells lead to activation of phagocyte alpha5beta1 integrin and the engulfment of apoptotic Jurkat T lymphocytes via a fibronectin (Fn) "bridge." Although Fn and serum served as an opsonin for beta1 integrin-dependent phagocytosis of apoptotic leukemic T cells, they failed to do so for neutrophils. Given the complexities and inherent variability of working with primary cells, we have refined our model to show that ligation of CD31 on THP-1 macrophages also regulates beta1 integrin-dependent phagocytosis of Fn-coated Latex beads. Thus, selective "tethering" of apoptotic leukocytes by phagocyte CD31 not only discriminates dying from viable cells but also selectively activates phagocyte integrins for the engulfment of apoptotic cells. PMID:16551678

  7. 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.

  8. Loss of beta1-integrin enhances TGF-beta1-induced collagen expression in epithelial cells via increased alphavbeta3-integrin and Rac1 activity.

    PubMed

    Hayashida, Tomoko; Jones, Jonathan C R; Lee, Carrie K; Schnaper, H William

    2010-10-01

    Transforming growth factor β (TGF-β) promotes tissue fibrosis via the receptor-specific Smad pathway and non-canonical pathways. We recently reported that TGF-β1-stimulated collagen expression by cultured kidney cells requires integrin-dependent activation of focal adhesion kinase (FAK) and consequent ERK MAP kinase activity leading to Smad3 linker region phosphorylation. Here, we defined a role for αvβ3-integrin in this non-canonical pathway. A human kidney tubular cell line in which β1-integrin was knocked down (β1-k/d) demonstrated enhanced type I collagen mRNA expression and promoter activity. A second shRNA to either αv-integrin or β3-integrin, but not to another αv-binding partner, β6-integrin, abrogated the enhanced COL1A2 promoter activity in β1-k/d cells. Although αvβ3-integrin surface expression levels were not different, αvβ3-integrins colocalized with sites of focal adhesion significantly more in β1-k/d cells, and activated αvβ3-integrin was detected only in β1-k/d cells. Further, the collagen response was decreased by a function-blocking antibody or a peptide inhibitor of αvβ3-integrin. In cells lacking αvβ3-integrin, the responses were attenuated, whereas the response was enhanced in αvβ3-overexpressing cells. Rac1 and ERK, previously defined mediators for this non-canonical pathway, showed increased activities in β1-k/d cells. Finally, inhibition of αvβ3-integrin decreased Rac1 activity and COL1A2 promoter activity in β1-k/d cells. Together, our results indicate that decreasing β1 chain causes αvβ3-integrin to become functionally dominant and promotes renal cell fibrogenesis via Rac1-mediated ERK activity. PMID:20650890

  9. Adhesion and migration of avian neural crest cells on fibronectin require the cooperating activities of multiple integrins of the (beta)1 and (beta)3 families.

    PubMed

    Testaz, S; Delannet, M; Duband, J

    1999-12-01

    Based on genetic, functional and histological studies, the extracellular matrix molecule fibronectin has been proposed to play a key role in the migration of neural crest cells in the vertebrate embryo. In the present study, we have analyzed in vitro the repertoire and function of integrin receptors involved in the adhesive and locomotory responses of avian truncal neural crest cells to fibronectin. Immunoprecipitation experiments showed that neural crest cells express multiple integrins, namely (alpha)3(beta)1, (alpha)4(beta)1, (alpha)5(beta)1, (alpha)8(beta)1, (alpha)v(beta)1, (alpha)v(beta)3 and a (beta)8 integrin, as potential fibronectin receptors, and flow cytometry analyses revealed no major heterogeneity among the cell population for expression of integrin subunits. In addition, the integrin repertoire expressed by neural crest cells was found not to change dramatically during migration. At the cellular level, only (alpha)v(beta)1 and (alpha)v(beta)3 were concentrated in focal adhesion sites in connection with the actin microfilaments, whereas the other integrins were predominantly diffuse over the cell surface. In inhibition assays with function-perturbing antibodies, it appeared that complete abolition of cell spreading and migration could be achieved only by blocking multiple integrins of the (beta)1 and (beta)3 families, suggesting possible functional compensations between different integrins. In addition, these studies provided evidence for functional partitioning of integrins in cell adhesion and migration. While spreading was essentially mediated by (alpha)v(beta)1 and (alpha)8(beta)1, migration involved primarily (alpha)4(beta)1, (alpha)v(beta)3 and (alpha)8(beta)1 and, more indirectly, (alpha)3(beta)1. (alpha)5(beta)1 and the (beta)8 integrin were not found to play any major role in either adhesion or migration. Finally, consistent with the results of inhibition experiments, recruitment of (alpha)4(beta)1 and (alpha)v(beta)3, individually or in

  10. Spatiotemporal expression of alpha(1), alpha(3) and beta(1) integrin subunits is altered in rat myometrium during pregnancy and labour.

    PubMed

    Williams, S J; Shynlova, O; Lye, S J; MacPhee, D J

    2010-01-01

    Integrins are transmembrane extracellular matrix (ECM) receptors composed of alpha- and beta-subunits. Integrins can cluster to form focal adhesions and, because there is significant ECM remodelling and focal adhesion turnover in the rat myometrium during late pregnancy, we hypothesised that the expression of alpha(1), alpha(3) and beta(1) integrin subunits in the rat myometrium would be altered at this time to accommodate these processes. Expression of alpha(1) and beta(1) integrin subunit mRNA was significantly increased on Days 6-23 of pregnancy compared with non-pregnant (NP) and postpartum (PP) time points (P < 0.05). In contrast, alpha(3) integrin subunit mRNA expression was significantly increased on Days 14, 21 and 22 compared with NP, Day 10, 1 day PP and 4 days PP (P < 0.05). A relative gene expression study revealed that, of the integrins studied, the expression of beta(1) integrin mRNA was highest in pregnant rat myometrium. The alpha(1), alpha(3) and beta(1) integrin subunit proteins became immunolocalised to myocyte membranes in situ by late pregnancy and labour in both myometrial muscle layers. Increased alpha(1), alpha(3) and beta(1) integrin gene expression during gestation and the specific detection of these subunits in myocyte membranes during late pregnancy and labour may contribute to the cell-ECM interactions required for the development of a mechanical syncytium. PMID:20353731

  11. Function of glycoprotein VI and integrin alpha2beta1 in the procoagulant response of single, collagen-adherent platelets.

    PubMed

    Heemskerk, J W; Siljander, P; Vuist, W M; Breikers, G; Reutelingsperger, C P; Barnes, M J; Knight, C G; Lassila, R; Farndale, R W

    1999-05-01

    Various collagen-based materials were used to assess the structural requirements of collagen for inducing the procoagulant response of adhering platelets, as well as the collagen receptors involved. Cross-linked or monomeric collagen-related peptide (CRP), Gly-Cys-Hyp-(Gly-Pro-Hyp)10-Gly-Cys-Hyp-Gly was highly adhesive for platelets in a glycoprotein VI-(GpVI-)dependent manner. Adhesion was followed by a prolonged increase in cytosolic [Ca2+]i, formation of membrane blebs, exposure of phosphatidylserine (PS) and generation of prothrombinase-stimulating activity. Fibrils of type-I collagen were less adhesive but, once adhered, many of the platelets presented a full procoagulant response. Monomeric type-I collagen was unable to support adhesion, unless Mg(2+)-dependent integrin alpha2beta1 interactions were facilitated by omission of Ca2+ ions. With all surfaces, however, post-addition of CaCl2 to adhering platelets resulted in a potent Ca(2+)-influx signal, followed by PS exposure and bleb formation. The procoagulant response elicited by binding to CRP was inhibited by anti-GpVI Fab fragments, but not by impeding integrin alpha2beta1-mediated events. With fibrillar collagen, it was inhibited by blocking either the GpVI- or integrin alpha2beta1-mediated interactions. This suggests that the triple-helical Gly-Pro-Hyp repeat in CRP and analogous sequences in fibrillar collagen stimulate the procoagulant response of adherent platelets by acting as ligands for GpVI. Influx of Ca2+ is required for this response, and adhesion via integrin alpha2beta1 serves to potentiate the signaling effects of GpVI. PMID:10365754

  12. 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

  13. The integrin receptor alpha 8 beta 1 mediates interactions of embryonic chick motor and sensory neurons with tenascin-C.

    PubMed

    Varnum-Finney, B; Venstrom, K; Muller, U; Kypta, R; Backus, C; Chiquet, M; Reichardt, L F

    1995-06-01

    This paper identifies a neuronal receptor for tenascin-C (tenascin/cytotactin), an extracellular matrix protein that has previously been detected in developing sensory and motor neuron pathways and has been shown to regulate cell migration in the developing CNS. Antibodies specific for each subunit of the integrin alpha 8 beta 1 are used to demonstrate that alpha 8 beta 1 mediates neurite outgrowth of embryonic sensory and motor neurons on this extracellular matrix protein. In addition, expression of alpha 8 in K562 cells results in surface expression of alpha 8 beta 1 heterodimers that are shown to promote attachment of this cell line to tenascin. The major domain in tenascin that mediates neurite outgrowth is shown to be localized to fibronectin type III repeats 6-8. PMID:7541634

  14. Identification of amino acid residues that form part of the ligand-binding pocket of integrin alpha5 beta1.

    PubMed

    Mould, A P; Burrows, L; Humphries, M J

    1998-10-01

    Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA) is a novel ligand peptide for integrin alpha5 beta1, which blocks alpha5 beta1-mediated cell adhesion to fibronectin (Koivunen, E., Wang, B., and Ruoslahti, E. (1994) J. Cell Biol. 124, 373-380). Here we have localized the binding site for RRETAWA on alpha5 beta1 using inhibitory monoclonal antibodies (mAbs) and site-directed mutagenesis. A cyclic peptide containing this sequence (*CRRETAWAC*) had little effect on the binding of most anti-alpha5 and anti-beta1 mAbs to alpha5 beta1 but completely blocked binding of the anti-alpha5 mAb 16 in a directly competitive manner. Hence, the binding site of RRETAWA appears to closely overlap with the epitope of mAb 16. *CRRETAWAC* also acted as a direct competitive inhibitor of the binding of Arg-Gly-Asp (RGD)-containing fibronectin fragments to alpha5 beta1, suggesting that the binding site for RRETAWA is also closely overlapping with that for RGD. However, differences between the binding sites of RRETAWA and RGD were apparent in that (i) RGD peptides allosterically inhibited the binding of mAb 16 to alpha5 beta1, and (ii) several mAbs that perturbed binding of alpha5 beta1 to RGD had little effect on binding of alpha5 beta1 to RRETAWA. A double mutation in alpha5 (S156G/W157S) blocked the interaction of both RRETAWA and mAb 16 with alpha5 beta1 but had no effect on fibronectin binding or on the binding of other anti-alpha5 mAbs. Ser156-Trp157 is located near the apex of a putative loop region on the upper surface of a predicted beta-propeller structure formed by the NH2-terminal repeats of alpha5. Our findings suggest that this sequence forms part of the ligand-binding pocket of alpha5 beta1. Furthermore, as Ser156-Trp157 is unique to the alpha5 subunit, it may be responsible for the specific recognition of RRETAWA by alpha5 beta1. PMID:9748233

  15. Discoidin domain receptor 1 activation suppresses alpha2beta1 integrin-dependent cell spreading through inhibition of Cdc42 activity.

    PubMed

    Yeh, Yi-Chun; Wang, Chau-Zen; Tang, Ming-Jer

    2009-01-01

    Upregulation and overexpression of discoidin domain receptor 1 (DDR1) have been implied in the regulation of kidney development and progression of cancers. Our previous studies with Mardin-Darby canine kidney (MDCK) cells showed that overexpression of DDR1 inhibited cell spreading, whereas dominant negative DDR1 promoted cell spreading on collagen-coated dish. Cell spreading is an important characteristic for cell differentiation and survival. However, little is known about the molecular mechanisms underlying the role of DDR1 in cell spreading. We have found here a novel signaling pathway of DDR1 consisting of Cdc42 that regulates the assembly and disassembly of cytoskeleton and cell spreading in MDCK cells. Cell spreading involves the organization of cytoskeleton that is mainly regulated by Rho-family GTPases. We assessed the activity of Rho-family GTPases and transfected MDCK cells with constitutively active or dominant negative GTPases, and quantified the extent of cell spreading. These results showed that DDR1 decreased the filamentous actin ratio and Rac1/Cdc42 activities, but had no effects on RhoA activity. Neither constitutively active nor dominant negative Rac1 altered DDR1-inhibited cell spreading. Constitutively active Cdc42 could rescue the DDR1-inhibited cell spreading, whereas dominant negative Cdc42 inhibited cell spreading, indicating that DDR1-inhibited cell spreading is Cdc42 dependent. With the use of alpha(2)beta(1) integrin blocking antibody, we showed that collagen-induced Cdc42 activation was mediated by alpha(2)beta(1) integrin. Moreover, ectopic FAK expression enhanced the Cdc42 activity. Reducing FAK activity by dominant negative FAK (FRNK) markedly abolished the Cdc42 activity. These findings show that DDR1a/b activation inhibits cell spreading through suppressing alpha(2)beta(1) integrin-mediated Cdc42 activation. PMID:18780290

  16. 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.

  17. Distinct spatio-temporal Ca2+ signaling elicited by integrin alpha2beta1 and glycoprotein VI under flow.

    PubMed

    Mazzucato, Mario; Cozzi, Maria Rita; Battiston, Monica; Jandrot-Perrus, Martine; Mongiat, Maurizio; Marchese, Patrizia; Kunicki, Thomas J; Ruggeri, Zaverio M; De Marco, Luigi

    2009-09-24

    We studied how integrin alpha2beta1 and glycoprotein VI (GPVI) contribute to collagen-induced platelet activation under flow conditions by evaluating stable adhesion and intracellular Ca(2+) concentration ([Ca(2+)](i)) of FLUO 3-AM-labeled platelets perfused over acid-soluble type I or microfibrillar type VI collagen. Adhering platelets displayed 2 kinds of [Ca(2+)](i) oscillations. Rapid alpha-like peaks were unaffected by the membrane-impermeable Ca(2+) chelator ethyleneglycoltetraacetic acid but abolished by membrane-permeable BAPTA-AM. Longer-lasting gamma-like peaks were always preceded by at least one alpha-like peak and abolished by intracellular or extracellular Ca(2+) chelation. Inhibition of phosphatidylinositol 3-kinase or phospholipase C and modulation of cyclic nucleotides, but not blockage of adenosine diphosphate receptors, prevented both Ca(2+) responses. Human or mouse platelets lacking GPVI function exhibited alpha-like but not gamma-like Ca(2+) peaks, whereas those lacking alpha2beta1 showed markedly reduced to absent alpha-like and no gamma-like Ca(2+) peaks. Specific alpha2beta1 ligation induced alpha-like but not gamma-like peaks. Thus, alpha2beta1 may generate Ca(2+) signals that are reinforced by GPVI and required for subsequent longer-lasting Ca(2+) oscillation mediated by GPVI through transmembrane ion flux. Our results delineate a GPVI-independent signaling role of alpha2beta1 in response to collagen stimulation. PMID:19622836

  18. 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

  19. 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.

  20. 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.

  1. Integrin alpha1beta1 regulates epidermal growth factor receptor activation by controlling peroxisome proliferator-activated receptor gamma-dependent caveolin-1 expression.

    PubMed

    Chen, Xiwu; Whiting, Carrie; Borza, Corina; Hu, Wen; Mont, Stacey; Bulus, Nada; Zhang, Ming-Zhi; Harris, Raymond C; Zent, Roy; Pozzi, Ambra

    2010-06-01

    Integrin alpha1beta1 negatively regulates the generation of profibrotic reactive oxygen species (ROS) by inhibiting epidermal growth factor receptor (EGFR) activation; however, the mechanism by which it does this is unknown. In this study, we show that caveolin-1 (Cav-1), a scaffolding protein that binds integrins and controls growth factor receptor signaling, participates in integrin alpha1beta1-mediated EGFR activation. Integrin alpha1-null mesangial cells (MCs) have reduced Cav-1 levels, and reexpression of the integrin alpha1 subunit increases Cav-1 levels, decreases EGFR activation, and reduces ROS production. Downregulation of Cav-1 in wild-type MCs increases EGFR phosphorylation and ROS synthesis, while overexpression of Cav-1 in the integrin alpha1-null MCs decreases EGFR-mediated ROS production. We further show that integrin alpha1-null MCs have increased levels of activated extracellular signal-regulated kinase (ERK), which leads to reduced activation of peroxisome proliferator-activated receptor gamma (PPARgamma), a transcription factor that positively regulates Cav-1 expression. Moreover, activation of PPARgamma or inhibition of ERK increases Cav-1 levels in the integrin alpha1-null MCs. Finally, we show that glomeruli of integrin alpha1-null mice have reduced levels of Cav-1 and activated PPARgamma but increased levels of phosphorylated EGFR both at baseline and following injury. Thus, integrin alpha1beta1 negatively regulates EGFR activation by positively controlling Cav-1 levels, and the ERK/PPARgamma axis plays a key role in regulating integrin alpha1beta1-dependent Cav-1 expression and consequent EGFR-mediated ROS production. PMID:20368353

  2. 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.

  3. Rotavirus spike protein VP5* binds alpha2beta1 integrin on the cell surface and competes with virus for cell binding and infectivity.

    PubMed

    Graham, Kate L; Takada, Yoshikazu; Coulson, Barbara S

    2006-05-01

    Rotaviruses recognize several cell-surface molecules, including the alpha2beta1 integrin, and the processes of rotavirus cell attachment and entry appear to be multifactorial. The VP5* subunit of the rotavirus spike protein VP4 contains the alpha2beta1 ligand sequence Asp-Gly-Glu at residues 308-310. Binding to alpha2beta1 and infectivity of monkey rotavirus strain RRV and human rotavirus strain Wa, but not porcine rotavirus strain CRW-8, are inhibited by peptides containing Asp-Gly-Glu. Asp308 and Gly309 are necessary for the binding of RRV VP5* (aa 248-474) to expressed I domain of the alpha2 integrin subunit. Here, the ability of RRV VP5* to bind cells and affect rotavirus-integrin interactions was determined. Interestingly, VP5* bound to cells at 4 and 37 degrees C, both via alpha2beta1 and independently of this integrin. Prior VP5* binding at 37 degrees C eliminated RRV binding to cellular alpha2beta1 and reduced RRV and Wa infectivity in MA104 cells by 38-46 %. VP5* binding did not affect the infectivity of CRW-8. VP5* binding at 4 degrees C did not affect permissive-cell infection by RRV, indicating an energy requirement for VP5* competition with virus for infectivity. Mutagenesis of VP5* Asp308 and Gly309 eliminated VP5* binding to alpha2beta1 and the VP5* inhibition of rotavirus cell binding and infection, but not alpha2beta1-independent cell binding by VP5*. These studies show for the first time that expressed VP5* binds cell-surface alpha2beta1 using Asp308 and Gly309 and inhibits the infection of homologous and heterologous rotaviruses that use alpha2beta1 as a receptor. PMID:16603530

  4. 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.

  5. Integrins modulate the Egfr signaling pathway to regulate tendon cell differentiation in the Drosophila embryo.

    PubMed

    Martin-Bermudo, M D

    2000-06-01

    Changes in the extracellular matrix (ECM) govern the differentiation of many cell types during embryogenesis. Integrins are cell matrix receptors that play a major role in cell-ECM adhesion and in transmitting signals from the ECM inside the cell to regulate gene expression. In this paper, it is shown that the PS integrins are required at the muscle attachment sites of the Drosophila embryo to regulate tendon cell differentiation. The analysis of the requirements of the individual alpha subunits, alphaPS1 and alphaPS2, demonstrates that both PS1 and PS2 integrins are involved in this process. In the absence of PS integrin function, the expression of tendon cell-specific genes such as stripe and beta1 tubulin is not maintained. In addition, embryos lacking the PS integrins also exhibit reduced levels of activated MAPK. This reduction is probably due to a downregulation of the Epidermal Growth Factor receptor (Egfr) pathway, since an activated form of the Egfr can rescue the phenotype of embryos mutant for the PS integrins. Furthermore, the levels of the Egfr ligand Vein at the muscle attachment sites are reduced in PS mutant embryos. Altogether, these results lead to a model in which integrin-mediated adhesion plays a role in regulating tendon cell differentiation by modulating the activity of the Egfr pathway at the level of its ligand Vein. PMID:10821759

  6. 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

  7. 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.

  8. 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

  9. A monoclonal antibody to a carbohydrate epitope expressed on glycolipid and on alpha3beta1 integrin on human esophageal carcinoma.

    PubMed

    Jamasbi, Roudabeh J; Stoner, Gary D; Foote, Linda J; Lankford, Trish K; Davern, Sandra; Kennel, Stephen J

    2003-12-01

    A mouse monoclonal antibody (MAb-9) produced by immunization with a human esophageal carcinoma cell line, TE-2 (derived from undifferentiated squamous cell carcinoma) reacted specifically with about 30% of esophageal carcinoma cell lines and tissue sections from clinical samples. MAb-9 showed minimal reactivity with normal esophageal tissue. (125)I, fluorescent or gold particle labeled MAb-9 bound to TE-2 cell surfaces. (125)I-radiolabeled MAb-9 was used to detect reactive material from cell extracts in Western blot. Treatment of TE-2 membrane proteins with neuraminidase, N-glycanase or O-glycanase reduced antigen detection. Treatment of cells with periodic acid destroyed antibody binding in ELISA. Lipid extracts from cell membranes, containing glycolipids, also reacted with MAb-9. MAb-9 was used to purify target antigen from detergent solubilized membrane proteins and the prominent bands from subsequent gel electrophoresis were trypsin digested and analyzed by mass spectrometry. Peptides from alpha(3) and beta(1) integrin chains were identified. These data indicate that alpha3beta1integrin is prominently expressed on certain esophageal carcinomas and that a specific carbohydrate unit is selectively displayed on the alpha(3) integrin subunit as well as on glycolipid on the cell surface. The alpha3beta1 integrin expressed on A-431 carcinoma cells does not display this carbohydrate epitope and is not detected by MAb-9. Thus, expression of the carbohydrate epitope is the basis for the tumor selective reaction of MAb-9 with a subset of esophageal carcinomas. PMID:14683596

  10. Vascular cell adhesion molecule 1 and alpha 4 and beta 1 integrins in lymphocyte aggregates in Sjögren's syndrome and rheumatoid arthritis.

    PubMed Central

    Edwards, J C; Wilkinson, L S; Speight, P; Isenberg, D A

    1993-01-01

    OBJECTIVES--Interactions between vascular cell adhesion molecule 1 (VCAM-1) and its ligand, the alpha 4/beta 1 integrin, have been shown to be important in a number of cellular events in vitro. To assess the importance of such interactions in the development of lymphocytic infiltration in diseased tissue the distribution of the two ligands has been studied immunohistochemically. METHODS--Cryostat sections of labial tissue from patients with Sjögren's syndrome, normal labial tissues, rheumatoid synovia, and normal tonsils were stained using antibodies to VCAM-1, alpha 4 and beta 1 integrin chains, and markers for T cells, B cells, macrophages, and follicular dendritic reticulum cells (FDRCs), visualised using alkaline phosphatase and fast red. RESULTS--Staining patterns for VCAM-1 and integrin chains in lymphocyte aggregates in synovial and labial tissues were similar. VCAM-1 staining was found on both vascular and ramifying dendritic cells at the centre of large T cell aggregates and in all aggregates where there was a central clustering of B cells. VCAM-1 colocalised with, but also extended beyond, staining for the FDRC marker R4/23. Staining for the alpha 4 and beta 1 integrin chains was more widespread than staining for VCAM-1, with no significant increase in staining at sites of maximum VCAM-1 staining. In tonsils VCAM-1 and R4/23 codistributed in germinal centres, but staining for the alpha 4 and beta 1 integrin chains was chiefly seen in T lymphocyte areas. CONCLUSIONS--VCAM-1 may be more important in determining the distribution of B than T lymphocytes in lymphocytic infiltration of non-lymphoid tissue. Unlike the follicles of lymphoid tissue, ectopic follicle-like structures in non-lymphoid tissues may form by immigration of B cells via VCAM-1+ vessels at the centre of T cell aggregates. Images PMID:7504438

  11. 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.

  12. Metastatic dissemination of human ovarian epithelial carcinoma is promoted by alpha2beta1-integrin-mediated interaction with type I collagen.

    PubMed

    Fishman, D A; Kearns, A; Chilukuri, K; Bafetti, L M; O'Toole, E A; Georgacopoulos, J; Ravosa, M J; Stack, M S

    1998-01-01

    Metastatic dissemination of epithelial ovarian carcinoma is thought to be mediated via tumor cell exfoliation into the peritoneal cavity, followed by adhesion to and invasion through the mesothelium which overlies the contents of the peritoneal cavity. In this study, we have utilized short-term primary cultures to analyze the effect of specific extracellular matrix proteins on properties of human ovarian epithelial carcinoma cells which contribute to the invasive phenotype. Analysis of cell:matrix adhesive profiles indicated that ovarian carcinoma cells adhere preferentially to type I collagen. Immunoprecipitation analyses demonstrated the presence of the collagen-binding alpha2beta1 integrin in biotin-labeled ovarian carcinoma cell membranes, and cellular adhesion was inhibited by blocking antibodies directed against the alpha2 and beta1 integrin subunits. The alpha2beta1-binding peptide Asp-Gly-Glu-Ala (DGEA) was also moderately effective at blocking adhesion to collagen relative to the control peptide Ala-Gly-Glu-Ala (AGEA). Analysis of cell motility on protein-coated colloidal gold coverslips demonstrated that ovarian carcinoma cells migrate preferentially on type I collagen coated surfaces. Type I collagen promoted migration in a concentration-dependent, saturable manner, with maximal migration observed at a collagen-coating concentration of 50 microg/ml. Migration on collagen was inhibited by antibodies directed against the alpha2 and beta1 integrin subunits and by DGEA peptide, providing evidence for the role of the alpha2beta1 integrin in ovarian carcinoma cell motility. Culturing ovarian carcinoma cells on type I collagen gels led to a significant increase in conversion of the matrix metalloproteinase 2 zymogen to the 66-kD form, suggesting that adhesion to collagen also influences matrix-degrading proteinases. These data suggest that alpha2beta1-integrin-mediated interaction of ovarian carcinoma cells with type I collagen, a protein prevalent both in the

  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. Attachment of primary neonatal rat astrocytes to vitronectin is mediated by integrins alphavbeta5 and alpha8beta1: modulation by the type 1 plasminogen activator inhibitor.

    PubMed

    Gladson, C L; Stewart, J E; Olman, M A; Chang, P L; Schnapp, L M; Grammer, J R; Benveniste, E N

    2000-04-01

    Vitronectin is expressed in a cell-specific manner in the developing brain and concentrated in the brain during disease processes, such as germinal matrix hemorrhage and infarction, in which there is breakdown of the blood-brain barrier. In this study, we identified the integrin receptors that mediate attachment of primary neonatal rat astrocytes to vitronectin. Using fluorescent activated cell sorter and immunoprecipitation analyses, we established that the vitronectin receptor integrins alphavbeta5 and alpha8beta1, but not alphavbeta3, are expressed on neonatal rat astrocytes. Attachment of the neonatal astrocytes to vitronectin was inhibited (85%) in an additive manner by neutralizing anti-alphavbeta5 and anti-beta1 antibodies. Attachment to vitronectin was also inhibited in a dose-dependent manner by the type I plasminogen activator inhibitor (PAI-1), a serine protease inhibitor. Our data demonstrate that unstimulated primary neonatal rat astrocytes attach to vitronectin, utilizing integrins alphavbeta5 and alpha8beta1, and that this attachment is regulated by PAI-1. PMID:10739899

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

    PubMed Central

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

    2001-01-01

    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. PMID:11336631

  17. Production of IL1-beta by ovarian cancer cells induces mesothelial cell beta1-integrin expression facilitating peritoneal dissemination

    PubMed Central

    2012-01-01

    Background A crucial step in the metastatic spread of ovarian cancer (OC) is the adhesion and implantation of tumor cells to the peritoneal mesothelium. In order to study this step in the cascade, we derived a pro-metastatic human ovarian carcinoma cell line (MFOC3) from the non-metastatic FOC3 line. Methods Molecular profiling of the isogeneic lines identified differentially expressed genes, and investigation for a role in dissemination for specific factors was achieved by development of a co-culture adhesion assay utilizing monolayers of human mesothelial cells. Results After murine intraperitoneal inoculation, the FOC3 cell line formed no metastases, but the MFOC3 subline formed metastases in > 80% of SCID mice. MFOC3 cells also adhered 2-3 times more avidly to mesothelial monolayers. This adhesion was inhibited by neutralizing antibodies to IL-1β and enhanced by recombinant IL-1β (p < 0.01). IL-1β induced mesothelial cell β1-integrin, and an antibody to this subunit also inhibited the adhesion of MFOC3 to mesothelial cells in vitro and significantly reduced metastases in vivo. Immunohistochemical analysis of a cohort of 96 ovarian cancer cases showed that negative IL-1β expression was significantly associated with an improved overall survival rate. Conclusions These results suggest that a IL-1β/β1-integrin axis plays a role in ovarian tumor cell adhesion to mesothelia, a crucial step in ovarian cancer dissemination. PMID:22296757

  18. Integrin {alpha}1{beta}1 promotes caveolin-1 dephosphorylation by activating T cell protein-tyrosine phosphatase.

    PubMed

    Borza, Corina M; Chen, Xiwu; Mathew, Sijo; Mont, Stacey; Sanders, Charles R; Zent, Roy; Pozzi, Ambra

    2010-12-17

    Integrin α1β1 is a collagen receptor that down-regulates collagen and reactive oxygen species (ROS) production, and mice lacking this receptor show increased ROS levels and exacerbated glomerular sclerosis following injury. Caveolin-1 (Cav-1) is a multifunctional protein that is tyrosine-phosphorylated in response to injury and has been implicated in ROS-mediated injury. Cav-1 interacts with integrins, and integrin α1β1 binds/activates T cell protein-tyrosine phosphatase (TCPTP), which is homologous to the tyrosine phosphatase PTP1B known to dephosphorylate Cav-1. In this study, we analyzed whether phosphorylated Cav-1 (pCav-1) is a substrate of TCPTP and if integrin α1β1 is essential for promoting TCPTP-mediated Cav-1 dephosphorylation. We found that Cav-1 phosphorylation is significantly higher in cells lacking integrin α1β1 at base line and following oxidative stress. Overexpression of TCPTP leads to reduced pCav-1 levels only in cells expressing integrin α1β1. Using solid phase binding assays, we demonstrated that 1) purified Cav-1 directly interacts with TCPTP and the integrin α1 subunit, 2) pCav-1 is a substrate of TCPTP, and 3) TCPTP-mediated Cav-1 dephosphorylation is highly increased by the addition of purified integrin α1β1 or an integrin α1 cytoplasmic peptide to which TCPTP has been shown to bind. Thus, our results demonstrate that pCav-1 is a new substrate of TCPTP and that integrin α1β1 acts as a negative regulator of Cav-1 phosphorylation by activating TCPTP. This could explain the protective function of integrin α1β1 in oxidative stress-mediated damage and why integrin α1-null mice are more susceptible to fibrosis following injury. PMID:20940300

  19. 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

    2016-01-01

    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. PMID:27336951

  20. CD44/chondroitin sulfate proteoglycan and alpha 2 beta 1 integrin mediate human melanoma cell migration on type IV collagen and invasion of basement membranes.

    PubMed Central

    Knutson, J R; Iida, J; Fields, G B; McCarthy, J B

    1996-01-01

    Tumor cell invasion of basement membranes (BM) represents one of the critical steps in the metastatic process. Tumor cell recognition of individual BM matrix components may involve individual cell adhesion receptors, such as integrins or cell surface proteoglycans, or may involve a coordinate action of both types of receptors. In this study, we have focused on the identification of a cell surface CD44/chondroitin sulfate proteoglycan (CSPG) and alpha 2 beta 1 integrin on human melanoma cells that are both directly involved in the in vitro invasion of reconstituted BM via a type IV collagen-dependent mechanism. Interfering with cell surface expression of human melanoma CSPG with either p-nitro-phenyl-beta-D-xylopyranoside treatment or anti-CD44 monoclonal antibody (mAb) preincubation (mAb) preincubation inhibits melanoma cell invasion through reconstituted BM. These treatments also strongly inhibit melanoma cell migration on type IV collagen, however, they are ineffective at inhibiting cell adhesion to type IV collagen. Purified melanoma cell surface CD44/CSPG, or purified chondroitin sulfate, bind to type IV collagen affinity columns, consistent with a role for CD44/CSPG-type IV collagen interactions in mediating tumor cell invasion. In contrast, melanoma cell migration on laminin (LM) does not involve CD44/CSPG, nor does CD44/CSPG bind to LM, suggesting that CD44/CSPG-type IV collagen interactions are specific in nature. Additionally, anti-alpha 2 and anti-beta 1 integrin mAbs are capable of blocking melanoma cell invasion of reconstituted BM. Both of these anti-integrin mAbs inhibit melanoma cell adhesion and migration on type IV collagen, whereas only anti-beta 1 mAb inhibits cell adhesion to LM. Collectively, these results indicate that melanoma cell adhesion to type IV collagen is an important consideration in invasion of reconstituted BM in vitro, and suggest that CD44/CSPG and alpha 2 beta 1 integrin may collaborate to promote human melanoma cell adhesion

  1. 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.

  2. 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

  3. Overexpression of the beta 1 thyroid receptor induces differentiation in neuro-2a cells.

    PubMed Central

    Lebel, J M; Dussault, J H; Puymirat, J

    1994-01-01

    To determine the functions of the alpha 1 and beta 1 thyroid hormone receptors (TRs) in neural differentiation, we have established stable transfected neuronal cell lines (Neuro-2a) that overexpress either TR alpha 1 or TR beta 1. 3,5,3'-Triiodothyronine (T3) treatment of cells that overexpress TR beta 1 blocks proliferation by an arrest of cells in G0/G1 and induces morphological and functional differentiation of Neuro-2a cells as indicated by the marked increase in the number of perisomatal filopodia-like neurites and in acetylcholinesterase (AChE) activity. The effect on AChE activity was dose-dependent, and the time-course analysis reveals that this effect occurs after 24 hr of T3 treatment, with a maximal increase occurring after 48 hr of treatment. The increase of AChE activity is paralleled by an increase of AChE mRNAs. Last, we present evidence that shows that the effects of T3 on differentiation are independent of its effect on proliferation. T3 had no effect on the differentiation of Neuro-2a cells that overexpressed TR alpha 1. Our results indicate that TR beta 1 may play a key role in the effects of T3 in neuroblastoma cell differentiation. Images PMID:8146169

  4. Bone Regeneration using an Alpha 2 Beta 1 Integrin-Specific Hydrogel as a BMP-2 Delivery Vehicle

    PubMed Central

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

    2014-01-01

    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. PMID:24726536

  5. Regulation of ionizing radiation-induced adhesion of breast cancer cells to fibronectin by alpha5beta1 integrin.

    PubMed

    Lee, Shin Hee; Cheng, Huiwen; Yuan, Ye; Wu, Shiyong

    2014-06-01

    Ionizing radiation (IR) is commonly used for cancer therapy, however, its potential influence on cancer metastatic potential remains controversial. In this study, we elucidated the role of integrins in regulation of IR-altered adhesion between breast cancer cells and extracellular matrix (ECM) proteins, which is a key step in the initial phase of metastasis. Our data suggest that the extent of effect that ionizing radiation had on cell adhesion depended on the genetic background of the breast cancer cells. Ionizing radiation was a better adhesion inducer for p53-mutated cells, such as MDA-MB-231 cells, than for p53 wild-type cells, such as MCF-7 cells. While IR-induced adhesions between MDA-MB-231 cells to fibronectin, laminin, collagen I and collagen IV, only blocking of the adhesion between α5β1 integrin and fibronectin using anti-α5β1 integrin antibody could completely inhibit the radiation-induced adhesion of the cells. A soluble Arg-Gly-Asp peptide, the binding motif for fibronectin binding integrins, could also reduce the adhesion of the cells to fibronectin with or without ionizing radiation exposure. The inhibition of the cell-fibronectin interaction also affected, but did not always correlate with, transwell migration of the cancer cells. In addition, our data showed that the total expression of α5 integrin and surface expression of α5β1 integrin were increased in the cells treated with ionizing radiation. The increased surface expression of α5β1 integrin, along with the adhesion between the cells and fibronectin, could be inhibited by both ataxia telangiectasia mutated (ATM) and Rad3-related (ATR) kinase inhibitors. These results suggested that ATM/ATR-mediated surface expression of α5β1 integrin might play a central role in regulation of ionizing radiation-altered adhesion. PMID:24785587

  6. Distinct roles of beta1 metal ion-dependent adhesion site (MIDAS), adjacent to MIDAS (ADMIDAS), and ligand-associated metal-binding site (LIMBS) cation-binding sites in ligand recognition by integrin alpha2beta1.

    PubMed

    Valdramidou, Dimitra; Humphries, Martin J; Mould, A Paul

    2008-11-21

    Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding sites. In collagen-binding integrins, such as alpha2beta1, ligand recognition takes place exclusively at the alpha subunit I domain. However, activation of the alphaI domain depends on its interaction with a structurally similar domain in the beta subunit known as the I-like or betaI domain. The top face of the betaI domain contains three cation-binding sites: the metal-ion dependent adhesion site (MIDAS), the ADMIDAS (adjacent to MIDAS), and LIMBS (ligand-associated metal-binding site). The role of these sites in controlling ligand binding to the alphaI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to alpha2beta1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating monoclonal antibody TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between alphaI and betaI, whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of betaI. An activating mutation in the alpha2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca(2+), Mg(2+), and Mn(2+) on ligand binding to these mutants showed that the MIDAS is a ligand-competent site through which Mn(2+) stimulates ligand binding, whereas the LIMBS is a stimulatory Ca(2+)-binding site, occupancy of which increases the affinity of Mg(2+) for the MIDAS. PMID:18820259

  7. Cytoskeleton reorganization mediates alpha6beta1 integrin-associated actions of laminin on proliferation and survival, but not on steroidogenesis of ovine granulosa cells

    PubMed Central

    Bellego, Frédérique Le; Fabre, Stéphane; Pisselet, Claudine; Monniaux, Danielle

    2005-01-01

    Background Laminin (LN) is one of the most abundant extracellular matrix components of the basal lamina and granulosa cell layers of ovarian follicles. Culture of ovine granulosa cells (GC) on LN substratum induces cell spreading, enhances cell survival and proliferation, and promotes luteinization. Previous investigations have shown that these effects are mostly mediated by the alpha6beta1 integrin, but its signalization pathways have not been investigated. This study aimed to assess the importance of the cytoskeleton in the alpha6beta1 integrin-mediated actions of laminin on survival, proliferation and steroidogenesis of ovine GC. Methods The relationships between morphology and functions of ovine GC cultured on substrata containing LN or/and RGD peptides were investigated. The effects of (1) cytochalasin D, an actin cytoskeleton-disrupting drug, (2) a specific function-blocking antibody raised against alpha6 integrin subunit (anti-alpha6 IgG), and (3) an inhibitor of the ERK1/2 signalization pathway (PD98059) were assessed for GC shape, pyknosis and proliferation rates, oestradiol and progesterone secretions. Results Cytoskeleton disruption by cytochalasin D induced cell rounding, inhibited proliferation, promoted pyknosis, inhibited progesterone secretion and enhanced oestradiol secretion by GC cultured on LN. When GC were cultured on various substrata containing LN and/or RGD peptides in the presence or absence of anti-alpha6 IgG, both the existence of close correlations between the percentage of round cells, and the GC proliferation rate (r = -0.87) and pyknotic rate (r = 0.76) were established, but no relationship was found between cell shape and steroidogenesis. Inhibition of the ERK1/2 signalization pathway by PD98059 had no effect on GC shape, proliferation or pyknotic rates. However, it dramatically reduced progesterone secretion, expression of cytochrome P450 cholesterol side-chain cleavage and 3beta-hydroxysteroid deshydrogenase enzymes, and enhanced

  8. 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.

  9. The beta 1 integrin, very late activation antigen-4 on human neutrophils can contribute to neutrophil migration through connective tissue fibroblast barriers.

    PubMed Central

    Gao, J X; Issekutz, A C

    1997-01-01

    Polymorphonuclear leucocyte (PMNL) accumulation in extravascular tissues and inflammatory exudates is dependent on their migration through blood vessel endothelium and then through connective tissue. Previously we utilized a barrier of human synovial and dermal fibroblasts (HSF or HDF) grown on microporous filters, as a model of PMNL migration through connective tissue. Those studies showed that beta 2 (CD18) and the beta 1 integrins, very late activation antigen-5 (VLA-5) and VLA-6, in part mediate this PMNL migration. Here we report that VLA-4, which can also be expressed at low levels on activated PMNL, is also involved in PMNL migration induced by C5a through fibroblast (HSF and HDF) barriers, because monoclonal antibody (mAb) to VLA-4 significantly inhibited (by 20-30%) PMNL migration. Blocking the function of CD18, VLA-5 or VLA-6 was not required for detection of the VLA-4-mediated migration. Combination treatment with mAb to VLA-4 and with mAb to VLA-5 or to VLA-6 further inhibited PMNL migration, irrespective of whether CD11/CD18 mechanisms were blocked with anti-CD18 mAb or not. Treatment of PMNL with a peptide based on the VLA-4-binding domain in the CS-1 fragment of fibronectin, but not a control peptide, inhibited PMNL migration to a comparable extent to treatment with mAb to VLA-4. A low level of VLA-4 was expressed on C5a-activated PMNL, detected by immunofluorescence flow cytometry. These results suggest that VLA-4 can be mobilized by human peripheral blood PMNL and can, in addition to VLA-5, VLA-6 and CD11/CD18 integrins, mediate PMNL migration through connective tissue. This is in marked contrast to PMNL transendothelial migration, where beta 1 integrins appear to play no significant role. PMID:9155654

  10. beta 1-Integrin-mediated glioma cell adhesion and free radical-induced apoptosis are regulated by binding to a C-terminal domain of PG-M/versican.

    PubMed

    Wu, Yaojiong; Chen, Liwen; Zheng, Peng-Sheng; Yang, Burton B

    2002-04-01

    Integrins are cell-surface glycoproteins that mediate cell activities, including tissue morphogenesis, development, immune response, and cancer, through interaction with extracellular proteins. Here we report a novel means by which integrin signaling and functions are regulated. In pull-down assays and immunoprecipitation, beta(1)-integrin bound to the C-terminal domain of PG-M/versican, an extracellular chondroitin sulfate proteoglycan. This was confirmed by cell-surface binding assays. Binding was calcium- and manganese-dependent. Upon native gel electrophoresis, beta(1)-integrin comigrated with the C-terminal domain of PG-M/versican. The interaction of beta(1)-integrin with the C-terminal domain of PG-M/versican activated focal adhesion kinase, enhanced integrin expression, and promoted cell adhesion. As a result, cells expressing the C-terminal domain of PG-M/versican were resistant to free radical-induced apoptosis. As the PG-M/versican peptide used in this study does not contain the RGD consensus-binding motif for integrins, the mechanism of the observed binding represents an entirely new function. PMID:11805102

  11. 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. PMID:25770999

  12. 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

  13. 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. PMID:11850066

  14. Beta 2 (CD18) and beta 1 (CD29) integrin mechanisms in migration of human polymorphonuclear leucocytes and monocytes through lung fibroblast barriers: shared and distinct mechanisms.

    PubMed Central

    Shang, X Z; Issekutz, A C

    1997-01-01

    Accumulation of leucocytes in inflamed lung tissue and alveolar space involves their migration through vascular endothelium and then lung connective tissue. As a model of this process, we investigated human polymorphonuclear leucocyte (PMNL) and monocyte migration through a biological barrier of human lung fibroblasts (HLF) grown on polycarbonate filters. Very few PMNL (1-2%) or monocytes (3-8%) migrated through the HLF barriers spontaneously. Migration increased to 48-53% of added PMNL and 17-24% of added monocytes, when a C5a chemotactic gradient was present. The monocyte migration induced by C5a was not inhibited by monoclonal antibodies (mAb) to CD18 (beta 2 integrins). This CD18-independent migration was partially inhibited (35%) by mAb to gamma 5 of VLA-5 and completely inhibited by the combination of mAb to gamma 4 of VLA-4 with mAb to VLA-5, in the presence of mAb to CD18. In contrast, PMNL migration across HLF induced by C5a was partially inhibited by mAb to CD18 alone, but even with the addition of mAb to VLA-4, VLA-5 beta 1 and VLA-6, the greatest degree of inhibition was only 60%. Blocking the function of CD18 was not required to observe the inhibition by mAb to VLA-4, although the inhibitory effect of mAb to VLA-5 and VLA-6 alone or in combination was only observed when CD18 mechanisms were also blocked with anti-CD18 mAb. These results demonstrate that (a) both monocytes and PMNL can use either CD11/CD18 (beta 2 integrin) or beta 1 (CD49/CD29) integrins to migrate through HLF barriers; (b) in the case of monocytes, the VLA-4 and VLA-5 integrins account for essentially all the CD11/CD18-independent migration mechanisms; and (c) in contrast to monocytes, PMNL CD18-independent migration is mediated not only by VLA-4 and VLA-5, but also by VLA-6, and up to 40% of the migration appears to be via yet to be defined PMNL surface molecules. PMID:9497495

  15. 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

  16. Efficacy of Beta1 Integrin and EGFR Targeting in Sphere-Forming Human Head and Neck Cancer Cells

    PubMed Central

    Zscheppang, Katja; Kurth, Ina; Wachtel, Nicole; Dubrovska, Anna; Kunz-Schughart, Leoni A.; Cordes, Nils

    2016-01-01

    Background: Resistance to radiotherapy continues to be a limiting factor in the treatment of cancer including head and neck squamous cell carcinoma (HNSCC). Simultaneous targeting of β1 integrin and EGFR was shown to have a higher radiosensitizing potential than mono-targeting in the majority of tested HNSCC cancer models. As tumor-initiating cells (TIC) are thought to play a key role for therapy resistance and recurrence and can be enriched in sphere forming conditions, this study investigated the efficacy of β1 integrin/EGFR targeting without and in combination with X-ray irradiation on the behavior of sphere-forming cells (SFC). Methods: HNSCC cell lines (UTSCC15, UTSCC5, Cal33, SAS) were injected subcutaneously into nude mice for tumor up-take and plated for primary and secondary sphere formation under non-adhesive conditions which is thought to reflect the enrichment of SFC and their self-renewal capacity, respectively. Treatment was accomplished by inhibitory antibodies for β1 integrin (AIIB2) and EGFR (Cetuximab) as well as X-ray irradiation (2 - 6 Gy single doses). Further, flow cytometry for TIC marker expression and cell cycling as well as Western blotting for DNA repair protein expression and phosphorylation were employed. Results: We found higher primary and secondary sphere forming capacity of SAS cells relative to other HNSCC cell lines, which was in line with the tumor up-take rates of SAS versus UTSCC15 cells. AIIB2 and Cetuximab administration had minor cytotoxic and no radiosensitizing effects on SFC. Intriguingly, secondary SAS spheres, representing the fraction of surviving SFC upon passaging, showed greatly enhanced radiosensitivity compared to primary spheres. Intriguingly, neither AIIB2 nor Cetuximab significantly altered basal sphere forming capacity and radiosensitivity. While an increased accumulation of G0/G1 phase cells was observable in secondary SAS spheres, DNA double strand break repair indicated no difference on the basis of

  17. Transforming growth factor-beta1 incorporated during setting in calcium phosphate cement stimulates bone cell differentiation in vitro.

    PubMed

    Blom, E J; Klein-Nulend, J; Klein, C P; Kurashina, K; van Waas, M A; Burger, E H

    2000-04-01

    Growth stimulation of periimplant tissues by growth factors like transforming growth factor-beta1 (TGF-beta1) may increase the indication for and success of implant use. Calcium phosphate as a material for implants or for coating of implants is known for its good biologic interaction with bone. Therefore, calcium phosphate implants combined with TGF-beta1 might improve osseointegration. In this study we hypothesise that the addition of recombinant human TGF-beta1 (rhTGF-beta1) to calcium phosphate cement (CPC) affects the differentiation of bone cells growing on the cement layer. rhTGF-beta1 incorporated during setting in a CPC layer at 20 ng rhTGF-beta1/60 mg cement was found to be gradually released into tissue culturing medium leading to a 20% release after 24 h. Two cell populations were obtained from collagenase-treated fragments of adult rat long bones: preosteoblastic cells, which were released by the collagenase treatment, and osteoblastic cells, which grew from the collagenase-stripped bone fragments. Both cell populations were tested for their osteoblastic characteristic phenotype by measuring their alkaline phosphatase (ALP) activity after vitamin D treatment and cyclic AMP after parathyroid hormone stimulation. After preculture the cells were plated on a layer of CPC containing 0 (control), 10, or 20 ng rhTGF-beta1/60 mg CPC. Bone cell differentiation was analyzed after 10 days by measuring the ALP activity, as well as the protein content of the cell layer. Incorporation of rhTGF-beta1 in the CPC did not change the ALP activity in osteoblastic cells, but a significant (analyzed by multivariate analysis of variance) increase was observed in preosteoblastic cells. Incorporation of 10 ng of rhTGF-beta1 in 60 mg of CPC increased the ALP activity in preosteoblastic cells by threefold and 20 ng rhTGF-beta1/60 mg CPC increased it by fivefold. The total protein content was not affected by rhTGF-beta1 in either of the cell populations. We conclude that rhTGF-beta

  18. Integrin Signaling, Cell Survival, and Anoikis: Distinctions, Differences, and Differentiation

    PubMed Central

    Vachon, Pierre H.

    2011-01-01

    Cell survival and apoptosis implicate an increasing complexity of players and signaling pathways which regulate not only the decision-making process of surviving (or dying), but as well the execution of cell death proper. The same complex nature applies to anoikis, a form of caspase-dependent apoptosis that is largely regulated by integrin-mediated, cell-extracellular matrix interactions. Not surprisingly, the regulation of cell survival, apoptosis, and anoikis furthermore implicates additional mechanistic distinctions according to the specific tissue, cell type, and species. Incidentally, studies in recent years have unearthed yet another layer of complexity in the regulation of these cell processes, namely, the implication of cell differentiation state-specific mechanisms. Further analyses of such differentiation state-distinct mechanisms, either under normal or physiopathological contexts, should increase our understanding of diseases which implicate a deregulation of integrin function, cell survival, and anoikis. PMID:21785723

  19. Venom gland EST analysis of the saw-scaled viper, Echis ocellatus, reveals novel alpha9beta1 integrin-binding motifs in venom metalloproteinases and a new group of putative toxins, renin-like aspartic proteases.

    PubMed

    Wagstaff, Simon C; Harrison, Robert A

    2006-08-01

    Echis ocellatus is the most medically important snake in West Africa. However, the composition of its venom and the differential contribution of these venom components to the severe haemorrhagic and coagulopathic pathology of envenoming are poorly understood. To address this situation we assembled a toxin transcriptome based upon 1000 expressed sequence tags (EST) from a cDNA library constructed from pooled venom glands of 10 individual E. ocellatus. We used a variety of bioinformatic tools to construct a fully annotated venom-toxin transcriptome that was interrogated with a combination of BLAST annotation, gene ontology cataloguing and disintegrin-motif searching. The results of these analyses revealed an unusually abundant and diverse expression of snake venom metalloproteinases (SVMP) and a broad toxin-expression profile including several distinct isoforms of bradykinin-potentiating peptides, phospholipase A(2), C-type lectins, serine proteinases and l-amino oxidases. Most significantly, we identified for the first time a conserved alpha(9)beta(1) integrin-binding motif in several SVMPs, and a new group of putative venom toxins, renin-like aspartic proteases. PMID:16713134

  20. β1-integrin restricts astrocytic differentiation of adult hippocampal neural stem cells.

    PubMed

    Brooker, Sarah M; Bond, Allison M; Peng, Chian-Yu; Kessler, John A

    2016-07-01

    Integrins are transmembrane receptors that mediate cell-extracellular matrix and cell-cell interactions. The β1-integrin subunit is highly expressed by embryonic neural stem cells (NSCs) and is critical for NSC maintenance in the developing nervous system, but its role in the adult hippocampal niche remains unexplored. We show that β1-integrin expression in the adult mouse dentate gyrus (DG) is localized to radial NSCs and early progenitors, but is lost in more mature progeny. Although NSCs in the hippocampal subgranular zone (SGZ) normally only infrequently differentiate into astrocytes, deletion of β1-integrin significantly enhanced astrocyte differentiation. Ablation of β1-integrin also led to reduced neurogenesis as well as depletion of the radial NSC population. Activation of integrin-linked kinase (ILK) in cultured adult NSCs from β1-integrin knockout mice reduced astrocyte differentiation, suggesting that at least some of the inhibitory effects of β1-integrin on astrocytic differentiation are mediated through ILK. In addition, β1-integrin conditional knockout also resulted in extensive cellular disorganization of the SGZ as well as non-neurogenic regions of the DG. The effects of β1-integrin ablation on DG structure and astrogliogenesis show sex-specific differences, with the effects following a substantially slower time-course in males. β1-integrin thus plays a dual role in maintaining the adult hippocampal NSC population by supporting the structural integrity of the NSC niche and by inhibiting astrocytic lineage commitment. GLIA 2016;64:1235-1251. PMID:27145730

  1. 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.

  2. Plasticity of integrin expression by nerve-derived connective tissue cells. Human Schwann cells, perineurial cells, and fibroblasts express markedly different patterns of beta 1 integrins during nerve development, neoplasia, and in vitro.

    PubMed Central

    Hsiao, L L; Peltonen, J; Jaakkola, S; Gralnick, H; Uitto, J

    1991-01-01

    Strikingly selective expression patterns of beta 1, alpha 2, alpha 3, and alpha 5 integrin subunits were revealed in endoneurium, perineurium, and epineurium of fetal and adult human peripheral nerve by immunostaining with specific antibodies. The alpha 2 subunit was expressed only on Schwann cells both in fetal and adult nerve, whereas the alpha 3 epitopes were expressed exclusively in the adult tissue and were primarily present on perineurial cells. The alpha 5 epitopes were expressed only on the innermost cell layer of perineurium of fetal and adult nerve. The tumor cells within schwannomas and cutaneous neurofibromas expressed both alpha 2 and alpha 3 subunits, indicating that Schwann cells have the potential to express also the alpha 3 subunit in vivo. Cell cultures established from human fetal nerve and neurofibromas revealed expression of the alpha 2 and alpha 5 epitopes on Schwann cells, perineurial cells, and fibroblasts, whereas only Schwann cells contained the alpha 3 epitopes which were occasionally concentrated on the adjacent Schwann cells at cell-cell contacts. Our findings emphasize that nerve connective tissue cells change their profiles for expression of extracellular matrix receptors under conditions which have different regulatory control signals exerted by, for example, axons, humoral factors, or the extracellular matrix of the peripheral nerve. This plasticity may play an important role during nerve development and in neoplastic processes affecting the connective tissue compartments of peripheral nerve. Images PMID:1999496

  3. Integrin beta 1 enhances the epithelial-mesenchymal transition in association with gefitinib resistance of non-small cell lung cancer.

    PubMed

    Ju, Lixia; Zhou, Caicun

    2013-01-01

    We have previously shown that integrinβ1 associates with gefitinib resistance. As epithelial-mesenchymal transition (EMT) also induces gefitinib resistance in vitro, we wished to determine the relation of them in gefitinib resistance. In this study, we show that integrinβ1 induced epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) resistance in xenograft tumors and gefitinib-resistant NSCLC tumors acquired EMT phenotype. Furthermore, inhibition of integrinβ1 reverses EMT, meanwhile overexpression and activation of integrinβ1 aggravates EMT. Lastly, we further identified that integrinβ1 enhanced EMT via FAK-AKT signaling pathway. These findings highlight a novel relation of integrinβ1 and EMT in EGFR TKI resistant NSCLC. PMID:24440972

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

    PubMed Central

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

    1995-01-01

    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. Images PMID:7860768

  5. Polymerized collagen inhibits fibroblast proliferation via a mechanism involving the formation of a beta1 integrin-protein phosphatase 2A-tuberous sclerosis complex 2 complex that suppresses S6K1 activity.

    PubMed

    Xia, Hong; Nho, Richard; Kleidon, Jill; Kahm, Judy; Henke, Craig A

    2008-07-18

    Polymerized type I collagen suppresses fibroblast proliferation. Previous studies have implicated inhibition of fibroblast proliferation with polymerized collagen-mediated suppression of S6K1, but the molecular mechanism of the critical negative feedback loop has not yet been fully elucidated. Here, we demonstrate that polymerized collagen suppresses G(1)/S phase transition and fibroblast proliferation by a novel mechanism involving the formation of a beta1 integrin-protein phosphatase 2A (PP2A)-tuberous sclerosis complex 2 (TSC2) complex that represses S6K1 activity. In response to fibroblast interaction with polymerized collagen, beta1 integrin forms a complex with PP2A that targets TSC2 as a substrate. PP2A represses the level of TSC2 phosphorylation and maintains TSC2 in an activated state. Activated TSC2 negatively regulates the downstream kinase S6K1 and inhibits G(1)/S transit. Knockdown of TSC2 enables fibroblasts to overcome the anti-proliferative properties of polymerized collagen. Furthermore, we show that this reduction in TSC2 and S6K1 phosphorylation occurs largely independent of Akt. Although S6K1 activity was markedly suppressed by polymerized collagen, we found that minimal changes in Akt activity occurred. We demonstrate that up-regulation of Akt by overexpression of constitutively active phosphatidylinositol 3-kinase p110 subunit had minor effects on TSC2 and S6K1 phosphorylation. These findings demonstrate that polymerized collagen represses fibroblast proliferation by a mechanism involving the formation of a beta1 integrin-PP2A-TSC2 complex that negatively regulates S6K1 and inhibits G(1)/S phase transition. PMID:18487611

  6. CD9 of mouse brain is implicated in neurite outgrowth and cell migration in vitro and is associated with the alpha 6/beta 1 integrin and the neural adhesion molecule L1.

    PubMed

    Schmidt, C; Künemund, V; Wintergerst, E S; Schmitz, B; Schachner, M

    1996-01-01

    We describe here a novel monoclonal antibody (mab H6) which recognizes CD9, an integral cell surface constituent previously described in cells of the hematopoietic lineage and involved in the aggregation of platelets. Mab H6 was raised against membranes of immature mouse astrocytes and reacted with a protein of 25-27 kD in detergent extracts of adult mouse brain membranes. Sequence analysis of the N-terminal amino acids revealed an identity of 96% with CD9 from mouse kidney. CD9 was localized in the central and peripheral mouse nervous systems: in the spinal cord of 11-day-old mouse embryos, CD9 was strongly expressed in the floor and roof plates. In the adult mouse sciatic nerve, myelin sheaths were highly CD9-immunoreactive. Mab H6 reacted with the cell surfaces of both glial cells and neurons in culture and inhibited migration of neuronal cell bodies, neurite fasciculation and outgrowth of astrocytic processes from cerebellar microexplants. Neurite outgrowth from isolated small cerebellar neurons was increased in the presence of mab H6 on substrate-coated laminin, but not on substrate-coated poly-L-lysine. Addition of mab H6 elicited an increase in intracellular Ca2+ concentration in these cells on substrate-coated laminin. Immunoprecipitates of CD9 from cultured mouse neuroblastoma N2A cells contained the alpha 6/beta 1 integrin. Moreover, preparations of CD9 immunoaffinity-purified from adult mouse brain using a mab H6 column contained the neural adhesion molecule L1, but not other neural adhesion molecules. CD9 bound to L1, but not to NCAM or MAG. Both the alpha 6/beta 1 integrin and L1 could be induced to coredistribute with CD9 on the surface of cultured neuroblastoma N2A cells. The combined observations suggest that CD9 can associate with L1 and alpha 6/beta 1 integrin to influence neural cell interactions in vitro. PMID:8838570

  7. Amphiregulin enhances alpha6beta1 integrin expression and cell motility in human chondrosarcoma cells through Ras/Raf/MEK/ERK/AP-1 pathway.

    PubMed

    Chen, Jui-Chieh; Chen, Yu-Ju; Lin, Chih-Yang; Fong, Yi-Chin; Hsu, Chin-Jung; Tsai, Chun-Hao; Su, Jen-Liang; Tang, Chih-Hsin

    2015-05-10

    Chondrosarcoma is a malignant tumor that produces cartilage matrix. The most lethal aspect is its metastatic property. We demonstrated that amphiregulin (AR) is significantly upregulated in highly aggressive cells. AR silencing markedly suppressed cell migration. Exogenous AR markedly increased cell migration by transactivation of α6β1 integrin expression. A neutralizing α6β1 integrin antibody can abolish AR-induced cell motility. Knockdown of AR inhibits metastasis of cells to the lung in vivo. Furthermore, elevated AR expression is positively correlated with α6β1 integrin levels and higher grades in patients. These findings can potentially serve as biomarker and therapeutic approach for controlling chondrosarcoma metastasis. PMID:25825984

  8. Laminin alpha1 chain mediated reduction of laminin alpha2 chain deficient muscular dystrophy involves integrin alpha7beta1 and dystroglycan.

    PubMed

    Gawlik, Kinga I; Mayer, Ulrike; Blomberg, Kristina; Sonnenberg, Arnoud; Ekblom, Peter; Durbeej, Madeleine

    2006-03-20

    Transgenically introduced laminin (LN) alpha1 chain prevents muscular dystrophy in LNalpha2 chain deficient mice. We now report increased integrin alpha7Bbeta1D synthesis in dystrophic LNalpha2 chain deficient muscle. Yet, immunofluorescence demonstrated a reduced expression of integrin alpha7B subunit at the sarcolemma. Transgenic expression of LNalpha1 chain reconstituted integrin alpha7B at the sarcolemma. Expression of alpha- and beta-dystroglycan is enhanced in LNalpha2 chain deficient muscle and normalized by transgenic expression of LNalpha1 chain. We suggest that LNalpha1 chain in part ameliorates the development of LNalpha2 chain deficient muscular dystrophy by retaining the binding sites for integrin alpha7Bbeta1D and alpha-dystroglycan, respectively. PMID:16504180

  9. 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. PMID:18400984

  10. Integrin-fibronectin interactions at the cell-material interface: initial integrin binding and signaling.

    PubMed

    García, A J; Boettiger, D

    1999-12-01

    Integrin receptors mediate cell adhesion to extracellular matrices and provide signals that direct proliferation and differentiation. Integrin binding involves receptor-ligand interactions at the cell-substrate interface and assembly and reorganization of structural and signaling elements at the cytoplasmic face. Using a cross-linking/extraction/reversal method to quantify bound integrins, we demonstrate that the density of alpha5beta1 integrin-fibronectin bonds increases linearly with ligand density, as predicted by simple receptor-ligand equilibrium. This linear relationship is consistent with linear increases in cell adhesion strength with receptor and ligand surface densities. Furthermore, we show that phosphorylation of FAK, a tyrosine kinase involved in early integrin-mediated signaling, increases linearly with the number of integrin-Fn bonds. These linear relationships suggest the absence of cooperative effects in the initial stages of mechanical coupling and adhesion-mediated signaling. PMID:10614947

  11. Protein 4.1R regulates cell adhesion, spreading, migration and motility of mouse keratinocytes by modulating surface expression of beta1 integrin.

    PubMed

    Chen, Lixiang; Hughes, Richard A; Baines, Anthony J; Conboy, John; Mohandas, Narla; An, Xiuli

    2011-07-15

    Protein 4.1R is a membrane-cytoskeleton adaptor protein that has diverse roles in controlling the cell surface expression and/or function of transmembrane proteins, and in organizing F-actin. 4.1R is expressed in keratinocytes, but its role in these cells has not been explored. Here, we have investigated the role of 4.1R in skin using 4.1R(-/-) mice. Cell adhesion, spreading, migration and motility were significantly impaired in 4.1R(-/-) keratinocytes, and 4.1R(-/-) mice exhibited defective epidermal wound healing. Cultured 4.1R(-/-) keratinocytes on fibronectin failed to form actin stress fibres and focal adhesions. Furthermore, in the absence of 4.1R, the surface expression, and consequently the activity of β1 integrin were reduced. These data enabled the identification of a functional role for protein 4.1R in keratinocytes by modulating the surface expression of β1 integrin, possibly through a direct association between 4.1R and β1 integrin. PMID:21693581

  12. 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.

  13. Modulation of RGD sequence motifs regulates disintegrin recognition of alphaIIb beta3 and alpha5 beta1 integrin complexes. Replacement of elegantin alanine-50 with proline, N-terminal to the RGD sequence, diminishes recognition of the alpha5 beta1 complex with restoration induced by Mn2+ cation.

    PubMed Central

    Rahman, S; Aitken, A; Flynn, G; Formstone, C; Savidge, G F

    1998-01-01

    Several recent studies have demonstrated that the amino acid residues flanking the RGD sequence of high-affinity ligands modulate their specificity of interaction with integrin complexes. The present study has addressed the role of the residues flanking the RGD sequence in regulating the recognition by disintegrin of the alphaIIb beta3 and alpha5beta1 complexes by construction of a panel of recombinant molecules of Elegantin (the platelet aggregation inhibitor from the venom of Trimerasurus elegans) expressing specific RGD sequence motifs. Wild-type Elegantin (ARGDNP) and several variants including Eleg. AM (ARGDMP), Eleg. PM (PRGDMP) and Eleg. PN (PRGDNP) were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. The inhibitory efficacies of the panel of Elegantin variants were analysed in platelet adhesion assays with substrates immobilized with fibrinogen and fibronectin. Elegantin molecules containing an Ala residue N-terminal to the RGD sequence (wild-type Elegantin and Eleg. AM) showed strong inhibitory activity towards alphaIIbbeta3-dependent platelet adhesion on fibronectin, whereas a Pro residue in this position (Eleg. PM and Kistrin, the inhibitor from the venom of Calloselasma rhodostoma) engendered lower activity. The decreased activity could not be attributed to a decrease in the affinity of the disintegrin for the alphaIIb beta3 complex because both Eleg. AM and Eleg. PM had similar Kd (app) values. In contrast, Elegantin molecules into which a Met residue was introduced in place of the Asn residue C-terminal to the RGD sequence showed 10-13-fold elevated inhibitory activity towards platelet adhesion on fibrinogen and this was maintained with either a Pro or Ala residue N-terminal to the RGD sequence. In experiments with the alpha5 beta1 complex on K562 cells, the inhibitory efficacies of the panel of Elegantin molecules were analysed under two different cation conditions. First, in the presence of Ca2+/Mg2+, K562 cell

  14. Differential regulation of immature articular cartilage compressive moduli and Poisson's ratios by in vitro stimulation with IGF-1 and TGF-beta1.

    PubMed

    Williams, Gregory M; Dills, Kristin J; Flores, Christian R; Stender, Michael E; Stewart, Kevin M; Nelson, Lauren M; Chen, Albert C; Masuda, Koichi; Hazelwood, Scott J; Klisch, Stephen M; Sah, Robert L

    2010-09-17

    Mechanisms of articular cartilage growth and maturation have been elucidated by studying composition-function dynamics during in vivo development and in vitro culture with stimuli such as insulin-like growth factor-1 (IGF-1) and transforming growth factor-beta 1 (TGF-beta1). This study tested the hypothesis that IGF-1 and TGF-beta1 regulate immature cartilage compressive moduli and Poisson's ratios in a manner consistent with known effects on tensile properties. Bovine calf articular cartilage from superficial-articular (S) and middle-growth (M) regions were analyzed fresh or following culture in medium with IGF-1 or TGF-beta1. Mechanical properties in confined (CC) and unconfined (UCC) compression, cartilage matrix composition, and explant size were assessed. Culture with IGF-1 resulted in softening in CC and UCC, increased Poisson's ratios, substantially increased tissue volume, and accumulation of glycosaminoglycan (GAG) and collagen (COL). Culture with TGF-beta1 promoted maturational changes in the S layer, including stiffening in CC and UCC and increased concentrations of GAG, COL, and pyridinoline crosslinks (PYR), but little growth. Culture of M layer explants with TGF-beta1 was nearly homeostatic. Across treatment groups, compressive moduli in CC and UCC were positively related to GAG, COL, and PYR concentrations, while Poisson's ratios were negatively related to concentrations of these matrix components. Thus, IGF-1 and TGF-beta1 differentially regulate the compressive mechanical properties and size of immature articular cartilage in vitro. Prescribing tissue growth, maturation, or homeostasis by controlling the in vitro biochemical environment with such growth factors may have applications in cartilage repair and tissue engineering. PMID:20570267

  15. Transforming growth factors-beta 1, -beta 2, and -beta 3 stimulate fibroblast procollagen production in vitro but are differentially expressed during bleomycin-induced lung fibrosis.

    PubMed Central

    Coker, R. K.; Laurent, G. J.; Shahzeidi, S.; Lympany, P. A.; du Bois, R. M.; Jeffery, P. K.; McAnulty, R. J.

    1997-01-01

    Transforming growth factor (TGF)-beta 1 may potentiate wound healing and fibrosis by stimulating fibroblast collagen deposition. TGF-beta 1 is implicated in the pathogenesis of pulmonary fibrosis, but the role of TGF-beta 2 and TGF-beta 3 remains unclear. We examined their effects on lung fibroblast procollagen metabolism in vitro and localized their gene expression during bleomycin-induced lung fibrosis using in situ hybridization with digoxigenin-labeled riboprobes. All three isoforms stimulated fibroblast procollagen production. TGF-beta 3 was the most potent and also reduced procollagen degradation. In normal mouse lung, TGF-beta 1 and TGF-beta 3 mRNA transcripts were abundant in bronchiolar epithelium. After bleomycin, TGF-beta 1 gene expression was maximally enhanced at 10 days, with the signal being predominant in macrophages. Signal was also enhanced in mesenchymal, pulmonary endothelial, and mesothelial cells. After 35 days, the pattern of TGF-beta 1 gene expression returned to that of control lung. TGF-beta 3 gene expression remained unchanged throughout compared with controls. TGF-beta 2 mRNA was not detected with the antisense probe, but signal obtained with the sense probe suggests the presence of a naturally occurring antisense. This study demonstrates that TGF-beta 1, -beta 2, and -beta 3 all exert profibrotic effects in vitro. However, TGF-beta isoform gene expression is differentially controlled during experimental pulmonary fibrosis with TGF-beta 1 the predominant isoform expressed during pathogenesis. Images Figure 3 Figure 4 Figure 5 Figure 6 PMID:9060836

  16. 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

  17. Rho/MRTF-A-Induced Integrin Expression Regulates Angiogenesis in Differentiated Multipotent Mesenchymal Stem Cells

    PubMed Central

    Zhang, Rui; Wang, Nan; Zhang, Man; Zhang, Li-Nan; Guo, Zhi-Xia; Luo, Xue-Gang; Zhou, Hao; He, Hong-Peng; Zhang, Tong-Cun

    2015-01-01

    Mesenchymal stem cells (MSCs) are known to undergo endothelial differentiation in response to treatment with vascular endothelial growth factor (VEGF), but their angiogenic ability is poorly characterized. In the present study, we aimed to further investigate the role of Rho/MRTF-A in angiogenesis by MSCs and the effect of the Rho/MRTF-A pathway on the expression of integrins α1β1 and α5β1, which are known to mediate physiological and pathological angiogenesis. Our results showed that increased expression of α1, α5, and β1 was observed during angiogenesis of differentiated MSCs, and the Rho/MRTF-A signaling pathway was demonstrated to be involved in regulating the expression of integrins α1, α5, and β1. Luciferase reporter assay and ChIP assay determined that MRTF-A could bind to and transactivate the integrin α1 and α5 promoters. Treatment with the Rho inhibitor C3 transferase, the Rho-associated protein kinase (ROCK) inhibitor Y27632 or with shMRTF-A inhibited both the upregulation of α1, α5, and β1 as well as angiogenesis. Furthermore, in human umbilical vein endothelial cells (HUVECs), MRTF-A deletion led to marked reductions in cell migration and vessel network formation compared with the control. These data demonstrate that Rho/MRTF-A signaling is an important mediator that controls integrin gene expression during MSC-mediated angiogenic processes. PMID:25949242

  18. 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

  19. Comparative functional analysis of rat TGF-beta1 and Xenopus laevis TGF-beta5 promoters suggest differential regulations.

    PubMed

    Goswami, Moloy T; Desai, Kartiki V; Kondaiah, Paturu

    2003-07-01

    We have carried out a comparative functional analysis of the rat TGF-beta1 and Xenopus laevis TGF-beta5 promoters across several mammalian and amphibian cell lines. Progressive deletion constructs of both the promoters have been made using a PCR based approach and the basal promoter activities studied in Xenopus tadpole cell line (XTC), Xenopus adult kidney fibroblast cell line (A6), human hepatoma cell line (HepG2), normal rat kidney cell line (NRK), and Chinese hamster ovary cell line (CHO). Data suggests that the basal promoter activity of TGF-beta1 is low as compared to TGF-beta5 promoter in XTC cells but comparable in A6 cells, while TGF-beta5 promoter shows nearly negligible activity as compared to TGF-beta5 promoter in all the tested mammalian cell lines. Moreover, TGF-beta5 promoter is found to be repressed in XTC cells on treatment with TGF-beta5 protein. Thus, the regulation of TGF-beta1 and TGF-beta5 promoters is distinct in amphibian and mammalian species. We therefore suggest that contrary to the suggested functional equivalence of TGF-beta1 and TGF-beta5 proteins, TGF-beta1 and TGF-beta5 genes have distinct functions in their respective species. PMID:12962305

  20. 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. PMID:25693840

  1. Transforming growth factor-beta 1 differentially regulates proliferation, morphology, and extracellular matrix expression by three neural crest-derived neuroblastoma cell lines.

    PubMed

    Rogers, S L; Cutts, J L; Gegick, P J; McGuire, P G; Rosenberger, C; Krisinski, S

    1994-04-01

    We reported previously (S. L. Rogers, P. J. Gegick, S. M. Alexander, and P. G. McGuire, Dev. Biol. 151, 191-203, 1992) that transforming growth factor-beta 1 (TGF beta 1) inhibited proliferation, up-regulated fibronectin synthesis, and suppressed melanogenesis in a population of quail neural crest cells in vitro. Here, we report that cell lines derived from the parent SK-N-SH neuroblastoma line (R. A. Ross, B. A. Spengler, and J. L. Biedler, J. Natl. Cancer Inst. 71, 741-747, 1983) respond differentially to TGF beta 1, and their responses provide further insights into the actions of this growth factor on neural crest subpopulations. The SH-EP cell line exhibits primarily nonneuronal traits and responded to TGF beta 1 with increased thymidine uptake after 6 days of culture, increased expression of fibronectin mRNA and protein, and decreased laminin synthesis. Many SH-EP cells also acquired a dramatically elongated morphology, reminiscent of Schwann cells in culture. Thymidine uptake by the neuronal SY5Y cell line was not substantially altered. Neither fibronectin mRNA nor protein was detectable in either TGF beta 1-treated or untreated cultures, although laminin synthesis was upregulated by the growth factor. In TGF beta 1-treated cultures of the intermediate SH-IN cell line, which has been reported to display both neuronal and nonneuronal characteristics, there was marked flattening of many cells, a steady decrease in thymidine uptake, and increased expression of both fibronectin and laminin. The observed responses of SH-IN cells mimic those observed in primary neural crest cultures and appear to represent similar differentiation toward a mesenchymal phenotype. These results substantiate the idea that closely related but diverging neural crest-derived cell types respond selectively to TGF beta 1 and demonstrate that these SK-N-SH-derived cell lines will be useful in experimental approaches that will allow us to infer mechanisms underlying regulation of neural crest

  2. Integrins in Wound Healing

    PubMed Central

    Koivisto, Leeni; Heino, Jyrki; Häkkinen, Lari; Larjava, Hannu

    2014-01-01

    Significance: Regulation of cell adhesions during tissue repair is fundamentally important for cell migration, proliferation, and protein production. All cells interact with extracellular matrix proteins with cell surface integrin receptors that convey signals from the environment into the nucleus, regulating gene expression and cell behavior. Integrins also interact with a variety of other proteins, such as growth factors, their receptors, and proteolytic enzymes. Re-epithelialization and granulation tissue formation are crucially dependent on the temporospatial function of multiple integrins. This review explains how integrins function in wound repair. Recent Advances: Certain integrins can activate latent transforming growth factor beta-1 (TGF-β1) that modulates wound inflammation and granulation tissue formation. Dysregulation of TGF-β1 function is associated with scarring and fibrotic disorders. Therefore, these integrins represent targets for therapeutic intervention in fibrosis. Critical Issues: Integrins have multifaceted functions and extensive crosstalk with other cell surface receptors and molecules. Moreover, in aberrant healing, integrins may assume different functions, further increasing the complexity of their functionality. Discovering and understanding the role that integrins play in wound healing provides an opportunity to identify the mechanisms for medical conditions, such as excessive scarring, chronic wounds, and even cancer. Future Directions: Integrin functions in acute and chronic wounds should be further addressed in models better mimicking human wounds. Application of any products in acute or chronic wounds will potentially alter integrin functions that need to be carefully considered in the design. PMID:25493210

  3. Evaluation of β1-integrin expression on chondrogenically differentiating human adipose-derived stem cells using atomic force microscopy.

    PubMed

    Quisenberry, Chrystal R; Nazempour, Arshan; Van Wie, Bernard J; Abu-Lail, Nehal I

    2016-06-01

    The expression of β1-integrin on human adipose-derived stem cells, differentiating toward a chondrogenic lineage, is hypothesized to decrease when cells are grown under in vivo-like environments due to sufficient extracellular matrix (ECM) buildup in the engineered tissues. The opposite is true when cells are grown in static cultures such as in pellet or micromass. To probe β1-integrin distribution on cellular surfaces, atomic force microscopy cantilevers modified with anti-β1-integrin antibodies were used. Specific antibody-antigen adhesion forces were identified and indicated the locations of β1-integrins on cells. ECM properties were assessed by estimating the Young's modulus of the matrix. Specific single antibody-antigen interactions averaged 78 ± 10 pN with multiple bindings occurring at approximate multiples of 78 pN. The author's results show that upregulated β1-integrin expression coincided with a less robust ECM as assessed by mechanical properties of tissues. In micromass and pellet cultures, transforming growth factor β3(TGF-β3) elicited a decrease in Young's modulus by 3.7- and 4.4-fold while eliciting an increase in β1-integrin count by 1.1- and 1.3-fold, respectively. β1-integrin counts on cells grown in the presence of TGF-β3 with oscillating hydrostatic pressure decreased by a 1.1-fold while the Young's modulus increased by a 1.9-fold. Collectively, our results suggest that cells in insufficiently robust ECM express more integrin perhaps to facilitate cell-ECM adhesion and compensate for a looser less robust ECM. PMID:27106564

  4. 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.

  5. Expression of L1 protein correlates with cluster of differentiation 24 and integrin β1 expression in gastrointestinal stromal tumors

    PubMed Central

    DU, YUE; ZHANG, HAIHONG; JIANG, ZHONGMIN; HUANG, GUOWEI; LU, WENLI; WANG, HESHENG

    2015-01-01

    The present study examined 66 cases of gastrointestinal stromal tumors (GISTs), 20 cases of smooth muscle tumors, 20 cases of schwannomas and 20 cases of normal gastric tissues in order to analyze the expression of L1, cluster of differentiation (CD)24 and integrin β1 by immunohistochemical staining. Patients were subjected to follow-up, and survival data were evaluated. L1 expression was detected in 57.6% of GIST cases; this was a significantly higher percentage compared with that found in the smooth muscle tumor cases or the normal control group. CD24 and integrin β1 were also expressed at significantly higher levels in the GIST cases than in the normal control group, although no significant difference was found in the expression levels of these proteins in smooth muscle tumor or schwannoma cases. These higher levels of L1 and integrin β1 expression were associated with an increased risk of invasive GIST, and were significantly positively correlated with Ki-67 expression. CD24 expression was not associated with the risk of GIST invasion or Ki-67 expression. There were positive correlations between L1, CD24 and integrin β1 expression; however, these had no significant association with patient survival. Therefore, L1 alone or in conjunction with CD24 (L1 + CD24), or integrin β1 (L1 + integrin β1) can be considered a valuable indicator for the differential diagnosis of GIST. Furthermore, L1 and integrin β1 can be used alone or in combination to evaluate the biological behavior of GISTs. Future studies are required to evaluate the prognostic value of these markers. PMID:26137113

  6. 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

  7. Quantitative Phosphoproteomic Study Reveals that Protein Kinase A Regulates Neural Stem Cell Differentiation Through Phosphorylation of Catenin Beta-1 and Glycogen Synthase Kinase 3β.

    PubMed

    Wang, Shuxin; Li, Zheyi; Shen, Hongyan; Zhang, Zhong; Yin, Yuxin; Wang, Qingsong; Zhao, Xuyang; Ji, Jianguo

    2016-08-01

    Protein phosphorylation is central to the understanding of multiple cellular signaling pathways responsible for regulating the self-renewal and differentiation of neural stem cells (NSCs). Here we performed a large-scale phosphoproteomic analysis of rat fetal NSCs using strong cation exchange chromatography prefractionation and citric acid-assisted two-step enrichment with TiO2 strategy followed by nanoLC-MS/MS analysis. Totally we identified 32,546 phosphosites on 5,091 phosphoproteins, among which 23,945 were class I phosphosites, and quantified 16,000 sites during NSC differentiation. More than 65% of class I phosphosites were novel when compared with PhosphoSitePlus database. Quantification results showed that the early and late stage of NSC differentiation differ greatly. We mapped 69 changed phosphosites on 20 proteins involved in Wnt signaling pathway, including S552 on catenin beta-1 (Ctnnb1) and S9 on glycogen synthase kinase 3β (Gsk3β). Western blotting and real-time PCR results proved that Wnt signaling pathway plays critical roles in NSC fate determination. Furthermore, inhibition and activation of PKA dramatically affected the phosphorylation state of Ctnnb1 and Gsk3β, which regulates the differentiation of NSCs. Our data provides a valuable resource for studying the self-renewal and differentiation of NSCs. Stem Cells 2016;34:2090-2101. PMID:27097102

  8. Integrin-Linked Kinase Is Indispensable for Keratinocyte Differentiation and Epidermal Barrier Function.

    PubMed

    Sayedyahossein, Samar; Rudkouskaya, Alena; Leclerc, Valerie; Dagnino, Lina

    2016-02-01

    A functional permeability barrier is essential to prevent the passage of water and electrolytes, macromolecules, and pathogens through the epidermis. This is accomplished in terminally differentiated keratinocytes through formation of a cornified envelope and the assembly of tight intercellular junctions. Integrin-linked kinase (ILK) is a scaffold protein essential for hair follicle morphogenesis and epidermal attachment to the basement membrane. However, the biological functions of ILK in differentiated keratinocytes remain poorly understood. Furthermore, whether ILK is implicated in keratinocyte differentiation and intercellular junction formation has remained an unresolved issue. Here we describe a pivotal role for ILK in keratinocyte differentiation responses to increased extracellular Ca(2+), regulation of adherens and tight junction assembly, and the formation of an outside-in permeability barrier toward macromolecules. In the absence of ILK, the calcium sensing receptor, E-cadherin, and ZO-1 fail to translocate to the cell membrane, through mechanisms that involve abnormalities in microtubules and in RhoA activation. In situ, ILK-deficient epidermis exhibits reduced tight junction formation and increased outside-in permeability to a dextran tracer, indicating reduced barrier properties toward macromolecules. Therefore, ILK is an essential component of keratinocyte differentiation programs that contribute to epidermal integrity and the establishment of its barrier properties. PMID:26967476

  9. Differential effects of bone morphogenetic protein-2 and transforming growth factor-beta1 on gene expression of collagen-modifying enzymes in human adipose tissue-derived mesenchymal stem cells.

    PubMed

    Knippenberg, Marlene; Helder, Marco N; Doulabi, Behrouz Zandieh; Bank, Ruud A; Wuisman, Paul I J M; Klein-Nulend, Jenneke

    2009-08-01

    Adipose tissue-derived mesenchymal stem cells (AT-MSCs) in combination with bone morphogenetic protein-2 (BMP-2) or transforming growth factor-beta1 (TGF-beta1) are under evaluation for bone tissue engineering. Posttranslational modification of type I collagen is essential for functional bone tissue with adequate physical and mechanical properties. We investigated whether BMP-2 (10-100 ng/mL) and/or TGF-beta1 (1-10 ng/mL) affect gene expression of alpha2(I) procollagen and collagen-modifying enzymes, that is, lysyl oxidase and lysyl hydroxylases 1, 2, and 3 (encoded by PLOD1, 2, and 3), by human AT-MSCs. BMP-2, but not TGF-beta1, increased alkaline phosphatase activity after 28 days, indicating osteogenic differentiation of AT-MSCs. At day 4, both BMP-2 and TGF-beta1 upregulated alpha2(I) procollagen and PLOD1, which was downregulated at day 28. TGF-beta1, but not BMP-2, downregulated PLOD3 at day 28. Lysyl oxidase was upregulated by TGF-beta1 at day 4 and by BMP-2 at day 7. Neither BMP-2 nor TGF-beta1 affected PLOD2. In conclusion, these results suggest that AT-MSCs differentially respond to BMP-2 and TGF-beta1 with changes in gene expression of collagen-modifying enzymes. AT-MSCs may thus be able to appropriately modify type I collagen to form a functional bone extracellular matrix for tissue engineering, dependent on the growth factor added. PMID:19231972

  10. Controlling integrin specificity and stem cell differentiation in 2-D and 3-D environments through regulation of fibronectin domain stability

    PubMed Central

    Martino, Mikaël M.; Mochizuki, Mayumi; Rothenfluh, Dominique A.; Rempel, Sandra A.; Hubbell, Jeffrey A.; Barker, Thomas H.

    2009-01-01

    The extracellular matrix (ECM) exerts powerful control over many cellular phenomena, including stem cell differentiation. As such, design and modulation of ECM analogs to ligate specific integrin is a promising approach to control cellular processes in vitro and in vivo for regenerative medicine strategies. Although fibronectin (FN), a crucial ECM protein in tissue development and repair, and its RGD peptide are widely used for cell adhesion, the promiscuity with which they engage integrins leads to difficulty in control of receptor-specific interactions. Recent simulations of force-mediated unfolding of FN domains and sequences analysis of human versus mouse FN suggest that the structural stability of the FN’s central cell-binding domains (FN III9-10) affects its integrin specificity. Through production of FN III9-10 variants with variable stabilities, we obtained ligands that present different specificities for the integrin α5β1 and that can be covalently linked into fibrin matrices. Here, we demonstrate the capacity of α5β1 integrin-specific engagement to influence human mesenchymal stem cell (MSC) behavior in 2D and 3D environments. Our data indicate that α5β1 has an important role in the control of MSC osteogenic differentiation. FN fragments with increased specificity for α5β1 versus αvβ3 results in significantly enhanced osteogenic differentiation of MSCs in 2D and in a clinically relevant 3D fibrin matrix system, although attachment/spreading and proliferation were comparable with that on full-length FN. This work shows how integrin-dependant cellular interactions with the ECM can be engineered to control stem cell fate, within a system appropriate for both 3D cell culture and tissue engineering. PMID:19027948

  11. Normalization of keratinocyte-type integrins during the establishment of the oral mucosa phenotype in vitro.

    PubMed

    Tomakidi, P; Breitkreutz, D; Kohl, A; Komposch, G

    1999-01-01

    In stratified epithelia, integrins play a fundamental role in mediating basal cell attachment to a variety of extracellular matrix molecules. To assess whether keratinocyte-specific integrins are expressed in a similar way as in the normal situation also under in vivo related conditions, we processed oral mucosa equivalents consisting of keratinocytes and fibroblasts from non-cornified gingiva. In this model histomorphology, the expression of differentiation-specific keratins and keratinocyte-type integrins exhibited similarity to the tissue of origin. The stages of tissue normalization were assessed on frozen sections by indirect immunofluorescence. The initial activated stage (1 week) was characterized by (i) incomplete epithelial organization and a weak presence of the suprabasal mucosa type keratin K4, (ii) diffuse expression of the integrin chains beta 1 and alpha 6 and (iii) abundance of the wound healing-associated integrin alpha v throughout the whole epithelium. After 2 weeks, the increase in epithelial organization was characterized by (i) the presence of a basal and suprabasal cell compartment, (ii) extension of K4 in the suprabasal compartment, (iii) extended expression of the keratinocyte integrins beta 1 and alpha 6 and (iv) concentration of alpha v integrin underneath basal cells. Further normalization of tissue architecture was indicated by (i) a slight increase in K4 extension, (ii) appearance of keratinocyte integrins beta 1 and alpha 6 in basal and parabasal cells and (iii) interruption of the band-like alpha v integrin immunolocalization at the subepithelial site. The findings in the in vitro model system indicate that these oral mucosa equivalents exhibit similarities to the in vivo situation of non-cornified gingiva, thus rendering them a suitable model for the assessment of stages during epithelial reconstruction or in vivo relevant studies on material effects. PMID:10081576

  12. 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.

  13. 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

  14. Modulation of integrin and E-cadherin-mediated adhesions to spatially control heterogeneity in human pluripotent stem cell differentiation.

    PubMed

    Toh, Yi-Chin; Xing, Jiangwa; Yu, Hanry

    2015-05-01

    Heterogeneity in human pluripotent stem cell (PSC) fates is partially caused by mechanical asymmetry arising from spatial polarization of cell-cell and cell-matrix adhesions. Independent studies have shown that integrin and E-cadherin adhesions promote opposing differentiation and pluripotent fates respectively although their crosstalk mechanism in modulating cell fate heterogeneity remains unknown. Here, we demonstrated that spatial polarization of integrin and E-cadherin adhesions in a human PSC colony compete to recruit Rho-ROCK activated myosin II to different localities to pattern pluripotent-differentiation decisions, resulting in spatially heterogeneous colonies. Cell micropatterning was used to modulate the spatial polarization of cell adhesions, which enabled us to prospectively determine localization patterns of activated myosin II and mesoendoderm differentiation. Direct inhibition of Rho-ROCK-myosin II activation phenocopied E-cadherin rather than integrin inhibition to form uniformly differentiated colonies. This indicated that E-cadherin was the primary gatekeeper to differentiation progression. This insight allows for biomaterials to be tailored for human PSC maintenance or differentiation with minimal heterogeneity. PMID:25736499

  15. 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

  16. The Integrin-Mediated ILK-Parvin-αPix Signaling Axis Controls Differentiation in Mammary Epithelial Cells.

    PubMed

    Rooney, Nicholas; Wang, Pengbo; Brennan, Keith; Gilmore, Andrew P; Streuli, Charles H

    2016-11-01

    Epithelial cell adhesion to the surrounding extracellular matrix is necessary for their proper behavior and function. During pregnancy and lactation, mammary epithelial cells (MECs) receive signals from their interaction with laminin via β1-integrin (β1-itg) to establish apico-basal polarity and to differentiate in response to prolactin. Downstream of β1-itg, the scaffold protein Integrin Linked Kinase (ILK) has been identified as the key signal transducer that is required for both lactational differentiation and the establishment of apico-basal polarity. ILK is an adaptor protein that forms the IPP complex with PINCH and Parvins, which are central to its adaptor functions. However, it is not known how ILK and its interacting partners control tissue-specific gene expression. Expression of ILK mutants, which weaken the interaction between ILK and Parvin, revealed that Parvins have a role in mammary epithelial differentiation. This conclusion was supported by shRNA-mediated knockdown of the Parvins. In addition, shRNA knockdown of the Parvin-binding guanine nucleotide exchange factor αPix prevented prolactin-induced differentiation. αPix depletion did not disrupt focal adhesions, MEC proliferation, or polarity. This suggests that αPix represents a differentiation-specific bifurcation point in β1-itg-ILK adhesive signaling. In summary, this study has identified a new role for Parvin and αPix downstream of the integrin-ILK signaling axis for MEC differentiation. J. Cell. Physiol. 231: 2408-2417, 2016. © 2016 Wiley Periodicals, Inc. PMID:27019299

  17. Integrin signalling regulates the expansion of neuroepithelial progenitors and neurogenesis via Wnt7a and Decorin

    PubMed Central

    Long, K.; Moss, L.; Laursen, L.; Boulter, L.; ffrench-Constant, C.

    2016-01-01

    Development of the cerebral cortex requires regulation of proliferation and differentiation of neural stem cells and a diverse range of progenitors. Recent work suggests a role for extracellular matrix (ECM) and the major family of ECM receptors, the integrins. Here we show that enhancing integrin beta-1 signalling, by expressing a constitutively active integrin beta-1 (CA*β1) in the embryonic chick mesencephalon, enhances neurogenesis and increases the number of mitotic cells dividing away from the ventricular surface, analogous to sub-apical progenitors in mouse. Only non-integrin-expressing neighbouring cells (lacking CA*β1) contributed to the increased neurogenesis. Transcriptome analysis reveals upregulation of Wnt7a within the CA*β1 cells and upregulation of the ECM protein Decorin in the neighbouring non-expressing cells. Experiments using inhibitors in explant models and genetic knock-downs in vivo reveal an integrin-Wnt7a-Decorin pathway that promotes proliferation and differentiation of neuroepithelial cells, and identify Decorin as a novel neurogenic factor in the central nervous system. PMID:26838601

  18. Vitronectin expression in differentiating neuroblastic tumors: integrin alpha v beta 5 mediates vitronectin-dependent adhesion of retinoic-acid-differentiated neuroblastoma cells.

    PubMed Central

    Gladson, C. L.; Dennis, C.; Rotolo, T. C.; Kelly, D. R.; Grammer, J. R.

    1997-01-01

    The metastatic potential of undifferentiated neuroblastomas is typically lost when differentiation into ganglioneuroblastomas occurs spontaneously or is induced. Cell adhesion may play a role in metastasis, and we have shown recently that expression of integrin alpha v beta 5 protein and mRNA is up-regulated in ganglioneuroblastomas in vivo. To investigate whether interactions of alpha v beta 5 with matrix components play a role in the loss of metastatic potential, we used immunohistochemical and in situ hybridization to analyze neuroblastic tumors at various stages of differentiation for expression of the alpha v beta 5 ligands, vitronectin and osteopontin, and determined the ability of vitronectin to promote attachment and neurite outgrowth in vitro in a retinoic-acid-differentiated neuroblastoma cell model. We found that vitronectin, but not osteopontin, was expressed in 5 of 5 ganglioneuroblastomas but was absent or weakly expressed in 6 of 6 undifferentiated neuroblastomas. Neuronal cell vitronectin was detected in 7 of 9 ganglioneuromas, 5 of 8 peripheral ganglia, and 14 of 21 adrenal gland medullae, confirming expression of vitronectin in mature peripheral neurons. In vitro, vitronectin promoted attachment of both undifferentiated and retinoic-acid-differentiated neuroblastoma cells, which was inhibited 20 and 60%, respectively, by monoclonal antibody anti-integrin alpha v beta 5. Vitronectin-promoted neurite outgrowth of retinoic-acid-differentiated neuroblastoma cells was not inhibited by monoclonal antibody anti-alpha v beta 5. These data suggest that the synthesis of vitronectin and the ability of integrin alpha v beta 5 to mediate vitronectin adhesion on retinoic-acid-differentiated neuroblastoma cells may promote differentiation of neuroblastoma cells in vivo. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 8 PMID:9137089

  19. 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

  20. Stage-specific expression of integrin alphaVbeta3 in neuroblastic tumors.

    PubMed Central

    Gladson, C. L.; Hancock, S.; Arnold, M. M.; Faye-Petersen, O. M.; Castleberry, R. P.; Kelly, D. R.

    1996-01-01

    The ligand specificity of the integrin cell adhesion receptors probably determines the ability of specific integrins to promote tumor cell proliferation and metastasis. Therefore, we compared the expression of integrin alphaVbeta3, a promiscuous receptor that binds with high affinity to numerous cell matrix proteins, with the expression of integrin alphaVbeta5 and the integrin beta 1 subunit (which pairs with multiple alpha subunits) in neuroblastic tumors at various stages of differentiation. Undifferentiated neuroblastoma tumors rapidly invade and metastasize, whereas ganglioneuroblastomas rarely metastasize. Differentiating neuroblastomas are associated with an intermediate prognosis. Paraffin sections of neuroblastic tumors at various stages of differentiation obtained at biopsy from 17 patients were hybridized with antisense integrin subunit-specific alphaV, beta3, beta1, and beta5 riboprobes. All neuroblastic tumors and seven adrenal glands obtained at autopsy were analyzed immunohistochemically with antibodies directed toward the alphaV, beta3, beta1, and beta5 subunits. The alphaV subunit was expressed in neuroblastic tumors independent of the stage of differentiation, although mRNA and protein expression were generally weak in ganglioneuroblastomas, and was also detected in adrenal gland medullae. The beta1 subunit was detected in most neuroblastic tumors independent of the stage of differentiation as well as in adrenal gland medullae. In contrast, the beta3 subunit, which was not expressed in adrenal gland medullae, was expressed at the protein and mRNA levels in undifferentiated neuroblastomas (six of seven and seven of seven, respectively) but was not expressed in neuroblasts or ganglion cells in ganglioneuroblastomas (one case weakly positive out of five). The beta 5 subunit was expressed at the protein (five of five) and mRNA (four of five) levels in the ganglion cells of ganglioneuroblastomas and, although mRNA for this subunit was detectable in

  1. Differential patterns of activity displayed by two exo-beta-1,3-glucanases associated with the Aspergillus fumigatus cell wall.

    PubMed Central

    Fontaine, T; Hartland, R P; Diaquin, M; Simenel, C; Latgé, J P

    1997-01-01

    Two exo-beta-1,3-glucanases (herein designated exoG-I and exoG-II) were isolated from the cell wall autolysate of the filamentous fungus Aspergillus fumigatus and purified by ion-exchange, hydrophobic-interaction, and gel filtration chromatographies. Molecular masses estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography were 82 kDa for the monomeric exoG-I and 230 kDa for the dimeric exoG-II. exoG-I and exoG-II were glycosylated, and N glycans accounted, respectively, for 2 and 44 kDa. Their pH optimum is 5.0. Their optimum temperatures are 55 degrees C for exoG-I and 65 degrees C for exoG-II. By a sensitive colorimetric method and high-performance anion-exchange chromatography for product analysis, two patterns of exo-beta-1,3-glucanase activities were found. The 230-kDa exoG-II enzyme acts on p-nitrophenyl-beta-D-glucoside, beta-1,6-glucan, and beta-1,3-glucan. This activity, which retains the anomeric configuration of glucose released, presented a multichain pattern of attack of the glucan chains and a decrease in the maximum initial velocity (Vm) with the increasing size of the substrate. In contrast, the 82-kDa exoG-I, which inverts the anomeric configuration of the glucose released, hydrolyzed exclusively the beta-1,3-glucan chain with a minimal substrate size of 4 glucose residues. This enzyme presented a repetitive-attack pattern, characterized by an increase in Vm with an increase in substrate size and by a degradation of the glucan chain until it reached laminaritetraose, the limit substrate size. The 82-kDa exoG-I and 230-kDa exoG-II enzymes correspond to a beta-1,3-glucan-glucohydrolase (EC 3.2.1.58) and to a beta-D-glucoside-glucohydrolase (EC 3.2.1.21), respectively. The occurrence and functions of these two classes of exo-beta-1,3-glucanases in other fungal species are discussed. PMID:9150209

  2. RhoA-dependent Switch between α2β1 and α3β1 Integrins Is Induced by Laminin-5 during Early Stage of HT-29 Cell Differentiation

    PubMed Central

    Gout, Stéphanie P.; Jacquier-Sarlin, Muriel R.; Rouard-Talbot, Laurence; Rousselle, Patricia; Block, Marc R.

    2001-01-01

    Integrin-mediated interactions between the basement membrane and epithelial cells control the differentiation of epithelia. We characterized the modulation of adhesive behaviors to basement membrane proteins and of integrin function in the human colon adenocarcinoma HT-29 cell line, which differentiates into enterocytes after the substitution of galactose for glucose in the medium. We demonstrate an increased capability of these cells to adhere to collagen type IV during the early stage of differentiation. This effect occurs without any changes in integrin cell surface expression but rather results from an α2β1/α3β1 integrin switch, α3β1 integrin becoming the major collagen receptor. The increase in laminin-5 secretion and deposit on the matrix is a key factor in the mechanism regulating cell adhesion, because it is responsible for the activation of α3β1 integrin. Furthermore, down-regulation of RhoA GTPase activity occurs during HT-29 cell differentiation and correlates with the activation of the integrin α3β1. Indeed, C3 transferase, a RhoA GTPase inhibitor, induces a similar α2β1/α3β1 switch in undifferentiated HT-29 cells. These results indicate that the decrease in RhoA activation is the biochemical mechanism underlying this integrin switch observed during cell differentiation. The physiological relevance of such modulation of integrin activity in the functioning of the crypt-villus axis is discussed. PMID:11598208

  3. Integrin activation

    PubMed Central

    Ginsberg, Mark H.

    2014-01-01

    Integrin-mediated cell adhesion is important for development, immune responses, hemostasis and wound healing. Integrins also function as signal transducing receptors that can control intracellular pathways that regulate cell survival, proliferation, and cell fate. Conversely, cells can modulate the affinity of integrins for their ligands a process operationally defined as integrin activation. Analysis of activation of integrins has now provided a detailed molecular understanding of this unique form of “inside-out” signal transduction and revealed new paradigms of how transmembrane domains (TMD) can transmit long range allosteric changes in transmembrane proteins. Here, we will review how talin and mediates integrin activation and how the integrin TMD can transmit these inside out signals. [BMB Reports 2014; 47(12): 655-659] PMID:25388208

  4. Differential effects of amyloid-beta 1-40 and 1-42 fibrils on 5-HT1A serotonin receptors in rat brain.

    PubMed

    Verdurand, Mathieu; Chauveau, Fabien; Daoust, Alexia; Morel, Anne-Laure; Bonnefoi, Frédéric; Liger, François; Bérod, Anne; Zimmer, Luc

    2016-04-01

    Evidence accumulates suggesting a complex interplay between neurodegenerative processes and serotonergic neurotransmission. We have previously reported an overexpression of serotonin 5-HT1A receptors (5-HT(1A)R) after intrahippocampal injections of amyloid-beta 1-40 (Aβ40) fibrils in rats. This serotonergic reactivity paralleled results from clinical positron emission tomography studies with [(18)F]MPPF revealing an overexpression of 5-HT(1A)R in the hippocampus of patients with mild cognitive impairment. Because Aβ40 and Aβ42 isoforms are found in amyloid plaques, we tested in this study the hypothesis of a peptide- and region-specific 5-HT(1A)R reactivity by injecting them, separately, into the hippocampus or striatum of rats. [(18)F]MPPF in vitro autoradiography revealed that Aβ40 fibrils, but not Aβ42, were triggering an overexpression of 5-HT(1A)R in the hippocampus and striatum of rat brains after 7 days. Immunohistochemical approaches targeting neuronal precursor cells, mature neurons, and astrocytes showed that Aβ42 fibrils caused more pathophysiological damages than Aβ40 fibrils. The mechanisms of Aβ40 fibrils-induced 5-HT(1A)R expression remains unknown, but hypotheses including neurogenesis, glial expression, and axonal sprouting are discussed. PMID:26973100

  5. Targeted disruption of the LAMA3 gene in mice reveals abnormalities in survival and late stage differentiation of epithelial cells.

    PubMed

    Ryan, M C; Lee, K; Miyashita, Y; Carter, W G

    1999-06-14

    Laminin 5 regulates anchorage and motility of epithelial cells through integrins alpha6beta4 and alpha3beta1, respectively. We used targeted disruption of the LAMA3 gene, which encodes the alpha3 subunit of laminin 5 and other isoforms, to examine developmental functions that are regulated by adhesion to the basement membrane (BM). In homozygous null animals, profound epithelial abnormalities were detected that resulted in neonatal lethality, consistent with removal of all alpha3-laminin isoforms from epithelial BMs. Alterations in three different cellular functions were identified. First, using a novel tissue adhesion assay, we found that the mutant BM could not induce stable adhesion by integrin alpha6beta4, consistent with the presence of junctional blisters and abnormal hemidesmosomes. In the absence of laminin 5 function, we were able to detect a new ligand for integrin alpha3beta1 in the epidermal BM, suggesting that basal keratinocytes can utilize integrin alpha3beta1 to interact with an alternative ligand. Second, we identified a survival defect in mutant epithelial cells that could be rescued by exogenous laminin 5, collagen, or an antibody against integrin alpha6beta4, suggesting that signaling through beta1 or beta4 integrins is sufficient for survival. Third, we detected abnormalities in ameloblast differentiation in developing mutant incisors indicating that events downstream of adhesion are affected in mutant animals. These results indicate that laminin 5 has an important role in regulating tissue organization, gene expression, and survival of epithelium. PMID:10366601

  6. Differential Expression Levels of Integrin α6 Enable the Selective Identification and Isolation of Atrial and Ventricular Cardiomyocytes

    PubMed Central

    Wiencierz, Anne Maria; Kernbach, Manuel; Ecklebe, Josephine; Monnerat, Gustavo; Tomiuk, Stefan; Raulf, Alexandra; Christalla, Peter; Malan, Daniela; Hesse, Michael; Bosio, Andreas; Fleischmann, Bernd K.; Eckardt, Dominik

    2015-01-01

    Rationale Central questions such as cardiomyocyte subtype emergence during cardiogenesis or the availability of cardiomyocyte subtypes for cell replacement therapy require selective identification and purification of atrial and ventricular cardiomyocytes. However, current methodologies do not allow for a transgene-free selective isolation of atrial or ventricular cardiomyocytes due to the lack of subtype specific cell surface markers. Methods and Results In order to develop cell surface marker-based isolation procedures for cardiomyocyte subtypes, we performed an antibody-based screening on embryonic mouse hearts. Our data indicate that atrial and ventricular cardiomyocytes are characterized by differential expression of integrin α6 (ITGA6) throughout development and in the adult heart. We discovered that the expression level of this surface marker correlates with the intracellular subtype-specific expression of MLC-2a and MLC-2v on the single cell level and thereby enables the discrimination of cardiomyocyte subtypes by flow cytometry. Based on the differential expression of ITGA6 in atria and ventricles during cardiogenesis, we developed purification protocols for atrial and ventricular cardiomyocytes from mouse hearts. Atrial and ventricular identities of sorted cells were confirmed by expression profiling and patch clamp analysis. Conclusion Here, we introduce a non-genetic, antibody-based approach to specifically isolate highly pure and viable atrial and ventricular cardiomyocytes from mouse hearts of various developmental stages. This will facilitate in-depth characterization of the individual cellular subsets and support translational research applications. PMID:26618511

  7. Interferon Beta-1b Injection

    MedlinePlus

    ... course of disease where symptoms flare up from time to time) of multiple sclerosis (MS, a disease in which ... interferon beta-1b injection at around the same time of day each time you inject it. Follow ...

  8. Peginterferon Beta-1a Injection

    MedlinePlus

    ... course of disease where symptoms flare up from time to time) of multiple sclerosis (MS, a disease in which ... peginterferon beta-1a injection at around the same time of day each time you inject it. Follow ...

  9. 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.

  10. Role of integrin α2 β1 in mediating osteoblastic differentiation on three-dimensional titanium scaffolds with submicron-scale texture.

    PubMed

    Wang, Xiaokun; Schwartz, Zvi; Gittens, Rolando A; Cheng, Alice; Olivares-Navarrete, Rene; Chen, Haifeng; Boyan, Barbara D

    2015-06-01

    Hierarchical surface roughness of titanium and titanium alloy implants plays an important role in osseointegration. In vitro and in vivo studies show greater osteoblast differentiation and bone formation when implants have submicron-scale textured surfaces. In this study, we tested the potential benefit of combining a submicron-scale textured surface with three-dimensional (3D) structure on osteoblast differentiation and the involvement of an integrin-driven mechanism. 3D titanium scaffolds were made using orderly oriented titanium meshes and microroughness was added to the wire surface by acid-etching. MG63 and human osteoblasts were seeded on 3D scaffolds and 2D surfaces with or without acid etching. At confluence, increased osteocalcin, vascular endothelial growth factor, osteoprotegerin (OPG), and alkaline phosphatase (ALP) activity were observed in MG63 and human osteoblasts on 3D scaffolds in comparison to 2D surfaces at the protein level, indicating enhanced osteoblast differentiation. To further investigate the mechanism of osteoblast-3D scaffold interaction, the role of integrin α2β1 was examined. The results showed β1 and α2β1 integrin silencing abolished the increase in osteoblastic differentiation markers on 3D scaffolds. Time course studies showed osteoblasts matured faster in the 3D environment in the early stage of culture, while as cells proliferated, the maturation slowed down to a comparative level as 2D surfaces. After 12 days of postconfluent culture, osteoblasts on 3D scaffolds showed a second-phase increase in ALP activity. This study shows that osteoblastic differentiation is improved on 3D scaffolds with submicron-scale texture and is mediated by integrin α2β1. PMID:25203434

  11. Role of integrin α2β1 in mediating osteoblastic differentiation on three-dimensional titanium scaffolds with submicron-scale texture

    PubMed Central

    Wang, Xiaokun; Schwartz, Zvi; Gittens, Rolando A.; Cheng, Alice; Olivares-Navarrete, Rene; Chen, Haifeng; Boyan, Barbara D.

    2014-01-01

    Hierarchical surface roughness of titanium and titanium alloy implants plays an important role in osseointegration. In vitro and in vivo studies show greater osteoblast differentiation and bone formation when implants have submicron-scale textured surfaces. In this study, we tested the potential benefit of combining a submicron-scale textured surface with three-dimensional (3D) structure on osteoblast differentiation and the involvement of an integrin-driven mechanism. 3D titanium scaffolds were made using orderly oriented titanium meshes and microroughness was added to the wire surface by acid-etching. MG63 and human osteoblasts were seeded on 3D scaffolds and 2D surfaces with or without acid etching. At confluence, increased osteocalcin, vascular endothelial growth factor, osteoprotegerin (OPG), and alkaline phosphatase (ALP) activity were observed in MG63 and human osteoblasts on 3D scaffolds in comparison to 2D surfaces at the protein level, indicating enhanced osteoblast differentiation. To further investigate the mechanism of osteoblast-3D scaffold interaction, the role of integrin α2β1 was examined. The results showed β1 and α2β1 integrin silencing abolished the increase in osteoblastic differentiation markers on 3D scaffolds. Time course studies showed osteoblasts matured faster in the 3D environment in the early stage of culture, while as cells proliferated, the maturation slowed down to a comparative level as 2D surfaces. After 12 days of postconfluent culture, osteoblasts on 3D scaffolds showed a second-phase increase in ALP activity. This study shows that osteoblastic differentiation is improved on 3D scaffolds with submicron-scale texture and is mediated by integrin α2β1. PMID:25203434

  12. Molecular characterization and differential expression of beta-1,3-glucanase during ripening in banana fruit in response to ethylene, auxin, ABA, wounding, cold and light-dark cycles.

    PubMed

    Roy Choudhury, Swarup; Roy, Sujit; Singh, Sanjay Kumar; Sengupta, Dibyendu N

    2010-08-01

    beta-1,3-Glucanases (E.C. 3.2.1.39) are widely distributed enzyme among bacteria, fungi, and higher plants. Analyses of accumulation levels of beta-1,3-glucanase protein in various tissues in banana have clearly indicated abundance of beta-1,3-glucanase protein accumulation in ripe pulp tissue. After cloning of beta-1,3-glucanase from banana pulp (cultivar Cavendish), we have carried out an in silico analysis to investigate the sequential, structural, and phylogenetic characteristics of the putative banana beta-1,3-glucanase protein. As like other ripening specific genes, beta-1,3-glucanase is regulated in response to a wide variety of factors. Therefore, we have analyzed the transcript accumulation pattern and protein levels of beta-1,3-glucanase in response to ethylene, auxin, ABA, wounding and, low temperature in preclimacteric banana fruit. Expression profile analyses have indicated that whereas exogenous application of ethylene strongly stimulated beta-1,3-glucanase transcript accumulation, ABA partially induced the expression of the gene. On the other hand, wound treatment did not induce beta-1,3-glucanase expression. Conversely, auxin and cold treatment negatively regulated beta-1,3-glucanase gene expression and thus inhibited glucanase activity. In addition, beta-1,3-glucanase transcript level was markedly decreased by constant exposure to white light. Protein level and enzymatic activity of beta-1,3-glucanase were substantially increased with considerable decrease in fruit firmness by ethylene treatment and reduced exposure to white light conditions as compared with other treatments. Together, the overall study of beta-1,3-glucanase expression pattern, glucanase activity, and changes in fruit firmness during ripening in various conditions suggest the possible physiological function of beta-1,3-glucanase in fruit pulp softening. PMID:20467747

  13. Profiles of activation, differentiation-markers, or β-integrins on T cells contribute to predict T cells' antileukemic responses after stimulation with leukemia-derived dendritic cells.

    PubMed

    Vogt, Valentin; Schick, Julia; Ansprenger, Christian; Braeu, Marion; Kroell, Tanja; Kraemer, Doris; Köhne, Claus-Henning; Hausmann, Andreas; Buhmann, Raymund; Tischer, Johanna; Schmetzer, Helga

    2014-01-01

    Stem cell transplantations and donor lymphocyte infusions are promising immunotherapies to cure acute myeloid leukemia (AML). Leukemia-derived dendritic cells are known to improve antileukemic functionality of T cells. We evaluated the composition and development of distinct T-cell subtypes in AML patients (n=12) compared with healthy probands (n=5) before and during stimulation with leukemia-derived dendritic cells-containing DC (DC) or blast-containing mononuclear cells (MNC) in 0-7 days mixed lymphocyte cultures (MLC) by flow cytometry. AML patients' T-cell subgroups were correlated with antileukemic functionality before and after DC/MNC stimulation by functional fluorolysis assays. (1) Unstimulated T cells from AML patients presented with significantly lower proportions of activated, Tcm, CD137, and β-integrin T cells, and significantly higher proportions of Tnaive and Teff compared with healthy probands. (2) After 7 days of DC or MNC stimulation, T-cell profiles were characterized by (significantly) increased proportions of activated T cells with effector function and significantly decreased proportions of β-integrin T cells. (3) Antileukemic cytotoxicity was achieved in 40% of T cells after MNC stimulation compared with 64% after DC stimulation. Antileukemic activity after DC stimulation but not after MNC stimulation correlated with higher proportions of Tcm and Tnaive before stimulation, as well as with significantly higher proportions of activated and β-integrin T cells. Furthermore, cutoff values for defined T-cell activation/differentiation markers and β-integrin T cells could be defined, allowing a prediction of antileukemic reactivity. We could demonstrate the potential of the composition of unstimulated/DC-stimulated T cells for the lysis of AML blasts. Especially, AML patients with high numbers of Tnaive and Tcm could benefit from DC stimulation; proportions of activated and β-integrin T cells correlated with increased antileukemic functionality

  14. Absence of alphavbeta6 integrin is linked to initiation and progression of periodontal disease.

    PubMed

    Ghannad, Farzin; Nica, Daniela; Fulle, Maria I Garcia; Grenier, Daniel; Putnins, Edward E; Johnston, Sarah; Eslami, Ameneh; Koivisto, Leeni; Jiang, Guoqiao; McKee, Marc D; Häkkinen, Lari; Larjava, Hannu

    2008-05-01

    Integrin alphavbeta6 is generally not expressed in adult epithelia but is induced in wound healing, cancer, and certain fibrotic disorders. Despite this generalized absence, we observed that alphavbeta6 integrin is constitutively expressed in the healthy junctional epithelium linking the gingiva to tooth enamel. Moreover, expression of alphavbeta6 integrin was down-regulated in human periodontal disease, a common medical condition causing tooth loss and also contributing to the development of cardiovascular diseases by increasing the total systemic inflammatory burden. Remarkably, integrin beta6 knockout mice developed classic signs of spontaneous, chronic periodontal disease with characteristic inflammation, epithelial down-growth, pocket formation, and bone loss around the teeth. Integrin alphavbeta6 acts as a major activator of transforming growth factor-beta1 (TGF-beta1), a key anti-inflammatory regulator in the immune system. Co-expression of TGF-beta1 and alphavbeta6 integrin was observed in the healthy junctional epithelium. Moreover, an antibody that blocks alphavbeta6 integrin-mediated activation of TGF-beta1 initiated inflammatory periodontal disease in a rat model of gingival inflammation. Thus, alphavbeta6 integrin is constitutively expressed in the epithelium sealing the gingiva to the tooth and plays a central role in protection against inflammatory periodontal disease through activation of TGF-beta1. PMID:18385522

  15. Differential β3 Integrin Expression Regulates the Response of Human Lung and Cardiac Fibroblasts to Extracellular Matrix and Its Components.

    PubMed

    Merna, Nick; Fung, Kelsey M; Wang, Jean J; King, Cristi R; Hansen, Kirk C; Christman, Karen L; George, Steven C

    2015-08-01

    Extracellular matrix (ECM) derived from whole organ decellularization has been successfully used in a variety of tissue engineering applications. ECM contains a complex mixture of functional and structural molecules that are ideally suited for the tissue from which the ECM is harvested. However, decellularization disrupts the structural properties and protein composition of the ECM, which may impact function when cells such as the fibroblast are reintroduced during recellularization. We hypothesized that the ECM structure and composition, fibroblast source, and integrin expression would influence the fibroblast phenotype. Human cardiac fibroblasts (HCFs) and normal human lung fibroblasts (NHLFs) were cultured on intact cardiac ECM, collagen gels, and coatings composed of cardiac ECM, lung ECM, and individual ECM components (collagen and fibronectin [FN]) for 48 h. COL1A expression of HCFs and NHLFs cultured on ECM and FN coatings decreased to <50% of that of untreated cells; COL1A expression for HCFs cultured on ECM coatings was one- to twofold higher than HCFs cultured on intact ECM. NHLFs cultured on ECM and FN coatings expressed 12- to 31-fold more alpha-smooth muscle actin (αSMA) than HCFs; the αSMA expression for HCFs and NHLFs cultured on ECM coatings was ∼2- to 5-fold higher than HCFs and NHLFs cultured on intact ECM. HCFs expressed significantly higher levels of β3 and β4 integrins when compared to NHLFs. Inhibition of the β3 integrin, but not β4, resulted in a 16- to 26-fold increase in αSMA expression in HCFs cultured on ECM coatings and FN. Our results demonstrate that β3 integrin expression depends on the source of the fibroblast and that its expression inhibits αSMA expression (and thus the myofibroblast phenotype). We conclude that the fibroblast source and integrin expression play important roles in regulating the fibroblast phenotype. PMID:25926101

  16. Differential β3 Integrin Expression Regulates the Response of Human Lung and Cardiac Fibroblasts to Extracellular Matrix and Its Components

    PubMed Central

    Merna, Nick; Fung, Kelsey M.; Wang, Jean J.; King, Cristi R.; Hansen, Kirk C.; Christman, Karen L.

    2015-01-01

    Extracellular matrix (ECM) derived from whole organ decellularization has been successfully used in a variety of tissue engineering applications. ECM contains a complex mixture of functional and structural molecules that are ideally suited for the tissue from which the ECM is harvested. However, decellularization disrupts the structural properties and protein composition of the ECM, which may impact function when cells such as the fibroblast are reintroduced during recellularization. We hypothesized that the ECM structure and composition, fibroblast source, and integrin expression would influence the fibroblast phenotype. Human cardiac fibroblasts (HCFs) and normal human lung fibroblasts (NHLFs) were cultured on intact cardiac ECM, collagen gels, and coatings composed of cardiac ECM, lung ECM, and individual ECM components (collagen and fibronectin [FN]) for 48 h. COL1A expression of HCFs and NHLFs cultured on ECM and FN coatings decreased to <50% of that of untreated cells; COL1A expression for HCFs cultured on ECM coatings was one- to twofold higher than HCFs cultured on intact ECM. NHLFs cultured on ECM and FN coatings expressed 12- to 31-fold more alpha-smooth muscle actin (αSMA) than HCFs; the αSMA expression for HCFs and NHLFs cultured on ECM coatings was ∼2- to 5-fold higher than HCFs and NHLFs cultured on intact ECM. HCFs expressed significantly higher levels of β3 and β4 integrins when compared to NHLFs. Inhibition of the β3 integrin, but not β4, resulted in a 16- to 26-fold increase in αSMA expression in HCFs cultured on ECM coatings and FN. Our results demonstrate that β3 integrin expression depends on the source of the fibroblast and that its expression inhibits αSMA expression (and thus the myofibroblast phenotype). We conclude that the fibroblast source and integrin expression play important roles in regulating the fibroblast phenotype. PMID:25926101

  17. EDA-containing cellular fibronectin induces fibroblast differentiation through binding to alpha4beta7 integrin receptor and MAPK/Erk 1/2-dependent signaling.

    PubMed

    Kohan, Martin; Muro, Andres F; White, Eric S; Berkman, Neville

    2010-11-01

    Fibroblast differentiation is an essential step during wound healing and fibrosis. Fibronectin (FN) is a major component of the extracellular matrix and occurs in two main forms: plasma and cellular FN. The latter includes the alternatively spliced domain A (EDA). Although EDA-containing cellular fibronectin (EDA-FN) is associated with fibroblast differentiation, how EDA-FN promotes differentiation is incompletely understood. In this study, we investigate the mechanism by which EDA-FN contributes to fibroblast differentiation with emphasis on the characterization of the EDA-FN receptor. We show that EDA-FN increases α-SMA expression (immunofluorescence), collagen deposition, cell contractility, and focal adhesion kinase (FAK) activation (immunoblotting); whereas plasma FN, a form lacking EDA, shows no effect. Primary lung fibroblasts constitutively express α(4)β(7) integrin receptor (FACS and RT-PCR). Blocking of α(4)β(7) reduces fibroblast adhesion to EDA-FN and inhibits α-SMA expression, collagen deposition, and FAK activation induced by EDA-FN. Using recombinant EDA-containing peptides, we demonstrate that the EDA segment is sufficient to induce fibroblast differentiation via binding to α(4)β(7). EDA-FN induces MAPK-Erk1/2 activation and inhibition of MEK1/2 attenuates EDA-FN-induced α-SMA expression. Our findings demonstrate that EDA-FN induces fibroblast differentiation by a mechanism that involves binding of EDA to α(4)β(7) integrin followed by activation of FAK and MAPK-associated signaling pathways. PMID:20643910

  18. Expression of epithelial adhesion proteins and integrins in chronic inflammation.

    PubMed Central

    Haapasalmi, K.; Mäkelä, M.; Oksala, O.; Heino, J.; Yamada, K. M.; Uitto, V. J.; Larjava, H.

    1995-01-01

    Epithelial cell behavior in chronic inflammation is poorly characterized. During inflammation of tooth-supporting structures (periodontal disease), increased proliferation of epithelial cells into the inflamed connective tissue stroma is commonly seen. In some areas ulceration and degeneration take place. We studied alterations in the expression of adhesion molecules and integrins during chronic periodontal inflammation. In inflamed tissue, laminin-1 and type IV collagen were still present in the basement membrane and surrounding blood vessels, but they were also found extravascularly in inflamed connective tissue stroma. Type VII collagen and laminin-5 (also known as kalinin, epiligrin, or nicein) were poorly preserved in the basement membrane zone, but both were found in unusual streak-like distributions in the subepithelial connective tissue stroma in inflamed tissue. Both fibronectin and tenascin were substantially decreased in chronically inflamed connective tissue, showing only punctate staining at the basement membrane zone. Integrins of the beta 1 family showed two distinct staining patterns in epithelial cells during chronic inflammation; focal losses of beta 1 integrins (alpha 2 beta 1 and alpha 3 beta 1) were found in most areas, while in other areas the entire pocket epithelium was found to be strongly positive for beta 1 integrins. No members of the alpha v integrin family were found in any epithelia studied. Expression of the alpha 6 beta 4 integrin was high in basal cells of healthy tissue, but weak in epithelium associated with chronic inflammation. Chronic inflammation therefore involves alterations in both adhesion proteins and integrins expressed by epithelial cells. Basement membrane components found at abnormal sites in stroma in chronic inflammation might serve as new adhesive ligands for various cell types in inflamed stroma. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:7541610

  19. Strain-induced Differentiation of Fetal Type II Epithelial Cells Is Mediated via the Integrin α6β1-ADAM17/Tumor Necrosis Factor-α-converting Enzyme (TACE) Signaling Pathway*

    PubMed Central

    Wang, Yulian; Huang, Zheping; Nayak, Pritha S.; Matthews, Benjamin D.; Warburton, David; Shi, Wei; Sanchez-Esteban, Juan

    2013-01-01

    Mechanical forces are critical for normal fetal lung development. However, the mechanisms regulating this process are not well-characterized. We hypothesized that strain-induced release of HB-EGF and TGF-α is mediated via integrin-ADAM17/TACE interactions. Employing an in vitro system to simulate mechanical forces in fetal lung development, we showed that mechanical strain of fetal epithelial cells actives TACE, releases HB-EGF and TGF-α, and promotes differentiation. In contrast, in samples incubated with the TACE inhibitor IC-3 or in cells isolated from TACE knock-out mice, mechanical strain did not release ligands or promote cell differentiation, which were both rescued after transfection of ADAM17. Cell adhesion assay and co-immunoprecipitation experiments in wild-type and TACE knock-out cells using several TACE constructs demonstrated not only that integrins α6 and β1 bind to TACE via the disintegrin domain but also that mechanical strain enhances these interactions. Furthermore, force applied to these integrin receptors by magnetic beads activated TACE and shed HB-EGF and TGF-α. The contribution of integrins α6 and β1 to differentiation of fetal epithelial cells by strain was demonstrated by blocking their binding site with specific antibodies and by culturing the cells on membranes coated with anti-integrin α6 and β1 antibodies. In conclusion, mechanical strain releases HB-EGF and TGF-α and promotes fetal type II cell differentiation via α6β1 integrin-ADAM17/TACE signaling pathway. These investigations provide novel mechanistic information on how mechanical forces promote fetal lung development and specifically differentiation of epithelial cells. This information could be also relevant to other tissues exposed to mechanical forces. PMID:23888051

  20. 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. PMID:24951930

  1. Roles of integrin activation in eosinophil function and the eosinophilic inflammation of asthma

    PubMed Central

    Barthel, Steven R.; Johansson, Mats W.; McNamee, Dawn M.; Mosher, Deane F.

    2010-01-01

    Eosinophilic inflammation is a characteristic feature of asthma. Integrins are highly versatile cellular receptors that regulate extravasation of eosinophils from the postcapillary segment of the bronchial circulation to the airway wall and airspace. Such movement into the asthmatic lung is described as a sequential, multistep paradigm, whereby integrins on circulating eosinophils become activated, eosinophils tether in flow and roll on bronchial endothelial cells, integrins on rolling eosinophils become further activated as a result of exposure to cytokines, eosinophils arrest firmly to adhesive ligands on activated endothelium, and eosinophils transmigrate to the airway in response to chemoattractants. Eosinophils express seven integrin heterodimeric adhesion molecules: alpha4beta1 (CD49d/29), alpha6beta1 (CD49f/29), alphaMbeta2 (CD11b/18), alphaLbeta2 (CD11a/18), alphaXbeta2 (CD11c/18), alphaDbeta2 (CD11d/18), and alpha4beta7 (CD49d/beta7). The role of these integrins in eosinophil recruitment has been elucidated by major advances in the understanding of integrin structure, integrin function, and modulators of integrins. Such findings have been facilitated by cellular experiments of eosinophils in vitro, studies of allergic asthma in humans and animal models in vivo, and crystal structures of integrins. Here, we elaborate on how integrins cooperate to mediate eosinophil movement to the asthmatic airway. Antagonists that target integrins or the effectors that regulate integrins of eosinophils represent potentially promising therapies in the treatment of asthma. PMID:17906117

  2. Pathological integrin signaling enhances proliferation of primary lung fibroblasts from patients with idiopathic pulmonary fibrosis.

    PubMed

    Xia, Hong; Diebold, Deanna; Nho, Richard; Perlman, David; Kleidon, Jill; Kahm, Judy; Avdulov, Svetlana; Peterson, Mark; Nerva, John; Bitterman, Peter; Henke, Craig

    2008-07-01

    Idiopathic pulmonary fibrosis (IPF) is a relentlessly progressive lung disease in which fibroblasts accumulate in the alveolar wall within a type I collagen-rich matrix. Although lung fibroblasts derived from patients with IPF display durable pathological alterations in proliferative function, the molecular mechanisms differentiating IPF fibroblasts from their normal counterparts remain unknown. Polymerized type I collagen normally inhibits fibroblast proliferation, providing a physiological mechanism to limit fibroproliferation after tissue injury. We demonstrate that beta1 integrin interaction with polymerized collagen inhibits normal fibroblast proliferation by suppression of the phosphoinositide 3-kinase (PI3K)-Akt-S6K1 signal pathway due to maintenance of high phosphatase activity of the tumor suppressor phosphatase and tensin homologue (PTEN). In contrast, IPF fibroblasts eluded this restraint, displaying a pathological pattern of beta1 integrin signaling in response to polymerized collagen that leads to aberrant activation of the PI3K-Akt-S6K1 signal pathway caused by inappropriately low PTEN activity. Mice deficient in PTEN showed a prolonged fibroproliferative response after tissue injury, and immunohistochemical analysis of IPF lung tissue demonstrates activation of Akt in cells within fibrotic foci. These results provide direct evidence for defective negative regulation of the proliferative pathway in IPF fibroblasts and support the theory that the pathogenesis of IPF involves an intrinsic fibroblast defect. PMID:18541712

  3. 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.

  4. Shear stress-induced release of basic fibroblast growth factor from endothelial cells is mediated by matrix interaction via integrin alpha(v)beta3.

    PubMed

    Gloe, Torsten; Sohn, Hae Young; Meininger, Gerald A; Pohl, Ulrich

    2002-06-28

    Considering that chronic elevation of shear stress results in remodeling of the vasculature, we analyzed whether mechanical load could mediate basic fibroblast growth factor (bFGF) release and whether bFGF would act as mediator of shear stress-induced endothelial proliferation and differentiation. Supernatant media of shear stress-exposed endothelial cells (EC) contained significantly higher amounts of bFGF than medium from static cells. Released bFGF was fully intact with regard to its function as an inductor of proliferation and differentiation. Shear stress-conditioned media induced capillary-like structure formation, whereas static control medium did not. Likewise, only shear stress-conditioned medium induced proliferation of serum starved EC. Both capillary-like structure formation and proliferation could be inhibited by neutralization of bFGF or its receptor. The release of bFGF was subject to specific, integrin-mediated control, since inhibition of alpha(v)beta(3) integrin prevented it, whereas inhibition of alpha(5)beta(1) integrin had no effect. We conclude that shear stress induces the release of bFGF from EC in a tightly controlled manner. The release is dependent on specific cell-matrix interactions via alpha(v)beta(3) integrins. The effects on cell proliferation and differentiation suggest that release of bFGF is functionally significant and may represent a necessary initial step in adaptive remodeling processes induced by shear stress. PMID:11976347

  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. Interferon Beta-1a Intramuscular Injection

    MedlinePlus

    ... symptoms flare up from time to time) of multiple sclerosis (MS, a disease in which the nerves do not ... known how interferon beta-1a works to treat MS. ... doctor.Interferon beta-1a controls the symptoms of MS but does not cure it. Continue to use ...

  7. [Transforming growth factor beta-1: structure, function, and regulation mechanisms in cancer].

    PubMed

    Peralta-Zaragoza, O; Lagunas-Martínez, A; Madrid-Marina, V

    2001-01-01

    Transforming growth factor beta-1 (TGF-beta 1) is produced by several cell lineages such as lymphocytes, macrophages, and dendritic cells, and its expression serves in both autocrine and paracrine modes to control the differentiation, proliferation, and state of activation of these and other cells. In general, TGF-beta 1 has pleiotropic properties on the immune response during the development of infection diseases and cancer; however, the mechanisms of action and regulation of gene expression of this cytokine are poorly understood, in this review, the biological properties and the molecular mechanisms that regulate TGF-beta 1 gene expression are described, to understand the role of this cytokine in growth and cell differentiation. The knowledge of molecular mechanisms of gene expression of TGF-beta 1 may serve to develop new cancer therapies. The English version of this paper is available at: http://www.insp.mx/salud/index.html PMID:11547595

  8. Direct transfer of transforming growth factor beta 1 gene into arteries stimulates fibrocellular hyperplasia.

    PubMed Central

    Nabel, E G; Shum, L; Pompili, V J; Yang, Z Y; San, H; Shu, H B; Liptay, S; Gold, L; Gordon, D; Derynck, R

    1993-01-01

    The arterial wall responds to thrombosis or mechanical injury through the induction of specific gene products that increase cellular proliferation and connective tissue formation. These changes result in intimal hyperplasia that is observed in restenosis and the early phases of atherosclerosis. Transforming growth factor beta 1 (TGF-beta 1) is a secreted multi-functional protein that plays an important role in embryonal development and in repair following tissue injury. However, the function of TGF-beta 1 in vascular cell growth in vivo has not been defined. In this report, we have evaluated the role of TGF-beta 1 in the pathophysiology of intimal and medial hyperplasia by gene transfer of an expression plasmid encoding active TGF-beta 1 into porcine arteries. Expression of TGF-beta 1 in normal arteries resulted in substantial extracellular matrix production accompanied by intimal and medial hyperplasia. Increased procollagen, collagen, and proteoglycan synthesis in the neointima was demonstrated by immunohistochemistry relative to control transfected arteries. Expression of TGF-beta 1 induced a distinctly different program of gene expression and biologic response from the platelet-derived growth factor B (PDGF B) gene: procollagen synthesis induced by TGF-beta 1 was greater, and cellular proliferation was less prominent. These findings show that TGF-beta 1 differentially modulates extracellular matrix production and cellular proliferation in the arterial wall in vivo and could play a reparative role in the response to arterial injury. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8248168

  9. Interferon Beta-1a Intramuscular Injection

    MedlinePlus

    ... course of disease where symptoms flare up from time to time) of multiple sclerosis (MS, a disease in which ... interferon beta-1a intramuscular at around the same time of day on your injection days. Follow the ...

  10. Transforming growth factor-beta1 upregulates myostatin expression in mouse C2C12 myoblasts.

    PubMed

    Budasz-Rwiderska, M; Jank, M; Motyl, T

    2005-06-01

    Myostatin (MSTN) and transforming growth factor-beta1 (TGF-beta1) belong to the same TGF-beta superfamily of proteins. They are involved in regulation of skeletal muscle growth and development as well as muscle catabolism. The aim of the present study was to investigate the relationship between MSTN and TGF-beta1 expression in proliferating and differentiating mouse C2C12 myoblasts cultured in normal and catabolic conditions and to evaluate the effect of exogenous TGF-beta1 as well as "knock down" of TGF-beta1 receptor type II on MSTN expression in proliferating and differentiating myogenic cells. The direct effect of TGF-beta1 on myostatin was also examined. Myostatin expression increased gradually with cell confluency in proliferating cultures, while the level of TGF-beta1, detected in the form of a 100 kDa small latent complex diminished. Myostatin expression was accompanied by a partial cell cycle arrest. Three forms of myostatin were found: a 52 kDa precursor, a 40 kDa latency associated propeptide, and a 26 kDa active peptide. A decrease in myostatin and TGF-beta1 levels was observed during the first three days of differentiation, which was subsequently followed by significant increase of their expression during next three to four days of differentiation. Catabolic state induced by dexamethasone significantly increased the level of all forms of myostatin as well as latent (100 kDa) and active (25 kDa) forms of TGF-beta1 in differentiating myoblasts in a dose dependent manner. Exogenous TGF-beta1 (2 ng/ml) significantly increased myostatin levels both in proliferating and differentiating C2C12 myoblasts, whereas silencing of the TGF-beta1 receptor II gene significantly lowered myostatin level in examined cells. The presented results indicate that TGF-beta1 may control myostatin-related regulation of myogenesis through up-regulation of myostatin, predominantly in the course of terminal differentiation and glucocorticoid-dependent catabolic stimulation. PMID

  11. Integrins mediate adhesion of medulloblastoma cells to tenascin and activate pathways associated with survival and proliferation.

    PubMed

    Fiorilli, Paul; Partridge, Darren; Staniszewska, Izabela; Wang, Jin Y; Grabacka, Maja; So, Kelvin; Marcinkiewicz, Cezary; Reiss, Krzysztof; Khalili, Kamel; Croul, Sidney E

    2008-11-01

    Medulloblastoma spreads by leptomeningeal dissemination rather than by infiltration that characterizes other CNS tumors, eg, gliomas. This study represents an initial attempt to identify both the molecules that mediate medulloblastoma adhesion to leptomeninges and the pathways that are key to survival and proliferation of tumor following adhesion. As a first step in molecule identification, we produced adhesion of D283 medulloblastoma cells to the extracellular matrix (ECM) of H4 glioma cells in vitro. Within this context, D283 cells preferentially expressed the alpha9 and beta1 integrin subunits; antibody and disintegrin blockade of alpha9 and beta1 binding eliminated the adhesion. The H4 ECM was enriched in tenascin, a binding partner for the alpha9beta1 integrin heterodimer. Purified tenascin-C supported D283 cell adhesion. The adhesion was blocked by antibodies to alpha9 and beta1 integrin. In vivo data were similar; immunohistochemistry of primary human medulloblastomas with leptomeningeal extension demonstrated increased expression of alpha9 and beta1 integrins as well as tenascin at the interface of brain and leptomeningeal tumor. These data suggest that tumor-cell expressions of alpha9 and beta1 integrins in combination with extracellular tenascin are necessary for medulloblastoma adhesion to the leptomeninges. As a first step in the identification of pathways that mediate survival and proliferation of tumor following adhesion, we demonstrated that adhesion to H4 ECM was associated with survival and proliferation of D283 cells as well as activation of the MAPK pathway in a growth factor deficient environment. Antibody blockade of alpha9 and beta1 integrin binding that eliminated adhesion also eliminated the in vitro survival benefit. These data suggest that adhesion of medulloblastoma to the meninges is necessary for the survival and proliferation of these tumor cells at the secondary site. PMID:18794852

  12. 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.

  13. The Biomechanical Integrin

    PubMed Central

    Baker, Erin L.; Zaman, Muhammad H.

    2009-01-01

    The integrin lies at the center of our efforts to understand mechanotransduction in the human body. Over the past two decades, a wealth of information has yielded important insights into integrin structure and functioning in biochemical pathways; however, relatively little emphasis has been placed on mechanics. In this article, we review the current knowledge base of integrin mechanobiology by examining the role of integrins in stabilizing tissue structure, the mechanisms of integrin force transfer, the process of cell migration, and the pathology of cancer. In order to successfully address the gaps in cancer and other disease research going forward, future efforts of integrin mechanobiology must focus on examining cells in 3D environments and integrating our current understanding into computational models that predict the behavior of integrins in non-equilibrium interactions. PMID:19811786

  14. Down-regulated expression of transforming growth factor beta 1 mRNA in endometrial carcinoma.

    PubMed Central

    Perlino, E.; Loverro, G.; Maiorano, E.; Giannini, T.; Cazzolla, A.; Napoli, A.; Fiore, M. G.; Ricco, R.; Marra, E.; Selvaggi, L.

    1998-01-01

    Transforming growth factor beta1 (TGF-beta1) is a potent modulator of cell proliferation in vitro, and recent studies have demonstrated its overexpression in several different tumours; nevertheless, the molecular mechanisms of TGF-beta1 action on cell growth and differentiation have not been fully elucidated. To clarify the role of TGF-beta and its receptor in human endometrial proliferation and differentiation, TGF-beta1 expression at both the mRNA and protein levels has been evaluated by using Northern blotting and immunohistochemistry, in both normal (atrophic, proliferative and secretory) and neoplastic (adenocarcinoma) endometrial samples. This study demonstrates that TGF-beta1 mRNA expression is dramatically reduced in endometrial carcinomas with respect to non-neoplastic tissues, whereas the immunohistochemical expression of TGF-beta1 is enhanced in the epithelial component of endometrial carcinomas compared with non-neoplastic tissues. These data suggest that TGF-beta1 acts as a paracrine regulator of endometrial cell proliferation and that it may contribute to the carcinogenic mechanisms of endometrial carcinoma. Images Figure 1 Figure 5 Figure 6 Figure 8 PMID:9579831

  15. Down-regulated expression of transforming growth factor beta 1 mRNA in endometrial carcinoma.

    PubMed

    Perlino, E; Loverro, G; Maiorano, E; Giannini, T; Cazzolla, A; Napoli, A; Fiore, M G; Ricco, R; Marra, E; Selvaggi, L

    1998-04-01

    Transforming growth factor beta1 (TGF-beta1) is a potent modulator of cell proliferation in vitro, and recent studies have demonstrated its overexpression in several different tumours; nevertheless, the molecular mechanisms of TGF-beta1 action on cell growth and differentiation have not been fully elucidated. To clarify the role of TGF-beta and its receptor in human endometrial proliferation and differentiation, TGF-beta1 expression at both the mRNA and protein levels has been evaluated by using Northern blotting and immunohistochemistry, in both normal (atrophic, proliferative and secretory) and neoplastic (adenocarcinoma) endometrial samples. This study demonstrates that TGF-beta1 mRNA expression is dramatically reduced in endometrial carcinomas with respect to non-neoplastic tissues, whereas the immunohistochemical expression of TGF-beta1 is enhanced in the epithelial component of endometrial carcinomas compared with non-neoplastic tissues. These data suggest that TGF-beta1 acts as a paracrine regulator of endometrial cell proliferation and that it may contribute to the carcinogenic mechanisms of endometrial carcinoma. PMID:9579831

  16. Phorbol esters alter alpha4 and alphad integrin usage during eosinophil adhesion to VCAM-1.

    PubMed

    Kikuchi, Matsuo; Tachimoto, Hiroshi; Nutku, Esra; Hudson, Sherry A; Bochner, Bruce S

    2003-01-01

    We examined the effect of the protein kinase C activator phorbol-12-myristate-13-acetate (PMA) on the human eosinophil adhesion molecule phenotype and attachment to VCAM-1 via alpha4 and alphad integrins under static and flow conditions. PMA increased surface expression of alphad integrins and decreased alpha4 integrin expression. Under static conditions, eosinophils bound well to VCAM-1, primarily via alpha4beta1 integrins, with a minor alphadbeta2 integrin component. Unexpectedly, PMA-stimulated eosinophils bound equally well to VCAM-1 and albumin in a temperature- and divalent cation-dependent manner, yet adhesion was independent of beta1 and beta2 integrins. Under flow conditions, eosinophils readily attached to VCAM-1, and adhesion was inhibited by both alpha4 and alphad mAbs (95 and 50% inhibition, respectively). Many fewer PMA-stimulated eosinophils bound to VCAM-1 under flow conditions, but both alpha4 and alphad mAbs inhibited adhesion equally. Thus, PMA alters eosinophil integrin expression and the relative contributions of alpha4 and alphad integrins during attachment to VCAM-1. PMID:14668059

  17. Human liver alcohol dehydrogenase. 1. The primary structure of the beta 1 beta 1 isoenzyme.

    PubMed

    Hempel, J; Bühler, R; Kaiser, R; Holmquist, B; de Zalenski, C; von Wartburg, J P; Vallee, B; Jörnvall, H

    1984-12-17

    Determination of the amino acid sequence of the beta 1 subunit from the class I (pyrazole-sensitive) human liver alcohol dehydrogenase isoenzyme beta 1 beta 1 revealed a 373-residue structure differing at 48 positions (including a gap) from that of the subunit of the well studied horse liver alcohol dehydrogenase EE isoenzyme. The structure deduced is compatible with known differences in composition, ultraviolet absorbance, electrophoretic mobility and catalytic properties between the horse and human enzymes. All zinc-liganding residues of the horse E subunit are strictly conserved in the human beta 1 subunit, despite an earlier report of a mutation involving Cys-46. This residue therefore remains conserved in all known alcohol dehydrogenase structures. However, the total cysteine content of the beta 1 structure is raised from 14 in the subunit of the horse enzyme to 15 by a Tyr----Cys exchange. Most exchanges are on the surface of the molecule and of a well conserved nature. Substitutions close to the catalytic centre are of interest to explain the altered substrate specificity and different catalytic activity of the beta 1 homodimer. Functionally, a Ser----Thr exchange at position 48 appears to be of special importance, since Thr-48 in beta 1 instead of Ser-48 in the horse enzyme can restrict available space. Four other substitutions also line the active-site pocket, and appear to constitute partly compensated exchanges. PMID:6391920

  18. The role of integrin alpha D beta2 (CD11d/CD18) in monocyte/macrophage migration.

    PubMed

    Yakubenko, Valentin P; Belevych, Nataly; Mishchuk, Daria; Schurin, Aleksey; Lam, Stephen C-T; Ugarova, Tatiana P

    2008-08-15

    Integrin alpha(D)beta(2) (CD11d/CD18) is a multiligand macrophage receptor with recognition specificity identical to that of the major myeloid cell-specific integrin alpha(M)beta(2) (CD11b/CD18, Mac-1). Despite its prominent upregulation on inflammatory macrophages, the role of alpha(D)beta(2) in monocyte and macrophage migration is unknown. In this study, we have generated model and natural cell lines expressing different densities of alpha(D)beta(2) and examined their migration to various extracellular matrix proteins. When expressed at a low density, alpha(D)beta(2) on the surface of recombinant HEK293 cells and murine IC-21 macrophages cooperates with beta(1)/beta(3) integrins to support cell migration. However, its increased expression on the alpha(D)beta(2)-expressing HEK293 cells and its upregulation by PMA on the IC-21 macrophages result in increased cell adhesiveness and inhibition of cell migration. Furthermore, ligation of alpha(D)beta(2) with anti-alpha(D) blocking antibodies restores beta(1)/beta(3)-driven cell migration by removing the excess alpha(D)beta(2)-mediated adhesive bonds. Consistent with in vitro data, increased numbers of inflammatory macrophages were recovered from the inflamed peritoneum of mice after the administration of anti-alpha(D) antibody. These results demonstrate that the density of alpha(D)beta(2) is critically involved in modulating macrophage adhesiveness and their migration, and suggest that low levels of alpha(D)beta(2) contribute to monocyte migration while alpha(D)beta(2) upregulation on differentiated macrophages may facilitate their retention at sites of inflammation. PMID:18621369

  19. 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.

  20. IGF-I and TGF-beta1 have distinct effects on phenotype and proliferation of intestinal fibroblasts.

    PubMed

    Simmons, James G; Pucilowska, Jolanta B; Keku, Temitope O; Lund, P Kay

    2002-09-01

    Insulin-like growth factor I (IGF-I) and transforming growth factor-beta1 (TGF-beta1) are upregulated in myofibroblasts at sites of fibrosis in experimental enterocolitis and in Crohn's disease (CD). We compared the sites of expression of IGF-I and TGF-beta1 in a rat peptidoglycan-polysaccharide (PG-PS) model of chronic granulomatous enterocolitis and fibrosis. We used the human colonic CCD-18Co fibroblast/myofibroblast cell line to test the hypothesis that TGF-beta1 and IGF-I interact to regulate proliferation, collagen synthesis, and activated phenotype typified by expression of alpha-smooth muscle actin and organization into stress fibers. IGF-I potently stimulated while TGF-beta1 inhibited basal DNA synthesis. TGF-beta1 and IGF-I each had similar but not additive effects to induce type I collagen. TGF-beta1 but not IGF-I potently stimulated expression of alpha-smooth muscle actin and stress fiber formation. IGF-I in combination with TGF-beta1 attenuated stress fiber formation without reducing alpha-smooth muscle actin expression. Stress fibers were not a prerequisite for increased collagen synthesis. TGF-beta1 upregulated IGF-I mRNA, which led us to examine the effects of IGF-I in cells previously activated by TGF-beta1 pretreatment. IGF-I potently stimulated proliferation of TGF-beta1-activated myofibroblasts without reversing activated fibrogenic phenotype. We conclude that TGF-beta1 and IGF-I both stimulate type I collagen synthesis but have differential effects on activated phenotype and proliferation. We propose that during intestinal inflammation, regulation of activated phenotype and proliferation may require sequential actions of TGF-beta1 and IGF-I, but they may act in concert to increase collagen deposition. PMID:12181198

  1. Protection of transforming growth factor-beta 1 activity by heparin and fucoidan.

    PubMed

    McCaffrey, T A; Falcone, D J; Vicente, D; Du, B; Consigli, S; Borth, W

    1994-04-01

    The transforming growth factor-beta (TGF-beta) family of proteins exert diverse and potent effects on proliferation, differentiation, and extracellular matrix synthesis. However, relatively little is known about the stability or processing of endogenous TGF-beta activity in vitro or in vivo. Our previous work indicated that 1) TGF-beta 1 has strong heparin-binding properties that were not previously recognized because of neutralization by iodination, and 2) heparin, and certain other polyanions, could block the binding of TGF-beta 1 to alpha 2-macroglobulin (alpha 2-M). The present studies investigated the influence of heparin-like molecules on the stability of the TGF-beta 1 signal in the pericellular environment. The results indicate that heparin and fucoidan, a naturally occurring sulfated L-fucose polymer, suppress the formation of an initial non-covalent interaction between 125I-TGF-beta 1 and activated alpha 2-M. Electrophoresis of 125I-TGF-beta 1 showed that fucoidan protects TGF-beta 1 from proteolytic degradation by plasmin and trypsin. While plasmin caused little, if any, activation of latent TGF-beta derived from vascular smooth muscle cells (SMC), plasmin degraded acid-activated TGF-beta, and purified TGF-beta 1, and this degradation was inhibited by fucoidan. In vitro, heparin and fucoidan tripled the half-life of 125I-TGF-beta 1 and doubled the amount of cell-associated 125I-TGF-beta 1. Consistent with this protective effect, heparin- and fucoidan-treated SMC demonstrated elevated levels of active, but not latent, TGF-beta activity. PMID:7511146

  2. 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

  3. 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

  4. Transendothelial migration enhances integrin-dependent human neutrophil chemokinesis.

    PubMed

    Gonzalez, Anjelica L; El-Bjeirami, Wafa; West, Jennifer L; McIntire, Larry V; Smith, C Wayne

    2007-03-01

    Transendothelial migration of neutrophils induces phenotypic changes that influence the interactions of neutrophils with extravascular tissue components. To assess the influence of transmigration on neutrophil chemokinetic motility, we used polyethylene glycol hydrogels covalently modified with specific peptide sequences relevant to extracellular matrix proteins. We evaluated fMLP-stimulated human neutrophil motility on peptides Arg-Gly-Asp-Ser (RGDS) and TMKIIPFNRTLIGG (P2), alone and in combination. RGDS is a bioactive sequence found in a number of proteins, and P2 is a membrane-activated complex-1 (Mac-1) ligand located in the gamma-chain of the fibrinogen protein. We evaluated, via video microscopy, cell motility by measuring cell displacement from origin and total accumulated distance traveled and then calculated average velocity. Results indicate that although adhesion and shape change were supported by hydrogels containing RGD alone, motility was not. Mac-1-dependent motility was supported on hydrogels containing P2 alone. Motility was enhanced through combined presentation of RGD and P2, engaging Mac-1, alpha(V)beta(3), and beta(1) integrins. Naïve neutrophil motility on combined peptide substrates was dependent on Mac-1, and alpha(4)beta(1) while alpha(6)beta(1) contributed to speed and linear movement. Transmigrated neutrophil motility was dependent on alpha(v)beta(3) and alpha(5)beta(1), and alpha(4)beta(1), alpha(6)beta(1), and Mac-1 contributed to speed and linear motion. Together, the data demonstrate that efficient neutrophil migration, dependent on multi-integrin interaction, is enhanced after transendothelial migration. PMID:17164427

  5. Integrin endosomal signalling suppresses anoikis.

    PubMed

    Alanko, Jonna; Mai, Anja; Jacquemet, Guillaume; Schauer, Kristine; Kaukonen, Riina; Saari, Markku; Goud, Bruno; Ivaska, Johanna

    2015-11-01

    Integrin-containing focal adhesions transmit extracellular signals across the plasma membrane to modulate cell adhesion, signalling and survival. Although integrins are known to undergo continuous endo/exocytic traffic, the 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, localizes with active integrins on endosomes. Integrin endocytosis positively regulates adhesion-induced FAK activation, which is early endosome antigen-1 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. PMID:26436690

  6. 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.

  7. Extraocular muscle is spared upon complete laminin alpha2 chain deficiency: comparative expression of laminin and integrin isoforms.

    PubMed

    Nyström, Alexander; Holmblad, Johanna; Pedrosa-Domellöf, Fatima; Sasaki, Takako; Durbeej, Madeleine

    2006-08-01

    Mutations in the gene encoding laminin (LM) alpha2 chain cause congenital muscular dystrophy. Here, we show that extraocular muscle (EOM) is spared upon complete LMalpha2 chain absence. The major LM chains in limb muscle basement membranes are alpha2, beta1, beta2 and gamma1 whereas alpha2, alpha4, beta1, beta2 and gamma1 chains are expressed in EOM. Expression of LMalpha4 chain mRNA is further increased in LMalpha2 chain deficient EOM. Mainly integrin alpha7X1 subunit, which binds to laminin-411, is expressed in EOM and in contrast to dystrophic limb muscle, sustained integrin alpha7B expression is seen in LMalpha2 chain deficient EOM. We propose that LMalpha4 chain, possibly by binding to integrin alpha7BX1beta1D, protects EOM in LMalpha2 chain deficient muscular dystrophy. PMID:16782317

  8. 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.

  9. 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.

  10. Human smooth muscle VLA-1 integrin: purification, substrate specificity, localization in aorta, and expression during development.

    PubMed

    Belkin, V M; Belkin, A M; Koteliansky, V E

    1990-11-01

    A membrane glycoprotein complex was isolated and purified from human smooth muscle by detergent solubilization and affinity chromatography on collagen-Sepharose. The complex was identified as VLA-1 integrin and consisted of two subunits of 195 and 130 kD in SDS-PAGE. Liposomes containing the VLA-1 integrin adhered to surfaces coated with type I, II, III, and IV collagens, Clq subcomponent of the first component of the complement, and laminin. The liposomes specifically adhered to these proteins in a Ca2+, Mg2(+)-dependent manner, but did not bind to gelatin, fibronectin, and thrombospondin substrates. The expression of VLA-1 integrin in different human tissues and cell types, and during aorta smooth muscle development was studied by SDS-PAGE, and subsequent quantitative immunoblotting was performed with antibodies recognizing alpha 1 and beta 1 subunits of the VLA-1 integrin. A high level of VLA-1 integrin expression was an exceptional feature of smooth muscles. Fibroblasts, endothelial cells, keratinocytes, striated muscles, and platelets contained trace amounts of VLA-1 integrin. In the 10-wk-old human fetal aorta, VLA-1 integrin was found only in smooth muscle cells whereas mesenchymal cells, surrounding aortic smooth muscle cells, were VLA-1 integrin negative. By the 24th wk of gestation, the amount of VLA-1 integrin was significantly reduced in the aortic media (4.3-fold for alpha 1 subunit and 2.5-fold for beta 1 subunit) compared with that in the 10-wk-old aortic smooth muscle cells. After birth, the expression of VLA-1 integrin increased and in the 1.5-yr-old child aorta the VLA-1 integrin level was almost the same as in adult aortic media. Smooth muscle cells from intimal thickening of adult aorta express five times less alpha 1 subunit of VLA integrin that smooth muscle cells from adult aortic media. In primary culture of aortic smooth muscle cells, the content of the VLA-1 integrin was dramatically reduced and subcultured cells did not contain VLA-1

  11. 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.

  12. Transforming growth factor beta1 (TGF-beta1) is a preoperative prognostic indicator in advanced gastric carcinoma.

    PubMed Central

    Nakamura, M.; Katano, M.; Kuwahara, A.; Fujimoto, K.; Miyazaki, K.; Morisaki, T.; Mori, M.

    1998-01-01

    It has been generally accepted that transforming growth factor beta1 (TGF-beta1) has both negative and positive effects on tumour growth and progression. This study analysed the prognostic value of TGF-beta1 mRNA in advanced gastric carcinoma. A reverse transcriptase-polymerase chain reaction analysis (RT-PCR) was used for TGF-beta1 in endoscopic biopsy specimens from 42 advanced gastric carcinomas. Thirty specimens expressed TGF-beta1 mRNA while 12 specimens did not. The follow-up duration ranged from 4 to 37 months (mean 22.8 months). TGF-beta1-positive group demonstrated a shorter overall survival compared with the TGF-beta1 -negative group (P=0.0014). A significant correlation was also found in the 32 patients who underwent curative resection (P=0.0048). Significant correlations were found between TGF-beta1 mRNA expression and both stage (P=0.0015) and nodal involvement (P=0.0060). Multivariate analysis demonstrated that only TGF-beta1 mRNA expression (P=0.0306) was an independent prognostic factor. All of ten patients who underwent non-curative resection expressed TGF-beta1 mRNA. Expression of TGF-beta1 mRNA in gastric biopsy specimens may be an important preoperative prognostic variable for advanced gastric carcinoma. Images Figure 1 PMID:9823982

  13. 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

  14. 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

  15. 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). PMID:12794117

  16. Integrin cytoplasmic domain-associated protein 1alpha (ICAP-1alpha ) interacts directly with the metastasis suppressor nm23-H2, and both proteins are targeted to newly formed cell adhesion sites upon integrin engagement.

    PubMed

    Fournier, Henri-Noël; Dupé-Manet, Sandra; Bouvard, Daniel; Lacombe, Marie-Lise; Marie, Christiane; Block, Marc R; Albiges-Rizo, Corinne

    2002-06-01

    Cell adhesion-dependent signaling implicates cytoplasmic proteins interacting with the intracellular tails of integrins. Among those, the integrin cytoplasmic domain-associated protein 1alpha (ICAP-1alpha) has been shown to interact specifically with the beta(1) integrin cytoplasmic domain. Although it is likely that this protein plays an important role in controlling cell adhesion and migration, little is known about its actual function. To search for potential ICAP-1alpha-binding proteins, we used a yeast two-hybrid screen and identified the human metastatic suppressor protein nm23-H2 as a new partner of ICAP-1alpha. This direct interaction was confirmed in vitro, using purified recombinant ICAP-1alpha and nm23-H2, and by co-immunoprecipitation from CHO cell lysates over-expressing ICAP-1alpha. The physiological relevance of this interaction is provided by confocal fluorescence microscopy, which shows that ICAP-1alpha and nm23-H2 are co-localized in lamellipodia during the early stages of cell spreading. These adhesion sites are enriched in occupied beta(1) integrins and precede the formation of focal adhesions devoid of ICAP-1alpha and nm23-H2, indicating the dynamic segregation of components of matrix adhesions. This peripheral staining of ICAP-1alpha and nm23-H2 is only observed in cells spreading on fibronectin and collagen and is absent in cells spreading on poly-l-lysine, vitronectin, or laminin. This is consistent with the fact that targeting of both ICAP-1alpha and nm23-H2 to the cell periphery is dependent on beta(1) integrin engagement rather than being a consequence of cell adhesion. This finding represents the first evidence that the tumor suppressor nm23-H2 could act on beta(1) integrin-mediated cell adhesion by interacting with one of the integrin partners, ICAP-1alpha. PMID:11919189

  17. 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

  18. 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.

  19. Functional analysis of the cytoplasmic domain of the integrin {alpha}1 subunit in endothelial cells.

    PubMed

    Abair, Tristin D; Bulus, Nada; Borza, Corina; Sundaramoorthy, Munirathinam; Zent, Roy; Pozzi, Ambra

    2008-10-15

    Integrin alpha1beta1, the major collagen type IV receptor, is expressed by endothelial cells and plays a role in both physiologic and pathologic angiogenesis. Because the molecular mechanisms whereby this collagen IV receptor mediates endothelial cell functions are poorly understood, truncation and point mutants of the integrin alpha1 subunit cytoplasmic tail (amino acids 1137-1151) were generated and expressed into alpha1-null endothelial cells. We show that alpha1-null endothelial cells expressing the alpha1 subunit, which lacks the entire cytoplasmic tail (mutant alpha1-1136) or expresses all the amino acids up to the highly conserved GFFKR motif (mutant alpha1-1143), have a similar phenotype to parental alpha1-null cells. Pro(1144) and Leu(1145) were shown to be necessary for alpha1beta1-mediated endothelial cell proliferation; Lys(1146) for adhesion, migration, and tubulogenesis and Lys(1147) for tubulogenesis. Integrin alpha1beta1-dependent endothelial cell proliferation is primarily mediated by ERK activation, whereas migration and tubulogenesis require both p38 MAPK and PI3K/Akt activation. Thus, distinct amino acids distal to the GFFKR motif of the alpha1 integrin cytoplasmic tail mediate activation of selective downstream signaling pathways and specific endothelial cell functions. PMID:18647959

  20. 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

  1. 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.

  2. Integrin activation state determines selectivity for novel recognition sites in fibrillar collagens.

    PubMed

    Siljander, Pia R-M; Hamaia, Samir; Peachey, Anthony R; Slatter, David A; Smethurst, Peter A; Ouwehand, Willem H; Knight, C Graham; Farndale, Richard W

    2004-11-12

    Only three recognition motifs, GFOGER, GLOGER, and GASGER, all present in type I collagen, have been identified to date for collagen-binding integrins, such as alpha(2)beta(1). Sequence alignment was used to investigate the occurrence of related motifs in other human fibrillar collagens, and located a conserved array of novel GER motifs within their triple helical domains. We compared the integrin binding properties of synthetic triple helical peptides containing examples of such sequences (GLSGER, GMOGER, GAOGER, and GQRGER) or the previously identified motifs. Recombinant inserted (I) domains of integrin subunits alpha(1), alpha(2) and alpha(11) all bound poorly to all motifs other than GFOGER and GLOGER. Similarly, alpha(2)beta(1) -containing resting platelets adhered well only to GFOGER and GLOGER, while ADP-activated platelets, HT1080 cells and two active alpha(2)I domain mutants (E318W, locked open) bound all motifs well, indicating that affinity modulation determines the sequence selectivity of integrins. GxO/SGER peptides inhibited platelet adhesion to collagen monomers with order of potency F >/= L >/= M > A. These results establish GFOGER as a high affinity sequence, which can interact with the alpha(2)I domain in the absence of activation and suggest that integrin reactivity of collagens may be predicted from their GER content. PMID:15345717

  3. Expression of fibronectin, fibronectin isoforms and integrin receptors in melanocytic lesions.

    PubMed Central

    Natali, P. G.; Nicotra, M. R.; Di Filippo, F.; Bigotti, A.

    1995-01-01

    In vitro studies have demonstrated that fibronectin (FN) can deliver a mitogenic signal to quiescent human melanoma cells and that the alpha 5/beta 1-integrin receptor mediates this stimulus. In view of this finding we have analysed the in vivo expression of FN, and of ED-A and ED-B FN isoforms, in benign and malignant lesions of melanocyte origin. In the same specimens the expression of fibronectin integrin receptors was evaluated. The results demonstrate that, while detection of FN does not correlate with transformation and tumour progression, the expression of the two isoforms is associated with transformation and that only the ED-A variant is found in metastases. Integrin phenotyping disclosed that alpha 3/beta 1 expression is associated with tumour progression, alpha v/beta 3 is a marker of transformation, alpha 4 is rarely expressed and alpha 5 is expressed by about 50% and 30% of the primary and metastatic lesions respectively. Taken together, the results of this study demonstrate that transformation and tumour progression of the melanocyte lineage are associated with modulation of expression of FN isoforms and FN integrin receptors. Furthermore, the expression of alpha 5-integrin in a considerable percentage of primary and metastatic lesions indicates that FN may deliver a proliferative stimulus to melanoma cells in vivo. Images Figure 1 PMID:7779718

  4. Immunolocalization of beta-1-4-galactan and its relationship with lignin distribution in developing compression wood of Cryptomeria japonica.

    PubMed

    Kim, Jong Sik; Awano, Tatsuya; Yoshinaga, Arata; Takabe, Keiji

    2010-06-01

    Compression wood (CW) contains higher quantities of beta-1-4-galactan than does normal wood (NW). However, the physiological roles and ultrastructural distribution of beta-1-4-galactan during CW formation are still not well understood. The present work investigated deposition of beta-1-4-galactan in differentiating tracheids of Cryptomeria japonica during CW formation using an immunological probe (LM5) combined with immunomicroscopy. Our immunolabeling studies clearly showed that differences in the distribution of beta-1-4-galactan between NW (and opposite wood, OW) and CW are initiated during the formation of the S(1) layer. At this stage, CW was strongly labeled in the S(1) layer, whereas no label was observed in the S(1) layer of NW and OW. Immunogold labeling showed that beta-1-4-galactan in the S(1) layer of CW tracheids significantly decreased during the formation of the S(2) layer. Most beta-1-4-galactan labeling was present in the outer S(2) region in mature CW tracheids, and was absent in the inner S(2) layer that contained helical cavities in the cell wall. In addition, delignified CW tracheids showed significantly more labeling of beta-1-4-galactan in the secondary cell wall, suggesting that lignin is likely to mask beta-1-4-galactan epitopes. The study clearly showed that beta-1-4-galactan in CW was mainly deposited in the outer portion of the secondary cell wall, indicating that its distribution may be spatially consistent with lignin distribution in CW tracheids of Cryptomeria japonica. PMID:20376677

  5. 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.

  6. Elicitor and resistance-inducing activities of beta-1,4 cellodextrins in grapevine, comparison with beta-1,3 glucans and alpha-1,4 oligogalacturonides.

    PubMed

    Aziz, Aziz; Gauthier, Adrien; Bézier, Annie; Poinssot, Benoît; Joubert, Jean-Marie; Pugin, Alain; Heyraud, Alain; Baillieul, Fabienne

    2007-01-01

    Cellodextrins (CD), water-soluble derivatives of cellulose composed of beta-1,4 glucoside residues, have been shown to induce a variety of defence responses in grapevine (Vitis vinifera L.) cells. The larger oligomers of CD rapidly induced transient generation of H2O2 and elevation in free cytosolic calcium, followed by a differential expression of genes encoding key enzymes of the phenylpropanoid pathway and pathogenesis-related (PR) proteins as well as stimulation of chitinase and beta-1,3 glucanase activities. Most of these defence reactions were also induced by linear beta-1,3 glucans (betaGlu) and alpha-1,4 oligogalacturonides (OGA) of different degree of polymerization (DP), but the intensity of some reactions induced by CD was different when compared with betaGlu and OGA effects. Moreover, desensitization assays using H2O2 production showed that cells treated with CD remained fully responsive to a second application of OGA, suggesting a different mode of perception of these oligosaccharides by grape cells. None of CD, betaGlu, or OGA induced HSR gene expression nor did they induce cell death. In accordance with elicitor activity in grapevine cells, CD-incubated leaves challenged with Botrytis cinerea also resulted in a significant reduction of the disease. Data suggest that CD could operate via other distinct reaction pathways than betaGlu and OGA. They also highlight the requirement of a specific DP for each oligosaccharide to induce the defence response. PMID:17322548

  7. Characterization of integrin expression in islets isolated from hamster, canine, porcine, and human pancreas.

    PubMed

    Wang, R N; Paraskevas, S; Rosenberg, L

    1999-04-01

    The reasons for the failure of clinical islet transplantation remain obscure. Islet isolation, however, exposes the islet to variety of cellular stresses, including disruption of the cell-matrix relationship, an event associated with apoptosis. The cell-matrix relationship is characterized by an interaction between cell surface integrin receptors and matrix molecules of the surrounding basement membrane (BM). The purpose of this study was to characterize integrin expression and the distribution of the peri-insular BM in human, porcine, canine, and hamster pancreas, and after routine islet isolation. Whereas islets in the porcine pancreas do not have a demonstrable BM, islets in the human, canine, and hamster pancreas have an almost continuous BM with very little direct exocrine to endocrine cell-cell contact. After islet isolation, the BM was destroyed, only to be reestablished during the period of culture. In the pancreas of all four species, integrin alpha3 was expressed only on islet cells, and integrin alpha5 was present on islet cells as well as on acinar, centroacinar, and duct cells. Integrin alphaV was detected only in human and canine pancreas. Integrin beta1 was demonstrated only in the human pancreas. In isolated islets, integrin alpha3, alpha5, and alphaV expression decreased during the culture period and the intensity of the staining was observed to be coincident with the distribution of the BM. In summary, this is the first report of integrin expression in hamster, canine, porcine, and human islets. After islet isolation, the altered islet cell-matrix relationship is reflected both in the decrease in integrin expression and in the destruction of the peri-insular BM. These profound changes will need to be considered as the process of islet isolation for transplantation is refined. (J Histochem Cytochem 47:499-506, 1999) PMID:10082751

  8. 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 accompanied by significant increases in cell motility and proliferation in the presence of its major ligand vitronectin. In the present study, we characterized integrin {alpha}v{beta}3-mediated tumor biological effects as a function of cellular KAI1 restoration and proved for the first time that KAI1, besides its already known physical crosstalk with {beta}1-integrins, also colocalizes with integrin {alpha}v{beta}3. Functionally, elevated KAI1 levels drastically increased integrin {alpha}v{beta}3/vitronectin-dependent ovarian cancer cell adhesion. Since an intermediate level of cell adhesive strength is required for optimal cell migration, we next studied ovarian cancer cell motility as a function of KAI1 restoration. By time lapse video microscopy, we found impaired integrin {alpha}v{beta}3/vitronectin-mediated cell migration most probably due to strongly enhanced cellular immobilization onto the adhesion-supporting matrix. Moreover, KAI1 reexpression significantly diminished cell proliferation. These data strongly indicate that KAI1 may suppress ovarian cancer progression by inhibiting integrin {alpha}v{beta}3/vitronectin-provoked tumor cell motility and proliferation as important hallmarks of the oncogenic process.

  9. Assessment of zona pellucida glycoprotein and integrin transcript contents in porcine oocytes.

    PubMed

    Kempisty, Bartosz; Antosik, Paweł; Bukowska, Dorota; Jackowska, Marta; Lianeri, Margarita; Jaśkowski, Jedrzej M; Jagodziński, Paweł P

    2009-03-01

    Using reverse transcription and real-time quantitative PCR analysis we evaluated the transcript levels of integrins (alphaL, alphaM, beta1, and beta6), CD9 and CD18 antigens as well as zona pellucida glycoproteins (pZP1, pZP2, pZP3 and pZP3alpha) in oocytes isolated from puberal gilts (n=20) and multiparous sows (n=20). We found significantly (p<0.05) higher transcript contents of alphaL, alphaM, beta1, and beta integrins, CD9 antigen, and pZP2 and pZP3 in puberal gilt oocytes compared to multiparous sow oocytes. Our results suggest that a decrease in the level of oocyte transcripts encoding essential proteins involved in oocyte fertilization may be associated with increased porcine female age. PMID:19352419

  10. 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.

  11. Identification of novel inhibitors of the transforming growth factor beta1 (TGF-beta1) type 1 receptor (ALK5).

    PubMed

    Callahan, James F; Burgess, Joelle L; Fornwald, James A; Gaster, Laramie M; Harling, John D; Harrington, Frank P; Heer, Jag; Kwon, Chet; Lehr, Ruth; Mathur, A; Olson, Barbara A; Weinstock, Joseph; Laping, Nicholas J

    2002-02-28

    Screening of our internal compound collection for inhibitors of the transforming growth factor beta1 (TGF-beta1) type I receptor (ALK5) identified several hits. Optimization of the dihydropyrroloimidazole hit 2 by introduction of a 2-pyridine and 3,4-methylenedioxyphenyl group gave 7, a selective ALK5 inhibitor. With this information, optimization of the triarylimidazole hit 8 gave the selective inhibitor 14, which inhibits TGF-beta1-induced fibronectin mRNA formation while displaying no measurable cytotoxicity in the 48 h XTT assay. PMID:11855979

  12. A novel integrin function in innate immunity from Chinese mitten crab (Eriocheir sinensis).

    PubMed

    Huang, Ying; Zhao, Ling-Ling; Feng, Jin-Ling; Zhu, Huan-Xi; Huang, Xin; Ren, Qian; Wang, Wen

    2015-10-01

    Integrins belong to a superfamily of conserved α β heterodimeric cell surface receptors that have critical function in cell migration, differentiation, and survival. In this study, an integrin called EsIntegrin was identified from Chinese mitten crab Eriocheir sinensis. EsIntegrin cDNA is 4415 bp long with a 2457 bp open reading frame that encodes an 818 amino acid protein. EsIntegrin contains a signal peptide, an integrin beta subunit (N-terminal portion of extracellular region) INB domain, an epidermal growth factor (hEGF) domain, an integrin B tail domain, a transmembrane region, and an integrin b cyt domain. EsIntegrin was mainly expressed in hemocytes and the heart, with a relatively lower expression level in gills, nerves, intestine, hepatopancreas, muscles, and eyestalk. When healthy crabs were challenged with LPS, PGN, Staphyloccocus aureus, or Vibrio parahaemolyticus, EsIntegrin expression level was upregulated significantly. Recombinant EsIntegrin has agglutination activity to Gram-positive (e.g., S. aureus and Bacillus subtilis) and Gram-negative bacteria (e.g., V. parahaemolyticus and Aeromonas hydrophila) in the presence of calcium. Furthermore, rEsIntegrin could not only bind to various bacteria such as S. aureus, Micrococcus luteus, B. subtilis, Bacillus megaterium, Bacillus thuringiensis, V. parahaemolyticus, Vibrio anguillarum, A. hydrophila, Vibrio natriegens, and Escherichia coli, but this compound also helped crabs in clearing virulent Gram-negative bacterium, V. parahaemolyticus, in vivo. These data suggested that EsIntegrin might function as cellular receptor that is involved in anti-bacterial immunity from E. sinensis. PMID:26004499

  13. Utilizing Fibronectin Integrin-Binding Specificity to Control Cellular Responses

    PubMed Central

    Bachman, Haylee; Nicosia, John; Dysart, Marilyn; Barker, Thomas H.

    2015-01-01

    Significance: Cells communicate with the extracellular matrix (ECM) protein fibronectin (Fn) through integrin receptors on the cell surface. Controlling integrin–Fn interactions offers a promising approach to directing cell behavior, such as adhesion, migration, and differentiation, as well as coordinated tissue behaviors such as morphogenesis and wound healing. Recent Advances: Several different groups have developed recombinant fragments of Fn that can control epithelial to mesenchymal transition, sequester growth factors, and promote bone and wound healing. It is thought that these physiological responses are, in part, due to specific integrin engagement. Furthermore, it has been postulated that the integrin-binding domain of Fn is a mechanically sensitive switch that drives binding of one integrin heterodimer over another. Critical Issues: Although computational simulations have predicted the mechano-switch hypothesis and recent evidence supports the existence of varying strain states of Fn in vivo, experimental evidence of the Fn integrin switch is still lacking. Future Directions: Evidence of the integrin mechano-switch will enable the development of new Fn-based peptides in tissue engineering and wound healing, as well as deepen our understanding of ECM pathologies, such as fibrosis. PMID:26244106

  14. Role of Integrin in Mechanical Loading of Osteoblasts

    NASA Technical Reports Server (NTRS)

    Globus, Ruth; Demsky, Caroline

    2000-01-01

    Mechanical forces generated by gravity, weightbearing, and muscle contraction play a key role in the genesis and maintenance of skeletal structure. The molecular mechanisms that mediate changes in osteoblast activity in response to altered patterns of skeletal loading are not known, and a better understanding of these processes may be essential for developing effective treatment strategies to prevent disuse osteoporosis. We have elucidated specific integrin/ECM (extracellular matrix) interactions that are required for osteoblast differentiation and survival and have developed a useful loading system to further explore the molecular basis of mechano-sensitivity of osteoblasts. The long term goal of our collaborative research is to understand how the ECM and cell adhesion proteins and integrins interaction to mediate the response of osteoblasts and their progenitors to mechanical loading. We suggest that integrin/ECM interactions are crucial for basic cellular processes, including differentiation and survival, as well as to participate in detecting and mediating cellular responses to mechanical stimuli.

  15. Osteopontin involvement in integrin-mediated cell signaling and regulation of expression of alkaline phosphatase during early differentiation of UMR cells.

    PubMed

    Liu, Y K; Uemura, T; Nemoto, A; Yabe, T; Fujii, N; Ushida, T; Tateishi, T

    1997-12-22

    To clarify the function of osteopontin in osteoblast differentiation, we have examined the signal transduction pathway in an osteoblastic cell line (UMR106-6) bound to osteopontin, fibronectin, vitronectin and collagen type I surfaces. This was done by investigating the production and autophosphorylation of focal adhesion kinase (FAK) and the expression of alkaline phosphatase (ALP) at the transcription level. Results suggest that osteopontin was not only responsible for the autophosphorylation of FAK but regulated the expression of ALP, which was strongly correlated with FAK activity. These results suggest that osteopontin might act as a trigger in the early differentiation of osteoblasts. PMID:9450560

  16. Integrin receptors play a role in the internalin B-dependent entry of Listeria monocytogenes into host cells.

    PubMed

    Auriemma, Clementina; Viscardi, Maurizio; Tafuri, Simona; Pavone, Luigi Michele; Capuano, Federico; Rinaldi, Laura; Della Morte, Rossella; Iovane, Giuseppe; Staiano, Norma

    2010-09-01

    Listeria monocytogenes enters non-phagocytic cells by binding its surface proteins inlA (internalin) and inlB to the host's E-cadherin and Met, respectively. The two internalins play either separate or cooperative roles in the colonization of infected tissues. Here, we studied bacterial uptake into HeLa cells using an L. monocytogenes mutant strain (DeltainlA) carrying a deletion in the gene coding for inlA. The DeltainlA mutant strain showed the capability to invade HeLa cells. The monoclonal anti-beta(3)- and anti-beta(1)-integrin subunit antibodies prevented bacterial uptake into the cells, while the anti-beta(2)- and anti-beta(4)-integrin subunit antibodies failed to affect L. monocytogenes entry into HeLa cells. Three structurally distinct disintegrins (kistrin, echistatin and flavoridin) also inhibited bacterial uptake, showing different potencies correlated to their selective affinity for the beta(3)- and beta(1)-integrin subunits. In addition to inducing Met phosphorylation, infection of cells by the L. monocytogenes DeltainlA mutant strain promoted the tyrosine phosphorylation of the focal adhesion-associated proteins FAK and paxillin. Our findings provide the first evidence that beta(3)- and beta(1)-integrin receptors play a role in the inlB-dependent internalization of L. monocytogenes into host cells. PMID:20526749

  17. Direct binding of F actin to the cytoplasmic domain of the alpha 2 integrin chain in vitro

    NASA Technical Reports Server (NTRS)

    Kieffer, J. D.; Plopper, G.; Ingber, D. E.; Hartwig, J. H.; Kupper, T. S.

    1995-01-01

    The transmembrane integrins have been shown to interact with the cytoskeleton via noncovalent binding between cytoplasmic domains (CDs) of integrin beta chains and various actin binding proteins within the focal adhesion complex. Direct or indirect integrin alpha chain CD binding to the actin cytoskeleton has not been reported. We show here that actin, as an abundant constituent of focal adhesion complex proteins isolated from fibroblasts, binds strongly and specifically to alpha 2 CD, but not to alpha 1 CD peptide. Similar specific binding to alpha 2 CD peptide was seen for highly purified F actin, free of putative actin-binding proteins. The bound complex of actin and peptide was visualized directly by coprecipitation, and actin binding was abrogated by removal of a five amino acid sequence from the alpha 2 CD peptide. Our findings may explain the earlier observation that, while integrins alpha 2 beta 1 and alpha 1 beta 1 both bind to collagen, only alpha 2 beta 1 can mediate contraction of extracellular collagen matrices.

  18. Low-shear modelled microgravity environment maintains morphology and differentiated functionality of primary porcine hepatocyte cultures.

    PubMed

    Nelson, Leonard J; Walker, Simon W; Hayes, Peter C; Plevris, John N

    2010-01-01

    Hepatocytes cultured in conventional static culture rapidly lose polarity and differentiated function. This could be explained by gravity-induced sedimentation, which prevents formation of complete three-dimensional (3D) cell-cell/cell-matrix interactions and disrupts integrin-mediated signals (including the most abundant hepatic integrin alpha(5)beta(1)), important for cellular polarity and differentiation. Cell culture in a low fluid shear modelled microgravity (about 10(-2) g) environment promotes spatial colocation/self-aggregation of dissociated cells and induction of 3D differentiated liver morphology. Previously, we demonstrated the utility of a NASA rotary bioreactor in maintaining key metabolic functions and 3D aggregate formation of high-density primary porcine hepatocyte cultures over 21 days. Using serum-free chemically defined medium, without confounding interactions of exogenous bioscaffolding or bioenhancing surface materials, we investigated features of hepatic cellular polarity and differentiated functionality, including expression of hepatic integrin alpha(5), as markers of functional morphology. We report here that in the absence of exogenous biomatrix scaffolding, hepatocytes cultured in serum-free chemically defined medium in a microgravity environment rapidly (<24 h) form macroscopic (2-5 mm), compacted 3D hepatospheroid structures consisting of a shell of glycogen-positive viable cells circumscribing a core of eosinophilic cells. The spheroid shell layers exhibited ultrastructural, morphological and functional features of differentiated, polarized hepatic tissue including strong expression of the integrin alpha(5) subunit, functional bile canaliculi, albumin synthesis, and fine ultrastructure reminiscent of in vivo hepatic tissue. The low fluid shear microgravity environment may promote tissue-like self-organization of dissociated cells, and offer advantages over spheroids cultured in conventional formats to delineate optimal conditions for

  19. Id-1 promotes TGF-{beta}1-induced cell motility through HSP27 activation and disassembly of adherens junction in prostate epithelial cells

    SciTech Connect

    Di Kaijun; Wong, Y.C. Wang Xianghong

    2007-11-15

    Id-1 (inhibitor of differentiation or DNA binding-1) has been positively associated with cell proliferation, cell cycle progression, and invasiveness during tumorigenesis. In addition, Id-1 has been shown to modulate cellular sensitivity to TGF-{beta}1 (transforming growth factor {beta}1). Here we demonstrate a novel role of Id-1 in promoting TGF-{beta}1-induced cell motility in a non-malignant prostate epithelial cell line, NPTX. We found that Id-1 promoted F-actin stress fiber formation in response to TGF-{beta}1, which was associated with increased cell-substrate adhesion and cell migration in NPTX cells. In addition, this positive effect of Id-1 on TGF-{beta}1-induced cell motility was mediated through activation of MEK-ERK signaling pathway and subsequent phosphorylation of HSP27 (heat shock protein 27). Furthermore, Id-1 disrupted the adherens junction complex in TGF-{beta}1-treated cells through down-regulation of E-cadherin, redistribution of {beta}-catenin, along with up-regulation of N-cadherin. These lines of evidence reveal a novel tumorigenic role of Id-1 through reorganization of actin cytoskeleton and disassembly of cell-cell adhesion in response to TGF-{beta}1 in human prostate epithelial cells, and suggest that intracellular Id-1 levels might be a determining factor for switching TGF-{beta}1 from a growth inhibitor to a tumor promoter during prostate carcinogenesis.

  20. Extracellular matrix receptors and the differentiation of human megakaryocytes in vitro.

    PubMed

    Molla, A; Mossuz, P; Berthier, R

    1999-03-01

    We investigated the expression and functions of extracellular matrix receptors (or integrins) in the course of the differentiation of human megakaryocytes (Mks) leading to the formation of platelets. Integrins beta1 or Very Late Antigens (VLA) are specialized transmembrane receptors allowing the attachment of the cells to collagen (VLA-2), fibronectin (VLA-4 and -5) and laminin (VLA-6). A proportion of committed megakaryocytic progenitor cells (CFU-MK) adhere to fibronectin but not to collagen or laminin. The early immature Mks are retained on fibronectin (30%) and laminin (12%) but not on collagen whereas large mature Mks are still adherent to fibronectin and laminin and also acquired the capacity to adhere to collagen. The expression of the different VLA in the maturation of Mks correlates well with their adhesive properties. Hence, VLA-2 is not expressed on immature Mks but is present on the mature polyploid cells. VLA-4 is detected only on immature Mks which do not seem to bear VLA-5, while this last integrin appears on late Mks. VLA-6 showed a broad distribution from the early to late stages of Mks differentiation. Integrins beta3 of the cytoadhesin family are represented by alphaIIb beta3 that is the receptor for fibrinogen and alphaV beta3 which mediates adhesion to vitronectin. AlphaIIb beta3 is present on the CFU-MK and highly expressed throughout the Mks maturation stages while alphaV beta3 expression is much lower and seems to be detected only on the late Mks. The regulation of the expression of these receptors by cytokines and their respective roles in the maturation of Mks and the final production of platelets, are discussed. The development of efficient culture systems of human Mks in the presence of the recently cloned thrombopoietin will undoubtedly help to shed more light on the molecular mechanisms of their interactions via integrins with the BM microenvironment. PMID:10194117

  1. Integrin α5 promotes tumor progression and is an independent unfavorable prognostic factor in esophageal squamous cell carcinoma.

    PubMed

    Xie, Jian-Jun; Guo, Jin-Cheng; Wu, Zhi-Yong; Xu, Xiu-E; Wu, Jian-Yi; Chen, Bo; Ran, Li-Qiang; Liao, Lian-Di; Li, En-Min; Xu, Li-Yan

    2016-02-01

    The integrin family plays a major role in complex biological events such as differentiation, development, wound healing, and the altered adhesive and invasive properties of tumor cells. The expression and function of integrin α5 in esophageal squamous cell carcinoma (ESCC) are not clear. Here, by using tissue microarrays and immunohistochemical method, integrin α5 expression was retrospectively evaluated in 147 samples of human ESCC. Results showed that expression of integrin α5 was heterogeneous and varied from negative to intense expression in a membrane and cytoplasmic distribution manner. High expression of integrin α5 was significantly correlated with lymph node metastasis (P = .042) and tumor size (P = .042). Kaplan-Meier analysis revealed that high expression of integrin α5 was related to poor overall survival of ESCC patients (P = .018). Multivariate analysis suggested that integrin α5 expression status was an independent prognostic factor for ESCC (P = .003). Moreover, integrin α5 expression was associated with the survival of patients with lymph node metastasis (P = .020), but did not influence the survival of patients without lymph node metastasis. Finally, we found that RNAi-mediated knockdown of integrin α5 led to decreased growth, migration, and invasion of ESCC cells. Combined, integrin α5 might play important roles in the progression of ESCC. Integrin α5 is a novel biomarker to predict the prognosis of ESCC patients. PMID:26772401

  2. Integrin Targeted Delivery of Chemotherapeutics

    PubMed Central

    Chen, Kai; Chen, Xiaoyuan

    2011-01-01

    Targeted delivery of chemotherapeutics is defined in the sense, that is, to maximize the therapeutic index of a chemotherapeutic agent by strictly localizing its pharmacological activity to the site or tissue of action. Integrins are a family of heterodimeric transmembrane glycoproteins involved in a wide range of cell-to-extracellular matrix (ECM) and cell-to-cell interactions. As cell surface receptors, integrins readily interact with extracellular ligands and play a vital role in angiogenesis, leukocytes function and tumor development, which sets up integrins as an excellent target for chemotherapy treatment. The peptide ligands containing the arginine-glycine-aspartic acid (RGD), which displays a strong binding affinity and selectivity to integrins, particularly to integrin αvβ3, have been developed to conjugate with various conventional chemotherapeutic agents, such as small molecules, peptides and proteins, and nanoparticle-carried drugs for integtrin targeted therapeutic studies. This review highlights the recent advances in integrin targeted delivery of chemotherapeutic agents with emphasis on target of integrin αvβ3, and describes the considerations for the design of the diverse RGD peptide-chemotherapeutics conjugates and their major applications. PMID:21547159

  3. Epithelial β1 integrin is required for lung branching morphogenesis and alveolarization.

    PubMed

    Plosa, Erin J; Young, Lisa R; Gulleman, Peter M; Polosukhin, Vasiliy V; Zaynagetdinov, Rinat; Benjamin, John T; Im, Amanda M; van der Meer, Riet; Gleaves, Linda A; Bulus, Nada; Han, Wei; Prince, Lawrence S; Blackwell, Timothy S; Zent, Roy

    2014-12-01

    Integrin-dependent interactions between cells and extracellular matrix regulate lung development; however, specific roles for β1-containing integrins in individual cell types, including epithelial cells, remain incompletely understood. In this study, the functional importance of β1 integrin in lung epithelium during mouse lung development was investigated by deleting the integrin from E10.5 onwards using surfactant protein C promoter-driven Cre. These mutant mice appeared normal at birth but failed to gain weight appropriately and died by 4 months of age with severe hypoxemia. Defects in airway branching morphogenesis in association with impaired epithelial cell adhesion and migration, as well as alveolarization defects and persistent macrophage-mediated inflammation were identified. Using an inducible system to delete β1 integrin after completion of airway branching, we showed that alveolarization defects, characterized by disrupted secondary septation, abnormal alveolar epithelial cell differentiation, excessive collagen I and elastin deposition, and hypercellularity of the mesenchyme occurred independently of airway branching defects. By depleting macrophages using liposomal clodronate, we found that alveolarization defects were secondary to persistent alveolar inflammation. β1 integrin-deficient alveolar epithelial cells produced excessive monocyte chemoattractant protein 1 and reactive oxygen species, suggesting a direct role for β1 integrin in regulating alveolar homeostasis. Taken together, these studies define distinct functions of epithelial β1 integrin during both early and late lung development that affect airway branching morphogenesis, epithelial cell differentiation, alveolar septation and regulation of alveolar homeostasis. PMID:25395457

  4. Role of plasminogen activator inhibitor in the reciprocal regulation of bovine aortic endothelial and smooth muscle cell migration by TGF-beta 1.

    PubMed Central

    Petzelbauer, E.; Springhorn, J. P.; Tucker, A. M.; Madri, J. A.

    1996-01-01

    Vascular endothelial and smooth muscle cells exhibit reciprocal migratory responses after transforming growth factor (TGF)-beta 1 treatment. Endothelial cells exhibit a decreased migratory rate and smooth muscle cells exhibit an increased migratory rate. Previous studies have demonstrated increases in extracellular matrix and integrin synthesis and expression in response to TGF-beta 1. In this report, we illustrate the roles of plasminogen activator inhibitor in modulating the migratory rates in these two cell types. Endothelial cells appear to require a proteolytic phenotype for rapid migration, whereas vascular smooth muscle cells appear to require an anti-proteolytic phenotype. Modulation of proteinase/anti-proteinase activity ratios was accomplished via TGF-beta 1 induction, addition of exogenous plasminogen activator inhibitor, addition of anti-catalytic antibodies directed against urokinase plasminogen activator, overexpression of plasminogen activator inhibitor utilizing stable transfectants, and the use of vitronectin as a substratum. The reciprocal migratory behaviors exhibited by these two vascular cell types in response to TGF-beta 1 is discussed in the context that these two vascular cell types utilize distinct adhesive and signaling pathways in their interactions with extracellular matrix components and responsiveness to proteolytic activity. Images Figure 1 Figure 2 Figure 3 PMID:8780396

  5. The role of integrins in the development and homeostasis of the epidermis and skin appendages.

    PubMed

    Rippa, A L; Vorotelyak, E A; Vasiliev, A V; Terskikh, V V

    2013-10-01

    Integrins play a critical role in the regulation of adhesion, migration, proliferation, and differentiation of cells. Because of the variety of the functions they play in the cell, they are necessary for the formation and maintenance of tissue structure integrity. The trove of data accumulated by researchers suggests that integrins participate in the morphogenesis of the epidermis and its appendages. The development of mice with tissue-specific integrin genes knockout and determination of the genetic basis for a number of skin diseases in humans showed the significance of integrins in the biology, physiology, and morphogenesis of the epidermis and hair follicles. This review discusses the data on the role of different classes of integrin receptors in the biology of epidermal cells, as well as the development of the epidermis and hair follicles. PMID:24455180

  6. The Role of Integrins in the Development and Homeostasis of the Epidermis and Skin Appendages

    PubMed Central

    Rippa, A.L.; Vorotelyak, E.A.; Vasiliev, A.V.; Terskikh, V.V.

    2013-01-01

    Integrins play a critical role in the regulation of adhesion, migration, proliferation, and differentiation of cells. Because of the variety of the functions they play in the cell, they are necessary for the formation and maintenance of tissue structure integrity. The trove of data accumulated by researchers suggests that integrins participate in the morphogenesis of the epidermis and its appendages. The development of mice with tissue-specific integrin genes knockout and determination of the genetic basis for a number of skin diseases in humans showed the significance of integrins in the biology, physiology, and morphogenesis of the epidermis and hair follicles. This review discusses the data on the role of different classes of integrin receptors in the biology of epidermal cells, as well as the development of the epidermis and hair follicles. PMID:24455180

  7. Transforming growth factor-beta 1 (TGF-beta 1)- and beta 2-like activities in malignant pleural effusions caused by malignant mesothelioma or primary lung cancer.

    PubMed Central

    Maeda, J; Ueki, N; Ohkawa, T; Iwahashi, N; Nakano, T; Hada, T; Higashino, K

    1994-01-01

    We investigated the levels of TGF-beta in malignant pleural effusions (MPE) caused by malignant mesothelioma (MESO) or primary lung cancer. TGF-beta levels in MPE caused by MESO were 283.9 +/- 219.2 pm (mean +/- s.d.) and were three to six times higher than those due to primary lung cancers (P < 0.01 or P < 0.05). We also evaluated TGF-beta 1- and beta 2-like activities in MPE using specific polyclonal antibodies. Although TGF-beta 1-like activity could be detected in all cases, TGF-beta 2-like activities were detected in five of seven in MESO and in a few cases with primary lung cancer. These results demonstrate that the levels of total TGF-beta and TGF-beta 2-like activity may be clinically useful to differentiate MESO from primary lung cancer. Our data also suggest that TGF-beta may help further characterize the clinical features of MESO. PMID:7955539

  8. High-mobility group box 1 activates integrin-dependent homing of endothelial progenitor cells.

    PubMed

    Chavakis, Emmanouil; Hain, Andreas; Vinci, Maria; Carmona, Guillaume; Bianchi, Marco E; Vajkoczy, Peter; Zeiher, Andreas M; Chavakis, Triantafyllos; Dimmeler, Stefanie

    2007-02-01

    Endothelial progenitor cells (EPCs) are recruited to ischemic regions and improve neovascularization. Integrins contribute to EPC homing. High-mobility group box 1 (HMGB1) is a nuclear protein that is released extracellularly on cell necrosis and tissue damage, eliciting a proinflammatory response and stimulating tissue repair. In the present study, we investigated the effects of HMGB1 on EPC homing. EPCs express the HMGB1 receptors RAGE (receptor for advanced glycation end products) and TLR2 (Toll-like receptor 2). EPC migration was stimulated by HMGB1 in a RAGE-dependent manner. In addition, the HMGB1-induced migration of EPCs on fibronectin and fibrinogen was significantly inhibited by antibodies against beta1 and beta2 integrins, respectively. Short-term prestimulation of EPCs with HMGB1 also increased EPC adhesion to endothelial cell monolayers, and this effect was blocked by antibodies to beta2 integrins or RAGE. HMGB1 increased EPC adhesion to the immobilized integrin ligands intercellular adhesion molecule-1 and fibronectin in a RAGE-dependent manner. Strikingly, HMGB1 rapidly increased integrin affinity and induced integrin polarization. Using intravital microscopy in a tumor model of neovascularization, prestimulation of EPCs with HMGB1 enhanced the initial in vivo adhesion of EPCs to microvessels and the recruitment of EPCs in the tumor tissue. In addition, prestimulation of EPCs with HMGB1 increased the homing of EPCs to ischemic muscles. In conclusion, these data represent a link between HMGB1 and integrin functions of EPCs and demonstrate that HMGB1 stimulates EPC homing to ischemic tissues. These results may provide a platform for the development of novel therapeutic approaches to improve EPC homing. PMID:17218606

  9. Integrins, tensegrity, and mechanotransduction

    NASA Technical Reports Server (NTRS)

    Ingber, D. E.

    1997-01-01

    Physical forces, such as those due to gravity, play an important role in tissue development and remodeling. Yet, little is known about how individual cells sense mechanical signals or how they transduce them into a chemical response. Rather than listing the numerous signal pathways that have been found to be sensitive to mechanical stimulation, we need to place potential molecular signaling mechanisms within the context of the entire cell. The model presented is based on the concept that cells use tensegrity architecture to organize their cytoskeleton and stabilize their form. Studies with stick and string tensegrity cell models predict that living cells are hard-wired to respond immediately to external mechanical stresses. This hard-wiring exists in the form of discrete cytoskeletal filament networks that mechanically couple specific cell surface receptors, such as integrins, to nuclear matrix scaffolds and to potential transducing molecules that physically associate with the cytoskeleton. If these signaling molecules do function in a "solid-state", then mechanical stresses may be transduced into biochemical responses through force-dependent changes in cytoskeletal geometry or through local alterations in thermodynamic or kinetic parameters. Changes in cytoskeletal tension (prestress) also may play a role in signal amplification and adaptation. Recent experimental results are described which provide direct support for the tensegrity theory.

  10. The effect of transforming growth factor beta1 (TGF-beta1) on the regenerate bone in distraction osteogenesis.

    PubMed

    Ozkan, Korhan; Eralp, Levent; Kocaoglu, Mehmet; Ahishali, Bulent; Bilgic, Bilge; Mutlu, Zihni; Turker, Mehmet; Ozkan, Feyza Unlu; Sahin, Kemal; Guven, Melih

    2007-04-01

    Distraction osteogenesis is a well established clinical treatment for limb length discrepancy and skeletal deformities. Transforming growth factor beta 1 (TGF-beta1) is a multifunctional peptide which controls proliferation and expression of cells specific to bone like chondrocytes, osteoblasts, osteoclasts including mesenchymal precursor cells. To decrease the external fixation time with increasing the strength of regenerate (newly formed bone after distraction) we tested the effect of locally applied transforming growth factor beta 1 on distraction osteogenesis. A total of 28 mature female white New zealand rabbits weighing 3,5 kg-4,5 kg were studied. 10 animals were belonging to biomechanical testing group (5 for the study and 5 for the control subgroups), and the others were to histology group. In biomechanical group after tibial osteotomy TGF-beta1 was applied subperiosteally for 5 days just proximal to osteotomy site. Control group received only the solvent. Seven days after tibial osteotomy distraction was started at a rate of 0.25 mm/12 hours for 3 weeks with a unilateral fixator. Rabbits were sacrificed at the end of a consolidation period 8 week after tibial osteotomy. We assessed density of the elongation zone of rabbit tibial bones with the computed tomography. Then biomechanical parametres were assessed using the torsional testing using the material testing machine. In histology group rabbits were classified as control and study (rabbits that were given TGF-beta1). Rabbits were sacrificed at the end of first week, second week and fourth week also at the end of consolidation period 8 week after tibial osteotomy. Immunohistochemical and histologic parameters were examined. Biomechanical testing was applied as torsional testing. These values are used in determination of maximal loading, stiffness and energy absorbed during testing (brittleness). The histomorphometric examination looked for the differences between the study and control groups in terms of

  11. Shear stress activation of SREBP1 in endothelial cells is mediated by integrins.

    PubMed

    Liu, Yi; Chen, Benjamin P-C; Lu, Min; Zhu, Yi; Stemerman, Michael B; Chien, Shu; Shyy, John Y-J

    2002-01-01

    We investigated the effect of shear stress on the sterol regulatory element-binding protein 1 (SREBP1) in vascular endothelial cells (ECs) and the mechanotransduction mechanism involved. Application of a shear stress (12 dyn/cm(2)) caused the proteolytic cleavage of SREBP1 and the ensuing translocation of its transcription factor domain into the nucleus. As a result, shear stress increased the mRNAs encoding the low density lipoprotein receptor (LDLR), as well as the binding of (125)I-LDL. Using a step flow channel, we showed that SREBP1 activation in ECs under laminar flow is transient, but disturbed flow causes sustained activation. In studying the shear stress-elicited molecular signaling that activates SREBP1, we found that blocking the beta(1)-integrin with the AIIB2 blocking-type monoclonal antibody inhibited SREBP1 activation induced by shear stress. EC attachment to fibronectin or the activation of beta(1)-integrin in the suspended ECs by the TS2/16 monoclonal antibody was sufficient for SREBP1 activation. Furthermore, transient transfection assays showed that dominant-negative mutants of focal adhesion kinase and c-Src attenuated the shear stress-increased LDLR promoter activity. These results demonstrate that integrin signaling plays a critical role in the modulation of SREBP in ECs in response to shear stress. PMID:11788464

  12. Characterization of cultivar differences in beta-1,3 glucanase gene expression, glucanase activity and fruit pulp softening rates during fruit ripening in three naturally occurring banana cultivars.

    PubMed

    Roy Choudhury, Swarup; Roy, Sujit; Sengupta, Dibyendu N

    2009-11-01

    beta-1,3 glucanase (E.C.3.2.1.39) is the key enzyme involved in the hydrolytic cleavage of 1,3 beta-D glucosidic linkages in beta-1,3 glucans. This work describes a comparative analysis of expression patterns of beta-1,3 glucanase gene in relation to changes in fruit pulp softening rates in three banana cultivars, Rasthali (AAB), Kanthali (AB), and Monthan (ABB). Analysis of transcript and protein levels of beta-1,3 glucanase gene during ripening revealed differential timing in expression of the gene which correlated well with the variation in enzymatic activity of glucanase and fruit pulp softening rates in the three cultivars. Exogenously applied ethylene strongly induced beta-1,3 glucanase expression during the early ripening days in Rasthali, while the expression of the gene was marginally stimulated following ethylene treatment in preclimacteric Kanthali fruit. Conversely, in Monthan, beta-1,3 glucanase expression was very low throughout the ripening stages, and ethylene treatment did not induce the expression of the gene in this cultivar. Analysis of glucanase activity using protein extracts from unripe and ripe fruit of Monthan with crude cell wall polysaccharide fractions (used as substrate) indicated that the natural substrate for glucanase remained almost unutilized in this cultivar due to low in vivo glucanase activity. Furthermore, the recombinant beta-1,3 glucanase protein, overexpressed in E. coli, showed requirement for substrates with contiguous beta-1,3 linkages for optimal activity. Overall, our results provide new information on the expression profile of beta-1,3 glucanase gene in connection with the pattern of changes in fruit firmness at the physiological and molecular levels during ripening in three banana cultivars. PMID:19697038

  13. Regulation of integrin and growth factor signaling in biomaterials for osteodifferentiation

    PubMed Central

    Wei, Qiang; Pohl, Theresa L M; Seckinger, Anja

    2015-01-01

    Summary Stem cells respond to the microenvironment (niche) they are located in. Under natural conditions, the extracellular matrix (ECM) is the essential component the in stem cell niche, in which both integrin ligands and growth factors are important regulators to directly or indirectly modulate the cell behavior. In this review, we summarize the current knowledge about the potential of integrin ligands and growth factors to induce osteogenic differentiation of stem cells, and discuss the signaling pathways that are initiated by both individual and cooperative parameters. The joint effect of integrin ligands and growth factors is highlighted as the multivalent interactions for bone therapy. PMID:26124879

  14. Localization of integrin receptors for fibronectin, collagen, and laminin in human skin. Variable expression in basal and squamous cell carcinomas.

    PubMed Central

    Peltonen, J; Larjava, H; Jaakkola, S; Gralnick, H; Akiyama, S K; Yamada, S S; Yamada, K M; Uitto, J

    1989-01-01

    VLA integrins in human skin were examined by indirect immunofluorescence utilizing antibodies recognizing the beta 1, alpha 2, alpha 3, or alpha 5 subunits. Staining of fetal, newborn, or adult skin with antibodies to beta 1, alpha 2, or alpha 3 subunits gave essentially similar staining patterns: intense staining was associated with the basal layer of the epidermis, hair follicles, and blood vessel walls. The alpha 5 subunit could be detected only in epidermis and the inner root sheath of hair follicles in fetal skin. In epidermis, the staining reaction for the beta 1 subunit was not only found in sites interfacing with the basement membrane zone, but also around the entire periphery of these cells. We speculate that these receptors might have previously unrecognized functions in cell-cell interactions or that these findings may suggest the presence of previously unrecognized ligands in the intercellular spaces of keratinocytes. Examination of nine nodular basal cell carcinomas revealed a prominent staining reaction with anti-beta 1 and anti-alpha 3 antibodies at the periphery of the tumor islands. In contrast, staining of five squamous cell carcinomas revealed either the absence of integrins or altered and variable expression. Thus, matrix components and their receptors may participate in modulation of growth, development, and organization of human skin. Images PMID:2556449

  15. Hypergravity Stimulates Osteoblast Proliferation Via Matrix-Integrin-Signaling Pathways

    NASA Technical Reports Server (NTRS)

    Vercoutere, W.; Parra, M.; Roden, C.; DaCosta, M.; Wing, A.; Damsky, C.; Holton, E.; Searby, N.; Globus, R.; Almeida, E.

    2003-01-01

    Extensive characterizations of the physiologic consequences of microgravity and gravity indicate that lack of weight-bearing may cause tissue atrophy through cellular and subcellular level mechanisms. We hypothesize that gravity is needed for the efficient transduction of cell growth and survival signals from the extra-cellular matrix (ECM) in mechanosensitive tissues. Recent work from our laboratory and from others shows that an increase of gravity increases bone cell growth and survival. We found that 50-g hypergravity stimulation increased osteoblast proliferation for cells grown on Collagen Type I and Fibronectin, but not on Laminin or uncoated plastic. This may be a tissue-specific response, because 50-g hypergravity stimulation caused no increase in proliferation for primary rat fibroblasts. These results combined with RT-PCR for all possible integrins indicate that beta1 integrin subunit may be involved. The osteoblast proliferation response on Collagen Type I was greater at 25-g than at 10-g or 50-g; 24-h duration of hypergravity was necessary to see an increase in proliferation. Survival was enhanced during hypergravity stimulation by the presence of matrix. Flow cytometry analysis indicated that cell cycle may be altered; BrdU incorporation in proliferating cells showed an increase in the number of actively dividing cells from about 60% at 1-g to over 90% at 25-g. To further investigate the molecular components involved, we applied fluorescence labeling of cytoskeletal and signaling molecules to cells after 2 to 30 minutes of hypergravity stimulation. While structural components did not appear to be altered, phosphorylation increased, indicating that signaling pathways may be activated. These data indicate that gravity mechanostimulation of osteoblast proliferation involves specific matrix-integrin signaling pathways which are sensitive to duration and g-level.

  16. Transforming growth factor-beta 1 and fibroblast growth factors in rat growth plate.

    PubMed

    Jingushi, S; Scully, S P; Joyce, M E; Sugioka, Y; Bolander, M E

    1995-09-01

    Chondrocytes in the growth plate progress in an orderly fashion from resting through proliferating to hypertrophic cells. In the region of hypertrophic chondrocytes, the cartilage is invaded by capillary loops and endochondral ossification is initiated. It is currently believed that growth factors may regulate the proliferation and maturation of chondrocytes and the synthesis of extracellular matrix in the growth plate. The ordered sequence of proliferation and differentiation observed in the growth plate provides a unique opportunity to study the role of acidic fibroblast growth factor, basic fibroblast growth factor, and transforming growth factor-beta 1 in the regulation of these processes. In this study, expression of the mRNA of these growth factors was examined using total RNA extracted from the physis and epiphysis of rat tibias. Transforming growth factor-beta 1 mRNA was detected by Northern hybridization. Expression of the genes encoding acidic and basic fibroblast growth factors was demonstrated by polymerase chain reaction amplification. In addition, using polyclonal antibodies against these growth factors, we localized them by immunohistochemical analysis. Strong intracellular staining with a predominantly nuclear pattern was observed in chondrocytes from the proliferating and upper hypertrophic zones. In contrast, chondrocytes in the resting zone stained only faintly for the presence of these growth factors. Some chondrocytes in the resting zone adjacent to the proliferating zone stained with these antibodies, and the antibodies also stained cells in the zone of Ranvier, which regulates latitudinal bone growth. Lastly, the location of transforming growth factor-beta 1 was examined further with use of a polyclonal antipeptide antibody specific for its extracellular epitope.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7472755

  17. Expression of α5 integrin rescues fibronectin responsiveness in NT2N CNS neuronal cells

    PubMed Central

    Meland, Marit N.; Herndon, Mary E.; Stipp, Christopher S.

    2010-01-01

    The extracellular matrix protein fibronectin is implicated in neuronal regeneration in the peripheral nervous system. In the central nervous system (CNS), fibronectin is upregulated at sites of penetrating injuries and stroke; however, CNS neurons downregulate the fibronectin receptor, α5β1 integrin, during differentiation and generally respond poorly to fibronectin. NT2N CNS neuron-like cells (derived from NT2 precursor cells) have been used in pre-clinical and clinical studies for treatment of stroke and a variety of CNS injury and disease models. Here we show that, like primary CNS neurons, NT2N cells downregulate α5β1 integrin during differentiation and respond poorly to fibronectin. The poor neurite outgrowth by NT2N cells on fibronectin can be rescued by transducing NT2 precursors with a retroviral vector expressing α5 integrin under the control of the Murine Stem Cell Virus 5′ long terminal repeat. Sustained α5 integrin expression is compatible with the CNS-like neuronal differentiation of NT2N cells and does not prevent robust neurite outgrowth on other integrin ligands. Thus, α5 integrin expression in CNS neuronal precursor cells may provide a strategy for enhancing the outgrowth and survival of implanted cells in cell replacement therapies for CNS injury and disease. PMID:19598247

  18. Human-restricted bacterial pathogens block shedding of epithelial cells by stimulating integrin activation.

    PubMed

    Muenzner, Petra; Bachmann, Verena; Zimmermann, Wolfgang; Hentschel, Jochen; Hauck, Christof R

    2010-09-01

    Colonization of mucosal surfaces is the key initial step in most bacterial infections. One mechanism protecting the mucosa is the rapid shedding of epithelial cells, also termed exfoliation, but it is unclear how pathogens counteract this process. We found that carcinoembryonic antigen (CEA)-binding bacteria colonized the urogenital tract of CEA transgenic mice, but not of wild-type mice, by suppressing exfoliation of mucosal cells. CEA binding triggered de novo expression of the transforming growth factor receptor CD105, changing focal adhesion composition and activating beta1 integrins. This manipulation of integrin inside-out signaling promotes efficient mucosal colonization and represents a potential target to prevent or cure bacterial infections. PMID:20813953

  19. Beta 8 integrins mediate interactions of chick sensory neurons with laminin-1, collagen IV, and fibronectin.

    PubMed Central

    Venstrom, K; Reichardt, L

    1995-01-01

    Integrins are major receptors used by cells to interact with extracellular matrices. In this paper, we identify the first ligands for the beta 8 family of integrins, presenting evidence that integrin heterodimers containing the beta 8 subunit mediate interactions of chick sensory neurons with laminin-1, collagen IV, and fibronectin. A polyclonal antibody, anti-beta 8-Ex, was prepared to a bacterial fusion protein expressing an extracellular portion of the chicken beta 8 subunit. In nonreducing conditions, this antibody immunoprecipitated from surface-labeled embryonic dorsal root ganglia neurons a M(r) 100 k protein, the expected M(r) of the beta 8 subunit, and putative alpha subunit(s) of M(r) 120 k. Affinity-purified anti-beta 8-Ex strongly inhibited sensory neurite outgrowth on laminin-1, collagen IV, and fibronectin-coated substrata. Binding sites were identified in a heat-resistant domain in laminin-1 and in the carboxyl terminal, 40-kDa fibronectin fragment. On substrates coated with the carboxyl terminal fragment of fibronectin, antibodies to beta 1 and beta 8 were only partially effective alone, but were completely effective in combination, at inhibiting neurite outgrowth. Results thus indicate that the integrin beta 8 subunit in association with one or more alpha subunits forms an important set of extracellular matrix receptors on sensory neurons. Images PMID:7542940

  20. Emerging Putative Biomarkers: The Role of Alpha 2 and 6 Integrins in Susceptibility, Treatment, and Prognosis

    PubMed Central

    Marthick, James R.; Dickinson, Joanne L.

    2012-01-01

    The genetic architecture underpinning prostate cancer is complex, polygenic and despite recent significant advances many questions remain. Advances in genetic technologies have greatly improved our ability to identify genetic variants associated with complex disease including prostate cancer. Genome-wide association studies (GWASs) and microarray gene expression studies have identified genetic associations with prostate cancer susceptibility and tumour development. The integrins feature prominently in both studies examining the underlying genetic susceptibility and mechanisms driving prostate tumour development. Integrins are cell adhesion molecules involved in extracellular and intracellular signalling and are imperative for tumour development, migration, and angiogenesis. Although several integrins have been implicated in tumour development, the roles of integrin α2 and integrin α6 are the focus of this paper as evidence is now emerging that these integrins are implicit in prostate cancer susceptibility, cancer stem cell biology, angiogenesis, cell migration, and metastases to bone and represent potential biomarkers and therapeutic targets. There currently exists an urgent need to develop tools that differentiate indolent from aggressive prostate cancers and predict how patients will respond to treatment. This paper outlines the evidence supporting the use of α2 and α6 integrins in clinical applications for tailored patient treatment. PMID:22900191

  1. Arginine-Glycine-Aspartate-Binding Integrins as Therapeutic and Diagnostic Targets.

    PubMed

    Sun, Cui-Cui; Qu, Xian-Jun; Gao, Zu-Hua

    2016-01-01

    Arginine-glycine-aspartate (RGD)-binding integrins, including αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, α5β1, αIIbβ3, and α8β1, recognize the tripeptide motif RGD in their ligands. RGD-binding integrins are involved in various cell functions, including cell proliferation, survival, differentiation, and motility that are critically important to both health and disease. The diagnostic and therapeutic value of some RGD-binding integrin inhibitors are either clinically proven or at different stages of development. In this review, we first summarized the structure and signaling characteristics of RGD-binding integrins. We then discussed the functions of RGD-binding integrins and their association with human disease. Finally, we recapitulated the research efforts and clinical trials of targeting RGD-binding integrins for the diagnosis and treatment of human disease. This comprehensive review of the current advances in RGD-binding integrins could assist scientists and clinicians in gaining a complete understanding of this group of molecules. It can also contribute to the design of new projects to further advance this field of research and to better apply the research results to benefit patients in clinical practice. PMID:24621642

  2. Tumor Targeting via Integrin Ligands

    PubMed Central

    Marelli, Udaya Kiran; Rechenmacher, Florian; Sobahi, Tariq Rashad Ali; Mas-Moruno, Carlos; Kessler, Horst

    2013-01-01

    Selective and targeted delivery of drugs to tumors is a major challenge for an effective cancer therapy and also to overcome the side-effects associated with current treatments. Overexpression of various receptors on tumor cells is a characteristic structural and biochemical aspect of tumors and distinguishes them from physiologically normal cells. This abnormal feature is therefore suitable for selectively directing anticancer molecules to tumors by using ligands that can preferentially recognize such receptors. Several subtypes of integrin receptors that are crucial for cell adhesion, cell signaling, cell viability, and motility have been shown to have an upregulated expression on cancer cells. Thus, ligands that recognize specific integrin subtypes represent excellent candidates to be conjugated to drugs or drug carrier systems and be targeted to tumors. In this regard, integrins recognizing the RGD cell adhesive sequence have been extensively targeted for tumor-specific drug delivery. Here we review key recent examples on the presentation of RGD-based integrin ligands by means of distinct drug-delivery systems, and discuss the prospects of such therapies to specifically target tumor cells. PMID:24010121

  3. D-galactosyl-beta1-1'-sphingosine and D-glucosyl-beta1-1'-sphingosine induce human natural killer cell apoptosis.

    PubMed

    Maghazachi, Azzam A; Knudsen, Eirunn; Jin, Yixin; Jenstad, Monica; Chaudhry, Farrukh A

    2004-07-30

    Natural killer (NK) cells perform multiple biological functions including tumor cell lysis and eradicating virally infected cells. Here, we report for the first time that D-galactosyl-beta1-1' sphingosine and D-glucosyl-beta1- 1' sphingosine damage human NK cells. We show that these cells express T-cell-associated gene-8, the receptor for glycosphingolipids. D-galactosyl-beta1-1' sphingosine and D-glucosyl-beta1-1' sphingosine induce the in vitro chemotaxis of human NK cells. Both D-galactosyl-beta1-1' sphingosine and D-glucosyl-beta1-1' sphingosine inhibit the cytotoxicity and IFN-gamma secretion by these cells. Further analysis shows that the glycosphingolipids D-galactosyl-beta1-1' sphingosine and D-glucosyl-beta1-1' sphingosine but not any other lipid examined, which include D-lactosyl-beta1-1' sphingosine, sphingosine 1-phosphate, sphingosine, lysophosphatidic acid, and phosphatidic acid, induce the apoptosis, globoid-like formation, and multinucleation in human NK cells. These results may have important implications on diseases where glycosphingolipids accumulate. PMID:15240120

  4. Glomerular injury is exacerbated in diabetic integrin alpha1-null mice.

    PubMed

    Zent, R; Yan, X; Su, Y; Hudson, B G; Borza, D-B; Moeckel, G W; Qi, Z; Sado, Y; Breyer, M D; Voziyan, P; Pozzi, A

    2006-08-01

    Excessive glomerular collagen IV and reactive oxygen species (ROS) production are key factors in the development of diabetic nephropathy. Integrin alpha1beta1, the major collagen IV receptor, dowregulates collagen IV and ROS production, suggesting this integrin might determine the severity of diabetic nephropathy. To test this possibility, wild-type and integrin alpha1-null mice were rendered diabetic with streptozotocin (STZ) (100 mg/kg single intraperitoneal injection), after which glomerular filtration rate (GFR), glomerular collagen deposition, and glomerular basement membrane (GBM) thickening were evaluated. In addition, ROS and collagen IV production by mesangial cells as well as their proliferation was measured in vitro. Diabetic alpha1-null mice developed worse renal disease than diabetic wild-type mice. A significant increase in GFR was evident in the alpha1-null mice at 6 weeks after the STZ injection; it started to decrease by week 24 and reached levels of non-diabetic mice by week 36. In contrast, GFR only increased in wild-type mice at week 12 and its elevation persisted throughout the study. Diabetic mutant mice also showed increased glomerular deposition of collagen IV and GBM thickening compared to diabetic wild-type mice. Primary alpha1-null mesangial cells exposed to high glucose produced more ROS than wild-type cells, which led to decreased proliferation and increased collagen IV synthesis, thus mimicking the in vivo finding. In conclusion, this study suggests that lack of integrin alpha1beta1 exacerbates the glomerular injury in a mouse model of diabetes by modulating GFR, ROS production, cell proliferation, and collagen deposition. PMID:16775606

  5. Axonal Localization of Integrins in the CNS Is Neuronal Type and Age Dependent

    PubMed Central

    Soleman, Sara; Mason, Matthew R. J.; Verhaagen, Joost; Bensadoun, Jean-Charles; Aebischer, Patrick

    2016-01-01

    The regenerative ability of CNS axons decreases with age, however, this ability remains largely intact in PNS axons throughout adulthood. These differences are likely to correspond with age-related silencing of proteins necessary for axon growth and elongation. In previous studies, it has been shown that reintroduction of the α9 integrin subunit (tenascin-C receptor, α9) that is downregulated in adult CNS can improve neurite outgrowth and sensory axon regeneration after a dorsal rhizotomy or a dorsal column crush spinal cord lesion. In the current study, we demonstrate that virally expressed integrins (α9, α6, or β1 integrin) in the adult rat sensorimotor cortex and adult red nucleus are excluded from axons following neuronal transduction. Attempts to stimulate transport by inclusion of a cervical spinal injury and thus an upregulation of extracellular matrix molecules at the lesion site, or cotransduction with its binding partner, β1 integrin, did not induce integrin localization within axons. In contrast, virally expressed α9 integrin in developing rat cortex (postnatal day 5 or 10) demonstrated clear localization of integrins in cortical axons revealed by the presence of integrin in the axons of the corpus callosum and internal capsule, as well as in the neuronal cell body. Furthermore, examination of dorsal root ganglia neurons and retinal ganglion cells demonstrated integrin localization both within peripheral nerve as well as dorsal root axons and within optic nerve axons, respectively. Together, our results suggest a differential ability for in vivo axonal transport of transmembrane proteins dependent on neuronal age and subtype.

  6. A NPxY-independent {beta}5 integrin activation signal regulates phagocytosis of apoptotic cells

    SciTech Connect

    Singh, Sukhwinder; D'mello, Veera; Henegouwen, Paul van Bergen en; Birge, Raymond B.

    2007-12-21

    Integrin receptors are heterodimeric transmembrane receptors with critical functions in cell adhesion and migration, cell cycle progression, differentiation, apoptosis, and phagocytosis of apoptotic cells. Integrins are activated by intracellular signaling that alter the binding affinity for extracellular ligands, so-called inside to outside signaling. A common element for integrin activation involves binding of the cytoskeletal protein talin, via its FERM domain, to a highly conserved NPxY motif in the {beta} chain cytoplasmic tails, which is involved in long-range conformation changes to the extracellular domain that impinges on ligand affinity. When the human beta-5 ({beta}5) integrin cDNA was expressed in {alpha}v positive, {beta}5 and {beta}3 negative hamster CS-1 cells, it promoted NPxY-dependent adhesion to VTN-coated surfaces, phosphorylation of FAK, and concomitantly, {beta}5 integrin-EGFP protein was recruited into talin and paxillin-containing focal adhesions. Expression of a NPxY destabilizing {beta}5 mutant (Y750A) abrogated adhesion and {beta}5-Y750A-EGFP was excluded from focal adhesions at the tips of stress fibers. Surprisingly, expression of {beta}5 Y750A integrin had a potent gain-of-function effect on apoptotic cell phagocytosis, and further, a {beta}5-Y750A-EGFP fusion integrin readily bound MFG-E8-coated 10 {mu}m diameter microspheres developed as apoptotic cell mimetics. The critical sequences in {beta}5 integrin were mapped to a YEMAS motif just proximal to the NPxY motif. Our studies suggest that the phagocytic function of {beta}5 integrin is regulated by an unconventional NPxY-talin-independent activation signal and argue for the existence of molecular switches in the {beta}5 cytoplasmic tail for adhesion and phagocytosis.

  7. Axonal Localization of Integrins in the CNS Is Neuronal Type and Age Dependent.

    PubMed

    Andrews, Melissa R; Soleman, Sara; Cheah, Menghon; Tumbarello, David A; Mason, Matthew R J; Moloney, Elizabeth; Verhaagen, Joost; Bensadoun, Jean-Charles; Schneider, Bernard; Aebischer, Patrick; Fawcett, James W

    2016-01-01

    The regenerative ability of CNS axons decreases with age, however, this ability remains largely intact in PNS axons throughout adulthood. These differences are likely to correspond with age-related silencing of proteins necessary for axon growth and elongation. In previous studies, it has been shown that reintroduction of the α9 integrin subunit (tenascin-C receptor, α9) that is downregulated in adult CNS can improve neurite outgrowth and sensory axon regeneration after a dorsal rhizotomy or a dorsal column crush spinal cord lesion. In the current study, we demonstrate that virally expressed integrins (α9, α6, or β1 integrin) in the adult rat sensorimotor cortex and adult red nucleus are excluded from axons following neuronal transduction. Attempts to stimulate transport by inclusion of a cervical spinal injury and thus an upregulation of extracellular matrix molecules at the lesion site, or cotransduction with its binding partner, β1 integrin, did not induce integrin localization within axons. In contrast, virally expressed α9 integrin in developing rat cortex (postnatal day 5 or 10) demonstrated clear localization of integrins in cortical axons revealed by the presence of integrin in the axons of the corpus callosum and internal capsule, as well as in the neuronal cell body. Furthermore, examination of dorsal root ganglia neurons and retinal ganglion cells demonstrated integrin localization both within peripheral nerve as well as dorsal root axons and within optic nerve axons, respectively. Together, our results suggest a differential ability for in vivo axonal transport of transmembrane proteins dependent on neuronal age and subtype. PMID:27570822

  8. α6-integrin is required for the adhesion and vasculogenic potential of hemangioma stem cells

    PubMed Central

    Smadja, David M.; Guerin, Coralie L.; Boscolo, Elisa; Bieche, Ivan; Mulliken, John B.; Bischoff, Joyce

    2013-01-01

    Background Infantile hemangioma (IH) is the most common tumor of infancy. Hemangioma stem cells (HemSC) are a mesenchymal subpopulation isolated from IH CD133+ cells. HemSC can differentiate into endothelial and pericyte/smooth muscle cells and form vascular networks when injected in immune-deficient mice. α6-Integrin subunit has been implicated in the tumorgenicity of glioblastoma stem cells and the homing properties of hematopoietic, endothelial and mesenchymal progenitor cells. Therefore, we investigated the possible function(s) of α6-integrin in HemSC. Methods/Results We documented α6-integrin expression in IH tumor specimens and HemSC by RT-qPCR and flow cytometry. We examined the effect of blocking or silencing α6-integrin on the adhesive and proliferative properties of HemSCin vitro and the vasculogenic and homing properties of HemSCin vivo. Targeting α6-integrin in cultured HemSC inhibited adhesion to laminin but had no effect on proliferation. Vessel-forming ability in Matrigel implants and hepatic homing after intravenous delivery were significantly decreased in α6-integrin siRNA transfected HemSC. Conclusion α6-Integrin is required for HemSC adherence to laminin, vessel formation in vivo and for homing to the liver. Thus, we uncovered an important role for α6 integrin in the vasculogenic properties of HemSC. Our results suggest that α6-integrin expression on HemSC could be a new target for anti-hemangioma therapy. PMID:24022922

  9. Integrins and epithelial cell polarity

    PubMed Central

    Lee, Jessica L.; Streuli, Charles H.

    2014-01-01

    ABSTRACT Cell polarity is characterised by differences in structure, composition and function between at least two poles of a cell. In epithelial cells, these spatial differences allow for the formation of defined apical and basal membranes. It has been increasingly recognised that cell–matrix interactions and integrins play an essential role in creating epithelial cell polarity, although key gaps in our knowledge remain. This Commentary will discuss the mounting evidence for the role of integrins in polarising epithelial cells. We build a model in which both inside-out signals to polarise basement membrane assembly at the basal surface, and outside-in signals to control microtubule apical–basal orientation and vesicular trafficking are required for establishing and maintaining the orientation of epithelial cell polarity. Finally, we discuss the relevance of the basal integrin polarity axis to cancer. This article is part of a Minifocus on Establishing polarity. For further reading, please see related articles: ‘ERM proteins at a glance’ by Andrea McClatchey (J. Cell Sci. 127, 3199–3204). ‘Establishment of epithelial polarity – GEF who's minding the GAP?’ by Siu Ngok et al. (J. Cell Sci. 127, 3205–3215). PMID:24994933

  10. Tissue localization of transforming growth factor-beta1 in pulmonary eosinophilic granuloma.

    PubMed

    Asakura, S; Colby, T V; Limper, A H

    1996-11-01

    Pulmonary eosinophilic granuloma is characterized by infiltration of the lungs with fibronodular lesions containing specialized Langerhans' cells. In some patients, progressive pulmonary fibrosis leads to significant respiratory impairment. Transforming growth factor-beta1 (TGF-beta1) promotes fibrosis by enhancing the synthesis of extracellular matrix components. The role of TGF-beta1 in promoting fibrosis in the setting of pulmonary eosinophilic granuloma is currently unknown. We used immunohistochemistry to evaluate the extent and distribution of TGF-beta1 and the extracellular matrix components type I collagen and decorin, a TGF-beta1-binding proteoglycan. Lung biopsies from 11 patients with pulmonary eosinophilic granuloma were evaluated. In biopsies with active inflammatory lesions containing Langerhans' cells, hyperplastic type 2 pneumocytes and alveolar macrophages within and surrounding the fibronodular lesions contained abundant TGF-beta1. Langerhans' cells were consistently devoid of immunoreactive TGF-beta1. Active inflammatory lesions also exhibited staining for decorin, in a loosely organized distribution. Advanced fibrotic lesions of eosinophilic granuloma, containing minimal inflammatory cells and few or no Langerhans' cells, exhibited weak or absent staining for TGF-beta1 within either hyperplastic type 2 pneumocytes or alveolar macrophages. The fibroconnective tissues of these advanced fibrotic lesions consistently revealed dense staining for decorin. Through their actions on extracellular matrix protein accumulation, TGF-beta1 and the TGF-beta1-binding proteoglycan decorin may modulate fibrotic repair accompanying pulmonary eosinophilic granuloma. PMID:8912775

  11. Glioma cell integrin expression and their interactions with integrin antagonists

    PubMed Central

    Mattern, Ralph-Heiko; Read, Susana B.; Pierschbacher, Michael D.; Sze, Chun-I; Eliceiri, Brian P.; Kruse, Carol A.

    2005-01-01

    Summary A panel of human glioma cell explants was screened for integrin expression by flow cytometry using ανβ-specific antibodies. A lower percentage of the glioma cells were positive for the ανβ3 (mean % positive = 20.8%) integrin, whereas higher percentages were positive for the ανβ5 (mean % positive = 72.7%), VLA5α (mean % positive = 87%) and VLAβ1 (mean % positive = 41.7%) integrins. A series of RGD peptides was designed, synthesized and tested for binding to integrin receptors. Based on the results of the binding to the isolated integrin receptors and the expression of integrins on glioma cell lines, a peptide that binds potently to the ανβ3, ανβ5 and α5β1 was selected for further investigations with regards to its effect on glioma cells. The peptide, Ac-c[(Pen)-Tyr(Me)-Ala-Arg-Gly-Asp-Asn-Tic-Cys]NH2 (RGD peptide), exhibited high potential for use in clinical intracranial administration since it had good stability in rat brain cell homogenates placed into artificial cerebrospinal fluid. Using an HPLC method for quantification of peptides in rat brain cell homogenates, we could demonstrate the half-life of the RGD peptide approximated 20 hr. Relative to a scrambled peptide control (non-RGD sequence, same amino acids), the experimental RGD peptide significantly decreased glioma cell proliferation of the entire panel of rat and human glioma cells tested. Adhesion of recently passaged glioma cells to glioma-derived extracellular matrix protein-coated plates was inhibited significantly by the RGD peptide. The peptide also reversed attachment of plated glioma cells. The RGD peptide caused some, but not substantial, glioma cell injury, as evidenced by a quantitative in vitro nuclear DNA morphologic assay and by a flow cytometric assay employing 7-amino actinomycin D (7AAD). We histologically monitored for toxicity caused by various doses of the RGD peptide infused repeatedly into normal cannulated rat brain. At safe doses, the experimental RGD

  12. Laminin-5 and modulation of keratin cytoskeleton arrangement in FG pancreatic carcinoma cells: involvement of IFAP300 and evidence that laminin-5/cell interactions correlate with a dephosphorylation of alpha 6A integrin.

    PubMed

    Baker, S E; Skalli, O; Goldman, R D; Jones, J C

    1997-01-01

    Under normal culture conditions, epithelial cells of the FG line, derived from a pancreatic tumor, characteristically grow in mounds and fail to flatten efficiently onto their substrate. In such cells, keratin intermediate filaments (IFs) are concentrated in the perinuclear region. Furthermore, the IF associated protein, IFAP300, primarily localizes along these keratin bundles. Additionally, alpha 6 beta 4 integrin heterodimers localize in streaks or spots towards the edges of cells while alpha 3 beta 1 integrin is predominantly at cell-cell surfaces. Neither show any obvious interaction with IF. Remarkably, upon plating FG cells into medium containing soluble rat laminin-5, FG cells rapidly adhere and spread onto their substrate. Moreover, FG cells "capture" rat laminin-5 and place it basally in circles or arcs at areas of cell-substrate interaction. Double label immunofluorescence microscopy reveals colocalization of IFAP300 as well as alpha 6 beta 4 and alpha 3 beta 1 integrin with the polarized laminin-5. Concomitantly, alpha 6 integrin undergoes dephosphorylation on serine residue 1041. Laminin-5-induced rapid adhesion can be blocked by antibodies against the alpha 3 integrin subunit. In contrast, while alpha 6 integrin antibodies do not block laminin-5-induced rapid adhesion, they prevent FG cells from assuming an epithelial-like morphology. Keratin IF bundles associate with IFAP300-alpha 6 beta 4/alpha 3 beta 1 integrin complexes along the cell-substratum-attached surface of FG cells coincubated in laminin-5-containing medium. Coprecipitation results suggest that in these complexes, IFAP300 may associate with the alpha 6 beta 4 integrin heterodimer. Based on our results and published evidence that IFAP300 binds keratin in vitro [Skalli et al., 1994; J. Cell Biol. 125:159-170], we propose that laminin-5/FG cell interaction results in a novel integrin dephosphorylation event, which subsequently induces IFAP300 association with alpha 6 beta 4 integrin. IFAP300

  13. Rat beta 1-adrenergic receptor regulatory region containing consensus AP-2 elements recognizes novel transactivator proteins.

    PubMed

    Kirigiti, P; Yang, Y F; Li, X; Li, B; Midson, C N; Machida, C A

    2000-03-01

    beta 1-Adrenergic receptors (beta1-ARs) serve as important regulators of central nervous system (CNS)-mediated behavior and several neural functions, including mood, memory, neuroendocrine control, and stimulation of autonomic function. Using beta 1-AR-luciferase reporter recombinants, we have previously determined that important beta 1-AR genetic elements controlling expression within the C6 glioma cell line are contained within the region -396 to -299, relative to the translational start site. By conducting progressive internal deletions of the rat beta 1-AR 5' flanking region and with the use of beta 1-AR-luciferase recombinants, we have verified that this region contains the primary beta 1-AR promoter and/or major regulatory elements. To begin the identification of protein factors involved in beta 1-AR transcriptional activity conferred by this beta 1-AR region and flanking sequences, we conducted electrophoretic mobility shift assays using defined beta 1-AR DNA subregion probes. One probe (GS-1), encompassing the region -396 to -367, was found to produce two major and two minor mobility shift complexes when bound to nuclear extracts from the beta 1-AR expresser C6 cell line. UV-crosslinking of DNA-protein complexes, coupled with DNase I digestion, indicated that this beta 1-AR region interacts with one major protein of approximately 117 kDa molecular weight and additional minor proteins. GS-1 DNA-protein complexes were observed using beta 1-AR expresser tissues in the CNS, including cortex, hippocampus, and olfactory bulb. No DNA-protein complexes were observed when using nuclear extracts from beta 1-AR nonexpresser tissues; in some cases, using L6 cells, previously characterized to express little or no beta1-ARs, a reduction in intensities of the DNA-protein complexes was observed. Competition experiments indicate that nuclear protein binds to one of two subregions within the GS-1 sequence that contain AP-2-like consensus elements. Recombinant AP-2 protein

  14. Butyrate downregulates α2β1 integrin: a possible role in the induction of apoptosis in colorectal cancer cell lines

    PubMed Central

    Buda, A; Qualtrough, D; Jepson, M A; Martines, D; Paraskeva, C; Pignatelli, M

    2003-01-01

    Background: Integrins mediate cell matrix adhesion and regulate cell growth and survival. In colonic epithelial cells, α2β1 integrin controls glandular differentiation and proliferation. Butyrate stimulates differentiation and induces apoptosis in vitro. Aims: We investigated whether butyrate induction of apoptosis was associated with perturbation of integrin mediated cell matrix adhesion. Methods: Three colonic cancer cell lines (SW1222, SW620, LS174T) were studied. Adhesion to extracellular matrix proteins, expression of α2β1 integrin, and apoptosis were studied in adherent cells after treatment with 4 mM butyrate. Results: Butyrate decreased the attachment to type I collagen in SW620 cells and type I and IV collagen in LS174T cells. The decreased cell attachment was associated with downregulation of α2β1 integrin and increased apoptosis in adherent cells. No changes in α2β1 expression or matrix adhesion were seen in SW1222 cells, which were also found to be less sensitive to butyrate induction of apoptosis. Downregulation of α2β1 integrin preceded the detection of apoptosis. Conclusion: Apoptosis induced by butyrate is associated with downregulation of expression and functional activity of α2β1 integrin. Perturbation of cell matrix adhesion may be a novel mechanism by which butyrate induces apoptosis in colorectal cancer cells. PMID:12692060

  15. In situ validation of VEGFR-2 and α v ß 3 integrin as targets for breast lesion characterization.

    PubMed

    Ehling, Josef; Misiewicz, Matthias; von Stillfried, Saskia; Möckel, Diana; Bzyl, Jessica; Pochon, Sibylle; Lederle, Wiltrud; Knuechel, Ruth; Lammers, Twan; Palmowski, Moritz; Kiessling, Fabian

    2016-04-01

    Vascular endothelial growth factor receptor 2 (VEGFR-2) and α v ß 3 integrin are the most frequently addressed targets in molecular imaging of tumor angiogenesis. In preclinical studies, molecular imaging of angiogenesis has shown potential to detect and differentiate benign and malignant lesions of the breast. Thus, in this retrospective clinical study employing patient tissues, the diagnostic value of VEGFR-2, α v ß 3 integrin and vascular area fraction for the diagnosis and differentiation of breast neoplasia was evaluated. To this end, tissue sections of breast cancer (n = 40), pre-invasive ductal carcinoma in situ (DCIS; n = 8), fibroadenoma (n = 40), radial scar (n = 6) and normal breast tissue (n = 40) were used to quantify (1) endothelial VEGFR-2, (2) endothelial α v ß 3 integrin and (3) total α v ß 3 integrin expression, as well as (4) the vascular area fraction. Sensitivity and specificity to differentiate benign from malignant lesions were calculated for each marker by receiver operating characteristics (ROC) analyses. Whereas vessel density, as commonly used, did not significantly differ between benign and malignant lesions (AUROC: 0.54), VEGFR-2 and α v ß 3 integrin levels were gradually up-regulated in carcinoma versus fibroadenoma versus healthy tissue. The highest diagnostic accuracy for differentiating carcinoma from fibroadenoma was found for total α v ß 3 integrin expression (AUROC: 0.76), followed by VEGFR-2 (AUROC: 0.71) and endothelial α v ß 3 integrin expression (AUROC: 0.68). In conclusion, total α v ß 3 integrin expression is the best discriminator between breast cancer, fibroadenoma and normal breast tissue. With respect to vascular targeting and molecular imaging of angiogenesis, endothelial VEGFR-2 appeared to be slightly superior to endothelial α v ß 3 for differentiating benign from cancerous lesions. PMID:26902100

  16. Modulation of the skeletal muscle sodium channel alpha-subunit by the beta 1-subunit.

    PubMed

    Wallner, M; Weigl, L; Meera, P; Lotan, I

    1993-12-28

    Co-expression of cloned sodium channel beta 1-subunit with the rat skeletal muscle-subunit (alpha microI) accelerated the macroscopic current decay, enhanced the current amplitude, shifted the steady state inactivation curve to more negative potentials and decreased the time required for complete recovery from inactivation. Sodium channels expressed from skeletal muscle mRNA showed a similar behaviour to that observed from alpha microI/beta 1, indicating that beta 1 restores 'physiological' behaviour. Northern blot analysis revealed that the Na+ channel beta 1-subunit is present in high abundance (about 0.1%) in rat heart, brain and skeletal muscle, and the hybridization with untranslated region of the 'brain' beta 1 cDNA to skeletal muscle and heart mRNA indicated that the different Na+ channel alpha-subunits in brain, skeletal muscle and heart may share a common beta 1-subunit. PMID:8282123

  17. Localization of insulin-like growth factor (IGFBP)-3 in cultured porcine embryonic myogenic cells before and after TGF-beta1 treatment.

    PubMed

    Xi, G; Hathaway, M R; White, M E; Dayton, W R

    2007-11-01

    Insulin-like growth factor binding protein (IGFBP)-3 binds IGFs with high affinity and affects their biological activity. IGFBP-3 that is not bound to IGF also affects cells via mechanisms involving binding to specific cell surface receptors and/or transport into the cell. IGFBP-3 is produced by porcine embryonic myogenic cell (PEMC) cultures. Additionally, IGFBP-3 facilitates the proliferation-suppressing actions of TGF-beta(1) and myostatin in PEMC cultures via mechanisms that do not involve IGF binding. Moreover, these mechanisms do not involve preventing myostatin or TGF-beta(1)-induced increases in phosphosmad2 or phosphosmad3 level. Consequently, the mechanism(s) by which IGFBP-3 facilitates the proliferation-suppressing actions of TGF-beta(1) and myostatin in PEMC is unclear. Since IGFBP-3 reportedly interacts with nuclear proteins that regulate transcription, TGF-beta(1) or myostatin-induced translocation of IGFBP-3 into the nucleus may facilitate the proliferation-suppressing actions of these cytokines. Here, we show that IGFBP-3 is localized in cells containing the muscle specific protein desmin, thus establishing the presence of this IGFBP in myogenic cells. IGFBP-3 is present in the cytoplasm of all myogenic cells and approximately 50% of the nuclei of proliferating PEMC. IGFBP-3 is also detectable in fused myotubes. IGFBP-3 suppresses IGF-I-stimulated differentiation of PEMC but has no affect on Long-R3-IGF-I-stimulated differentiation of PEMC. Treatment of PEMC for 24h with TGF-beta(1) (20 ng/ml) results in a 78% (p<0.01) increase in the number of nuclei that contain detectable IGFBP-3. These results suggest that translocation of IGFBP-3 into the nucleus of PEMC could play a role in mediating the proliferation-suppressing action of TGF-beta(1). PMID:17049199

  18. Lactate adversely affects the in vitro formation of endothelial cell tubular structures through the action of TGF-{beta}1

    SciTech Connect

    Schmid, Stephan A. . E-mail: leoni.kunz-schughart@oncoray.de; Gaumann, Andreas; Wondrak, Marit; Eckermann, Christoph; Schulte, Stephanie; Mueller-Klieser, Wolfgang; Wheatley, Denys N.; Kunz-Schughart, Leoni A.

    2007-07-15

    When lactate accumulation in a tumor microenvironment reaches an average concentration of 10-20 mM, it tends to reflect a high degree of malignancy. However, the hypothesis that tumor-derived lactate has a number of partially adverse biological effects on malignant and tumor-associated host cells requires further evidence. The present study attempted to evaluate the impact of lactate on the process of angiogenesis, in particular on the formation of tubular structures. The endothelial cell (EC) network in desmoplastic breast tumors is primarily located in areas of reactive fibroblastic stroma. We employed a fibroblast-endothelial cell co-culture model as in vitro angiogenesis system normally producing florid in vitro tubule formation to analyze this situation. In contrast to previous studies, we found that lactate significantly reduces EC network formation in a dose-dependent manner as quantified by semi-automated morphometric analyses following immunohistochemical staining. The decrease in CD31-positive tubular structures and the number of intersections was independent of VEGF supplementation and became more pronounced in the presence of protons. The number of cells, primarily of the fibroblast population, was reduced but cell loss could not be attributed to a decrease in proliferative activity or pronounced apoptotic cell death. Treatment with 10 mM lactate was accompanied by enhanced mRNA expression and release of TGF-{beta}1, which also shows anti-angiogenic activity in the model. Both TGF-{beta}1 and lactate induced myofibroblastic differentiation adjacent to the EC tubular structures. The lactate response on the EC network was diminished by TGF-{beta}1 neutralization, indicating a causal relationship between lactate and TGF-{beta}1 in the finely tuned processes of vessel formation and maturation which may also occur in vivo within tumor tissue.

  19. Hypertrophic stimuli induce transforming growth factor-beta 1 expression in rat ventricular myocytes.

    PubMed Central

    Takahashi, N; Calderone, A; Izzo, N J; Mäki, T M; Marsh, J D; Colucci, W S

    1994-01-01

    Transforming growth factor-beta 1 (TGF-beta 1) is a peptide growth factor that may play a role in the myocardial response to hypertrophic stimuli. However, the cellular distribution, mechanism of induction, and source of increased TGF-beta 1 in response to hypertrophic stimuli are not known. We tested the hypothesis that the cardiac myocyte responds to hypertrophic stimuli with the increased expression of TGF-beta 1. In adult rat ventricular myocardium freshly dissociated into myocyte and nonmyocyte cellular fractions, the preponderance of TGF-beta 1 mRNA visualized by Northern hybridization was in the nonmyocyte fraction. Abdominal aortic constriction (7 d) and subcutaneous norepinephrine infusion (36 h) each caused ventricular hypertrophy associated with 3.1-fold and 3.8-fold increases, respectively, in TGF-beta 1 mRNA in the myocyte fraction, but had no effect on the level of TGF-beta 1 mRNA in the nonmyocyte fraction. In ventricular myocytes, norepinephrine likewise caused a 4.1-fold increase in TGF-beta 1 mRNA associated with an increase in TGF-beta bioactivity. This induction of TGF-beta 1 mRNA occurred at norepinephrine concentrations as low as 1 nM and was blocked by prazosin, but not propranolol. NE did not increase the TGF-beta 1 mRNA level in nonmyocytes, primarily fibroblasts, cultured from neonatal rat ventricle. Thus, the cardiac myocyte responds to two hypertrophic stimuli, pressure overload and norepinephrine, with the induction of TGF-beta 1. These data support the view that TGF-beta 1, released by myocytes and acting in an autocrine and/or paracrine manner, is involved in myocardial remodeling by hypertrophic stimuli. Images PMID:7929822

  20. Specific binding of endocrine transforming growth factor-beta 1 to vascular endothelium.

    PubMed Central

    Dickson, K; Philip, A; Warshawsky, H; O'Connor-McCourt, M; Bergeron, J J

    1995-01-01

    The presentation of recombinant biologically active 125I-TGF-beta 1 via the bloodstream to potential target cells in mice and rats was evaluated by quantitative light and electron microscope radioautography. Specificity was evaluated by in vivo competition with excess unlabeled TGF-beta 1, and integrity of the ligand at the binding site was demonstrated by trichloroacetic acid precipitation after extraction from tissues. The distribution of radiolabel at 2.5, 15, 30, 45, and 60 min after 125I-TGF-beta 1 injection revealed radiolabel principally over microvasculature endothelium but at times > 2.5 min over endothelial endocytic components indicative of internalization. Nonspecific binding of 125I-TGF-beta 1 to the apex of the proximal convoluted tubule of the kidney indicated it as the likely site of rapid clearance of TGF-beta 1 from the circulation, while a comparison of the binding of 125I-TGF-beta 1 (endothelial) to that of 125I-TGF-beta 1 complexed with alpha 2-macroglobulin-methylamine (liver parenchyma) indicated that clearance of TGF-beta 1 complexed alpha 2-macroglobulin was likely via the hepatic alpha 2-macroglobulin receptor. The endothelial TGF-beta receptors uncovered here are likely involved in the local regulatory mechanism of leukocyte and monocyte adhesion and tissue infiltration regulated by endocrine TGF-beta 1. Images PMID:7539454

  1. Beta 1,4-oligoglucosides inhibit the binding of Aspergillus fumigatus conidia to human monocytes.

    PubMed

    Kan, V L; Bennett, J E

    1991-05-01

    The binding of Aspergillus fumigatus conidia to human monocytes is mediated by a barley beta-glucan-inhibitable receptor. The simplest linkages in this glucan are present in the disaccharides laminaribiose (beta 1,3) and cellobiose (beta 1,4). Although laminaribiose gave strong inhibition of conidial binding to monocytes, cellobiose and oligosaccharides with beta 1,4-linked glucose residues were more potent as specific inhibitors of this binding over similar concentrations. Increasing the number of beta 1,4-linked glucose residues led to greater inhibition of conidial binding by human monocytes. PMID:2019764

  2. The action modes of an extracellular beta-1,3-glucanase isolated from Bacillus clausii NM-1 on beta-1,3-glucooligosaccharides.

    PubMed

    Miyanishi, Nobumitsu; Matsubara, Yasuhito; Hamada, Naoko; Kobayashi, Takeshi; Imada, Chiaki; Watanabe, Etsuo

    2003-01-01

    The mode of action of an extracellular -1,3-glucanase from Bacillus clausii NM-1 on beta-1,3-3glucooligosaccharides and their alditols was studied. The enzyme could not hydrolyze laminaribiose or laminaritriose. beta-1,3-Glucooligosaccharides higher than laminarihexaose were rapidly hydrolyzed, while laminaritetraose was slowly hydrolyzed. The k(cat)/K(m) ratios for a series of beta-1,3-glucooligosaccharides from laminaritetraose to laminariheptaose showed that the substrate binding site of the enzyme covered a wide range of beta-1,3-glucooligosaccharides having six glucose residues. The action pattern of the enzyme on the alditols corresponding to each laminarioligosaccharide suggested that the catalytic site of the enzyme existed between the third and fourth glucose residue from the non-reducing terminal. The value of k(cat)/K(m) also suggested that the sixth binding position contributed to the catalytic efficiency and stability. PMID:16233479

  3. Extracellular matrix stiffness dictates Wnt expression through integrin pathway

    PubMed Central

    Du, Jing; Zu, Yan; Li, Jing; Du, Shuyuan; Xu, Yipu; Zhang, Lang; Jiang, Li; Wang, Zhao; Chien, Shu; Yang, Chun

    2016-01-01

    It is well established that extracellular matrix (ECM) stiffness plays a significant role in regulating the phenotypes and behaviors of many cell types. However, the mechanism underlying the sensing of mechanical cues and subsequent elasticity-triggered pathways remains largely unknown. We observed that stiff ECM significantly enhanced the expression level of several members of the Wnt/β-catenin pathway in both bone marrow mesenchymal stem cells and primary chondrocytes. The activation of β-catenin by stiff ECM is not dependent on Wnt signals but is elevated by the activation of integrin/ focal adhesion kinase (FAK) pathway. The accumulated β-catenin then bound to the wnt1 promoter region to up-regulate the gene transcription, thus constituting a positive feedback of the Wnt/β-catenin pathway. With the amplifying effect of positive feedback, this integrin-activated β-catenin/Wnt pathway plays significant roles in mediating the enhancement of Wnt signal on stiff ECM and contributes to the regulation of mesenchymal stem cell differentiation and primary chondrocyte phenotype maintenance. The present integrin-regulated Wnt1 expression and signaling contributes to the understanding of the molecular mechanisms underlying the regulation of cell behaviors by ECM elasticity. PMID:26854061

  4. Extracellular matrix stiffness dictates Wnt expression through integrin pathway.

    PubMed

    Du, Jing; Zu, Yan; Li, Jing; Du, Shuyuan; Xu, Yipu; Zhang, Lang; Jiang, Li; Wang, Zhao; Chien, Shu; Yang, Chun

    2016-01-01

    It is well established that extracellular matrix (ECM) stiffness plays a significant role in regulating the phenotypes and behaviors of many cell types. However, the mechanism underlying the sensing of mechanical cues and subsequent elasticity-triggered pathways remains largely unknown. We observed that stiff ECM significantly enhanced the expression level of several members of the Wnt/β-catenin pathway in both bone marrow mesenchymal stem cells and primary chondrocytes. The activation of β-catenin by stiff ECM is not dependent on Wnt signals but is elevated by the activation of integrin/ focal adhesion kinase (FAK) pathway. The accumulated β-catenin then bound to the wnt1 promoter region to up-regulate the gene transcription, thus constituting a positive feedback of the Wnt/β-catenin pathway. With the amplifying effect of positive feedback, this integrin-activated β-catenin/Wnt pathway plays significant roles in mediating the enhancement of Wnt signal on stiff ECM and contributes to the regulation of mesenchymal stem cell differentiation and primary chondrocyte phenotype maintenance. The present integrin-regulated Wnt1 expression and signaling contributes to the understanding of the molecular mechanisms underlying the regulation of cell behaviors by ECM elasticity. PMID:26854061

  5. β Integrin-like protein-mediated adhesion and its disturbances during cell cultivation of the mussel Mytilus trossulus.

    PubMed

    Maiorova, Mariia A; Odintsova, Nelly A

    2015-08-01

    In this study, we focus on the specific contribution of β integrin-like protein to adhesion-mediated events in molluscan larval cells in culture that could not have been investigated within the whole animal. An analysis of disturbances to cell-substratum adhesion, caused by the integrin receptor inhibiting Arg-Gly-Asp-Ser (RGDS)-peptide, the Ca(2+)/Mg(2+)-chelators and the stress influence of freezing-thawing, reveals that all these factors resulted in the partial destruction of the integrin-extracellular matrix (ECM) interaction in culture and, in particular, changes in the distribution and relative abundance of β integrin-positive cells. The experiments, carried out on selected substrates, found that β integrin-positive cells demonstrate different affinities for the substrates. This finding further supports the assumption that epithelial differentiation in cultivated cells of larval Mytilus may be mediated by β integrin-like proteins via binding to laminin; direct binding to other components of the ECM could not be demonstrated. The mussel β integrin-positive cells are not involved in myogenic or neuronal differentiation on any of the substrates but part of them has tubulin-positive cilia, forming some epithelia-like structures. Our data indicate that β integrin-positive cells are able to proliferate in vitro which suggests that they could participate in renewing the digestive epithelium in larvae. The findings provide evidence that the distribution pattern of β integrin-like protein depends on the cell type and the factors influencing the adhesion. PMID:25673210

  6. Ligand-specific, transient interaction between integrins and calreticulin during cell adhesion to extracellular matrix proteins is dependent upon phosphorylation/dephosphorylation events.

    PubMed Central

    Coppolino, M G; Dedhar, S

    1999-01-01

    As transmembrane heterodimers, integrins bind to both extracellular ligands and intracellular proteins. We are currently investigating the interaction between integrins and the intracellular protein calreticulin. A prostatic carcinoma cell line (PC-3) was used to demonstrate that calreticulin can be found in the alpha3 immunoprecipitates of cells plated on collagen type IV, but not when plated on vitronectin. Conversely, alphav immunoprecipitates contained calreticulin only when cells were plated on vitronectin, i. e. not when plated on collagen IV. The interactions between these integrins and calreticulin were independent of actin cytoskeleton assembly and were transient, being maximal approx. 10-30 min after the cells came into contact with the substrates prior to complete cell spreading and formation of firm adhesive contacts. We demonstrate that okadaic acid, an inhibitor of intracellular serine/threonine protein phosphatases, inhibited the alpha3beta1-mediated adhesion of PC-3 cells to collagen IV and the alpha2beta1-mediated attachment of Jurkat cells to collagen I. This inhibition by okadaic acid was accompanied by inhibition of the ligand-specific interaction of calreticulin with the respective integrins in the two cell types. Additionally, we found that pharmacological inhibition of mitogen-activated protein kinase kinase (MEK) resulted in prolongation of the calreticulin-integrin interaction, and enhancement of PC-3 cell attachment to collagen IV. We conclude that calreticulin interacts transiently with integrins during cell attachment and spreading. This interaction depends on receptor occupation, is ligand-specific, and can be modulated by protein phosphatase and MEK activity. PMID:10229657

  7. Mesodermal expression of integrin α5β1 regulates neural crest development and cardiovascular morphogenesis

    PubMed Central

    Liang, Dong; Wang, Xia; Mittal, Ashok; Dhiman, Sonam; Hou, Shuan-Yu; Degenhardt, Karl; Astrof, Sophie

    2014-01-01

    Integrin α5-null embryos die in mid-gestation from severe defects in cardiovascular morphogenesis, which stem from defective development of the neural crest, heart and vasculature. To investigate the role of integrin α5β1 in cardiovascular development, we used the Mesp1Cre knock-in strain of mice to ablate integrin α5 in the anterior mesoderm, which gives rise to all of the cardiac and many of the vascular and muscle lineages in the anterior portion of the embryo. Surprisingly, we found that mutant embryos displayed numerous defects related to the abnormal development of the neural crest such as cleft palate, ventricular septal defect, abnormal development of hypoglossal nerves, and defective remodeling of the aortic arch arteries. We found that defects in arch artery remodeling stem from the role of mesodermal integrin α5β1 in neural crest proliferation and differentiation into vascular smooth muscle cells, while proliferation of pharyngeal mesoderm and differentiation of mesodermal derivatives into vascular smooth muscle cells was not defective. Taken together our studies demonstrate a requisite role for mesodermal integrin α5β1 in signaling between the mesoderm and the neural crest, thereby regulating neural crest-dependent morphogenesis of essential embryonic structures. PMID:25242040

  8. Molecular cloning and characterisation of a pattern recognition protein, lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) from Chinese shrimp Fenneropenaeus chinensis.

    PubMed

    Liu, Fengsong; Li, Fuhua; Dong, Bo; Wang, Xiaomei; Xiang, Jianhai

    2009-03-01

    A pattern recognition protein (PRP), lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) cDNA was cloned from the haemocyte of Chinese shrimp Fenneropenaeus chinensis by the techniques of homology cloning and RACE. Analysis of nucleotide sequence revealed that the full-length cDNA of 1,275 bp has an open reading frame of 1,098 bp encoding a protein of 366 amino acids including a 17 amino acid signal peptide. Sequence comparison of the deduced amino acid sequence of F. chinensis LGBP showed a high identity of 94%, 90%, 87%, 72% and 63% with Penaeus monodon BGBP, Litopenaeus stylirostris LGBP, Marsupenaeu japonicus BGBP, Homarus gammarus BGBP and Pacifastacus leniusculus LGBP, respectively. The calculated molecular mass of the mature protein is 39,857 Da with a deduced pI of 4.39. Two putative integrin binding motifs, RGD (Arg-Gly-Asp) and a potential recognition motif for beta-1,3-linkage of polysaccharides were observed in LGBP sequence. RT-PCR analysis showed that LGBP gene expresses in haemocyte and hepatopancreas only, but not in other tissues. Capillary electrophoresis RT-PCR method was used to quantify the variation of mRNA transcription level during artificial infection with heat-killed Vibrio anguillarum and Staphylococcus aureusin. A significant enhancement of LGBP transcription was appeared at 6 h post-injection in response to bacterial infection. These results have provided useful information to understand the function of LGBP in shrimp. PMID:18163220

  9. Molecular cloning and characterisation of a pattern recognition molecule, lipopolysaccharide- and beta-1,3-glucan binding protein (LGBP) from the white shrimp Litopenaeus vannamei.

    PubMed

    Cheng, Winton; Liu, Chun-Hung; Tsai, Chiung-Hui; Chen, Jiann-Chu

    2005-04-01

    A lipopolysaccharide- and beta-1,3-glucan binding protein (LGBP) cDNA was cloned from the haemocyte and hepatopancreas of white shrimp Litopenaeus vannamei using oligonucleotide primers and RT-PCR. Both 3'- and 5'-regions were isolated by rapid amplification of cDNA end RACE method. Analysis of nucleotide sequence revealed that the cDNA clone has an open reading frame of 1101 bp encoding a protein of 367 amino acids including a 17 amino acid signal peptide. The calculated molecular mass of the mature proteins (350 amino acids) is 39.92 kDa with an estimated pI of 4.37. Two putative integrin binding motifs (cell adhesion site), RGD (Arg-Gly-Asp) and a potential recognition motif for beta- (1-->3) linkage of polysaccharides were observed in the LGBP. Sequence comparison showed that LGBP deduced amino acid of L. vannamei has an overall similarity of 95%, 92% and 61% to that of blue shrimp Litopenaeus stylirostris LGBP, tiger shrimp Penaeus monodon BGBP and crayfish Pacifastacus leniusculus LGBP, respectively. Quantitative real-time RT-PCR analysis showed that LGBP transcript in haemocyte of L. vannamei increased in 3- and 6-h post Vibrio alginolyticus injection. PMID:15561560

  10. Calpains promote α2β1 integrin turnover in nonrecycling integrin pathway

    PubMed Central

    Rintanen, Nina; Karjalainen, Mikko; Alanko, Jonna; Paavolainen, Lassi; Mäki, Anita; Nissinen, Liisa; Lehkonen, Moona; Kallio, Katri; Cheng, R. Holland; Upla, Paula; Ivaska, Johanna; Marjomäki, Varpu

    2012-01-01

    Collagen receptor integrins recycle between the plasma membrane and endosomes and facilitate formation and turnover of focal adhesions. In contrast, clustering of α2β1 integrin with antibodies or the human pathogen echovirus 1 (EV1) causes redistribution of α2 integrin to perinuclear multivesicular bodies, α2-MVBs. We show here that the internalized clustered α2 integrin remains in α2-MVBs and is not recycled back to the plasma membrane. Instead, receptor clustering and internalization lead to an accelerated down-regulation of α2β1 integrin compared to the slow turnover of unclustered α2 integrin. EV1 infection or integrin degradation is not associated with proteasomal or autophagosomal processes and shows no significant association with lysosomal pathway. In contrast, degradation is dependent on calpains, such that it is blocked by calpain inhibitors. We show that active calpain is present in α2-MVBs, internalized clustered α2β1 integrin coprecipitates with calpain-1, and calpain enzymes can degrade α2β1 integrin. In conclusion, we identified a novel virus- and clustering-specific pathway that diverts α2β1 integrin from its normal endo/exocytic traffic to a nonrecycling, calpain-dependent degradative endosomal route. PMID:22160595

  11. B-Raf Regulation of Integrin α4β1-mediated Resistance to Shear Stress through Changes in Cell Spreading and Cytoskeletal Association in T Cells*

    PubMed Central

    Brown, Wells S.; Khalili, Jahan S.; Rodriguez-Cruz, Tania G.; Lizee, Greg; McIntyre, Bradley W.

    2014-01-01

    The regulation of integrin-mediated adhesion is of vital importance to adaptive and innate immunity. Integrins are versatile proteins and mediate T cell migration and trafficking by binding to extracellular matrix or other cells as well as initiating intracellular signaling cascades promoting survival or activation. The MAPK pathway is known to be downstream from integrins and to regulate survival, differentiation, and motility. However, secondary roles for canonical MAPK pathway members are being discovered. We show that chemical inhibition of RAF by sorafenib or shRNA-mediated knockdown of B-Raf reduces T cell resistance to shear stress to α4β1 integrin ligands vascular cell adhesion molecule 1 (VCAM-1) and fibronectin, whereas inhibition of MEK/ERK by U0126 had no effect. Microscopy showed that RAF inhibition leads to significant inhibition of T cell spreading on VCAM-1. The association of α4β1 integrin with the actin cytoskeleton was shown to be dependent on B-Raf activity or expression, whereas α4β1 integrin affinity for soluble VCAM-1 was not. These effects were shown to be specific for α4β1 integrin and not other integrins, such as α5β1 or LFA-1, or a variety of membrane proteins. We demonstrate a novel role for B-Raf in the selective regulation of α4β1 integrin-mediated adhesion. PMID:24936068

  12. Integrin αvβ3 expression in tongue squamous carcinoma cells Cal27 confers anticancer drug resistance through loss of pSrc(Y418).

    PubMed

    Stojanović, Nikolina; Brozovic, Anamaria; Majhen, Dragomira; Bosnar, Maja Herak; Fritz, Gerhard; Osmak, Maja; Ambriović-Ristov, Andreja

    2016-08-01

    Integrins play key roles in the regulation of tumor cell adhesion, migration, invasion and sensitivity to anticancer drugs. In the present study we investigate the mechanism of resistance of tongue squamous carcinoma cells Cal27 with de novo integrin αvβ3 expression to anticancer drugs. Cal27-derived cell clones, obtained by transfection of plasmid containing integrin subunit β3 cDNA, as compared to control cells demonstrate: expression of integrin αvβ3; increased expression of integrin αvβ5; increased adhesion to fibronectin and vitronectin; resistance to cisplatin, mitomycin C, doxorubicin and 5-fluorouracil; increased migration and invasion, increased amount of integrin-linked kinase (ILK) and decreased amounts of non-receptor tyrosine kinase (Src) and pSrc(Y418). Knockdown of ILK and integrin β5 in cells expressing integrin αvβ3 ruled out their involvement in drug resistance. Opposite, Src knockdown in Cal27 cells which led to a reduction in pSrc(Y418), as well as treatment with the pSrc(Y418) inhibitors dasatinib and PP2, conferred resistance to all four anticancer drugs, indicating that the loss of pSrc(Y418) is responsible for the observed effect. We identified differential integrin signaling between Cal27 and integrin αvβ3-expressing cells. In Cal27 cells integrin αv heterodimers signal through pSrc(Y418) while this is not the case in integrin αvβ3-expressing cells. Finally, we show that dasatinib counteracts the effect of cisplatin in two additional head and neck squamous cell carcinoma (HNSCC) cell lines Cal33 and Detroit562. Our results suggest that pSrc(Y418) inhibitors, potential drugs for cancer therapy, may reduce therapeutic efficacy if combined with chemotherapeutics, and might not be recommended for HNSCC treatment. PMID:27108184

  13. Transforming growth factor-beta 1 in experimental autoimmune neuritis. Cellular localization and time course.

    PubMed Central

    Kiefer, R.; Funa, K.; Schweitzer, T.; Jung, S.; Bourde, O.; Toyka, K. V.; Hartung, H. P.

    1996-01-01

    Experimental autoimmune neuritis (EAN) is a monophasic inflammatory disorder of the peripheral nervous system that resolves spontaneously by molecular mechanisms as yet unknown. We have investigated whether the immunosuppressive cytokine transforming growth factor-beta 1 (TGF-beta 1) might be endogenously expressed in the peripheral nervous system of Lewis rats with actively induced and adoptive transfer EAN. TGF-beta 1 mRNA was upregulated to high levels in sensory and motor roots, spinal ganglia, and sciatic nerve as revealed by quantitative Northern blot analysis and in situ hybridization histochemistry, with peak levels just preceding the first signs of clinical recovery. TGF-beta 1 mRNA was localized to scattered round cells and dense cellular infiltrates, but only rarely to Schwann cell profiles. Double labeling studies revealed macrophages and subpopulations of T cells as the major cellular source of TGF-beta 1 mRNA. TGF-beta 1 protein was visualized immunocytochemically and localized to infiltrating mononuclear cells with peak expression around the same time as mRNA, in addition to some constitutive expression in axons and Schwann cells. Our studies suggest that the spontaneous recovery observed in Lewis rat EAN might be mediated by the endogenous elaboration of TGF-beta 1 within the peripheral nerve, and that macrophages might control their own cytotoxicity by expressing TGF-beta 1. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:8546208

  14. Transforming growth factor-beta1 mediates cellular response to DNA damage in situ

    NASA Technical Reports Server (NTRS)

    Ewan, Kenneth B.; Henshall-Powell, Rhonda L.; Ravani, Shraddha A.; Pajares, Maria Jose; Arteaga, Carlos; Warters, Ray; Akhurst, Rosemary J.; Barcellos-Hoff, Mary Helen

    2002-01-01

    Transforming growth factor (TGF)-beta1 is rapidly activated after ionizing radiation, but its specific role in cellular responses to DNA damage is not known. Here we use Tgfbeta1 knockout mice to show that radiation-induced apoptotic response is TGF-beta1 dependent in the mammary epithelium, and that both apoptosis and inhibition of proliferation in response to DNA damage decrease as a function of TGF-beta1 gene dose in embryonic epithelial tissues. Because apoptosis in these tissues has been shown previously to be p53 dependent, we then examined p53 protein activation. TGF-beta1 depletion, by either gene knockout or by using TGF-beta neutralizing antibodies, resulted in decreased p53 Ser-18 phosphorylation in irradiated mammary gland. These data indicate that TGF-beta1 is essential for rapid p53-mediated cellular responses that mediate cell fate decisions in situ.

  15. Prognostic value of β1 integrin expression in colorectal liver metastases

    PubMed Central

    Vassos, Nikolaos; Rau, Tilman; Merkel, Susanne; Feiersinger, Fabian; Geppert, Carol I; Stürzl, Michael; Hohenberger, Werner; Croner, Roland S

    2014-01-01

    Integrins are cell surface adhesion molecules (CAM) that regulate via intercellular and cell-matrix signaling various cellular processes including wound healing, cell differentiation, division, growth, migration and metastatic dissemination. Although a correlation between carcinogenesis and changes in integrin expression, especially β1 integrin, has been reported, its role in colorectal liver metastases remains unclear. This study aimed to evaluate the expression of β1 integrin in colorectal liver metastases and to correlate the pattern of expression with clinicopathological features and to investigate the putative role of β1 integrin expression on survival of these patients. Methods: Formalin-fixed, paraffin-embedded (FFPE) tumor samples of 81 patients who were operated because of colorectal liver metastases without any neoadjuvant therapy were obtained and stained with hematoxylin and eosin (H & E). An immunohistochemical examination was performed using Dako, Peroxidase/DAB kit and a primary monoclonal β1 integrin (CD29, fibronectin receptor subunit beta; ab3167, Abcam plc). β1 integrin expression was evaluated according to the immunoreactive score of Remmele and Stegner and was related with clinicopathological features of prognostic significance and with disease-free and overall survival as well. Statistical analysis was performed using SPSS version 21.0. Results: β1 integrin was overexpressed in tumor cells in 37 (48%) patients and in stromal cell in 27 (33%) patients. The β1 expression was not statistically correlated with clinicopathological features of the primary tumors but it was statistically correlated (p=0.03) with the histological grading of liver metastases. Kaplan-Meier survival analysis showed that there is a tendency but no statistically significant correlation in disease-free and overall survival. Conclusion: Considering that expression of β1 integrin in colorectal liver metastases remains controversial, specially its relation with

  16. Ameloblastin modulates osteoclastogenesis through the integrin/ERK pathway

    PubMed Central

    Atsawasuwan, Phimon; Dangaria, Smit; Yan, Xiulin; Wu, Tuojiang; Evans, Carla A.; Luan, Xianghong

    2014-01-01

    Proteins of the extracellular matrix often have multiple functions to facilitate complex tasks ranging from signaling to structural support. Here we have focused on the function of one of the matrix proteins expressed in bones and teeth, the matrix adhesion protein ameloblastin (AMBN). Transgenic mice with 5-fold elevated AMBN levels in mandibles suffered from root cementum resorption, delamination, and reduced alveolar bone thickness. AMBN gain of function also resulted in a significant reduction in trabecular bone volume and bone mass dentistry in 42 days postnatal mouse jaws. In an in vitro model of osteoclastogenesis, AMBN modulated osteoclast differentiation from bone marrow derived monocytes (BMMCs), and dramatically increased osteoclast numbers and resorption pits. Furthermore, AMBN more than doubled BMMC adhesion, accelerated cell spreading, and promoted podosome belt and actin ring formation. These effects were associated with elevated ERK1/2 and AKT phosphorylation as well as higher expression of osteoclast activation related genes. Blocking integrin α2β1 and ERK 1/2 pathways alleviated the effects of AMBN on osteoclast differentiation. Together, our data indicate that AMBN increases osteoclast number and differentiation as well as mineralized tissue resorption by regulating cell adhesion and actin cytoskeleton polymerization, initiating integrin-dependent extracellular matrix signaling cascades and enhancing osteoclastogenesis. PMID:23385480

  17. Ameloblastin modulates osteoclastogenesis through the integrin/ERK pathway.

    PubMed

    Lu, Xuanyu; Ito, Yoshihiro; Atsawasuwan, Phimon; Dangaria, Smit; Yan, Xiulin; Wu, Tuojiang; Evans, Carla A; Luan, Xianghong

    2013-05-01

    Proteins of the extracellular matrix often have multiple functions to facilitate complex tasks ranging from signaling to structural support. Here we have focused on the function of one of the matrix proteins expressed in bones and teeth, the matrix adhesion protein ameloblastin (AMBN). Transgenic mice with 5-fold elevated AMBN levels in mandibles suffered from root cementum resorption, delamination, and reduced alveolar bone thickness. AMBN gain of function also resulted in a significant reduction in trabecular bone volume and bone mass dentistry in 42 days postnatal mouse jaws. In an in vitro model of osteoclastogenesis, AMBN modulated osteoclast differentiation from bone marrow derived monocytes (BMMCs), and dramatically increased osteoclast numbers and resorption pits. Furthermore, AMBN more than doubled BMMC adhesion, accelerated cell spreading, and promoted podosome belt and actin ring formation. These effects were associated with elevated ERK1/2 and AKT phosphorylation as well as higher expression of osteoclast activation related genes. Blocking integrin α2β1 and ERK 1/2 pathways alleviated the effects of AMBN on osteoclast differentiation. Together, our data indicate that AMBN increases osteoclast number and differentiation as well as mineralized tissue resorption by regulating cell adhesion and actin cytoskeleton polymerization, initiating integrin-dependent extracellular matrix signaling cascades and enhancing osteoclastogenesis. PMID:23385480

  18. Selective integrin endocytosis is driven by interactions between the integrin α-chain and AP2.

    PubMed

    De Franceschi, Nicola; Arjonen, Antti; Elkhatib, Nadia; Denessiouk, Konstantin; Wrobel, Antoni G; Wilson, Thomas A; Pouwels, Jeroen; Montagnac, Guillaume; Owen, David J; Ivaska, Johanna

    2016-02-01

    Integrins are heterodimeric cell-surface adhesion molecules comprising one of 18 possible α-chains and one of eight possible β-chains. They control a range of cell functions in a matrix- and ligand-specific manner. Integrins can be internalized by clathrin-mediated endocytosis (CME) through β subunit-based motifs found in all integrin heterodimers. However, whether specific integrin heterodimers can be selectively endocytosed was unknown. Here, we found that a subset of α subunits contain an evolutionarily conserved and functional YxxΦ motif directing integrins to selective internalization by the most abundant endocytic clathrin adaptor, AP2. We determined the structure of the human integrin α4-tail motif in complex with the AP2 C-μ2 subunit and confirmed the interaction by isothermal titration calorimetry. Mutagenesis of the motif impaired selective heterodimer endocytosis and attenuated integrin-mediated cell migration. We propose that integrins evolved to enable selective integrin-receptor turnover in response to changing matrix conditions. PMID:26779610

  19. SNP analyses of growth factor genes EGF, TGF{beta}-1, and HGF reveal haplotypic association of EGF with autism

    SciTech Connect

    Toyoda, Takao; Thanseem, Ismail; Kawai, Masayoshi; Sekine, Yoshimoto; Nakamura, Kazuhiko; Anitha, Ayyappan; Suda, Shiro . E-mail: nakamura@hama-med.ac.jp; Yamada, Kazuo; Tsujii, Masatsugu |; Iwayama, Yoshimi; Hattori, Eiji; Toyota, Tomoko; Yoshikawa, Takeo; Miyachi, Taishi; Tsuchiya, Kenji; Sugihara, Gen-ichi; Matsuzaki, Hideo; Iwata, Yasuhide; Suzuki, Katsuaki; Mori, Norio |; Ouchi, Yasuomi |; Sugiyama, Toshiro; Takei, Nori

    2007-09-07

    Autism is a pervasive neurodevelopmental disorder diagnosed in early childhood. Growth factors have been found to play a key role in the cellular differentiation and proliferation of the central and peripheral nervous systems. Epidermal growth factor (EGF) is detected in several regions of the developing and adult brain, where, it enhances the differentiation, maturation, and survival of a variety of neurons. Transforming growth factor-{beta} (TGF{beta}) isoforms play an important role in neuronal survival, and the hepatocyte growth factor (HGF) has been shown to exhibit neurotrophic activity. We examined the association of EGF, TGF{beta}1, and HGF genes with autism, in a trio association study, using DNA samples from families recruited to the Autism Genetic Resource Exchange; 252 trios with a male offspring scored for autism were selected for the study. Transmission disequilibrium test revealed significant haplotypic association of EGF with autism. No significant SNP or haplotypic associations were observed for TGF{beta}1 or HGF. Given the role of EGF in brain and neuronal development, we suggest a possible role of EGF in the pathogenesis of autism.

  20. Curcumin inhibits cellular condensation and alters microfilament organization during chondrogenic differentiation of limb bud mesenchymal cells.

    PubMed

    Kim, Dong Kyun; Kim, Song Ja; Kang, Shin Sung; Jin, Eun Jung

    2009-09-30

    Curcumin is a well known natural polyphenol product isolated from the rhizome of the plant Curcuma longa, anti-inflammatory agent for arthritis by inhibiting synthesis of inflammatory prostaglandins. However, the mechanisms by which curcumin regulates the functions of chondroprogenitor, such as proliferation, precartilage condensation, cytoskeletal organization or overall chondrogenic behavior, are largely unknown. In the present report, we investigated the effects and signaling mechanism of curcumin on the regulation of chondrogenesis. Treating chick limb bud mesenchymal cells with curcumin suppressed chondrogenesis by stimulating apoptotic cell death. It also inhibited reorganization of the actin cytoskeleton into a cortical pattern concomitant with rounding of chondrogenic competent cells and down-regulation of integrin beta1 and focal adhesion kinase (FAK) phosphorylation. Curcumin suppressed the phosphorylation of Akt leading to Akt inactivation. Activation of Akt by introducing a myristoylated, constitutively active form of Akt reversed the inhibitory actions of curcumin during chondrogenesis. In summary, for the first time, we describe biological properties of curcumin during chondrogenic differentiation of chick limb bud mesenchymal cells. Curcumin suppressed chondrogenesis by stimulating apoptotic cell death and down-regulating integrin-mediated reorganization of actin cytoskeleton via modulation of Akt signaling. PMID:19478554

  1. Interaction between Fibronectin and β1 Integrin Is Essential for Tooth Development

    PubMed Central

    Yamada, Aya; Yuasa, Kenji; Yoshizaki, Keigo; Iwamoto, Tsutomu; Saito, Masahiro; Nakamura, Takashi; Fukumoto, Satoshi

    2015-01-01

    The dental epithelium and extracellular matrix interact to ensure that cell growth and differentiation lead to the formation of teeth of appropriate size and quality. To determine the role of fibronectin in differentiation of the dental epithelium and tooth formation, we analyzed its expression in developing incisors. Fibronectin mRNA was expressed during the presecretory stage in developing dental epithelium, decreased in the secretory and early maturation stages, and then reappeared during the late maturation stage. The binding of dental epithelial cells derived from postnatal day-1 molars to a fibronectin-coated dish was inhibited by the RGD but not RAD peptide, and by a β1 integrin-neutralizing antibody, suggesting that fibronectin-β1 integrin interactions contribute to dental epithelial-cell binding. Because fibronectin and β1 integrin are highly expressed in the dental mesenchyme, it is difficult to determine precisely how their interactions influence dental epithelial differentiation in vivo. Therefore, we analyzed β1 integrin conditional knockout mice (Intβ1lox-/lox-/K14-Cre) and found that they exhibited partial enamel hypoplasia, and delayed eruption of molars and differentiation of ameloblasts, but not of odontoblasts. Furthermore, a cyst-like structure was observed during late ameloblast maturation. Dental epithelial cells from knockout mice did not bind to fibronectin, and induction of ameloblastin expression in these cells by neurotrophic factor-4 was inhibited by treatment with RGD peptide or a fibronectin siRNA, suggesting that the epithelial interaction between fibronectin and β1 integrin is important for ameloblast differentiation and enamel formation. PMID:25830530

  2. Covisualization in living onion cells of putative integrin, putative spectrin, actin, putative intermediate filaments, and other proteins at the cell membrane and in an endomembrane sheath

    NASA Technical Reports Server (NTRS)

    Reuzeau, C.; Doolittle, K. W.; McNally, J. G.; Pickard, B. G.; Evans, M. L. (Principal Investigator)

    1997-01-01

    Covisualizations with wide-field computational optical-sectioning microscopy of living epidermal cells of the onion bulb scale have evidenced two major new cellular features. First, a sheath of cytoskeletal elements clads the endomembrane system. Similar elements clad the inner faces of punctate plasmalemmal sites interpreted as plasmalemmal control centers. One component of the endomembrane sheath and plasmalemmal control center cladding is anti-genicity-recognized by two injected antibodies against animal spectrin. Immunoblots of separated epidermal protein also showed bands recognized by these antibodies. Injected phalloidin identified F-actin with the same cellular distribution pattern, as did antibodies against intermediate-filament protein and other cytoskeletal elements known from animal cells. Injection of general protein stains demonstrated the abundance of endomembrane sheath protein. Second, the endomembrane system, like the plasmalemmal puncta, contains antigen recognized by an anti-beta 1 integrin injected into the cytoplasm. Previously, immunoblots of separated epidermal protein were shown to have a major band recognized both by this antibody prepared against a peptide representing the cytosolic region of beta 1 integrin and an antibody against the matrix region of beta 1 integrin. The latter antiboby also identified puncta at the external face of protoplasts. It is proposed that integrin and associated transmembrane proteins secure the endomembrane sheath and transmit signals between it and the lumen or matrix of the endoplasmic reticulum and organellar matrices. This function is comparable to that proposed for such transmembrane linkers in the plasmalemmal control centers, which also appear to bind cytoskeleton and a host of related molecules and transmit signals between them and the wall matrix. It is at the plasmalemmal control centers that the endoplasmic reticulum, a major component of the endomembrane system, attaches to the plasma membrane.

  3. Inhibition of vimentin or B1 integrin reverts morphology of prostate tumor cells grown in laminin-rich extracellular matrix gels and reduces tumor growth in vivo

    SciTech Connect

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

    2008-06-12

    Prostate epithelial cells grown embedded in laminin-rich extracellular matrix (lrECM) undergo morphologic 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 lrECM gels. M12, a highly tumorigenic and metastatic subline, was derived from the immortalized, prostate epithelial P69 cell line by selection in athymic, 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 parental, nontumorigenic P69 line produced acini with clearly defined lumena. Immunostaining with antibodies to {beta}-catenin, E-cadherin, or {alpha}6 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 small interfering RNA interference or the expression of {alpha}6 and {beta}1 integrins by the addition of blocking antibodies, reorganized the M12 subline into forming polarized acini. The loss of vimentin significantly reduced M12-Vim tumor growth when assessed by s.c. injection in athymic mice. Thus, tumorigenicity in vivo correlated with disorganized growth in three-dimensional lrECM gels. These studies suggest that the levels of vimentin and {beta}1 integrin play a key role in the homeostasis of the normal acinus in prostate and that their dysregulation may lead to tumorigenesis. [Mol Cancer Ther 2009;8(3):499-508].

  4. Only Specific Tobacco (Nicotiana tabacum) Chitinases and [beta]-1,3-Glucanases Exhibit Antifungal Activity.

    PubMed Central

    Sela-Buurlage, M. B.; Ponstein, A. S.; Bres-Vloemans, S. A.; Melchers, L. S.; Van Den Elzen, PJM.; Cornelissen, BJC.

    1993-01-01

    Different isoforms of chitinases and [beta]-1,3-glucanases of tobacco (Nicotiana tabacum cv Samsun NN) were tested for their antifungal activities. The class I, vacuolar chitinase and [beta]-1,3-glucanase isoforms were the most active against Fusarium solani germlings, resulting in lysis of the hyphal tips and in growth inhibition. In additon, we observed that the class I chitinase and [beta]-1,3-glucanase acted synergistically. The class II isoforms of the two hydrolases exhibited no antifungal activity. However, the class II chitinases showed limited growth inhibitory activity in combination with higher amounts of class I [beta]-1,3-glucanase. The class II [beta]-1,3-glucanases showed no inhibitory activity in any combination. In transgenic tobacco plants producing modified forms of either a class I chitinase or a class I [beta]-1,3-glucanase, or both, these proteins were targeted extracellularly. Both modified proteins lack their C-terminal propeptide, which functions as a vacuolar targeting signal. Extracellular targeting had no effect on the specific activities of the chitinase and [beta]-1,3-glucanase enzymes. Furthermore, the extracellular washing fluid (EF) from leaves of transgenic plants expressing either of the secreted class I enzymes exhibited antifungal activity on F. solani germlings in vitro comparable to that of the purified vacuolar class I proteins. Mixing EF fractions from these plants revealed synergism in inhibitory activity against F. solani; the mixed fractions exhibited inhibitory activity similar to that of EF from plants expressing both secreted enzymes. PMID:12231736

  5. Two pine endo-beta-1,4-glucanases are associated with rapidly growing reproductive structures.

    PubMed

    Loopstra, C A; Mouradov, A; Vivian-Smith, A; Glassick, T V; Gale, B V; Southerton, S G; Marshall, H; Teasdale, R D

    1998-03-01

    Two cDNA clones encoding endo-beta-1,4-glucanases (EGases) were isolated from a radiata pine (Pinus radiata) cDNA library prepared from immature female strobili. The cDNAs PrCel1 (inus adiata cellulase ) and PrCel2 encode proteins 509 and 515 amino acids in length, respectively, including putative signal peptides. Both proteins contain domains conserved in plant and bacterial EGases. The proteins PRCEL1 and PRCEL2 showed strong similarity to each other (76% amino acid identity), and higher similarity to TPP18 (73 and 67%, respectively), an EGase cloned from tomato (Lycopersicon esculentum) pistils, than to any other reported EGases. Northern-blot analyses indicated that both genes displayed a similar pattern of expression. The only significant difference was in the level of expression. In situ hybridizations were used to demonstrate that, within differentiating pine reproductive structures, PrCel1 expression was greatest in microsporangia in pollen strobili and near the developing ovule in the seed strobili. Expression was also found in vegetative tissues, especially in regions experiencing cell elongation, such as the elongating region of root tips. Both proteins have an ability to degrade carboxymethylcellulose in vitro. Genomic-blot analysis indicated the presence of a family of EGase genes in the radiata pine genome, and that PrCel1 and PrCel2 are transcribed from distinct one-copy genes. PMID:9501128

  6. Changes in Maternal Serum Transforming Growth Factor Beta-1 during Pregnancy: A Cross-Sectional Study

    PubMed Central

    Orazulike, Ngozi C.; Ashmore, Jill; Konje, Justin C.

    2013-01-01

    Changes in circulating levels of maternal serum transforming growth factor beta-1 (TGF-β1), collected from 98 women (AGA) at different gestational ages (10–38 weeks) were measured and comparisons were made between levels in pregnant and nonpregnant controls and also between 10 women with small-for-gestational age (SGA) and 7 with appropriate-for-gestational age (AGA) fetuses. Maternal serum TGF-β1 levels at all stages of pregnancy were higher than those in normal healthy nonpregnant adults. The mean TGF-β1 levels in SGA pregnancies at 34-week gestation (32.5 + 3.2 ng/mL) were significantly less than those in AGA pregnancies (39.2 + 9.8 ng/mL) while at 38-week gestation, the levels were similar in the two groups (36.04 + 4.3 versus 36.7 + 7.0 ng/mL). This differential change in TGF-β1 levels is probably an important modulating factor in the aetiopathogenesis of abnormal intrauterine fetal growth. PMID:24350258

  7. Integrin Targeting for Tumor Optical Imaging

    PubMed Central

    Ye, Yunpeng; Chen, Xiaoyuan

    2011-01-01

    Optical imaging has emerged as a powerful modality for studying molecular recognitions and molecular imaging in a noninvasive, sensitive, and real-time way. Some advantages of optical imaging include cost-effectiveness, convenience, and non-ionization safety as well as complementation with other imaging modalities such as positron emission tomography (PET), single-photon emission computed tomography (SPECT), and magnetic resonance imaging (MRI). Over the past decade, considerable advances have been made in tumor optical imaging by targeting integrin receptors in preclinical studies. This review has emphasized the construction and evaluation of diverse integrin targeting agents for optical imaging of tumors in mouse models. They mainly include some near-infrared fluorescent dye-RGD peptide conjugates, their multivalent analogs, and nanoparticle conjugates for targeting integrin αvβ3. Some compounds targeting other integrin subtypes such as α4β1 and α3 for tumor optical imaging have also been included. Both in vitro and in vivo studies have revealed some promising integrin-targeting optical agents which have further enhanced our understanding of integrin expression and targeting in cancer biology as well as related anticancer drug discovery. Especially, some integrin-targeted multifunctional optical agents including nanoparticle-based optical agents can multiplex optical imaging with other imaging modalities and targeted therapy, serving as an attractive type of theranostics for simultaneous imaging and targeted therapy. Continued efforts to discover and develop novel, innovative integrin-based optical agents with improved targeting specificity and imaging sensitivity hold great promises for improving cancer early detection, diagnosis, and targeted therapy in clinic. PMID:21546996

  8. Tumour exosome integrins determine organotropic metastasis.

    PubMed

    Hoshino, Ayuko; Costa-Silva, Bruno; Shen, Tang-Long; Rodrigues, Goncalo; Hashimoto, Ayako; Tesic Mark, Milica; Molina, Henrik; Kohsaka, Shinji; Di Giannatale, Angela; Ceder, Sophia; Singh, Swarnima; Williams, Caitlin; Soplop, Nadine; Uryu, Kunihiro; Pharmer, Lindsay; King, Tari; Bojmar, Linda; Davies, Alexander E; Ararso, Yonathan; Zhang, Tuo; Zhang, Haiying; Hernandez, Jonathan; Weiss, Joshua M; Dumont-Cole, Vanessa D; Kramer, Kimberly; Wexler, Leonard H; Narendran, Aru; Schwartz, Gary K; Healey, John H; Sandstrom, Per; Labori, Knut Jørgen; Kure, Elin H; Grandgenett, Paul M; Hollingsworth, Michael A; de Sousa, Maria; Kaur, Sukhwinder; Jain, Maneesh; Mallya, Kavita; Batra, Surinder K; Jarnagin, William R; Brady, Mary S; Fodstad, Oystein; Muller, Volkmar; Pantel, Klaus; Minn, Andy J; Bissell, Mina J; Garcia, Benjamin A; Kang, Yibin; Rajasekhar, Vinagolu K; Ghajar, Cyrus M; Matei, Irina; Peinado, Hector; Bromberg, Jacqueline; Lyden, David

    2015-11-19

    Ever since Stephen Paget's 1889 hypothesis, metastatic organotropism has remained one of cancer's greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6β4 and α6β1 were associated with lung metastasis, while exosomal integrin αvβ5 was linked to liver metastasis. Targeting the integrins α6β4 and αvβ5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis. PMID:26524530

  9. Tumour exosome integrins determine organotropic metastasis

    PubMed Central

    Hoshino, Ayuko; Costa-Silva, Bruno; Shen, Tang-Long; Rodrigues, Goncalo; Hashimoto, Ayako; Mark, Milica Tesic; Molina, Henrik; Kohsaka, Shinji; Di Giannatale, Angela; Ceder, Sophia; Singh, Swarnima; Williams, Caitlin; Soplop, Nadine; Uryu, Kunihiro; Pharmer, Lindsay; King, Tari; Bojmar, Linda; Davies, Alexander E.; Ararso, Yonathan; Zhang, Tuo; Zhang, Haiying; Hernandez, Jonathan; Weiss, Joshua M.; Dumont-Cole, Vanessa D.; Kramer, Kimberly; Wexler, Leonard H.; Narendran, Aru; Schwartz, Gary K.; Healey, John H.; Sandstrom, Per; Labori, Knut Jørgen; Kure, Elin H.; Grandgenett, Paul M.; Hollingsworth, Michael A.; de Sousa, Maria; Kaur, Sukhwinder; Jain, Maneesh; Mallya, Kavita; Batra, Surinder K.; Jarnagin, William R.; Brady, Mary S.; Fodstad, Oystein; Muller, Volkmar; Pantel, Klaus; Minn, Andy J.; Bissell, Mina J.; Garcia, Benjamin A.; Kang, Yibin; Rajasekhar, Vinagolu K.; Ghajar, Cyrus M.; Matei, Irina; Peinado, Hector; Bromberg, Jacqueline; Lyden, David

    2015-01-01

    Ever since Stephen Paget’s 1889 hypothesis, metastatic organotropism has remained one of cancer’s greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6β4 and α6β1 were associated with lung metastasis, while exosomal integrin αvβ5 was linked to liver metastasis. Targeting the integrins α6β4 and αvβ5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis. PMID:26524530

  10. Angiotensin II stimulated expression of transforming growth factor-beta1 in cardiac fibroblasts and myofibroblasts.

    PubMed

    Campbell, S E; Katwa, L C

    1997-07-01

    Angiotensin II (Ang II) stimulates pathologic myocardial fibrosis. Cardiac fibroblasts (CFb) and myofibroblasts mediate this response, perhaps in part by indirect production of specific cytokines. We sought to determine if Ang II could stimulate transforming growth factor-beta1 (TGF-beta1) gene expression and protein production in adult rat CFb and two cardiac myofibroblast cell types, scar myofibroblasts (MyoFb) and valvular interstitial cells (VIC). Confluent CFb, MyoFb, and VIC in serum-deprived (0.4% FCS) media were treated with Ang II (10(-7) m for CFb; 10(-9) m for MyoFb, VIC) for 24 h. Untreated cells served as controls. Culture media was collected and TGF-beta1 levels determined in triplicate using a sandwich ELISA. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed to determine TGF-beta1 mRNA expression. Ang II increased CFb (P<0.02) and VIC (P<0.04) TGF-beta1 mRNA expression, while the increase in MyoFb was not statistically significant. MyoFb produced the highest TGF-beta1 levels under control conditions compared to VIC and CFb. Ang II stimulated further TGF-beta1 secretion in VIC and CFb, but not MyoFb. The AT1 receptor antagonist Losartan (10(-7) m) greatly attenuated Ang II-stimulated TGF-B1 secretion and decreased TGF-beta1 immunostaining in VIC. The AT2 receptor antagonist PD123177 (10(-7) m) also decreased secretion and immunostaining of TGF-beta1 in VIC, but to a lesser extent than Losartan. TGF-beta1 secretion by MyoFb was unaffected by Losartan and PD123177, although TGF-B1 immunostaining was absent or greatly decreased, respectively, compared to Ang II-treated MyoFb. Ang II stimulates TGF-beta1 gene expression and/or protein production in cardiac fibroblast-like cells which may act as an autocrine/paracrine stimulus to collagen formation. Furthermore, TGF-beta1 production and secretion in these cells can be modulated by specific Ang II receptor antagonists, suggesting a potential benefit in preventing

  11. DARPP-32 binds to tra2-beta1 and influences alternative splicing

    PubMed Central

    Benderska, Natalya; Becker, Kristina; Girault, Jean-Antoine; Becker, Cord-Michael; Andreadis, Athena; Stamm, Stefan

    2010-01-01

    The majority of human genes undergo alternative splicing, which is frequently altered in response to physiological stimuli. DARPP-32 (Dopamine and cAMP regulated phosphoprotein, 32 kD) is a component of PKA-dependent signaling pathways. Here we show that DARPP-32 binds directly to the splicing factor tra2-beta1 (transformer 2). DARPP-32 changes the usage of tra2-beta1 dependent alternative exons in a concentration dependent manner, suggesting that the DARPP-32:tra2-beta1 interaction is a molecular link between signaling pathways and pre-mRNA processing. PMID:20074680

  12. The antifibrotic effects of TGF-{beta}1 siRNA on hepatic fibrosis in rats

    SciTech Connect

    Lang, Qing; Liu, Qi; Xu, Ning; Qian, Ke-Li; Qi, Jing-Hu; Sun, Yin-Chun; Xiao, Lang; Shi, Xiao-Feng

    2011-06-10

    Highlights: {yields} We constructed CCL4 induced liver fibrosis model successfully. {yields} We proofed that the TGF-{beta}1 siRNA had a definite therapy effect to CCL4 induced liver fibrosis. {yields} The therapy effect of TGF-{beta}1 siRNA had dose-dependent. -- Abstract: Background/aims: Hepatic fibrosis results from the excessive secretion of matrix proteins by hepatic stellate cells (HSCs), which proliferate during fibrotic liver injury. Transforming growth factor (TGF)-{beta}1 is the dominant stimulus for extracellular matrix (ECM) production by stellate cells. Our study was designed to investigate the antifibrotic effects of using short interference RNA (siRNA) to target TGF-{beta}1 in hepatic fibrosis and its mechanism in rats exposed to a high-fat diet and carbon tetrachloride (CCL4). Methods: A total of 40 healthy, male SD (Sprague-Dawley) rats were randomly divided into five even groups containing of eight rats each: normal group, model group, TGF-{beta}1 siRNA 0.125 mg/kg treatment group, TGF-{beta}1 siRNA 0.25 mg/kg treatment group and TGF-{beta}1 siRNA negative control group (0.25 mg/kg). CCL4 and a high-fat diet were used for 8 weeks to induce hepatic fibrosis. All the rats were then sacrificed to collect liver tissue samples. A portion of the liver samples were soaked in formalin for Hematoxylin-Eosin staining, classifying the degree of liver fibrosis, and detecting the expression of type I and III collagen and TGF-{beta}1; the remaining liver samples were stored in liquid nitrogen to be used for detecting TGF-{beta}1 by Western blotting and for measuring the mRNA expression of type I and III collagen and TGF-{beta}1 by quantitative real-time polymerase chain reaction. Results: Comparing the TGF-{beta}1 siRNA 0.25 mg/kg treatment group to the model group, the TGF-{beta}1 siRNA negative control group and the TGF-{beta}1 siRNA 0.125 mg/kg treatment group showed significantly reduced levels of pathological changes, protein expression and the m

  13. Evidence that cell surface beta 1,4-galactosyltransferase spontaneously galactosylates an underlying laminin substrate during fibroblast migration.

    PubMed

    Begovac, P C; Shi, Y X; Mansfield, D; Shur, B D

    1994-12-16

    beta 1,4-Galactosyltransferase is unusual among the glycosyltransferases in that a subpopulation exists on the cell surface in addition to its traditional biosynthetic location within the Golgi complex. On the cell surface, galactosyltransferase is expressed in spatially restricted, cell type-specific domains, where it functions as a receptor for extracellular oligosaccharide ligands during selected cellular interactions. For example, galactosyltransferase is found on the leading and trailing edges of migrating cells, where it facilitates lamellipodia formation and cell spreading by binding to specific N-linked oligosaccharides within laminin. Although the ability of galactosyltransferase to serve as a laminin receptor is well documented, it is unclear whether it functions solely in a lectin-like capacity to bind laminin glycoside ligands or uses its intrinsic catalytic activity to release itself from and modify its oligosaccharide substrate. In this study, we determined whether cell surface galactosyltransferase spontaneously galactosylates laminin matrices during cell migration using endogenous galactose donors. Cells were prelabeled with [3H]galactose, washed, and transferred in small clusters onto laminin matrices. The prelabeled cells migrated out from the cell cluster, during which time they deposited covalently bound [3H]galactose residues onto the laminin matrix. The degree of galactosylation was both laminin- and time-dependent and required actively migrating, intact cells. The radioactivity released from the 3H-galactosylated laminin by acid hydrolysis comigrated with authentic galactose standards on paper chromatography. In parallel assays, there was no radioactivity deposited on laminin matrices when cells were prelabeled with [3H]fucose or [3H]leucine. Furthermore, [3H]galactosylation was dependent upon galactosyltransferase-mediated cell migration, since prelabeled cells did not deposit [3H]galactose when migrating on fibronectin, upon which migration

  14. Novel α2β1 Integrin Inhibitors Reveal That Integrin Binding to Collagen under Shear Stress Conditions Does Not Require Receptor Preactivation*

    PubMed Central

    Nissinen, Liisa; Koivunen, Jarkko; Käpylä, Jarmo; Salmela, Maria; Nieminen, Jonna; Jokinen, Johanna; Sipilä, Kalle; Pihlavisto, Marjo; Pentikäinen, Olli T.; Marjamäki, Anne; Heino, Jyrki

    2012-01-01

    The interaction between α2β1 integrin (GPIa/IIa, VLA-2) and vascular collagen is one of the initiating events in thrombus formation. Here, we describe two structurally similar sulfonamide derivatives, BTT-3033 and BTT-3034, and show that, under static conditions, they have an almost identical effect on α2-expressing CHO cell adhesion to collagen I, but only BTT-3033 blocks platelet attachment under flow (90 dynes/cm2). Differential scanning fluorimetry showed that both molecules bind to the α2I domain of the recombinant α2 subunit. To further study integrin binding mechanism(s) of the two sulfonamides, we created an α2 Y285F mutant containing a substitution near the metal ion-dependent adhesion site motif in the α2I domain. The action of BTT-3033, unlike that of BTT-3034, was dependent on Tyr-285. In static conditions BTT-3034, but not BTT-3033, inhibited collagen binding by an α2 variant carrying a conformationally activating E318W mutation. Conversely, in under flow conditions (90 dynes/cm2) BTT-3033, but not BTT-3034, inhibited collagen binding by an α2 variant expressing E336A loss-of-function mutation. Thus, the binding sites for BTT-3033 and BTT-3034 are differentially available in distinct integrin conformations. Therefore, these sulfonamides can be used to study the biological role of different functional stages of α2β1. Furthermore, only the inhibitor that recognized the non-activated conformation of α2β1 integrin under shear stress conditions effectively blocked platelet adhesion, suggesting that the initial interaction between integrin and collagen takes place prior to receptor activation. PMID:23132859

  15. Nascent Integrin Adhesions Form on All Matrix Rigidities after Integrin Activation.

    PubMed

    Changede, Rishita; Xu, Xiaochun; Margadant, Felix; Sheetz, Michael P

    2015-12-01

    Integrin adhesions assemble and mature in response to ligand binding and mechanical factors, but the molecular-level organization is not known. We report that ∼100-nm clusters of ∼50 β3-activated integrins form very early adhesions under a wide variety of conditions on RGD surfaces. These adhesions form similarly on fluid and rigid substrates, but most adhesions are transient on rigid substrates. Without talin or actin polymerization, few early adhesions form, but expression of either the talin head or rod domain in talin-depleted cells restores early adhesion formation. Mutation of the integrin binding site in the talin rod decreases cluster size. We suggest that the integrin clusters constitute universal early adhesions and that they are the modular units of cell matrix adhesions. They require the association of activated integrins with cytoplasmic proteins, in particular talin and actin, and cytoskeletal contraction on them causes adhesion maturation for cell motility and growth. PMID:26625956

  16. [Interferon beta 1-a in multiple sclerosis: 1-year experience in 62 patients].

    PubMed

    Tilbery, C P; Felipe, E; Moreira, M A; Mendes, M F; França, A S

    2000-06-01

    We report the results of a trial of interferon beta 1-a in 62 ambulatory patients with relapsing-remitting multiple sclerosis. Entry criteria included EDSS of 0 to 5.5 and at least two exacerbations in the previous 2 years. The patients received 3 million international units by subcutaneous injections three times a week. The end points were differences in exacerbation rate and treatment effect on disease progression. The annual exacerbation rate for patients that did not take the interferon beta 1-a was 1.32 and for the patients under medication 0.63. The EDSS score in patients that did not take the mediaction was 4.7 and 2.0 for the patients with interferon beta 1-a. Interferon beta 1-a was well tolerated and 85% of patients completed 1 year treatment. PMID:10920406

  17. Protective effect of transforming growth factor beta 1 on experimental autoimmune diseases in mice.

    PubMed

    Kuruvilla, A P; Shah, R; Hochwald, G M; Liggitt, H D; Palladino, M A; Thorbecke, G J

    1991-04-01

    Interleukin 1 (IL-1) and tumor necrosis factor alpha are thought to contribute to the inflammatory response associated with autoimmune diseases. Transforming growth factor beta 1 (TGF-beta 1) counteracts many effects of these cytokines and has various immunosuppressive properties. In the present study, it is shown that microgram amounts of TGF-beta 1, injected daily for 1-2 weeks, protect against collagen-induced arthritis (CIA) and relapsing experimental allergic encephalomyelitis (REAE), the animal models for rheumatoid arthritis and multiple sclerosis, respectively. When administered during induction of the disease, TGF-beta 1 prevents CIA but only delays the onset of REAE by 2-3 days. However, when administered during a remission. TGF-beta 1 prevents the occurrence of relapses in REAE. The results suggest that TGF-beta 1 has powerful anti-inflammatory effects, mimicking in some respects the beneficial effects of immunosuppressive drugs in these experimental models of autoimmune disease, but without discernable adverse effects. PMID:2011600

  18. Antibodies to beta 1 and beta 2 adrenoreceptors in Chagas' disease.

    PubMed Central

    Sterin-Borda, L; Perez Leiros, C; Wald, M; Cremaschi, G; Borda, E

    1988-01-01

    Evidence accumulated over the last decade concerning human and experimental models suggests that an immunopathological mechanism may be involved in the pathogenesis of chronic Chagas' disease. In this paper we demonstrate the existence of two different circulating IgG in chagasic patients which bind with myocardial beta 1 and spleen cell beta 2 adrenoceptors, acting as non-competitive inhibitors. Both chagasic IgG against beta 1 and beta 2 adrenoceptor increased intracellular levels of cAMP that could be blocked by specific beta 1 and beta 2 adrenoceptor antagonists. The specificity for beta 1 and beta 2 adrenoceptors and the independence of other tissue reactive antibodies was demonstrated by IgG absorption with turkey red blood cell (TRBC), human lymphocytes (HL) or guinea pig red blood cells (GPRBC). The F(ab')2 fraction acted similarly. This supports the specificity of beta 1 and beta 2 adrenoceptors to the chagasic IgG and the independence of the other tissue reactive antibodies, such as EVI system. The probable pathogenic role of both beta 1 and beta 2 adrenergic chagasic antibody is discussed. PMID:2853016

  19. Transforming growth factor beta 1 suppresses acute and chronic arthritis in experimental animals.

    PubMed Central

    Brandes, M E; Allen, J B; Ogawa, Y; Wahl, S M

    1991-01-01

    Systemic administration of the cytokine, TGF beta 1, profoundly antagonized the development of polyarthritis in susceptible rats. TGF beta 1 administration (1 or 5 micrograms/animal), initiated one day before an arthritogenic dose of streptococcal cell wall (SCW) fragments, virtually eliminated the joint swelling and distortion typically observed during both the acute phase (articular index, AI = 2.5 vs. 11; P less than 0.025) and the chronic phase (AI = 0 vs. 12.5) of the disease. Moreover, TGF beta 1 suppressed the evolution of arthritis even when administration was begun after the acute phase of the disease. Histopathological examination of the joint revealed the systemic TGF beta 1 treatment greatly reduced inflammatory cell infiltration, pannus formation, and joint erosion. Consistent with the inhibition of inflammatory cell recruitment into the synovium, TGF beta 1 reversed the leukocytosis associated with the chronic phase of the arthritis. Control animals subjected to the same TGF beta 1 dosing regimen displayed no discernable immunosuppressive or toxic effects even after 4 wk of treatment. These observations not only provide insight into the immunoregulatory effects of TGF beta, but also implicate this cytokine as a potentially important therapeutic agent. Images PMID:1999490

  20. Quantitative relationship among integrin-ligand binding, adhesion, and signaling via focal adhesion kinase and extracellular signal-regulated kinase 2.

    PubMed

    Asthagiri, A R; Nelson, C M; Horwitz, A F; Lauffenburger, D A

    1999-09-17

    Because integrin-mediated signals are transferred through a physical architecture and synergistic biochemical network whose properties are not well defined, quantitative relationships between extracellular integrin-ligand binding events and key intracellular responses are poorly understood. We begin to address this by quantifying integrin-mediated FAK and ERK2 responses in CHO cells for varied alpha(5)beta(1) expression level and substratum fibronectin density. Plating cells on fibronectin-coated surfaces initiated a transient, biphasic ERK2 response, the magnitude and kinetics of which depended on integrin-ligand binding properties. Whereas ERK2 activity initially increased with a rate proportional to integrin-ligand bond number for low fibronectin density, the desensitization rate was independent of integrin and fibronectin amount but proportional to the ERK2 activity level with an exponential decay constant of 0.3 (+/- 0.08) min(-1). Unlike the ERK2 activation time course, FAK phosphorylation followed a superficially disparate time course. However, analysis of the early kinetics of the two signals revealed them to be correlated. The initial rates of FAK and ERK2 signal generation exhibited similar dependence on fibronectin surface density, with both rates monotonically increasing with fibronectin amount until saturating at high fibronectin density. Because of this similar initial rate dependence on integrin-ligand bond formation, the disparity in their time courses is attributed to differences in feedback regulation of these signals. Whereas FAK phosphorylation increased to a steady-state level as new integrin-ligand bond formation continued during cell spreading, ERK2 activity was decoupled from the integrin-ligand stimulus and decayed back to a basal level. Accordingly, we propose different functional metrics for representing these two disparate dynamic signals: the steady-state tyrosine phosphorylation level for FAK and the integral of the pulse response for

  1. Annexin V/beta5 integrin interactions regulate apoptosis of growth plate chondrocytes.

    PubMed

    Wang, Wei; Kirsch, Thorsten

    2006-10-13

    Apoptosis of terminally differentiated chondrocytes allows the replacement of growth plate cartilage by bone. Despite its importance, little is known about the regulation of chondrocyte apoptosis. We show that overexpression of annexin V, which binds to the cytoplasmic domain of beta5 integrin and protein kinase C alpha (PKCalpha), stimulates apoptotic events in hypertrophic growth plate chondrocytes. To determine whether the balance between the interactions of annexin V/beta5 integrin and annexin V/active PKCalpha play a role in the regulation of terminally differentiated growth plate chondrocyte apoptosis, a peptide mimic of annexin V (Penetratin (Pen)-VVISYSMPD) that binds to beta5 integrin but not to PKCalpha was used. This peptide stimulated apoptotic events in growth plate chondrocytes. Suppression of annexin V expression using small interfering ribonucleic acid decreased caspase-3 activity and increased cell viability in Pen-VVISYSMPD-treated growth plate chondrocytes. An activator of PKC resulted in a further decrease of cell viability and further increase of caspase-3 activity in Pen-VVISYSMPD-treated growth plate chondrocytes, whereas inhibitors of PKCalpha led to an increase of cell viability and decrease of caspase-3 activity of Pen-VVISYSMPD-treated cells. These findings suggest that binding of annexin V to active PKCalpha stimulates apoptotic events in growth plate chondrocytes and that binding of annexin Vto beta5 integrin controls these interactions and ultimately apoptosis. PMID:16914549

  2. TM4SF5 suppression disturbs integrin α5-related signalling and muscle development in zebrafish.

    PubMed

    Choi, Yoon-Ju; Kim, Hyun Ho; Kim, Jeong-Gyun; Kim, Hye-Jin; Kang, Minkyung; Lee, Mi-Sook; Ryu, Jihye; Song, Haeng Eun; Nam, Seo Hee; Lee, Doohyung; Kim, Kyu-Won; Lee, Jung Weon

    2014-08-15

    TM4SF5 (transmembrane 4 L six family member 5) is involved in EMT (epithelial-mesenchymal transition) for liver fibrosis and cancer metastasis; however, the function(s) of TM4SF5 during embryogenesis remains unknown. In the present study the effects of TM4SF5 on embryogenesis of zebrafish were investigated. tm4sf5 mRNA was expressed in the posterior somites during somitogenesis and in whole myotome 1 dpf (day post-fertilization). tm4sf5 suppression impaired development of the trunk with aberrant morphology of muscle fibres and altered expression of integrin α5. The arrangement and adhesion of muscle cells were abnormally disorganized in tm4sf5 morphants with reduced muscle fibre masses, where integrin α5-related signalling molecules, including fibronectin, FAK (focal adhesion kinase), vinculin and actin were aberrantly localized, compared with those in control fish. Aberrant muscle developments in tm4sf5 morphants were recovered by additional tm4sf5 or integrin α5 mRNA injection. Such a role for TM4SF5 was observed in the differentiation of C2C12 mouse myoblast cells to multinuclear muscle cells. Taken together, the results show that TM4SF5 controls muscle differentiation via co-operation with integrin α5-related signalling. PMID:24897542

  3. Identification of β integrin-like- and fibronectin-like proteins in the bivalve mollusk Mytilus trossulus.

    PubMed

    Dyachuk, Vyacheslav A; Maiorova, Maria A; Odintsova, Nelly A

    2015-09-01

    Integrins play a key role in the intermediation and coordination between cells and extracellular matrix components. In this study, we first determined the presence of the β integrin-like protein and its presumptive ligand, fibronectin-like protein, during development and in some adult tissues of the bivalve mollusc Mytilus trossulus. We found that β integrin-like protein expression correlated with the development and differentiation of the digestive system in larvae. Besides the presence of β integrin-like protein in the digestive epithelial larval cells, this protein was detected in the hemocytes and some adult tissues of M. trossulus. The fibronectin-like protein was detected firstly at the blastula stage and later, the FN-LP-immunoreactive cells were scattered in the trochophore larvae. The fibronectin-like protein was not expressed in the β integrin-positive cells of either the veliger stage larvae or the adult mussel tissues and the primary hemocyte cell culture. Despite the β integrin- and fibronectin-like proteins being expressed in different cell types of mussel larvae, we do not exclude the possibility of direct interaction between these two proteins during M. trossulus development or in adult tissues. PMID:26183371

  4. Human fallopian tube epithelium constitutively expresses integrin endometrial receptivity markers: no evidence for a tubal implantation window.

    PubMed

    Brown, J K; Shaw, J L V; Critchley, H O D; Horne, A W

    2012-03-01

    Understanding of ectopic implantation within the Fallopian tube (FT) is limited. In the human uterus, the putative 'window of implantation' in the mid-luteal phase of the menstrual cycle is accompanied by increased endometrial epithelial expression of the integrins α(1)β(1), α(4)β(1) and α(v)β(3) and its ligand osteopontin. Similar cyclical changes in FT integrin expression have been proposed to contribute to ectopic implantation, but supporting data are limited. In the current study, we present quantitative data on human FT transcription and translation of the integrin subunits α(1), α(4), α(V), β(1) and β(3) during the follicular and mid-luteal phases of the menstrual cycle, together with a supporting immuocytochemical analysis of their spatial distribution within the FT, and that of osteopontin. In contrast to previous studies, our data indicate that all five integrin receptivity markers are constitutively transcribed and translated in the FT, with no evidence for changes in their expression or distribution during the window of implantation in the mid-luteal phase of the cycle. Furthermore, we could find no evidence for cyclic redistribution of the integrin α(v)β(3) ligand osteopontin within the FT. Although we do not rule out the involvement of integrin endometrial receptivity markers in the establishment of ectopic pregnancy, our findings do not support their differential expression during a tubal implantation window. PMID:22002573

  5. Fibrin and Collagen Differentially but Synergistically Regulate Sprout Angiogenesis of Human Dermal Microvascular Endothelial Cells in 3-Dimensional Matrix

    PubMed Central

    Tonnesen, Marcia G.; Mousa, Shaker A.; Clark, Richard A. F.

    2013-01-01

    Angiogenesis is a highly regulated event involving complex, dynamic interactions between microvascular endothelial cells and extracellular matrix (ECM) proteins. Alteration of ECM composition and architecture is a hallmark feature of wound clot and tumor stroma. We previously reported that during angiogenesis, endothelial cell responses to growth factors are modulated by the compositional and mechanical properties of a surrounding three-dimensional (3D) extracellular matrix (ECM) that is dominated by either cross-linked fibrin or type I collagen. However, the role of 3D ECM in the regulation of angiogenesis associated with wound healing and tumor growth is not well defined. This study investigates the correlation of sprout angiogenesis and ECM microenvironment using in vivo and in vitro 3D angiogenesis models. It demonstrates that fibrin and type I collagen 3D matrices differentially but synergistically regulate sprout angiogenesis. Thus blocking both integrin alpha v beta 3 and integrin alpha 2 beta 1 might be a novel strategy to synergistically block sprout angiogenesis in solid tumors. PMID:23737792

  6. VLA-4 integrin can mediate CD11/CD18-independent transendothelial migration of human monocytes.

    PubMed Central

    Chuluyan, H E; Issekutz, A C

    1993-01-01

    The migration of human monocytes across unactivated and activated human umbilical vein endothelium (HUVE) in response to chemotactic factors was studied, and the adhesion molecules involved were characterized. Migration of blood monocytes or U937 cell line-derived monocytes across unactivated HUVE induced by C5a, was partially inhibited (by 75%) by mAbs (R15.7 or 60.3) to CD18 of the CD11/CD18 complex on the monocyte. However, when the HUVE was pretreated for 5 h with IL-1 alpha (0.1 ng/ml), TNF-alpha (100 U/ml), or LPS (1 ng/ml), migration induced by C5a was no longer inhibited; i.e., migration became CD18 independent. The monocyte CD18-independent migration was completely blocked by mAbs against alpha 4 or beta 1 integrin chains of VLA-4. This migration was also partially inhibited by mAbs against vascular cell adhesion molecule-1 (VCAM-1), a major counter-receptor on HUVE for VLA-4, but not by mAbs to E-selectin or intercellular adhesion molecule-1. The significant CD18-independent migration across "unactivated" HUVE was also inhibited by mAbs against alpha 4 or beta 1 chains of VLA-4, although mAbs against VCAM-1 did not inhibit under these conditions. Finally, considerable VLA-4-dependent transendothelial migration to C5a was also observed with monocytes from a patient with CD18 deficiency (leukocyte adhesion deficiency). These results suggest that (a) there is a major CD18-independent component in monocyte chemotactic factor-dependent migration across activated and unactivated endothelium; (b) that VLA-4 integrin on the monocyte has a major role in this migration; and (c) that VCAM-1 on activated endothelium functions as a counter-receptor in this process, but other ligands for VLA-4, especially on unactivated endothelium, may also be involved. Images PMID:7902847

  7. Discovery of Platyhelminth-Specific α/β-Integrin Families and Evidence for Their Role in Reproduction in Schistosoma mansoni

    PubMed Central

    Beckmann, Svenja; Quack, Thomas; Dissous, Colette; Cailliau, Katia; Lang, Gabriele; Grevelding, Christoph G.

    2012-01-01

    In all metazoa, the response of cells to molecular stimuli from their environment represents a fundamental principle of regulatory processes controlling cell growth and differentiation. Among the membrane-linked receptors mediating extracellular communication processes are integrin receptors. Besides managing adhesion to the extracellular matrix or to other cells, they arrange information flow into the cells by activating intracellular signaling pathways often acting synergistically through cooperation with growth factor receptors. Although a wealth of information exists on integrins in different model organisms, there is a big gap of knowledge for platyhelminths. Here we report on the in silico detection and reconstruction of α and β integrins from free-living and parasitic platyhelminths, which according to structural and phylogenetic analyses form specific clades separate from each other and from further metazoan integrins. As representative orthologs of parasitic platyhelminths we have cloned one beta-integrin (Smβ-Int1) and four alpha-integrins (Smα-Int1 - Smα-Int4) from Schistosoma mansoni; they were characterized by molecular and biochemical analyses. Evidence is provided that Smβ-Int1 interacts and co-localizes in the reproductive organs with known schistosome cellular tyrosine kinases (CTKs), of which the Syk kinase SmTK4 appeared to be the strongest interaction partner as shown by yeast two-hybrid analyses and coimmunoprecipitation experiments. By a novel RNAi approach with adult schistosomes in vitro we demonstrate for the first time multinucleated oocytes in treated females, indicating a decisive role Smβ-Int1 during oogenesis as phenotypically analyzed by confocal laser scanning microscopy (CLSM). Our findings provide a first comprehensive overview about platyhelminth integrins, of which the parasite group exhibits unique features allowing a clear distinction from the free-living groups. Furthermore, we shed first lights on the functions of

  8. The Role of Integrins in the Trabecular Meshwork

    PubMed Central

    Gagen, Debjani; Faralli, Jennifer A.; Filla, Mark S.

    2014-01-01

    Abstract Integrins are a family of heterodimeric transmembrane receptors that mediate adhesion to the extracellular matrix (ECM). However, integrins are not just adhesion receptors. They can act as “bidirectional signal transducers” that coordinate a large number of cellular activities in response to the extracellular environment and intracellular signaling events. Among the activities regulated by integrins are cell adhesion, assembly of the ECM, growth factor signaling, apoptosis, organization of the cytoskeleton, and cytoskeleton-mediated processes such as contraction, endocytosis, and phagocytosis. Integrins regulate these activities through a complex network of intracellular signaling kinases and adaptor proteins that associate with the transmembrane and cytoplasmic domains of the integrin subunits. In this review, we will discuss how some of the known integrin-mediated activities can control the function of the trabecular meshwork. We will also discuss how integrin activity is a tightly regulated process that involves conformation changes within the heterodimer which are mediated by specific integrin-binding proteins. PMID:24266581

  9. ITGAV and ITGA5 diversely regulate proliferation and adipogenic differentiation of human adipose derived stem cells.

    PubMed

    Morandi, E M; Verstappen, R; Zwierzina, M E; Geley, S; Pierer, G; Ploner, C

    2016-01-01

    The fate of human adipose tissue stem cells (ASCs) is largely determined by biochemical and mechanical cues from the extracellular matrix (ECM), which are sensed and transmitted by integrins. It is well known that specific ECM constituents influence ASC proliferation and differentiation. Nevertheless, knowledge on how individual integrins regulate distinct processes is still limited. We performed gene profiling of 18 alpha integrins in sorted ASCs and adipocytes, identifying downregulations of RGD-motif binding integrins integrin-alpha-V (ITGAV) and integrin-alpha-5 (ITGA5), upregulation of laminin binding and leukocyte-specific integrins and individual regulations of collagen and LDV-receptors in differentiated adipocytes in-vivo. Gene function analyses in in-vitro cultured ASCs unraveled differential functions of ITGA5 and ITGAV. Knockdown of ITGAV, but not ITGA5 reduced proliferation, caused p21(Cip1) induction, repression of survivin and specific regulation of Hippo pathway mediator TAZ. Gene knockdown of both integrins promoted adipogenic differentiation, while transgenic expression impaired adipogenesis. Inhibition of ITGAV using cilengitide resulted in a similar phenotype, mimicking loss of pan-ITGAV expression using RNAi. Herein we show ASC specific integrin expression patterns and demonstrate distinct regulating roles of both integrins in human ASCs and adipocyte physiology suggesting a negative impact of RDG-motif signaling on adipogenic differentiation of ASCs via ITGA5 and ITGAV. PMID:27363302

  10. ITGAV and ITGA5 diversely regulate proliferation and adipogenic differentiation of human adipose derived stem cells

    PubMed Central

    Morandi, E. M.; Verstappen, R.; Zwierzina, M. E.; Geley, S.; Pierer, G.; Ploner, C.

    2016-01-01

    The fate of human adipose tissue stem cells (ASCs) is largely determined by biochemical and mechanical cues from the extracellular matrix (ECM), which are sensed and transmitted by integrins. It is well known that specific ECM constituents influence ASC proliferation and differentiation. Nevertheless, knowledge on how individual integrins regulate distinct processes is still limited. We performed gene profiling of 18 alpha integrins in sorted ASCs and adipocytes, identifying downregulations of RGD-motif binding integrins integrin-alpha-V (ITGAV) and integrin-alpha-5 (ITGA5), upregulation of laminin binding and leukocyte-specific integrins and individual regulations of collagen and LDV-receptors in differentiated adipocytes in-vivo. Gene function analyses in in-vitro cultured ASCs unraveled differential functions of ITGA5 and ITGAV. Knockdown of ITGAV, but not ITGA5 reduced proliferation, caused p21Cip1 induction, repression of survivin and specific regulation of Hippo pathway mediator TAZ. Gene knockdown of both integrins promoted adipogenic differentiation, while transgenic expression impaired adipogenesis. Inhibition of ITGAV using cilengitide resulted in a similar phenotype, mimicking loss of pan-ITGAV expression using RNAi. Herein we show ASC specific integrin expression patterns and demonstrate distinct regulating roles of both integrins in human ASCs and adipocyte physiology suggesting a negative impact of RDG-motif signaling on adipogenic differentiation of ASCs via ITGA5 and ITGAV. PMID:27363302

  11. The immunomodulatory activity of human amniotic fluid can be correlated with transforming growth factor-beta 1 (TGF-beta 1) and beta 2 activity.

    PubMed Central

    Lang, A K; Searle, R F

    1994-01-01

    The role of alphafetoprotein (AFP) in the immunomodulatory activity of amniotic fluids (AF) from normally progressing human pregnancy (weeks 14-16) was investigated. A panel of 42 AF (25% v/v) reduced significantly phytohaemagglutinin (PHA)-induced peripheral blood mononuclear cell (PBMC) proliferation in serum-free cultures with a mean per cent inhibition of 68.4 +/- 5.5%. In contrast, AFP preparations, with one exception (U.AFP), failed to display inhibitory activity. Pretreatment of AF with anti-TGF-beta 1 and beta 2 antibodies used alone resulted in the mean per cent loss of inhibition of 33.1 +/- 3.9% and 52.3 +/- 7.5%, respectively. A summative loss of AF-mediated inhibition was detected when anti-TGF-beta 1 and beta 2 antibodies were used in combination, but immunomodulation was rarely abolished 100% by this treatment. Anti-TGF-beta 2 antibody treatment, unlike anti-TGF-beta 1 antibody treatment, reversed the inhibitory activity of U.AFP. The amount of TGF-beta 1 and beta 2 contained in human AF was studied by growth inhibition of Mv1 Lu cells. The mean levels of TGF-beta 1 and beta 2 in AF were 11 +/- 0.9 U/ml and 2.3 +/- 0.4 U/ml, respectively, which corresponds with a mean per cent inhibition of 49 +/- 4.7%. U.AFP also significantly inhibited Mv1 Lu cell growth. To investigate the mechanism of AF-mediated inhibition, the effect of AF and AFP on IL-2 production by concanavalin A (Con A)-stimulated PBMC blasts was determined by the CTLL-2 cell bioassay. IL-2 production was reduced 55.5% in AF-treated blasts and 61% in U.AFP-treated blasts compared with controls. Our findings indicate that the immunomodulatory activity of human AF can be correlated with TGF-beta 1 and beta 2 and not with AFP, the inhibitory activity of U.AFP preparation reflecting copurifying TGF-beta 2 activity. PMID:7518368

  12. Expression of Cyclooxygenase-2 and Transforming Growth Factor-beta1 in HCV-Induced Chronic Liver Disease and Hepatocellular Carcinoma

    PubMed Central

    El-Bassiouny, Azza E.I.; Zoheiry, Mona M.K.; Nosseir, Mona M.F.; El-Ahwany, Eman G.; Ibrahim, Raafat A.; El-Bassiouni, Nora E.I.

    2007-01-01

    Cyclooxygenase-2 (COX-2) and transforming growth factor-beta1 (TGF-beta1) were modulated in a variety of viral infections, but there is a paucity of data about their role in the pathologic process of cirrhosis and/or hepatocellular carcinoma (HCC) following chronic hepatitis C virus (HCV) infection. The material of the current study included 50 cases of chronic hepatitis C (CHC) without cirrhosis, 30 cases of CHC with cirrhosis, and 30 cases of HCC with HCV admitted to the Gastroenterology and Hepatology Department of Theodor Bilharz Research Institute, Giza, Egypt. Fifteen wedge liver biopsies, taken during laparoscopic cholecystectomy, were included in the study as normal controls. Laboratory investigations, serologic markers for viral hepatitis, and serum alpha fetoprotein levels (alpha-FP) were done for all cases of the study. Immunohistochemistry using primary antibodies against both factors revealed weak to faint immunoreactivity to COX-2 and TGF-beta1 in normal hepatic tissue (< 30% and < 50% of the cells, respectively). COX-2 expression was upregulated in patients with CHC with and without cirrhosis, yet 80% of positively stained cirrhotic cases showed marked staining intensity. Higher COX-2 expression was observed in well-differentiated HCC cases (80%) with marked staining intensity (75%) compared with advanced HCC tumors (P < .001). TGF-beta1 was expressed in the hepatocytes of all cases of CHC with and without cirrhosis as well as in 67% of HCC cases. Extensive cytoplasmic expression was detected in 52%, 93.3%, and 46.6% of CHC patients without cirrhosis, patients with cirrhosis, and patients with HCC, respectively. A positive correlation was observed between hepatic expression of COX-2 and TGF-beta1 (r = 0.67, P < .05); however, no correlation was detected between the latter and grade of HCC differentiation (r = 0.33, P > .05). Conclusion These findings may suggest that TGF-beta1 plays a role in hepatic cell damage following HCV infection thus stressing

  13. Tumour nuclear oestrogen receptor beta 1 correlates inversely with parathyroid tumour weight

    PubMed Central

    Haglund, Felix; Rosin, Gustaf; Nilsson, Inga-Lena; Juhlin, C Christofer; Pernow, Ylva; Norenstedt, Sophie; Dinets, Andrii; Larsson, Catharina; Hartman, Johan; Höög, Anders

    2015-01-01

    Primary hyperparathyroidism (PHPT) is a common endocrinopathy, frequently caused by a parathyroid adenoma, rarely by a parathyroid carcinoma that lacks effective oncological treatment. As the majority of cases are present in postmenopausal women, oestrogen signalling has been implicated in the tumourigenesis. Oestrogen receptor beta 1 (ERB1) and ERB2 have been recently identified in parathyroid adenomas, the former inducing genes coupled to tumour apoptosis. We applied immunohistochemistry and slide digitalisation to quantify nuclear ERB1 and ERB2 in 172 parathyroid adenomas, atypical adenomas and carcinomas, and ten normal parathyroid glands. All the normal parathyroid glands expressed ERB1 and ERB2. The majority of tumours expressed ERB1 (70.6%) at varying intensities, and ERB2 (96.5%) at strong intensities. Parathyroid carcinomas expressed ERB1 in three out of six cases and ERB2 in five out of six cases. The intensity of tumour nuclear ERB1 staining significantly correlated inversely with tumour weight (P=0.011), and patients whose tumours were classified as ERB1-negative had significantly greater tumour weight as well as higher serum calcium (P=0.002) and parathyroid hormone levels (P=0.003). Additionally, tumour nuclear ERB1 was not expressed differentially with respect to sex or age of the patient. Levels of tumour nuclear ERB2 did not correlate with clinical characteristics. In conclusion, decreased ERB1 immunoreactivity is associated with increased tumour weight in parathyroid adenomas. Given the previously reported correlation with tumour-suppressive signalling, selective oestrogen receptor modulation (SERMs) may play a role in the treatment of parathyroid carcinomas. Future studies of SERMs and oestrogen treatment in PHPT should consider tumour weight as a potential factor in pharmacological responsiveness. PMID:25648860

  14. Copper-GHK increases integrin expression and p63 positivity by keratinocytes.

    PubMed

    Kang, Youn-A; Choi, Hye-Ryung; Na, Jung-Im; Huh, Chang-Hun; Kim, Min-Ji; Youn, Sang-Woong; Kim, Kyu-Han; Park, Kyoung-Chan

    2009-04-01

    Glycyl-L-histidyl-L-lysyl (GHK) possesses a high affinity for copper(II) ions, with which it spontaneously forms a complex (copper-GHK). It is well known that copper-GHK plays a physiological role in the process of wound healing and tissue repair by stimulating collagen synthesis in fibroblasts. This study was conducted to investigate the effects of copper-GHK on keratinocytes. Proliferative effects were analyzed and hematoxylin and eosin staining and immunohistochemistry were conducted to evaluate the effects of copper-GHK in skin equivalent (SE) models. In addition, western blotting was performed. In monolayer cultured keratinocytes, copper-GHK increased the proliferation of keratinocytes. When the SE models were evaluated, basal cells became cuboidal when copper-GHK was added. Immunohistochemical analysis revealed that copper-GHK increased proliferating cell nuclear antigen (PCNA) and p63 positivity. Furthermore, the expression of integrin alpha6 and beta1 increased in SE models, and these results were confirmed by Western blotting. The results of this study indicate that treatment with copper-GHK may increase the proliferative potential of basal keratinocytes by modulating the expression of integrins, p63 and PCNA. In addition, increased levels of p63, a putative stem cell marker of the skin, suggests that copper-GHK promotes the survival of basal stem cells in the skin. PMID:19319546

  15. Identification of the integrin binding domain of the Yersinia pseudotuberculosis invasin protein.

    PubMed

    Leong, J M; Fournier, R S; Isberg, R R

    1990-06-01

    The invasin protein of the pathogenic Yersinia pseudotuberculosis mediates entry of the bacterium into cultured mammalian cells by binding several beta 1 chain integrins. In this study, we identified the region of invasin responsible for cell recognition. Thirty-two monoclonal antibodies directed against invasin were isolated, and of those, six blocked cell attachment to invasin. These six antibodies recognized epitopes within the last 192 amino acids of invasin. Deletion mutants of invasin and maltose-binding protein (MBP)--invasin fusion proteins were generated and tested for cell attachment. All of the invasin derivatives that carried the carboxyl-terminal 192 amino acids retained cell binding activity. One carboxyl-terminal invasin fragment and seven MBP--invasin fusion proteins were purified. The purified derivatives that retained binding activity inhibited bacterial entry into cultured mammalian cells. These results indicated that the carboxyl-terminal 192 amino acids of invasin contains the integrin-binding domain, even though this region does not contain the tripeptide sequence Arg-Gly-Asp. PMID:1693333

  16. Rapid isolation of integrin rich multipotent stem cell pool and reconstruction of mouse epidermis equivalent.

    PubMed

    Kumar, Sushil; Poojan, Shiv; Verma, Vikas; Verma, Mukesh K; Lohani, Mohatashim

    2014-01-01

    We describe here epidermis reconstruction using multipotent mouse epidermal stem cells (EpSCs) enriched from keratinocyte isolates exploting exclusively the stem cell-adhesive property. This method excluded flowcytometry and was swift. Percent enrichment was measured by the uptake of Propidium iodide and Hoechst-33342 dye using flowcytometry to determine EpSCs yield. The sorted cells were characterized by analysis of stem cell markers using immunocytochemistry and immunoblotting techniques. Epidermis was reconstructed using the identified seeding density of EpSCs and the airlift tissue culture. Histology of natural vs reconstructed mammalian epidermis was also compared. Results showed a radical improvement of near 99% in the yield of integrin overexpressing EpSCs. The enriched EpSCs tested positive for biomarkers namely cytokeratin K-15 and, K-14, p63, beta-1-integrin, CD34 and could be passaged for longer durations. Adhesion sorted cells reconstructed the epidermis. The process of tissue reconstruction was faster using the adhesion sorted cells than the FACS sorted EpSCs. The product bioengineered using multipotent EpSCs was histologically similar to normal epidermis. Features like strata basalae, spinosum, granulosum, and corneum were alike real epidermis. The reconstructed epidermis displayed normal homeostasis, which can be considered an approximating actual product for investigative dermatology, toxicology, therapeutic research, regenerative medicine, and tissue engineering. PMID:24660112

  17. Fluid shear-induced mechanical signaling in MC3T3-E1 osteoblasts requires cytoskeleton-integrin interactions

    NASA Technical Reports Server (NTRS)

    Pavalko, F. M.; Chen, N. X.; Turner, C. H.; Burr, D. B.; Atkinson, S.; Hsieh, Y. F.; Qiu, J.; Duncan, R. L.

    1998-01-01

    Mechanical stimulation of bone induces new bone formation in vivo and increases the metabolic activity and gene expression of osteoblasts in culture. We investigated the role of the actin cytoskeleton and actin-membrane interactions in the transmission of mechanical signals leading to altered gene expression in cultured MC3T3-E1 osteoblasts. Application of fluid shear to osteoblasts caused reorganization of actin filaments into contractile stress fibers and involved recruitment of beta1-integrins and alpha-actinin to focal adhesions. Fluid shear also increased expression of two proteins linked to mechanotransduction in vivo, cyclooxygenase-2 (COX-2) and the early response gene product c-fos. Inhibition of actin stress fiber development by treatment of cells with cytochalasin D, by expression of a dominant negative form of the small GTPase Rho, or by microinjection into cells of a proteolytic fragment of alpha-actinin that inhibits alpha-actinin-mediated anchoring of actin filaments to integrins at the plasma membrane each blocked fluid-shear-induced gene expression in osteoblasts. We conclude that fluid shear-induced mechanical signaling in osteoblasts leads to increased expression of COX-2 and c-Fos through a mechanism that involves reorganization of the actin cytoskeleton. Thus Rho-mediated stress fiber formation and the alpha-actinin-dependent anchorage of stress fibers to integrins in focal adhesions may promote fluid shear-induced metabolic changes in bone cells.

  18. Digestion of Yeasts and Beta-1,3-Glucanases in Mosquito Larvae: Physiological and Biochemical Considerations

    PubMed Central

    Souza, Raquel Santos; Diaz-Albiter, Hector Manuel; Dillon, Vivian Maureen; Dillon, Rod J.; Genta, Fernando Ariel

    2016-01-01

    Aedes aegypti larvae ingest several kinds of microorganisms. In spite of studies regarding mosquito digestion, little is known about the nutritional utilization of ingested cells by larvae. We investigated the effects of using yeasts as the sole nutrient source for A. aegypti larvae. We also assessed the role of beta-1,3-glucanases in digestion of live yeast cells. Beta-1,3-glucanases are enzymes which hydrolyze the cell wall beta-1,3-glucan polyssacharide. Larvae were fed with cat food (controls), live or autoclaved Saccharomyces cerevisiae cells and larval weight, time for pupation and adult emergence, larval and pupal mortality were measured. The presence of S. cerevisiae cells inside the larval gut was demonstrated by light microscopy. Beta-1,3-glucanase was measured in dissected larval samples. Viability assays were performed with live yeast cells and larval gut homogenates, with or without addition of competing beta-1,3-glucan. A. aegypti larvae fed with yeast cells were heavier at the 4th instar and showed complete development with normal mortality rates. Yeast cells were efficiently ingested by larvae and quickly killed (10% death in 2h, 100% in 48h). Larvae showed beta-1,3-glucanase in head, gut and rest of body. Gut beta-1,3-glucanase was not derived from ingested yeast cells. Gut and rest of body activity was not affected by the yeast diet, but head homogenates showed a lower activity in animals fed with autoclaved S. cerevisiae cells. The enzymatic lysis of live S. cerevisiae cells was demonstrated using gut homogenates, and this activity was abolished when excess beta-1,3-glucan was added to assays. These results show that live yeast cells are efficiently ingested and hydrolyzed by A. aegypti larvae, which are able to fully-develop on a diet based exclusively on these organisms. Beta-1,3-glucanase seems to be essential for yeast lytic activity of A. aegypti larvae, which possess significant amounts of these enzyme in all parts investigated. PMID

  19. Synthesis of glycerophosphorylated cyclic beta-(1,2)-glucans by Rhizobium meliloti ndv mutants.

    PubMed Central

    Breedveld, M W; Yoo, J S; Reinhold, V N; Miller, K J

    1994-01-01

    The periplasmic cyclic beta-(1,2)-glucans of Rhizobium spp. are believed to provide functions during hypoosmotic adaptation and legume nodulation. In Rhizobium meliloti, cyclic beta-(1,2)-glucans are synthesized at highest levels when cells are grown at low osmolarity, and a considerable fraction (> or = 35%) of these glucans may become substituted with phosphoglycerol moieties. Thus far, two chromosomally encoded proteins, NdvA and NdvB, have been shown to function during cyclic beta-(1,2)-glucan biosynthesis; however, the precise roles for these proteins remain unclear. In the present study, we show that R. meliloti mutants lacking up to one-third of the downstream region of ndvB synthesize cyclic beta-(1,2)-glucans similar to those produced by wild-type cells with respect to size and phosphoglycerol substituent profile. In contrast, no phosphoglycerol substituents were detected on the cyclic beta-(1,2)-glucans synthesized by an R. meliloti ndvA mutant. PMID:8106315

  20. Mammary tumor suppression by transforming growth factor beta 1 transgene expression.

    PubMed Central

    Pierce, D F; Gorska, A E; Chytil, A; Meise, K S; Page, D L; Coffey, R J; Moses, H L

    1995-01-01

    In cell culture, type alpha transforming growth factor (TGF-alpha) stimulates epithelial cell growth, whereas TGF-beta 1 overrides this stimulatory effect and is growth inhibitory. Transgenic mice that overexpress TGF-alpha under control of the mouse mammary tumor virus (MMTV) promoter/enhancer exhibit mammary ductal hyperplasia and stochastic development of mammary carcinomas, a process that can be accelerated by administration of the chemical carcinogen 7,12-dimethylbenz[a]anthracene. MMTV-TGF-beta 1 transgenic mice display mammary ductal hypoplasia and do not develop mammary tumors. We report that in crossbreeding experiments involving the production of mice carrying both the MMTV-TGF-beta 1 and MMTV-TGF-alpha transgenes, there is marked suppression of mammary tumor formation and that MMTV-TGF-beta 1 transgenic mice are resistant to 7,12-dimethylbenz[a]anthracene-induced mammary tumor formation. These data demonstrate that overexpression of TGF-beta 1 in vivo can markedly suppress mammary tumor development. Images Fig. 1 Fig. 3 PMID:7753792

  1. Modulation of sodium current in mammalian cells by an epilepsy-correlated beta 1-subunit mutation.

    PubMed

    Tammaro, Paolo; Conti, Franco; Moran, Oscar

    2002-03-01

    The syndrome of generalized epilepsy with febrile seizure plus (GEFS+) is associated with a single point mutation on the gene SCN1B that results in a substitution of the cysteine 121 with a tryptophane in the sodium channel beta 1-subunit protein. We have studied, in the HEK cells permanently transfected with the skeletal muscle sodium channel alpha-subunit (SkM1), the effects of a transient transfection of the wild type (WT) or C121W mutant beta 1-subunit. Coexpression of the WT beta 1 produces two effects on the sodium currents expressed in mammalian cells: the increase in the density of sodium channels, and the modulation of the inactivation of the sodium currents, inducing a hastening of the recovery from the inactivation. This modulation is less severe as observed when sodium channels are expressed in frog oocytes. We have observed that mutant C121W lacks this modulatory property, but maintains its property to increase the current density. Our observation suggests a possible involvement of this lack of modulation in the development of the GEFS+, providing the first hypothesis based on the observation of the functional properties of the beta 1-subunit C121W mutant in mammalian cells, which certainly represents a more physiological preparation, instead of in Xenopus oocytes, where the modulatory properties of the beta 1-subunit are artificially amplified. PMID:11866477

  2. Properties of a beta-(1-4)-glucan hydrolase from Aspergillus niger.

    PubMed

    Clarke, A E; Stone, B A

    1965-09-01

    1. A beta-(1-->4)-glucan hydrolase prepared from Aspergillus niger, as described by Clarke & Stone (1965a), showed a pH optimum in the range 4.5-6 and K(m) 0.25% when acting on a cellulose dextrin sulphate substrate. 2. The hydrolase rapidly decreased the specific viscosity of carboxymethylcellulose with a small increase in the production of reducing sugars. The identity of the products of hydrolysis of cellotetraose, cellopentaose and their reduced analogues indicate a preferential cleavage of non-terminal glucosidic linkages. The enzyme may be described as beta-(1-->4)-glucan 4-glucanohydrolase (EC 3.2.1.4). 3. In addition to carboxymethylcellulose, cellulose dextrins, cellopentaose and cellotetraose the enzyme fraction hydrolysed lichenin, oat and barley glucans, ivory-nut mannan and a glucomannan from Konjak flour. No hydrolysis of wheat-straw beta-(1-->4)-xylan, Lupinus albus beta-(1-->4)-galactan, pneumococcal type III polysaccharide, chitin, hyaluronic acid, laminarin, pachydextrins, carboxymethylpachyman or beta-(1-->3)-oligoglucosides was detected. 4. The hydrolase showed no transglycosylase activity from cellodextrin or cellopentaose substrates to glucose or methanol acceptors. 5. The hydrolysis of cellodextrins was inhibited completely by 1.0mm-Hg(2+), 0.7mm-phenylmercuric nitrate and 1.0mm-iodine. PMID:5862418

  3. Targeted disruption of the mouse beta1-adrenergic receptor gene: developmental and cardiovascular effects.

    PubMed Central

    Rohrer, D K; Desai, K H; Jasper, J R; Stevens, M E; Regula, D P; Barsh, G S; Bernstein, D; Kobilka, B K

    1996-01-01

    At least three distinct beta-adrenergic receptor (beta-AR) subtypes exist in mammals. These receptors modulate a wide variety of processes, from development and behavior, to cardiac function, metabolism, and smooth muscle tone. To understand the roles that individual beta-AR subtypes play in these processes, we have used the technique of gene targeting to create homozygous beta 1-AR null mutants (beta 1-AR -/-) in mice. The majority of beta 1-AR -/- mice die prenatally, and the penetrance of lethality shows strain dependence. Beta l-AR -/- mice that do survive to adulthood appear normal, but lack the chronotropic and inotropic responses seen in wild-type mice when beta-AR agonists such as isoproterenol are administered. Moreover, this lack of responsiveness is accompanied by markedly reduced stimulation of adenylate cyclase in cardiac membranes from beta 1-AR -/- mice. These findings occur despite persistent cardiac beta 2-AR expression, demonstrating the importance of beta 1-ARs for proper mouse development and cardiac function, while highlighting functional differences between beta-AR subtypes. Images Fig. 1 Fig. 3 PMID:8693001

  4. Effects of transforming growth factor beta 1 on the regulation of osteoclastic development and function

    SciTech Connect

    Hattersley, G.; Chambers, T.J. )

    1991-02-01

    Transforming growth factor (TGF) beta 1 is a multifunctional cytokine with powerful effects on osteoblastic cells. Its role in the regulation of osteoclast generation and function, however, is unclear. It has been reported both to stimulate and to inhibit resorption in organ culture and to inhibit multinuclear cell formation in bone marrow cultures. We tested the effects of TGF-beta 1 on bone resorption by osteoclasts isolated from neonatal rat long bones. We found potent stimulation of osteoclastic bone resorption, mediated by osteoblastic cells, with an EC50 of 10 pg/ml, considerably lower than that of well-documented osteotropic hormones. Stimulation was not mediated by Swiss mouse 3T3 cells, a nonosteoblastic cell line. TGF-beta 1 strongly inhibited the generation of calcitonin receptor (CTR)-positive cells in mouse bone marrow cultures, but as for isolated osteoclasts, bone resorption per CTR-positive cell was increased. The inhibition of CTR-positive cell formation was associated with suppression of maturation of other bone marrow derivatives and may be related more to the known ability of TGF-beta 1 to suppress the proliferation of primitive hematopoietic cells than to a specific role of TGF-beta 1 in osteoclast generation.

  5. IL-4 and TGF-beta 1 counterbalance one another while regulating mast cell homeostasis.

    PubMed

    Macey, Matthew R; Sturgill, Jamie L; Morales, Johanna K; Falanga, Yves T; Morales, Joshua; Norton, Sarah K; Yerram, Nitin; Shim, Hoon; Fernando, Josephine; Gifillan, Alasdair M; Gomez, Gregorio; Schwartz, Lawrence; Oskeritzian, Carole; Spiegel, Sarah; Conrad, Daniel; Ryan, John J

    2010-05-01

    Mast cell responses can be altered by cytokines, including those secreted by Th2 and regulatory T cells (Treg). Given the important role of mast cells in Th2-mediated inflammation and recent demonstrations of Treg-mast cell interactions, we examined the ability of IL-4 and TGF-beta1 to regulate mast cell homeostasis. Using in vitro and in vivo studies of mouse and human mast cells, we demonstrate that IL-4 suppresses TGF-beta1 receptor expression and signaling, and vice versa. In vitro studies demonstrated that IL-4 and TGF-beta1 had balancing effects on mast cell survival, migration, and FcepsilonRI expression, with each cytokine cancelling the effects of the other. However, in vivo analysis of peritoneal inflammation during Nippostrongylus brasiliensis infection in mice revealed a dominant suppressive function for TGF-beta1. These data support the existence of a cytokine network involving the Th2 cytokine IL-4 and the Treg cytokine TGF-beta1 that can regulate mast cell homeostasis. Dysregulation of this balance may impact allergic disease and be amenable to targeted therapy. PMID:20304823

  6. Altered extent of cross-linking of beta1,6-glucosylated mannoproteins to chitin in Saccharomyces cerevisiae mutants with reduced cell wall beta1,3-glucan content.

    PubMed Central

    Kapteyn, J C; Ram, A F; Groos, E M; Kollar, R; Montijn, R C; Van Den Ende, H; Llobell, A; Cabib, E; Klis, F M

    1997-01-01

    The yeast cell wall contains beta1,3-glucanase-extractable and beta1,3-glucanase-resistant mannoproteins. The beta1,3-glucanase-extractable proteins are retained in the cell wall by attachment to a beta1,6-glucan moiety, which in its turn is linked to beta1,3-glucan (J. C. Kapteyn, R. C. Montijn, E. Vink, J. De La Cruz, A. Llobell, J. E. Douwes, H. Shimoi, P. N. Lipke, and F. M. Klis, Glycobiology 6:337-345, 1996). The beta1,3-glucanase-resistant protein fraction could be largely released by exochitinase treatment and contained the same set of beta1,6-glucosylated proteins, including Cwp1p, as the B1,3-glucanase-extractable fraction. Chitin was linked to the proteins in the beta1,3-glucanase-resistant fraction through a beta1,6-glucan moiety. In wild-type cell walls, the beta1,3-glucanase-resistant protein fraction represented only 1 to 2% of the covalently linked cell wall proteins, whereas in cell walls of fks1 and gas1 deletion strains, which contain much less beta1,3-glucan but more chitin, beta1,3-glucanase-resistant proteins represented about 40% of the total. We propose that the increased cross-linking of cell wall proteins via beta1,6-glucan to chitin represents a cell wall repair mechanism in yeast, which is activated in response to cell wall weakening. PMID:9335273

  7. Tumor cell migration and invasion are regulated by expression of variant integrin glycoforms

    PubMed Central

    Shaikh, Faheem M; Seales, Eric C; Clem, William C; Hennessy, Kristin M; Zhuo, Ya; Bellis, Susan L

    2008-01-01

    The ST6Gal-I glycosyltransferase, which adds α2-6-linked sialic acids to glycoproteins, is overexpressed in colon adenocarcinoma, and enzyme activity is correlated with tumor cell invasiveness. Previously we reported that forced expression of oncogenic ras in HD3 colonocytes causes upregulation of ST6Gal-I, leading to increased α2-6 sialylation of β1 integrins. To determine whether ras-induced sialylation is involved in promoting the tumor cell phenotype, we used shRNA to downregulate ST6Gal-I in ras-expressors, and then monitored integrin-dependent responses. Here we show that forced ST6Gal-I downregulation, leading to diminished α2-6 sialylation of integrins, inhibits cell adhesion to collagen-I, a β1 ligand. Correspondingly, collagen binding is reduced by enzymatic removal of cell surface sialic acids from ras-expressors with high ST6Gal-I levels (i.e., no shRNA). Cells with forced ST6Gal-I downregulation also exhibit decreased migration on collagen-I and diminished invasion through Matrigel. Importantly, GD25 cells, which lack β1 integrins (and ST6Gal-I), do not demonstrate differential invasiveness when forced to express ST6Gal-I, suggesting that the effects of variant sialylation are mediated specifically by β1 integrins. The observation that cell migration and invasion can be blocked in oncogenic ras-expressing cells by forcing ST6Gal-I downregulation implicates differential sialylation as an important ras effector, and also suggests that ST6Gal-I is a promising therapeutic target. PMID:18703050

  8. Research advances on structure and biological functions of integrins.

    PubMed

    Pan, Li; Zhao, Yuan; Yuan, Zhijie; Qin, Guixin

    2016-01-01

    Integrins are an important family of adhesion molecules that were first discovered two decades ago. Integrins are transmembrane heterodimeric glycoprotein receptors consisting of α and β subunits, and are comprised of an extracellular domain, a transmembrane domain, and a cytoplasmic tail. Therein, integrin cytoplasmic domains may associate directly with numerous cytoskeletal proteins and intracellular signaling molecules, which are crucial for modulating fundamental cell processes and functions including cell adhesion, proliferation, migration, and survival. The purpose of this review is to describe the unique structure of each integrin subunit, primary cytoplasmic association proteins, and transduction signaling pathway of integrins, with an emphasis on their biological functions. PMID:27468395

  9. Upregulated Expression of Integrin α1 in Mesangial Cells and Integrin α3 and Vimentin in Podocytes of Col4a3-Null (Alport) Mice

    PubMed Central

    Steenhard, Brooke M.; Vanacore, Roberto; Friedman, David; Zelenchuk, Adrian; Stroganova, Larysa; Isom, Kathryn; St. John, Patricia L.; Hudson, Billy G.; Abrahamson, Dale R.

    2012-01-01

    Alport disease in humans, which usually results in proteinuria and kidney failure, is caused by mutations to the COL4A3, COL4A4, or COL4A5 genes, and absence of collagen α3α4α5(IV) networks found in mature kidney glomerular basement membrane (GBM). The Alport mouse harbors a deletion of the Col4a3 gene, which also results in the lack of GBM collagen α3α4α5(IV). This animal model shares many features with human Alport patients, including the retention of collagen α1α2α1(IV) in GBMs, effacement of podocyte foot processes, gradual loss of glomerular barrier properties, and progression to renal failure. To learn more about the pathogenesis of Alport disease, we undertook a discovery proteomics approach to identify proteins that were differentially expressed in glomeruli purified from Alport and wild-type mouse kidneys. Pairs of cy3- and cy5-labeled extracts from 5-week old Alport and wild-type glomeruli, respectively, underwent 2-dimensional difference gel electrophoresis. Differentially expressed proteins were digested with trypsin and prepared for mass spectrometry, peptide ion mapping/fingerprinting, and protein identification through database searching. The intermediate filament protein, vimentin, was upregulated ∼2.5 fold in Alport glomeruli compared to wild-type. Upregulation was confirmed by quantitative real time RT-PCR of isolated Alport glomeruli (5.4 fold over wild-type), and quantitative confocal immunofluorescence microscopy localized over-expressed vimentin specifically to Alport podocytes. We next hypothesized that increases in vimentin abundance might affect the basement membrane protein receptors, integrins, and screened Alport and wild-type glomeruli for expression of integrins likely to be the main receptors for GBM type IV collagen and laminin. Quantitative immunofluorescence showed an increase in integrin α1 expression in Alport mesangial cells and an increase in integrin α3 in Alport podocytes. We conclude that overexpression of

  10. Unusual Side Effects of Interferon Beta-1a in Patient with Multiple Sclerosis

    PubMed Central

    Tavakoli, Maryam; Pour Manshadi, Seyed Mohammad Yousof Mostafavi; Naderi, Nafiseh; Dehghan, Abolfazl; Azizi, Sanaz

    2012-01-01

    This is a descriptive case report of a 30-year-old man with massive epistaxis, echymosis on arms, abnormal CBC and increased plasma urea and creatinine level (i.e. above normal range). Probably, these are as side effects of interferon beta-1a injection. This is the first report according to our literature search (Pub Med, Google scholar, ISI web of knowledge, ProQuest, MD consult, Science Direct, and SCOPUS) about interferon beta-1a related abnormal kidney function tests hereafter. Abnormal kidney function tests (i.e. increased plasma urea and creatinie level) is not a known as side effect of interferon beta-1a. This case indicates, likely potential for development of these side effects with this medication. PMID:23922529

  11. Human mesenchymal stem cells in contact with their environment: surface characteristics and the integrin system

    PubMed Central

    Docheva, Denitsa; Popov, Cvetan; Mutschler, Wolf; Schieker, Matthias

    2007-01-01

    Abstract The identification of mesenchymal stem cells (MSCs) in adult human tissues and the disclosure of their self-renew-al and multi-lineage differentiation capabilities have provided exciting prospects for cell-based regeneration and tis-sue engineering. Although a considerable amount of data is available describing MSCs, there is still lack of information regarding the molecular mechanisms that govern their adhesion and migration. In this work, we will review the current state of knowledge on integrins and other adhesion molecules found to be expressed on MSCs. The dis-cussed topics include the characteristics of MSCs and their clinical applications, integrins and their central role in cell-matrix attachment and migration, and comments on mobilization, differentiation and contribution to tumour development. Finally, by understanding the complex and fundamental pathways by which MSCs attach and migrate, it might be possible to fine-tune the strategies for effective and safe use of MSCs in regenerative therapies. PMID:17367499

  12. Epithelial polarity--generating and integrating signals from the ECM with integrins.

    PubMed

    Manninen, Aki

    2015-06-10

    Epithelial cells are important building blocks of most tissues and the corner stone of tissue architectures that allow directional transport of nutrients, ions and waste products in and out of the body. In tissues composed of millions of cells every individual cell needs to make right decisions when to differentiate, migrate, divide or die. Tight control of such fundamental cell-level processes ensures proper tissue morphogenesis, homeostasis and function. Cellular decisions are guided by biochemical and mechanical cues from their immediate microenvironment that consists of the extracellular matrix (ECM), neighboring cells and soluble factors. Generation of two distinct surfaces one facing the outside world (the apical domain) and the other contacting the neighboring cells and basal ECM (basolateral domain) is the most fundamental property of epithelial cells. The cues from the ECM are of particular importance in this process and communication between the cells and the ECM is largely mediated by transmembrane ECM receptors. Integrins constitute the largest family of such receptors binding to the ECM. Integrins have been shown to be essential for the establishment of initial polarity cues that define the position of the basal domain and thereby govern the orientation of the forming apico-basal axis. In this review I will discuss the multifaceted roles of integrins in epithelial cells with a particular focus on recent developments unveiling the specific functions of the different integrin heterodimers in regulating epithelial cell polarization and morphogenesis. PMID:25597426

  13. SWiss Atorvastatin and Interferon Beta-1b Trial In Multiple Sclerosis (SWABIMS) - rationale, design and methodology

    PubMed Central

    2009-01-01

    Background Statins have anti-inflammatory and immunomodulatory properties in addition to their lipid-lowering effects. Currently, the effects of statins on multiple sclerosis are still controversial. Therefore, randomized clinical trials are needed to provide better evidence on the therapeutic potential of statins in multiple sclerosis. The SWiss Atorvastatin and Interferon Beta-1b trial in Multiple Sclerosis (SWABIMS) evaluates the efficacy, safety and tolerability of atorvastatin 40 mg per os daily and subcutaneous interferon beta-1b every other day compared to monotherapy with subcutaneous interferon beta-1b every other day in patients with relapsing-remitting multiple sclerosis. Methods/Design SWABIMS is a multi-centre, randomized, parallel-group, rater-blinded, Phase IIb-study conducted in eight hospitals in Switzerland. 80 treatment naïve patients with relapsing-remitting forms of multiple sclerosis will receive subcutaneous interferon beta-1b for three months. Afterwards, they are randomized into two equal-sized parallel arms, receiving atorvastatin 40 mg/d or not in addition to interferon beta-1b for another 12 months. Disease activity measured by the proportion of patients with new T2 lesions is the primary endpoint. Discussion SWABIMS is designed to give further information about the therapeutic effect of atorvastatin 40 mg per os daily as add-on therapy to interferon beta-1b in patients with relapsing-remitting multiple sclerosis. Furthermore important safety and tolerability data will be generated. Trial Registration http://www.clinicaltrials.gov. Identifier: NCT00942591; Swissmedic reference number: 2005DR2119 PMID:20003436

  14. ISOLATION OF INTEGRIN-BASED ADHESION COMPLEXES

    PubMed Central

    Jones, Matthew C.; Humphries, Jonathan D.; Byron, Adam; Millon-Frémillon, Angelique; Robertson, Joseph; Paul, Nikki R.; Ng, Daniel H. J.; Askari, Janet A.; Humphries, Martin J.

    2015-01-01

    The integration of cells with their extracellular environment is facilitated by cell surface adhesion receptors, such as integrins, which play important roles in both normal development and the onset of pathologies. Engagement of integrins with their ligands in the extracellular matrix, or counter receptors on other cells, initiates the intracellular assembly of a wide variety of proteins into adhesion complexes such as focal contacts, focal adhesions and fibrillar adhesions. The proteins recruited to these complexes mediate bidirectional signalling across the plasma membrane and as such help to coordinate and / or modulate the multitude of physical or chemical signals to which the cell is subjected. The protocols in this unit describe two approaches for the isolation or enrichment of proteins contained within integrin-associated adhesion complexes together with their local plasma membrane / cytosolic environments from cells in culture. In the first protocol integrin-associated adhesion structures are affinity isolated using microbeads coated with extracellular ligands or antibodies. The second protocol describes the isolation of ventral membrane preparations that are enriched for adhesion complex structures. The protocols permit the determination of adhesion complex components by subsequent downstream analysis by Western blotting or mass spectrometry. PMID:25727331

  15. Alterated integrin expression in lichen planopilaris

    PubMed Central

    d'Ovidio, Roberto; Sgarra, Concetta; Conserva, Anna; Angelotti, Umberto Filippo; Erriquez, Roberta; Foti, Caterina

    2007-01-01

    Background Lichen planopilaris (LPP) is an inflammatory disease characterized by a lymphomononuclear infiltrate surrounding the isthmus and infundibulum of the hair follicle of the scalp, that evolves into atrophic/scarring alopecia. In the active phase of the disease hairs are easily plucked with anagen-like hair-roots. In this study we focused on the expression of integrins and basement membrane components of the hair follicle in active LPP lesions. Methods Scalp biopsies were taken in 10 patients with LPP and in 5 normal controls. Using monoclonal antibodies against α3β1 and α6β4 integrins we showed the expression of these integrins and of the basement membrane components of the hair follicle in active LPP lesions and in healthy scalp skin. Results In the LPP involved areas, α3β1 was distributed in a pericellular pattern, the α6 subunit was present with a basolateral distribution while the β4 subunit showed discontinuous expression at the basal pole and occasionally, basolateral staining of the hair follicle. Conclusion: An altered distribution of the integrins in active LPP lesions can explain the phenomenon of easy pulling-out of the hair with a "gelatinous" root-sheath. PMID:17288588

  16. Expression of transforming growth factors beta-1, beta 2 and beta 3 in human bladder carcinomas.

    PubMed Central

    Eder, I. E.; Stenzl, A.; Hobisch, A.; Cronauer, M. V.; Bartsch, G.; Klocker, H.

    1997-01-01

    We previously detected elevated transforming growth factor beta-1 (TGF-beta1) serum levels in patients with invasive bladder carcinomas. In this study, we therefore investigated whether elevated serum levels correlate with enhanced TGF-beta expression in human bladder tumours. mRNA levels of TGF-beta1, -beta2 and -beta3 were reduced in bladder tumour tissue to 86%, 68% and 56%, respectively, of the levels in normal urothelium. On the other hand, TGF-beta1 protein levels were found to be higher in superficial tumours (Ta-T1) (mean level of 0.153 ng mg(-1)) and in invasive T2/T3 tumours (mean level of 0.104 ng mg(-1)) compared with normal urothelium (mean level of 0.065 ng mg(-1)). Invasive T4 tumours, however, contained only low amounts of TGF-beta1 (mean level of 0.02 ng mg(-1)). Neither in mean nor in individual patients were serum and tissue TGF-beta levels correlated with each other. Cell culture experiments on primary bladder cells revealed a 57% decrease in TGF-beta1 mRNA levels in tumour compared with normal epithelial cells. Tumour epithelial cells contained about two times higher levels of TGF-beta2 and TGF-beta3 mRNA than normal epithelial cells. Fibroblasts expressed about the same amount of TGF-beta1 or TGF-beta2 as epithelial cells. Yet, fibroblasts released only 19% and 13% of the amount secreted by tumour epithelial cells into the supernatant. TGF-beta3, on the other hand, was expressed by fibroblasts with higher levels than by epithelial cells. TGF-beta1 was the predominent isoform in bladder tissue and cells at protein as well as on mRNA levels indicating that TGFs-beta2 and -beta3 are of minor importance in bladder cancer. In summary, there is a lack of correlation between TGF-beta serum levels and TGF-beta expression in tumour tissue in bladder cancer. Images Figure 1 PMID:9192977

  17. Down-regulation of phospholipase C-beta1 following chronic muscarinic receptor activation.

    PubMed

    Sorensen, S D; Linseman, D A; Fisher, S K

    1998-04-01

    To determine whether prolonged activation of a phospholipase C-coupled receptor can lead to a down-regulation of its effector enzyme, SH-SY5Y neuroblastoma cells were incubated for 24 h with the muscarinic receptor agonist, oxotremorine-M. Under these conditions, significant reductions (46-53%) in muscarinic cholinergic receptor density, G(alphaq/11) and phospholipase C-beta1 (but not the beta3-or gamma1 isoforms) were observed. These results suggest that a selective down-regulation of phospholipase C-beta1 may play a role in adaptation to chronic muscarinic receptor activation. PMID:9617763

  18. The polymerization and thrombin-binding properties of des-(B beta 1-42)-fibrin.

    PubMed

    Siebenlist, K R; DiOrio, J P; Budzynski, A Z; Mosesson, M W

    1990-10-25

    Multiple factors affect the thrombin-catalyzed conversion of fibrinogen to fibrin, including: fibrinopeptide (FPA and FPB) release leading to exposure of two types of polymerization domains ("A" and "B," respectively) in the central portion of the molecule, and exposure of a noncatalytic "secondary" thrombin-binding site in fibrin. Fibrinogen containing the FPA sequence but lacking the B beta 1-42 sequence ("des-(B beta 1-42)-fibrinogen"), was compared to native fibrinogen (containing both FPA and FPB) to investigate the role played by B beta 1-42 in the polymerization of alpha-fibrin (i.e. fibrin lacking FPA), to compare reptilase and thrombin cleavage of FPA from fibrinogen, and to explore the location and function of the secondary thrombin-binding site. Electron microscopy of evolving polymer structures (mu, 0.14; pH 7.4) plus turbidity measurements, showed that early thin fibril formation as well as subsequent lateral fibril associations were impaired in des-(B beta 1-42)-alpha-fibrin, thus indicating that the B beta 1-42 sequence contributes to the A polymerization site. Reptilase-activated des-(B beta 1-42)-alpha-fibrin polymerized even more slowly than thrombin-activated des-(B beta 1-42)-alpha-fibrin, differences that disappeared when repolymerization of preformed fibrin monomers was carried out. Since existing data indicate that thrombin releases FPA in a concerted manner, resulting in relatively rapid evolution of fully functional divalent alpha-fibrin monomers, it can be inferred that delayed fibrin assembly of reptilase fibrin is due to slower formation of divalent alpha-fibrin monomers. Thrombin-activated des-(B beta 1-42)-alpha-fibrin polymerized more rapidly at low ionic strength (mu, 0.04) than did native alpha,beta-fibrin, a reversal of their behavior at physiological ionic strength (mu, 0.14). Concomitant measurement of FPA release revealed modest slowing of release at low ionic strength from des-(B beta 1-42)-fibrinogen (t1/2, 36.5 versus 21

  19. Effects of dietary beta-1,3-glucan on the growth, survival, physiological and immune response of marron, Cherax tenuimanus (smith, 1912).

    PubMed

    Sang, Huynh Minh; Fotedar, Ravi

    2010-01-01

    Six isonitrogenous and isocalorific diets supplemented with five different levels of beta-1,3-glucan (0.08%, 0.1%, 0.2%, 0.4% and 0.8%) were formulated and tested for marron (Cherax tenuimanus) growth, survival, organosomatic indices, osmoregulatory capacity and immunological parameters (total and differential haemocyte counts, haemolymph clotting time and bacteraemia). The sixth diet without any beta-1,3-glucan was used as a control. Each diet was provided to 18 marron (0.47 +/- 0.02 g initial weight) replicated 3 times in individual 250 L fiberglass cylindrical tanks. Each tank was provided with a biological filtration recirculating water system. After 84 days of culture, the survival and yield were higher in the marron fed 0.1% beta glucan supplemented diet. The different levels of beta glucan did not alter any of the physiological parameters of marron. However, dietary supplementation with beta glucan resulted in significantly higher (P < 0.05) total haemocyte count (THC) and granular cells. The bacteraemia rank was lower in all diets having beta glucan supplemented with more than or equal to 0.1% compared to the control and 0.08% beta glucan supplemented diets. Results suggest that dietary beta-1,3-glucan at a minimum concentration of 0.1-0.2% can improve the immune system of marron. PMID:20139008

  20. Monocyte-endothelial adhesion in chronic rheumatoid arthritis. In situ detection of selectin and integrin-dependent interactions.

    PubMed Central

    Grober, J S; Bowen, B L; Ebling, H; Athey, B; Thompson, C B; Fox, D A; Stoolman, L M

    1993-01-01

    Blood monocytes are the principal reservoir for tissue macrophages in rheumatoid synovitis. Receptor-mediated adhesive interactions between circulating cells and the synovial venules initiate recruitment. These interactions have been studied primarily in cultured endothelial cells. Thus the functional activities of specific adhesion receptors, such as the endothelial selectins and the leukocytic integrins, have not been evaluated directly in diseased tissues. We therefore examined monocyte-microvascular interactions in rheumatoid synovitis by modifying the Stamper-Woodruff frozen section binding assay initially developed to study lymphocyte homing. Specific binding of monocytes to venules lined by low or high endothelium occurred at concentrations as low as 5 x 10(5) cells/ml. mAbs specific for P-selectin (CD62, GMP-140/PADGEM) blocked adhesion by > 90% in all synovitis specimens examined. In contrast, P-selectin-mediated adhesion to the microvasculature was either lower or absent in frozen sections of normal foreskin and placenta. mAbs specific for E-selectin (ELAM-1) blocked 20-50% of monocyte attachment in several RA synovial specimens but had no effect in others. mAbs specific for LFA-1, Mo1/Mac 1, the integrin beta 2-chain, and L-selectin individually inhibited 30-40% of adhesion. An mAb specific for the integrin beta 1-chain inhibited the attachment of elutriated monocytes up to 20%. We conclude that P-selectin associated with the synovial microvasculature initiates shear-resistant adhesion of monocytes in the Stamper-Woodruff assay and stabilizes bonds formed by other selectins and the integrins. Thus the frozen section binding assay permits direct evaluation of leukocyte-microvascular adhesive interactions in inflamed tissues and suggests a prominent role for P-selectin in monocyte recruitment in vivo. Images PMID:7685772

  1. Alteration of Pituitary Tumor Transforming Gene-1 Regulates Trophoblast Invasion via the Integrin/Rho-Family Signaling Pathway

    PubMed Central

    Lim, Seung Mook; Jang, Hee Yeon; Lee, Ji Eun; Shin, Joong Sik; Park, Sun-Hwa; Yoon, Bo Hyun; Kim, Gi Jin

    2016-01-01

    Trophoblast invasion ability is an important factor in early implantation and placental development. Recently, pituitary tumor transforming gene 1 (PTTG1) was shown to be involved in invasion and proliferation of cancer. However, the role of PTTG1 in trophoblast invasion remains unknown. Thus, in this study we analyzed PTTG1 expression in trophoblasts and its effect on trophoblast invasion activity and determined the mechanism through which PTTG1 regulates trophoblast invasion. Trophoblast proliferation and invasion abilities, regardless of PTTG1 expression, were analyzed by quantitative real-time polymerase chain reaction, fluorescence-activated cell sorting analysis, invasion assay, western blot, and zymography after treatment with small interfering RNA against PTTG1 (siPTTG1). Additionally, integrin/Rho-family signaling in trophoblasts by PTTG1 alteration was analyzed. Furthermore, the effect of PTTG1 on trophoblast invasion was evaluated by microRNA (miRNA) mimic and inhibitor treatment. Trophoblast invasion was significantly reduced through decreased matrix metalloproteinase (MMP)-2 and MMP-9 expression when PTTG1 expression was inhibited by siPTTG1 (p < 0.05). Furthermore, knockdown of PTTG1 increased expression of integrin alpha 4 (ITGA4), ITGA5, and integrin beta 1 (ITGB1); otherwise, RhoA expression was significantly decreased (p < 0.05). Treatment of miRNA-186-5p mimic and inhibitor controlled trophoblast invasion ability by altering PTTG1 and MMP expression. PTTG1 can control trophoblast invasion ability via regulation of MMP expression through integrin/Rho-family signaling. In addition, PTTG1 expression and its function were regulated by miRNA-186-5p. These results help in understanding the mechanism through which PTTG1 regulates trophoblast invasion and thereby implantation and placental development. PMID:26900962

  2. Dynamic Structural Changes Are Observed upon Collagen and Metal Ion Binding to the Integrin α1 I Domain*

    PubMed Central

    Weinreb, Paul H.; Li, Sheng; Gao, Sharon X.; Liu, Tong; Pepinsky, R. Blake; Caravella, Justin A.; Lee, Jun H.; Woods, Virgil L.

    2012-01-01

    We have applied hydrogen-deuterium exchange mass spectrometry, in conjunction with differential scanning calorimetry and protein stability analysis, to examine solution dynamics of the integrin α1 I domain induced by the binding of divalent cations, full-length type IV collagen, or a function-blocking monoclonal antibody. These studies revealed features of integrin activation and α1I-ligand complexes that were not detected by static crystallographic data. Mg2+ and Mn2+ stabilized α1I but differed in their effects on exchange rates in the αC helix. Ca2+ impacted α1I conformational dynamics without altering its gross thermal stability. Interaction with collagen affected the exchange rates in just one of three metal ion-dependent adhesion site (MIDAS) loops, suggesting that MIDAS loop 2 plays a primary role in mediating ligand binding. Collagen also induced changes consistent with increased unfolding in both the αC and allosteric C-terminal helices of α1I. The antibody AQC2, which binds to α1I in a ligand-mimetic manner, also reduced exchange in MIDAS loop 2 and increased exchange in αC, but it did not impact the C-terminal region. This is the first study to directly demonstrate the conformational changes induced upon binding of an integrin I domain to a full-length collagen ligand, and it demonstrates the utility of the deuterium exchange mass spectrometry method to study the solution dynamics of integrin/ligand and integrin/metal ion interactions. Based on the ligand and metal ion binding data, we propose a model for collagen-binding integrin activation that explains the differing abilities of Mg2+, Mn2+, and Ca2+ to activate I domain-containing integrins. PMID:22847004

  3. Proinsulin C-peptide antagonizes the profibrotic effects of TGF-beta1 via up-regulation of retinoic acid and HGF-related signaling pathways.

    PubMed

    Hills, Claire E; Willars, Gary B; Brunskill, Nigel J

    2010-04-01

    Novel signaling roles for C-peptide have recently been discovered with evidence that it can ameliorate complications of type 1 diabetes. Here we sought to identify new pathways regulated by C-peptide of relevance to the pathophysiology of diabetic nephropathy. Microarray analysis was performed to identify genes regulated by either C-peptide and/or TGF-beta1 in a human proximal tubular cell line, HK-2. Expression of retinoic acid receptor beta (RARbeta), hepatocyte growth factor (HGF), cellular retinoic acid-binding protein II (CRABPII), vimentin, E-cadherin, Snail, and beta-catenin was assessed by immunoblotting. The cellular localization of vimentin and beta-catenin was determined by immunocytochemistry. Changes in cell morphology were assessed by phase contrast microscopy. Gene expression profiling demonstrated differential expression of 953 and 1458 genes after C-peptide exposure for 18 h or 48 h, respectively. From these, members of the antifibrotic retinoic acid (RA)- and HGF-signaling pathways were selected. Immunoblotting demonstrated that C-peptide increased RARbeta, CRABPII, and HGF. We confirmed a role for RA in reversal of TGF-beta1-induced changes associated with epithelial-mesenchymal transition, including expression changes in Snail, E-cadherin, vimetin, and redistribution of beta-catenin. Importantly, these TGF-beta1-induced changes were inhibited by C-peptide. Further, effects of TGF-beta1 on Snail and E-cadherin expression were blocked by HGF, and inhibitory effects of C-peptide were removed by blockade of HGF activity. This study identifies a novel role for HGF as an effector of C-peptide, possibly via an RA-signaling pathway, highlighting C-peptide as a potential therapy for diabetic nephropathy. PMID:20197308

  4. Heating-induced conformational change of a novel beta-(1-->3)-D-glucan from Pleurotus geestanus.

    PubMed

    Zhang, Mei

    2010-02-01

    Recently, we isolated and purified a neutral polysaccharide (PGN) from edible fungus Pleurotus geestanus. Its structure was characterized by a range of physical-chemical methods, including high performance anion exchange chromatography, uronic acid, and protein analyses, size exclusion chromatography with ultraviolet, refractive index and light scattering detectors, and nuclear magnetic resonance. Our results revealed that PGN is a novel beta-(1-->3)-D-glucan with glucose attached to every other sugar residues at Position 6 in the backbone. It has a degree of branching of 1/2. Such structure is different from typical beta-(1-->3)-D-glucans schizophyllan and lentinan in which DB is 1/3 and 2/5, respectively. Rheological study showed a very interesting melting behavior of PGN in water solution: heating PGN in water leads to two transitions, in the range of 8-12.5 degrees C and 25-60 degrees C, respectively. The melting behavior and conformational changes were characterized by rheometry, micro-differential scan calorimetry, atomic force microscopy, static and dynamic light scattering at different temperatures. The first heating-induced transition corresponds to the disintegration of polymer bundles into small helical clusters, resembling the heating-induced dissociation of SPG in water at 7 degrees C; the second one might correspond to the dissociation of helical strands to individual chains. The ability of PGN to undergo a conformation/viscosity transition in water upon heating is very valuable to immobilize cells or enzymes or therapeutic DNA/RNA, which makes PGN a potentially useful biomaterial. PMID:19768780

  5. Requirement of a novel splicing variant of human histone deacetylase 6 for TGF-{beta}1-mediated gene activation

    SciTech Connect

    Zhuang, Yan; Nguyen, Hong T.; Lasky, Joseph A.; Cao, Subing; Li, Cui; Hu, Jiyao; Guo, Xinyue; Burow, Matthew E.; Shan, Bin

    2010-02-19

    Histone deacetylase 6 (HDAC6) belongs to the family of class IIb HDACs and predominantly deacetylates non-histone proteins in the cytoplasm via the C-terminal deacetylase domain of its two tandem deacetylase domains. HDAC6 modulates fundamental cellular processes via deacetylation of {alpha}-tubulin, cortactin, molecular chaperones, and other peptides. Our previous study indicates that HDAC6 mediates TGF-{beta}1-induced epithelial-mesenchymal transition (EMT) in A549 cells. In the current study, we identify a novel splicing variant of human HDAC6, hHDAC6p114. The hHDAC6p114 mRNA arises from incomplete splicing and encodes a truncated isoform of the hHDAC6p114 protein of 114 kDa when compared to the major isoform hHDAC6p131. The hHDAC6p114 protein lacks the first 152 amino acids from N-terminus in the hHDAC6p131 protein, which harbors a nuclear export signal peptide and 76 amino acids of the N-terminal deacetylase domain. hHDAC6p114 is intact in its deacetylase activity against {alpha}-tubulin. The expression hHDAC6p114 is elevated in a MCF-7 derivative that exhibits an EMT-like phenotype. Moreover, hHDAC6p114 is required for TGF-{beta}1-activated gene expression associated with EMT in A549 cells. Taken together, our results implicate that expression and function of hHDAC6p114 is differentially regulated when compared to hHDAC6p131.

  6. A Putatively Functional Haplotype in the Gene Encoding Transforming Growth Factor Beta-1 as a Potential Biomarker for Radiosensitivity

    SciTech Connect

    Schirmer, Markus A.; Brockmoeller, Juergen; Rave-Fraenk, Margret; Virsik, Patricia; Wilken, Barbara; Kuehnle, Elna; Campean, Radu; Hoffmann, Arne O.; Mueller, Katarina; Goetze, Robert G.; Neumann, Michael; Janke, Joerg H.; Nasser, Fatima; Wolff, Hendrik A.; Ghadimi, B. Michael; Schmidberger, Heinz; Hess, Clemens F.; Christiansen, Hans; Hille, Andrea

    2011-03-01

    Purpose: To determine whether genetic variability in TGFB1 is related to circulating transforming growth factor-{beta}1 (TGF-{beta}1) plasma concentrations after radiotherapy and to radiosensitivity of lymphoid cells. Patients and Methods: Transforming growth factor-{beta}1 plasma concentrations (n = 79) were measured in patients 1 year after radiotherapy and chromosomal aberrations (n = 71) ex vivo before therapy start. Furthermore, TGF-{beta}1 secretion and apoptosis were measured in isolated peripheral blood mononuclear cells of 55 healthy volunteers. These phenotypes were analyzed in relation to five germline polymorphisms in the 5' region of the TGFB1 gene. Because of high linkage disequilibrium, these five polymorphisms reflect frequent genetic variation in this region. A presumed impact of TGF-{beta}1 on DNA damage or repair was measured as micronucleus formation in 30 lymphoblastoid cell lines. Results: We identified a hypofunctional genetic haplotype termed H3 tagging the 5' region of the TGFB1 gene encoding TGF-{beta}1. H3 was associated with lower TGF-{beta}1 plasma concentrations in patients (p = 0.01) and reduced TGF-{beta}1 secretion in irradiated peripheral blood mononuclear cells (p = 0.003). Furthermore, cells with H3 were less prone to induction of chromosomal aberrations (p = 0.001) and apoptosis (p = 0.003) by irradiation. The hypothesis that high TGF-{beta}1 could sensitize cells to DNA damage was further supported by increased micronuclei formation in 30 lymphoblastoid cell lines when preincubated with TGF-{beta}1 before irradiation (p = 0.04). Conclusions: On the basis of TGF-{beta}1 plasma levels and radiation sensitivity of lymphoid cells, this study revealed a putatively hypofunctional TGFB1 haplotype. The significance of this haplotype and the suggested link between TGF-{beta}1 function and DNA integrity should be further explored in other cell types, as well as other experimental and clinical conditions.

  7. Bacterial cyclic beta-(1,2)-glucan acts in systemic suppression of plant immune responses.

    PubMed

    Rigano, Luciano Ariel; Payette, Caroline; Brouillard, Geneviève; Marano, Maria Rosa; Abramowicz, Laura; Torres, Pablo Sebastián; Yun, Maximina; Castagnaro, Atilio Pedro; Oirdi, Mohamed El; Dufour, Vanessa; Malamud, Florencia; Dow, John Maxwell; Bouarab, Kamal; Vojnov, Adrian Alberto

    2007-06-01

    Although cyclic glucans have been shown to be important for a number of symbiotic and pathogenic bacterium-plant interactions, their precise roles are unclear. Here, we examined the role of cyclic beta-(1,2)-glucan in the virulence of the black rot pathogen Xanthomonas campestris pv campestris (Xcc). Disruption of the Xcc nodule development B (ndvB) gene, which encodes a glycosyltransferase required for cyclic glucan synthesis, generated a mutant that failed to synthesize extracellular cyclic beta-(1,2)-glucan and was compromised in virulence in the model plants Arabidopsis thaliana and Nicotiana benthamiana. Infection of the mutant bacterium in N. benthamiana was associated with enhanced callose deposition and earlier expression of the PATHOGENESIS-RELATED1 (PR-1) gene. Application of purified cyclic beta-(1,2)-glucan prior to inoculation of the ndvB mutant suppressed the accumulation of callose deposition and the expression of PR-1 in N. benthamiana and restored virulence in both N. benthamiana and Arabidopsis plants. These effects were seen when cyclic glucan and bacteria were applied either to the same or to different leaves. Cyclic beta-(1,2)-glucan-induced systemic suppression was associated with the transport of the molecule throughout the plant. Systemic suppression is a novel counterdefensive strategy that may facilitate pathogen spread in plants and may have important implications for the understanding of plant-pathogen coevolution and for the development of phytoprotection measures. PMID:17601826

  8. Cutaneous leukocytoclastic vasculitis in a child with interleukin-12 receptor beta-1 deficiency.

    PubMed

    Kutukculer, Necil; Genel, Ferah; Aksu, Guzide; Karapinar, Bulent; Ozturk, Can; Cavusoglu, Cengiz; Casanova, Jean-Laurent; Fieschi, Claire

    2006-03-01

    We report a patient with complete interleukin-12 receptor beta-1 deficiency associated with cutaneous leukocytoclastic vasculitis. The patient experienced Bacille Calmette Guérin, Mycobacterium chelonae, and Salmonella enteritidis infection. Vasculitis affecting both small arteries and postcapillary venules due to deposition of immune complexes was probably caused by S. enteritidis and/or M. chelonae infection. PMID:16615980

  9. The journey of integrins and partners in a complex interactions landscape studied by super-resolution microscopy and single protein tracking.

    PubMed

    Rossier, Olivier; Giannone, Grégory

    2016-04-10

    Cells adjust their adhesive and cytoskeletal organizations according to changes in the biochemical and physical nature of their surroundings. In return, by adhering and generating forces on the extracellular matrix (ECM) cells organize their microenvironment. Integrin-dependent focal adhesions (FAs) are the converging zones integrating biochemical and biomechanical signals arising from the ECM and the actin cytoskeleton. Thus, integrin-mediated adhesion and mechanotransduction, the conversion of mechanical forces into biochemical signals, are involved in critical cellular functions such as migration, proliferation and differentiation, and their deregulation contributes to pathologies including cancer. A challenging problem is to decipher how stochastic protein movements and interactions lead to formation of dynamic architecture such as integrin-dependent adhesive structures. In this review, we will describe recent advances made possible by super-resolution microscopies and single molecule tracking approaches that provided new understanding on the organization and the dynamics of integrins and intracellular regulators at the nanoscale in living cells. PMID:26571074

  10. Chemokines and the Signaling Modules Regulating Integrin Affinity

    PubMed Central

    Montresor, Alessio; Toffali, Lara; Constantin, Gabriela; Laudanna, Carlo

    2012-01-01

    Integrin-mediated adhesion is a general concept referring to a series of adhesive phenomena including tethering–rolling, affinity, valency, and binding stabilization altogether controlling cell avidity (adhesiveness) for the substrate. Arrest chemokines modulate each aspect of integrin activation, although integrin affinity regulation has been recognized as the prominent event in rapid leukocyte arrest induced by chemokines. A variety of inside-out and outside-in signaling mechanisms have been related to the process of integrin-mediated adhesion in different cellular models, but only few of them have been clearly contextualized to rapid integrin affinity modulation by arrest chemokines in primary leukocytes. Complex signaling processes triggered by arrest chemokines and controlling leukocyte integrin activation have been described for ras-related rap and for rho-related small GTPases. We summarize the role of rap and rho small GTPases in the regulation of rapid integrin affinity in primary leukocytes and provide a modular view of these pro-adhesive signaling events. A potential, albeit still speculative, mechanism of rho-mediated regulation of cytoskeletal proteins controlling the last step of integrin activation is also discussed. We also discuss data suggesting a functional integration between the rho- and rap-modules of integrin activation. Finally we examine the universality of signaling mechanisms regulating integrin triggering by arrest chemokines. PMID:22654882

  11. Peginterferon beta-1a in multiple sclerosis: 2-year results from ADVANCE

    PubMed Central

    Arnold, Douglas L; Balcer, Laura J; Boyko, Alexey A; Pelletier, Jean; Liu, Shifang; Zhu, Ying; Seddighzadeh, Ali; Hung, Serena; Deykin, Aaron; Sheikh, Sarah I; Calabresi, Peter A

    2015-01-01

    Objective: To evaluate the efficacy and safety of subcutaneous peginterferon beta-1a over 2 years in patients with relapsing–remitting multiple sclerosis in the ADVANCE study. Methods: Patients were randomized to placebo or 125 µg peginterferon beta-1a every 2 or 4 weeks. For Year 2 (Y2), patients originally randomized to placebo were re-randomized to peginterferon beta-1a every 2 weeks or every 4 weeks. Patients randomized to peginterferon beta-1a in Year 1 (Y1) remained on the same dosing regimen in Y2. Results: Compared with Y1, annualized relapse rate (ARR) was further reduced in Y2 with every 2 week dosing (Y1: 0.230 [95% CI 0.183–0.291], Y2: 0.178 [0.136–0.233]) and maintained with every 4 week dosing (Y1: 0.286 [0.231–0.355], Y2: 0.291 [0.231–0.368]). Patients starting peginterferon beta-1a from Y1 displayed improved efficacy versus patients initially assigned placebo, with reductions in ARR (every 2 weeks: 37%, p<0.0001; every 4 weeks: 17%, p=0.0906), risk of relapse (every 2 weeks: 39%, p<0.0001; every 4 weeks: 19%, p=0.0465), 12-week disability progression (every 2 weeks: 33%, p=0.0257; every 4 weeks: 25%, p=0.0960), and 24-week disability progression (every 2 weeks: 41%, p=0.0137; every 4 weeks: 9%, p=0.6243). Over 2 years, greater reductions were observed with every 2 week versus every 4 week dosing for all endpoints and peginterferon beta-1a was well tolerated. Conclusions: Peginterferon beta-1a efficacy is maintained beyond 1 year, with greater effects observed with every 2 week versus every 4 week dosing, and a similar safety profile to Y1. Clinicaltrials.gov Registration Number: NCT00906399. PMID:25432952

  12. Integrin β3 Haploinsufficiency Modulates Serotonin Transport and Antidepressant-Sensitive Behavior in Mice

    PubMed Central

    Mazalouskas, Matthew; Jessen, Tammy; Varney, Seth; Sutcliffe, James S; Veenstra-VanderWeele, Jeremy; Cook, Edwin H; Carneiro, Ana M D

    2015-01-01

    Converging lines of evidence have identified genetic interactions between the serotonin transporter (SERT) gene and ITGB3, which encodes the β3 subunit that forms the αIIbβ3 and αvβ3 integrin receptor complexes. Here we examine the consequences of haploinsufficiency in the mouse integrin β3 subunit gene (Itgb3) on SERT function and selective 5-hydroxytryptamine (5-HT) reuptake inhibitor (SSRI) effectiveness in vivo. Biochemical fractionation studies and immunofluorescent staining of murine brain slices reveal that αvβ3 receptors and SERTs are enriched in presynaptic membranes from several brain regions and that αvβ3 colocalizes with a subpopulation of SERT-containing synapses in raphe nuclei. Notably, we establish that loss of a single allele of Itgb3 in murine neurons is sufficient to decrease 5-HT uptake by SERT in midbrain synaptosomes. Pharmacological assays to elucidate the αvβ3-mediated mechanism of reduced SERT function indicate that decreased integrin β3 subunit expression scales down the population size of active SERT molecules and, as a consequence, lowers the effective dose of SSRIs. These data are consistent with the existence of a subpopulation of SERTs that are tightly modulated by integrin αvβ3 and significantly contribute to global SERT function at 5-HT synapses in the midbrain. Importantly, our screen of a normal human population for single nucleotide polymorphisms in human ITGB3 identified a variant associated with reductions in integrin β3 expression levels that parallel our mouse findings. Thus, polymorphisms in human ITGB3 may contribute to the differential responsiveness of select patients to SSRIs. PMID:25684064

  13. Collagen-Binding α11 Integrin Expression in Human Myometrium and Fibroids Utilizing a Novel RNA In Situ Probe

    PubMed Central

    Peavey, Mary; Salleh, Najah

    2014-01-01

    Objective: Integrin α11β1 is a collagen receptor specific to fibroblasts that regulates myofibroblast differentiation. We sought to determine whether α11β1 is expressed in myometrium and fibroids and whether tissue expression varies. Design: Comparison of α11 in human myometrium and fibroids using Western blot and RNA in situ hybridization. Materials and Methods: Specimens were obtained from fibroid and myometrium. For Western blots, we used a polyclonal antibody to integrin α11. RNA in situ hybridization was performed using a custom RNA probe for α11 subunit. Results: Myometrium and fibroids express α11 integrin, with expression 2-fold greater in fibroids. The RNA probe offers a more precise method compared to Western blot using polyclonal human antibody. Conclusions: The difference in expression in myometrium and fibroids suggests that α11 is involved in the formation of myofibroblasts and fibroid development. PMID:24520080

  14. α-Spectrin and integrins act together to regulate actomyosin and columnarization, and to maintain a monolayered follicular epithelium

    PubMed Central

    Ng, Bing Fu; Selvaraj, Gokul Kannan; Santa-Cruz Mateos, Carmen; Grosheva, Inna; Alvarez-Garcia, Ines; Martín-Bermudo, María Dolores; Palacios, Isabel M.

    2016-01-01

    The spectrin cytoskeleton crosslinks actin to the membrane, and although it has been greatly studied in erythrocytes, much is unknown about its function in epithelia. We have studied the role of spectrins during epithelia morphogenesis using the Drosophila follicular epithelium (FE). As previously described, we show that α-Spectrin and β-Spectrin are essential to maintain a monolayered FE, but, contrary to previous work, spectrins are not required to control proliferation. Furthermore, spectrin mutant cells show differentiation and polarity defects only in the ectopic layers of stratified epithelia, similar to integrin mutants. Our results identify α-Spectrin and integrins as novel regulators of apical constriction-independent cell elongation, as α-Spectrin and integrin mutant cells fail to columnarize. Finally, we show that increasing and reducing the activity of the Rho1-Myosin II pathway enhances and decreases multilayering of α-Spectrin cells, respectively. Similarly, higher Myosin II activity enhances the integrin multilayering phenotype. This work identifies a primary role for α-Spectrin in controlling cell shape, perhaps by modulating actomyosin. In summary, we suggest that a functional spectrin-integrin complex is essential to balance adequate forces, in order to maintain a monolayered epithelium. PMID:26952981

  15. α-Spectrin and integrins act together to regulate actomyosin and columnarization, and to maintain a monolayered follicular epithelium.

    PubMed

    Ng, Bing Fu; Selvaraj, Gokul Kannan; Santa-Cruz Mateos, Carmen; Grosheva, Inna; Alvarez-Garcia, Ines; Martín-Bermudo, María Dolores; Palacios, Isabel M

    2016-04-15

    The spectrin cytoskeleton crosslinks actin to the membrane, and although it has been greatly studied in erythrocytes, much is unknown about its function in epithelia. We have studied the role of spectrins during epithelia morphogenesis using theDrosophilafollicular epithelium (FE). As previously described, we show that α-Spectrin and β-Spectrin are essential to maintain a monolayered FE, but, contrary to previous work, spectrins are not required to control proliferation. Furthermore, spectrin mutant cells show differentiation and polarity defects only in the ectopic layers of stratified epithelia, similar to integrin mutants. Our results identify α-Spectrin and integrins as novel regulators of apical constriction-independent cell elongation, asα-Spectrinand integrin mutant cells fail to columnarize. Finally, we show that increasing and reducing the activity of the Rho1-Myosin II pathway enhances and decreases multilayering ofα-Spectrincells, respectively. Similarly, higher Myosin II activity enhances the integrin multilayering phenotype. This work identifies a primary role for α-Spectrin in controlling cell shape, perhaps by modulating actomyosin. In summary, we suggest that a functional spectrin-integrin complex is essential to balance adequate forces, in order to maintain a monolayered epithelium. PMID:26952981

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

    PubMed Central

    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

    2016-01-01

    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. PMID:27336951

  17. Regulation of the extracellular ligand binding activity of integrins.

    PubMed

    Fernandez, C; Clark, K; Burrows, L; Schofield, N R; Humphries, M J

    1998-07-01

    Integrins are a large heterodimeric family of cell surface adhesion receptors that bind extracellular matrix and cell surface ligands. The extracellular ligand binding activity of integrins is a dynamic and highly regulated event involving the induction of conformational changes within the integrin structure. The adhesive properties of integrins can be controlled by altering the activation state of the integrin, either through conformational change or receptor clustering, using mechanisms that are regulated by intracellular proteins. In this review, we will discuss what is currently known about integrin structure and the ligand binding sites present within the receptor. In addition, the mechanisms by which the ligand binding event is regulated through conformational change will be addressed, and the potential role of intracellular cytoplasmic proteins will be discussed. PMID:9637803

  18. Targeting the expression of integrin receptors in tumors

    NASA Astrophysics Data System (ADS)

    Bloch, Sharon; Liang, Kexian; Dorshow, Richard B.; Ye, Yunpeng; Achilefu, Samuel I.

    2004-06-01

    Expression of integrin αvβ3 is upregulated in a number of cancers including colon, pancreas, lung and breast. Additionally, αvβ3 integrin expression has been linked to tumor metastasis and targeting this cell surface protein could provide a viable approach to image and evaluate the metastatic potential of tumors. Accordingly, we evaluated the selective retention of some near infrared (NIR) fluorescent probes in nude mice bearing A549 lung cancer xenograft that express αvβ3 integrin. Our preliminary results indicate that a novel NIR probe designed to target this integrin selectively accumulated in A549 tumor while other non-integrin specific probes were not retained in the tumor. Blocking studies show that tumor uptake of the probe is mediated by αvβ3 integrin receptor.

  19. Endosomes: Emerging Platforms for Integrin-Mediated FAK Signalling.

    PubMed

    Alanko, Jonna; Ivaska, Johanna

    2016-06-01

    Integrins are vital cell adhesion receptors with the ability to transmit extracellular matrix (ECM) cues to intracellular signalling pathways. ECM-integrin signalling regulates various cellular functions such as cell survival and movement. Integrin signalling has been considered to occur exclusively from adhesion sites at the plasma membrane (PM). However, recent data demonstrates integrin signalling also from endosomes. Integrin-mediated focal adhesion kinase (FAK) signalling is strongly dependent on integrin endocytosis, and endosomal FAK signalling facilitates cancer metastasis by supporting anchorage-independent growth and anoikis resistance. Here we discuss the possible mechanisms and functions of endosomal FAK signalling compared with its previously known roles in other cellular locations and discuss the potential of endosomal FAK as novel target for future cancer therapies. PMID:26944773

  20. Integrins αvβ5 and αvβ3 promote latent TGF-β1 activation by human cardiac fibroblast contraction

    PubMed Central

    Sarrazy, Vincent; Koehler, Anne; Chow, Melissa L.; Zimina, Elena; Li, Chen X.; Kato, Hideyuki; Caldarone, Christopher A.; Hinz, Boris

    2014-01-01

    Aims Pathological tissue remodelling by myofibroblast contraction is a hallmark of cardiac fibrosis. Myofibroblasts differentiate from cardiac fibroblasts under the action of transforming growth factor-β1 (TGF-β1), which is secreted into the extracellular matrix as a large latent complex. Integrin-mediated traction forces activate TGF-β1 by inducing a conformational change in the latent complex. The mesenchymal integrins αvβ5 and αvβ3 are expressed in the heart, but their role in the activation of TGF-β1 remains elusive. Here, we test whether targeting αvβ5 and αvβ3 integrins reduces latent TGF-β1 activation by cardiac fibroblasts with the goal to prevent the formation of α-smooth muscle actin (α-SMA)-expressing cardiac myofibroblasts and their contribution to fibrosis. Methods and results Using a porcine model of induced right ventricular fibrosis and pro-fibrotic culture conditions, we show that integrins αvβ5 and αvβ3 are up-regulated in myofibroblast-enriched fibrotic lesions and differentiated cultured human cardiac myofibroblasts. Both integrins autonomously contribute to latent TGF-β1 activation and myofibroblast differentiation, as demonstrated by function-blocking peptides and antibodies. Acute blocking of both integrins leads to significantly reduced TGF-β1 activation by cardiac fibroblast contraction and loss of α-SMA expression, which is restored by adding active TGF-β1. Manipulating integrin protein levels in overexpression and shRNA experiments reveals that both integrins can compensate for each other with respect to TGF-β1 activation and induction of α-SMA expression. Conclusions Integrins αvβ5 and αvβ3 both control myofibroblast differentiation by activating latent TGF-β1. Pharmacological targeting of mesenchymal integrins is a possible strategy to selectively block TGF-β1 activation by cardiac myofibroblasts and progression of fibrosis in the heart. PMID:24639195

  1. Tenascin-C and integrins in cancer

    PubMed Central

    Yoshida, Toshimichi; Akatsuka, Tatsuya; Imanaka-Yoshida, Kyoko

    2015-01-01

    Tenascin-C (TNC) is highly expressed in cancer tissues. Its cellular sources are cancer and stromal cells, including fibroblasts/myofibroblasts, and also vascular cells. TNC expressed in cancer tissues dominantly contains large splice variants. Deposition of the stroma promotes the epithelial-mesenchymal transition, proliferation, and migration of cancer cells. It also facilitates the formation of cancer stroma including desmoplasia and angiogenesis. Integrin receptors that mediate the signals of TNC have also been discussed. PMID:25793576

  2. Tenascin-C and integrins in cancer.

    PubMed

    Yoshida, Toshimichi; Akatsuka, Tatsuya; Imanaka-Yoshida, Kyoko

    2015-01-01

    Tenascin-C (TNC) is highly expressed in cancer tissues. Its cellular sources are cancer and stromal cells, including fibroblasts/myofibroblasts, and also vascular cells. TNC expressed in cancer tissues dominantly contains large splice variants. Deposition of the stroma promotes the epithelial-mesenchymal transition, proliferation, and migration of cancer cells. It also facilitates the formation of cancer stroma including desmoplasia and angiogenesis. Integrin receptors that mediate the signals of TNC have also been discussed. PMID:25793576

  3. Migfilin, a Molecular Switch in Regulation of Integrin Activation*S⃞

    PubMed Central

    Ithychanda, Sujay Subbayya; Das, Mitali; Ma, Yan-Qing; Ding, Keyang; Wang, Xiaoxia; Gupta, Sudhiranjan; Wu, Chuanyue; Plow, Edward F.; Qin, Jun

    2009-01-01

    The linkage of heterodimeric (α/β) integrin receptors with their extracellular matrix ligands and intracellular actin cytoskeleton is a fundamental step for controlling cell adhesion and migration. Binding of the actin-linking protein, talin, to integrin β cytoplasmic tails (CTs) induces high affinity ligand binding (integrin activation), whereas binding of another actin-linking protein, filamin, to the integrin β CTs negatively regulates this process by blocking the talin-integrin interaction. Here we show structurally that migfilin, a novel cytoskeletal adaptor highly enriched in the integrin adhesion sites, strongly interacts with the same region in filamin where integrin β CTs bind. We further demonstrate that the migfilin interaction dissociates filamin from integrin and promotes the talin/integrin binding and integrin activation. Migfilin thus acts as a molecular switch to disconnect filamin from integrin for regulating integrin activation and dynamics of extracellular matrix-actin linkage. PMID:19074766

  4. Modulation of acute lung injury by integrins.

    PubMed

    Sheppard, Dean

    2012-07-01

    Acute lung injury is a common disorder with a high mortality rate, but previous efforts to develop drugs to treat this disorder have been unsuccessful. In an effort to develop more effective treatments, we have been studying the molecular pathways that regulate the dysfunction of alveolar epithelial cells and endothelial cells that serve as a final common pathway leading to alveolar flooding. Using integrin subunit knockout mice and antibodies we developed by immunizing these mice, we have found important and distinct roles for the αvβ6 integrin on epithelial cells and the αvβ5 integrin on endothelial cells in mediating increases in alveolar permeability in multiple models of acute lung injury. We have also found therapeutic effects of αvβ5 inhibition in two models of septic shock even when the antibody was administered to animals that were obviously ill. These results identify αvβ6 and αvβ5 as promising therapeutic targets for the treatment of acute lung injury and septic shock. PMID:22802286

  5. Traction forces mediated by integrin signaling are necessary for definitive endoderm specification.

    PubMed

    Taylor-Weiner, Hermes; Ravi, Neeraja; Engler, Adam J

    2015-05-15

    Pluripotent embryonic stem cells (ESCs) exert low-traction forces on their niche in vitro whereas specification to definitive endoderm in vivo coincides with force-mediated motility, suggesting a differentiation-mediated switch. However, the onset of contractility and extent to which force-mediated integrin signaling regulates fate choices is not understood. To address the requirement of tractions forces for differentiation, we examined mouse embryonic stem cell (ESC) specification towards definitive endoderm on fibrillar fibronectin containing a deformation-sensitive FRET probe. Inhibiting contractility resulted in an increase in the observed fibronectin FRET intensity ratio but also decreased the amount of phosphorylated nuclear SMAD2, leading to reduced expression of the definitive endoderm marker SOX17. By contrast ESCs maintained in pluripotency medium did not exert significant tractions against the fibronectin matrix. When laminin-111 was added to fibrillar matrices to improve the efficiency of definitive endoderm induction, ESCs decreased their fibronectin traction forces in a laminin-dependent manner; blocking the laminin-binding α3-integrin restored fibronectin matrix deformation and reduced SOX17 expression and SMAD2 phosphorylation, probably because of compensation of inhibitory signaling from SMAD7 after 5 days in culture. These data imply that traction forces and integrin signaling are important regulators of early fate decisions in ESCs. PMID:25908864

  6. Osteoblast mineralization requires β1 integrin/ICAP-1–dependent fibronectin deposition

    PubMed Central

    Brunner, Molly; Millon-Frémillon, Angélique; Chevalier, Genevieve; Nakchbandi, Inaam A.; Mosher, Deane; Block, Marc R.

    2011-01-01

    The morphogenetic and differentiation events required for bone formation are orchestrated by diffusible and insoluble factors that are localized within the extracellular matrix. In mice, the deletion of ICAP-1, a modulator of β1 integrin activation, leads to severe defects in osteoblast proliferation, differentiation, and mineralization and to a delay in bone formation. Deposition of fibronectin and maturation of fibrillar adhesions, adhesive structures that accompany fibronectin deposition, are impaired upon ICAP-1 loss, as are type I collagen deposition and mineralization. Expression of β1 integrin with a mutated binding site for ICAP-1 recapitulates the ICAP-1–null phenotype. Follow-up experiments demonstrated that ICAP-1 negatively regulates kindlin-2 recruitment onto the β1 integrin cytoplasmic domain, whereas an excess of kindlin-2 binding has a deleterious effect on fibrillar adhesion formation. These results suggest that ICAP-1 works in concert with kindlin-2 to control the dynamics of β1 integrin–containing fibrillar adhesions and, thereby, regulates fibronectin deposition and osteoblast mineralization. PMID:21768292

  7. TRPV4 channels mediate cyclic strain-induced endothelial cell reorientation through integrin to integrin signaling

    PubMed Central

    Thodeti, Charles K.; Matthews, Benjamin; Ravi, Arvind; Mammoto, Akiko; Ghosh, Kaustabh; Bracha, Abigail L.; Ingber, Donald E.

    2009-01-01

    Cyclic mechanical strain produced by pulsatile blood flow regulates the orientation of endothelial cells lining blood vessels, and influences critical processes such as angiogenesis. Mechanical stimulation of stretch-activated calcium channels is known to mediate this reorientation response, however, the molecular basis remains unknown. Here we show that cyclically stretching capillary endothelial cells adherent to flexible extracellular matrix substrates activates mechanosensitive TRPV4 ion channels that, in turn, stimulate phosphatidyl inositol-3-kinase-dependent activation and binding of additional ·1 integrin receptors, which promotes cytoskeletal remodeling and cell reorientation. Inhibition of integrin activation using blocking antibodies and knockdown of TRPV4 channels using specific siRNA suppress strain-induced capillary cell reorientation. Thus, mechanical forces that physically deform extracellular matrix may guide capillary cell reorientation through a strain-dependent ‘integrin to integrin’ signaling mechanism mediated by force-induced activation of mechanically-gated TRPV4 ion channels on the cell surface. PMID:19359599

  8. Effect of ECM Stiffness on Integrin-Ligand Binding Strength

    NASA Astrophysics Data System (ADS)

    Thomas, Gawain; Wen, Qi

    2014-03-01

    Many studies have shown that cells respond to the stiffness of their extracellular matrix (ECM). However, the mechanism of this stiffness sensing is not fully understood. We believe that cells probe stiffness by applying intracellular force to the ECM via the integrin-mediated adhesions. The linkage of integrins to the cytoskeleton has been modeled as a slip clutch, which has been shown to affect focal adhesion formation and hence force transmission in a stiffness dependent manner. In contrast, the bonds between integrins and ECM have been characterized as ``catch bonds.'' It is unclear how ECM viscoelasticity affects these catch bonds. We report, for the first time, the effects of ECM stiffness on the binding strength of integrins to ECM ligands by measuring the rupture force of individual integrin-ligand bonds of cells on collagen-coated polyacrylamide gels. Results show that the integrin-collagen bonds of 3T3 fibroblasts are nearly four times stronger on a stiff (30 kPa) gel than on a soft (3 kPa) gel. The stronger integrin bonds on stiffer substrates can promote focal adhesion formation. This suggests that the substrate stiffness regulates the cell-ECM adhesions not only by affecting the cytoskeleton-integrin links but also by modulating the binding of integrins to the ECM.

  9. Blocking neutrophil integrin activation prevents ischemia–reperfusion injury

    PubMed Central

    Yago, Tadayuki; Petrich, Brian G.; Zhang, Nan; Liu, Zhenghui; Shao, Bojing; Ginsberg, Mark H.

    2015-01-01

    Neutrophil recruitment, mediated by β2 integrins, combats pyogenic infections but also plays a key role in ischemia–reperfusion injury and other inflammatory disorders. Talin induces allosteric rearrangements in integrins that increase affinity for ligands (activation). Talin also links integrins to actin and other proteins that enable formation of adhesions. Structural studies have identified a talin1 mutant (L325R) that perturbs activation without impairing talin’s capacity to link integrins to actin and other proteins. Here, we found that mice engineered to express only talin1(L325R) in myeloid cells were protected from renal ischemia–reperfusion injury. Dissection of neutrophil function in vitro and in vivo revealed that talin1(L325R) neutrophils had markedly impaired chemokine-induced, β2 integrin–mediated arrest, spreading, and migration. Surprisingly, talin1(L325R) neutrophils exhibited normal selectin-induced, β2 integrin–mediated slow rolling, in sharp contrast to the defective slow rolling of neutrophils lacking talin1 or expressing a talin1 mutant (W359A) that blocks talin interaction with integrins. These studies reveal the importance of talin-mediated activation of integrins for renal ischemia–reperfusion injury. They further show that neutrophil arrest requires talin recruitment to and activation of integrins. However, although neutrophil slow rolling requires talin recruitment to integrins, talin-mediated integrin activation is dispensable. PMID:26169939

  10. Integrins are required for cardioblast polarisation in Drosophila

    PubMed Central

    2012-01-01

    Background The formation of a tubular organ, such as the heart, requires the communication of positional and polarity signals between migratory cells. Key to this process is the establishment of a new luminal domain on the cell surface, generally from the apical domain of a migratory cell. This domain will also acquire basal properties, as it will produce a luminal extracellular matrix. Integrin receptors are the primary means of cell adhesion and adhesion signaling with the extracellular matrix. Here we characterise the requirement of Integrins in a genetic model of vasculogenesis, the formation of the heart in Drosophila. Results As with vertebrates, the Drosophila heart arises from lateral mesoderm that migrates medially to meet their contralateral partners, to then assemble a midline vessel. During migration, Integrins are among the first proteins restricted to the presumptive luminal domain of cardioblasts. Integrins are required for normal levels of leading edge membrane motility. Apical accumulation of Integrins is enhanced by Robo, and reciprocally, apicalisation of luminal factors like Slit and Robo requires Integrin function. Integrins may provide a template for the formation of a lumen by stabilising lumen factors like Robo. Subsequent to migration, Integrin is required for normal cardioblast alignment and lumen formation. This phenotype is most readily modified by other mutations that affect adhesion, such as Talin and extracellular matrix ligands. Conclusion Our findings reveal an instructive role for Integrins in communicating polarising information to cells during migration, and during transition to an epithelial tube structure. PMID:22353787

  11. Inhibition of integrin α2β1 ameliorates glomerular injury.

    PubMed

    Borza, Corina M; Su, Yan; Chen, Xiwu; Yu, Ling; Mont, Stacey; Chetyrkin, Sergei; Voziyan, Paul; Hudson, Billy G; Billings, Paul C; Jo, Hyunil; Bennett, Joel S; Degrado, William F; Eckes, Beate; Zent, Roy; Pozzi, Ambra

    2012-06-01

    Mesangial cells and podocytes express integrins α1β1 and α2β1, which are the two major collagen receptors that regulate multiple cellular functions, including extracellular matrix homeostasis. Integrin α1β1 protects from glomerular injury by negatively regulating collagen production, but the role of integrin α2β1 in renal injury is unclear. Here, we subjected wild-type and integrin α2-null mice to injury with adriamycin or partial renal ablation. In both of these models, integrin α2-null mice developed significantly less proteinuria and glomerulosclerosis. In addition, selective pharmacological inhibition of integrin α2β1 significantly reduced adriamycin-induced proteinuria, glomerular injury, and collagen deposition in wild-type mice. This inhibitor significantly reduced collagen synthesis in wild-type, but not integrin α2-null, mesangial cells in vitro, demonstrating that its effects are integrin α2β1-dependent. Taken together, these results indicate that integrin α2β1 contributes to glomerular injury by positively regulating collagen synthesis and suggest that its inhibition may be a promising strategy to reduce glomerular injury and proteinuria. PMID:22440900

  12. Inhibition of Integrin α2β1 Ameliorates Glomerular Injury

    PubMed Central

    Borza, Corina M.; Su, Yan; Chen, Xiwu; Yu, Ling; Mont, Stacey; Chetyrkin, Sergei; Voziyan, Paul; Hudson, Billy G.; Billings, Paul C.; Jo, Hyunil; Bennett, Joel S.; DeGrado, William F.; Eckes, Beate; Zent, Roy

    2012-01-01

    Mesangial cells and podocytes express integrins α1β1 and α2β1, which are the two major collagen receptors that regulate multiple cellular functions, including extracellular matrix homeostasis. Integrin α1β1 protects from glomerular injury by negatively regulating collagen production, but the role of integrin α2β1 in renal injury is unclear. Here, we subjected wild-type and integrin α2-null mice to injury with adriamycin or partial renal ablation. In both of these models, integrin α2-null mice developed significantly less proteinuria and glomerulosclerosis. In addition, selective pharmacological inhibition of integrin α2β1 significantly reduced adriamycin-induced proteinuria, glomerular injury, and collagen deposition in wild-type mice. This inhibitor significantly reduced collagen synthesis in wild-type, but not integrin α2-null, mesangial cells in vitro, demonstrating that its effects are integrin α2β1-dependent. Taken together, these results indicate that integrin α2β1 contributes to glomerular injury by positively regulating collagen synthesis and suggest that its inhibition may be a promising strategy to reduce glomerular injury and proteinuria. PMID:22440900

  13. Antagonizing Integrin β3 Increases Immunosuppression in Cancer.

    PubMed

    Su, Xinming; Esser, Alison K; Amend, Sarah R; Xiang, Jingyu; Xu, Yalin; Ross, Michael H; Fox, Gregory C; Kobayashi, Takayuki; Steri, Veronica; Roomp, Kirsten; Fontana, Francesca; Hurchla, Michelle A; Knolhoff, Brett L; Meyer, Melissa A; Morgan, Elizabeth A; Tomasson, Julia C; Novack, Joshua S; Zou, Wei; Faccio, Roberta; Novack, Deborah V; Robinson, Stephen D; Teitelbaum, Steven L; DeNardo, David G; Schneider, Jochen G; Weilbaecher, Katherine N

    2016-06-15

    Integrin β3 is critical for tumor invasion, neoangiogenesis, and inflammation, making it a promising cancer target. However, preclinical and clinical data of integrin β3 antagonists have demonstrated no benefit or worse outcomes. We hypothesized that integrin β3 could affect tumor immunity and evaluated tumors in mice with deletion of integrin β3 in macrophage lineage cells (β3KOM). β3KOM mice had increased melanoma and breast cancer growth with increased tumor-promoting M2 macrophages and decreased CD8(+) T cells. Integrin β3 antagonist, cilengitide, also enhanced tumor growth and increased M2 function. We uncovered a negative feedback loop in M2 myeloid cells, wherein integrin β3 signaling favored STAT1 activation, an M1-polarizing signal, and suppressed M2-polarizing STAT6 activation. Finally, disruption of CD8(+) T cells, macrophages, or macrophage integrin β3 signaling blocked the tumor-promoting effects of integrin β3 antagonism. These results suggest that effects of integrin β3 therapies on immune cells should be considered to improve outcomes. Cancer Res; 76(12); 3484-95. ©2016 AACR. PMID:27216180

  14. Molecular Interplay between Endostatin, Integrins, and Heparan Sulfate*

    PubMed Central

    Faye, Clément; Moreau, Christophe; Chautard, Emilie; Jetne, Reidunn; Fukai, Naomi; Ruggiero, Florence; Humphries, Martin J.; Olsen, Bjorn R.; Ricard-Blum, Sylvie

    2009-01-01

    Endostatin is an endogenous inhibitor of angiogenesis. Although several endothelial cell surface molecules have been reported to interact with endostatin, its molecular mechanism of action is not fully elucidated. We used surface plasmon resonance assays to characterize interactions between endostatin, integrins, and heparin/heparan sulfate. α5β1 and αvβ3 integrins form stable complexes with immobilized endostatin (KD = ∼1.8 × 10−8 m, two-state model). Two arginine residues (Arg27 and Arg139) are crucial for the binding of endostatin to integrins and to heparin/heparan sulfate, suggesting that endostatin would not bind simultaneously to integrins and to heparan sulfate. Experimental data and molecular modeling support endostatin binding to the headpiece of the αvβ3 integrin at the interface between the β-propeller domain of the αv subunit and the βA domain of the β3 subunit. In addition, we report that α5β1 and αvβ3 integrins bind to heparin/heparan sulfate. The ectodomain of the α5β1 integrin binds to haparin with high affinity (KD = 15.5 nm). The direct binding between integrins and heparin/heparan sulfate might explain why both heparan sulfate and α5β1 integrin are required for the localization of endostatin in endothelial cell lipid rafts. PMID:19502598

  15. How Osteoblasts Sense their Environment: Integrin-Extracellular Matrix Interactions and Mechanical Loading of Bone

    NASA Technical Reports Server (NTRS)

    Globus, Ruth K.; Dalton, Bonnie (Technical Monitor)

    2002-01-01

    Osteoblasts are the cells responsible for forming and replacing bone throughout life. We know that mechanical stimulation through weight-bearing at I gravity on Earth is needed to maintain healthy bone, and that osteoblasts play a critical role in that process. Over the last 9 years in my laboratory at NASA ARC, we have studied the regulation of osteoblast function by interactions between the extracellular matrix and die cell. Using a cell culture approach, we defined the repertoire of adhesion receptors, called integrins, which are expressed on the osteoblast surface, as well as specific extracellular matrix proteins, which are needed for cellular differentiation and survival. We are now extending these observations to determine if integrin signaling is involved in the skeletal responses to disuse and recovery from disuse using the rodent model of hindlimb unloading by tail suspension. Together, our cell culture and animal studies are providing new insight into the regulation of osteoblast function in bone.

  16. Epidermal growth factor (EGF) antagonizes transforming growth factor (TGF)-beta1-induced collagen lattice contraction by human skin fibroblasts.

    PubMed

    Park, J S; Kim, J Y; Cho, J Y; Kang, J S; Yu, Y H

    2000-12-01

    Wound contraction plays an important role in healing, but in extreme conditions, it may lead to excessive scar formation and pathological wound contracture. To date, the key regulator of excessive contracture is known to be transforming growth factor-beta (TGF-beta1). In this study, we have evaluated epidermal growth factor (EGF) antagonism in fibroblast-populated collagen lattice (FPCL) gel contraction, which has been generally used as an in vitro model thought to mimic wound contraction in vivo. As expected, TGF-beta1 treatment enhanced normal fibroblast-induced collagen gel contraction in a dose-dependent manner. In contrast, EGF did not affect normal gel formation, but significantly antagonized TGF-beta1-induced gel formation (p<0.05 at 100 ng/ml), whereas the other growth factor, platelet-derived growth factor (PDGF), did not altered either normal or TGF-beta1-induced gel contractions. Similarly, EGF treatment, but not PDGF, also significantly suppressed TGF-beta1 release that was autologously elicited by TGF-beta1 treatment (p<0.01 at 100 ng/ml). Therefore, the results suggest that EGF may negatively regulate the role of TGF-beta1 through attenuating autologous release of TGF-beta1. PMID:11145189

  17. Hepatic expression of mature transforming growth factor beta 1 in transgenic mice results in multiple tissue lesions.

    PubMed

    Sanderson, N; Factor, V; Nagy, P; Kopp, J; Kondaiah, P; Wakefield, L; Roberts, A B; Sporn, M B; Thorgeirsson, S S

    1995-03-28

    Aberrant expression of transforming growth factor beta 1 (TGF-beta 1) has been implicated in a number of disease processes, particularly those involving fibrotic and inflammatory lesions. To determine the in vivo effects of overexpression of TGF-beta 1 on the function and structure of hepatic as well as extrahepatic tissues, transgenic mice were generated containing a fusion gene (Alb/TGF-beta 1) consisting of modified porcine TGF-beta 1 cDNA under the control of the regulatory elements of the mouse albumin gene. Five transgenic lines were developed, all of which expressed the Alb/TGF-beta 1 transgene selectively in hepatocytes. The transgenic line 25 expressing the highest level of the transgene in the liver also had high (> 10-fold over control) plasma levels of TGF-beta 1. Hepatic fibrosis and apoptotic death of hepatocytes developed in all the transgenic lines but was more pronounced in line 25. The fibrotic process was characterized by deposition of collagen around individual hepatocytes and within the space of Disse in a radiating linear pattern. Several extrahepatic lesions developed in line 25, including glomerulonephritis and renal failure, arteritis and myocarditis, as well as atrophic changes in pancreas and testis. The results from this transgenic model strongly support the proposed etiological role for TGF-beta 1 in a variety of fibrotic and inflammatory disorders. The transgenic model may also provide an appropriate paradigm for testing therapeutic interventions aimed at neutralizing the detrimental effects of this important cytokine. PMID:7708687

  18. Leukocyte arrest: Biomechanics and molecular mechanisms of β2 integrin activation

    PubMed Central

    Fan, Zhichao; Ley, Klaus

    2016-01-01

    Integrins are a group of heterodimeric transmembrane receptors that play essential roles in cell–cell and cell–matrix interaction. Integrins are important in many physiological processes and diseases. Integrins acquire affinity to their ligand by undergoing molecular conformational changes called activation. Here we review the molecular biomechanics during conformational changes of integrins, integrin functions in leukocyte biorheology (adhesive functions during rolling and arrest) and molecules involved in integrin activation. PMID:26684674

  19. Characterization of esterified hyaluronan-gelatin polymer composites suitable for chondrogenic differentiation of mesenchymal stem cells.

    PubMed

    Angele, Peter; Müller, Rainer; Schumann, Detlef; Englert, Carsten; Zellner, Johannes; Johnstone, Brian; Yoo, Jung; Hammer, Joachim; Fierlbeck, Johann; Angele, Martin K; Nerlich, Michael; Kujat, Richard

    2009-11-01

    Composite scaffolds of homogeneously mixed esterified hyaluronan (HY) and gelatin (G) were manufactured with variable component compositions (HY100%; HY95%/G5%; HY70%/G30%). The goals of this study were to analyze the produced composite scaffolds using physical and chemical methods, for example, scanning electron microscopy, IR-spectroscopy, water contact angle, protein assay, and tensile testing as well as to assess the effects of adding gelatin to the composite scaffolds on attachment, proliferation, and chondrogenic differentiation of human mesenchymal stem cells. Numbers of attached cells were significantly higher on the composite material compared to pure hyaluronan at different time points of two-dimensional or three-dimensional cell culture (p< 0.02). In composite scaffolds, a significantly greater amount of cartilage-specific extracellular matrix components was deposited after 28 days in culture (glycosaminoglycan: p < 0.001; collagen: p < 0.001) as compared with 100% hyaluronan scaffolds. Additionally, gelatin-containing composite scaffolds displayed stronger promotion of collagen type II expression than pure hyaluronan scaffolds. The mechanism, based on which gelatin influences cell adhesion, was examined. The effect was inhibited by collagenase treatment of the composites or by addition of alpha5beta1-integrin blocking antibodies to the cell suspension. In summary, the results describe the establishment of a class of composite polymer scaffolds, consisting of esterified hyaluronan and gelatin, which are potentially useful for cell-based tissue engineering approaches using mesenchymal stem cells for chondrogenic differentiation. PMID:18985778

  20. Integrin α4β7 expression increases HIV susceptibility in activated cervical CD4+ T cells via an HIV attachment-independent mechanism

    PubMed Central

    Ding, Jian; Tasker, Carley; Lespinasse, Pierre; Dai, Jihong; Fitzgerald-Bocarsly, Patricia; Lu, Wuyuan; Heller, Debra; Chang, Theresa L.

    2015-01-01

    Background CD4+ T cells, the principal target in acute SIV and HIV infection, are crucial for the establishment and dissemination of HIV infection in mucosal tissues. Studies indicate that α4β7 CD4+ T cells are preferentially infected by HIV in vitro and during acute SIV infection. The integrin α4β7 is thought to promote HIV capture by target cells; however, the role of integrin α4β7 in HIV transmission remains controversial. In this study, we characterized immune phenotypes of human cervical T cells and examined HIV preference in integrin α4β7+ CD4+ T cells. In vitro all-trans retinoic acid differentiated peripheral CD4+ T cells (at-RA differentiated cells) were included as a comparison. Results In both peripheral and cervical cells, the majority of HIV p24+ cells were activated CD4+ T cells expressing integrin α4β7. Among infected at-RA differentiated cells, the frequency of CCR5 expression was higher in HIV p24+ cells than in HIV p24- cells; no such difference was observed in cervical cells. Neither the cyclic hexapeptide CWLDVC nor a monoclonal antibody against integrin α4β7 blocked HIV attachment or gp120 binding to target cells regardless of the presence of CD4, indicating that integrin α4β7 did not facilitate HIV capture by target cells. Conclusion Integrin α4β7 expression increases HIV susceptibility, but the mechanism is not through promoting HIV binding to target cells. PMID:26167616

  1. Tumor cell surface beta 1-6 branched oligosaccharides and lung metastasis.

    PubMed

    Lu, Y; Pelling, J C; Chaney, W G

    1994-01-01

    NIH3T3 cells transfected with an activated Ha-ras oncogene were treated with L-PHA, the leukoagglutinin from red kidney beans. Cell lines resistant to L-PHA-mediated cytotoxicity were isolated and found to contain reduced levels of L-PHA-binding oligosaccharides. The levels of N-acetylglucosaminyltransferase V, the enzyme responsible for the initiation of the beta 1-6 branch, were reduced in L-PHA-resistant cells. Tumorigenicity in nude mice was unchanged by the change in oligosaccharide expression, but the ability to form lung tumors after intravenous injection was significantly reduced. These results demonstrate that the ability of NIH3T3 cells transfected with an activated Ha-ras oncogene to form lung tumors after intravenous injection into nude mice is reduced in all six L-PHA selected cell lines containing a reduction in beta 1-6 branched Asn-linked oligosaccharides. PMID:8287620

  2. Protective effects of melittin on transforming growth factor-{beta}1 injury to hepatocytes via anti-apoptotic mechanism

    SciTech Connect

    Lee, Woo-Ram; Park, Ji-Hyun; Kim, Kyung-Hyun; Park, Yoon-Yub; Han, Sang-Mi; Park, Kwan-kyu

    2011-10-15

    Melittin is a cationic, hemolytic peptide that is the main toxic component in the venom of the honey bee (Apis mellifera). Melittin has multiple effects, including anti-bacterial, anti-viral and anti-inflammatory, in various cell types. However, the anti-apoptotic mechanisms of melittin have not been fully elucidated in hepatocytes. Apoptosis contributes to liver inflammation and fibrosis. Knowledge of the apoptotic mechanisms is important to develop new and effective therapies for treatment of cirrhosis, portal hypertension, liver cancer, and other liver diseases. In the present study, we investigated the anti-apoptotic effect of melittin on transforming growth factor (TGF)-{beta}1-induced apoptosis in hepatocytes. TGF-{beta}1-treated hepatocytes were exposed to low doses (0.5 and 1 {mu}g/mL) and high dose (2 {mu}g/mL) of melittin. The low doses significantly protected these cells from DNA damage in TGF-{beta}1-induced apoptosis compared to the high dose. Also, melittin suppressed TGF-{beta}1-induced apoptotic activation of the Bcl-2 family and caspase family of proteins, which resulted in the inhibition of poly-ADP-ribose polymerase (PARP) cleavage. These results demonstrate that TGF-{beta}1 induces hepatocyte apoptosis and that an optimal dose of melittin exerts anti-apoptotic effects against TGF-{beta}1-induced injury to hepatocytes via the mitochondrial pathway. These results suggest that an optimal dose of melittin can serve to protect cells against TGF-{beta}1-mediated injury. - Highlights: > We investigated the anti-apoptotic effect of melittin on TGF-{beta}1-induced hepatocyte. > TGF-{beta}1 induces hepatocyte apoptosis. > TGF-{beta}1-treated hepatocytes were exposed to low doses and high dose of melittin. > Optimal dose of melittin exerts anti-apoptotic effects to hepatocytes.

  3. TGF{beta}1 polymorphisms and late clinical radiosensitivity in patients treated for gynecologic tumors

    SciTech Connect

    Ruyck, Kim de . E-mail: kim.deruyck@UGent.be; Van Eijkeren, Marc; Claes, Kathleen; Bacher, Klaus; Vral, Anne; Neve, Wilfried de; Thierens, Hubert

    2006-07-15

    Purpose: To investigate the association between six transforming growth factor {beta}1 gene (TGF{beta}1) polymorphisms (-1.552delAGG, -800G>A, -509C>T, Leu10Pro, Arg25Pro, Thr263Ile) and the occurrence of late normal tissue reactions after gynecologic radiotherapy (RT). Methods and Materials: Seventy-eight women with cervical or endometrial cancer and 140 control individuals were included in the study. According to the Common Terminology Criteria for Adverse Events version 3.0 (CTCAEv3.0) scale, 25 patients showed late adverse RT reactions (CTC2+), of whom 11 had severe complications (CTC3+). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), single base extension and genotyping assays were performed to examine the polymorphic sites in TGF{beta}1. Results: Homozygous variant -1.552delAGG, -509TT, and 10Pro genotypes were associated with the risk of developing late severe RT reactions. Triple (variant) homozygous patients had a 3.6 times increased risk to develop severe RT reactions (p = 0.26). Neither the -800A allele, nor the 25Pro allele or the 263Ile allele were associated with clinical radiosensitivity. There was perfect linkage disequilibrium (LD) between the -1.552delAGG and the -509C>T polymorphisms, and tight LD between the -1.552/-509 and the Leu10Pro polymorphisms. Haplotype analysis revealed two major haplotypes but could not distinguish radiosensitive from nonradiosensitive patients. Conclusions: The present study shows that homozygous variant TGF{beta}1 -1.552delAGG, -509TT, and 10Pro genotypes may be associated with severe clinical radiosensitivity after gynecologic RT.

  4. Anti-microRNA-378a Enhances Wound Healing Process by Upregulating Integrin Beta-3 and Vimentin

    PubMed Central

    Li, Haoran; Chang, Leslie; Du, William W; Gupta, Shaan; Khorshidi, Azam; Sefton, Michael; Yang, Burton B

    2014-01-01

    Delayed or impaired wound healing is a major public health issue worldwide, especially in patients with diabetes mellitus and vascular atherosclerosis. MicroRNAs have been identified as key regulators of wound healing. Here, we show that miR-Pirate378a transgenic mice (and thus have inhibited miR-378a-5p function) display enhanced wound healing. Expression of vimentin and β3 integrin, two important modulators of wound healing, is markedly elevated in the transgenic mice. MiR-Pirate378a-transfected cells display greater mobility during migration assays, which was hypothesized to be due to the upregulation of vimentin and β3 integrin. Both molecules were confirmed to be targets of miR-378a, and thus their expression could be rescued by miR-Pirate378a. Overexpression of vimentin also contributed to fibroblast differentiation, and upregulation of β3 integrin was responsible for increased angiogenesis. Mice treatment with miR-Pirate378a-conjugated nanoparticles displayed enhanced wound healing. Thus, we have demonstrated that knockdown of miR-378a increased the expression of its target proteins, vimentin, and β3 integrin, which accelerated fibroblast migration and differentiation in vitro and enhanced wound healing in vivo. PMID:24954475

  5. Excessive excretion of cyclic beta-(1,2)-glucan by Rhizobium trifolii TA-1.

    PubMed Central

    Breedveld, M W; Zevenhuizen, L P; Zehnder, A J

    1990-01-01

    At 25 degrees C, the optimal temperature for growth of Rhizobium trifolii TA-1, extracellular and capsular polysaccharide (EPS and CPS) were the main carbohydrate products synthesized in mannitol-rich medium (10 g of mannitol and 1 g of glutamic acid per liter). In the same medium at 33 degrees C, EPS and CPS production was inhibited, and up to 3.9 g of cyclic beta-(1,2)-glucan was produced during an incubation period of 20 days with a total biomass of 0.55 g of protein. In a medium containing 50 g of mannitol and 10 g of glutamic acid per liter, high cell densities (3.95 g of protein) were obtained at 25 degrees C. This biomass excreted 10.9 g of cyclic beta-(1,2)-glucan within 10 days. Concomitantly, 4.8 g of EPS were synthesized, while CPS production was strongly suppressed. The excreted cyclic beta-(1,2)-glucans were neutral and had degrees of polymerization ranging from 17 to 25, with a degree of polymerization of 19 as the major glucan cycle. PMID:2117876

  6. Induction of TNF-alpha production from human peripheral blood monocytes with beta-1,3-glucan oligomer prepared from laminarin with beta-1,3-glucanase from Bacillus clausii NM-1.

    PubMed

    Miyanishi, Nobumitsu; Iwamoto, Yoshiko; Watanabe, Etsuo; Odaz, Tatsuya

    2003-01-01

    We prepared a beta-1,3-glucan oligomer (DP> or = 4) from laminarin (DP: 25-30) derived from Laminaria digitata with beta-1,3-glucanase, and examined its effect on human peripheral blood monocytes. Conditioned medium prepared by incubating monocytes (MC-CM) with the beta-1,3-glucan oligomer showed strong inhibitory activity against the proliferation of human leukemic U937 cells. Since the beta-1,3-glucan oligomer had no direct cytotoxic effect on U937 cells up to 1000 microg/ml, the cytotoxicity of the MC-CM may be due to cytotoxic cytokines produced from monocytes stimulated by the beta-1,3-glucan oligomer. On the other hand, the MC-CM prepared with original laminarin had little effect on the growth of U937 cells. The cytotoxicity of the MC-CM prepared with the beta-1,3-glucan oligomer was significantly reduced by an anti-TNF-alpha antibody, but the anti-TNF-beta antibody had no effect. Our results suggest that the enzymatically depolymerized beta-1,3-glucan oligomer induces TNF-alpha production from human monocytes. PMID:16233391

  7. Simulated Microgravity Alters Actin Cytoskeleton and Integrin-Mediated Focal Adhesions of Cultured Human Mesenchymal Stromal Cells

    NASA Astrophysics Data System (ADS)

    Gershovich, P. M.; Gershovic, J. G.; Buravkova, L. B.

    2008-06-01

    Cytoskeletal alterations occur in several cell types including lymphocytes, glial cells, and osteoblasts, during spaceflight and under simulated microgravity (SMG) (3, 4). One potential mechanism for cytoskeletal gravisensitivity is disruption of extracellular matrix (ECM) and integrin interactions. Focal adhesions are specialized sites of cell-matrix interaction composed of integrins and the diversity of focal adhesion-associated cytoplasmic proteins including vinculin, talin, α-actinin, and actin filaments (4, 5). Integrins produce signals essential for proper cellular function, survival and differentiation. Therefore, we investigated the effects of SMG on F-actin cytoskeleton structure, vinculin focal adhesions, expression of some integrin subtypes and cellular adhesion molecules (CAMs) in mesenchymal stem cells derived from human bone marrow (hMSCs). Simulated microgravity was produced by 3D-clinostat (Dutch Space, Netherlands). Staining of actin fibers with TRITC-phalloidin showed reorganization even after 30 minutes of simulated microgravity. The increasing of cells number with abnormal F-actin was observed after subsequent terms of 3D-clinorotation (6, 24, 48, 120 hours). Randomization of gravity vector altered dimensional structure of stress fibers and resulted in remodeling of actin fibers inside the cells. In addition, we observed vinculin redistribution inside the cells after 6 hours and prolonged terms of clinorotation. Tubulin fibers in a contrast with F-actin and vinculin didn't show any reorganization even after long 3Dclinorotation (120 hours). The expression of integrin α2 increased 1,5-6-fold in clinorotated hMSCs. Also we observed decrease in number of VCAM-1-positive cells and changes in expression of ICAM-1. Taken together, our findings indicate that SMG leads to microfilament and adhesion alterations of hMSCs most probably associated with involvement of some integrin subtypes.

  8. Integrin signalling regulates YAP and TAZ to control skin homeostasis.

    PubMed

    Elbediwy, Ahmed; Vincent-Mistiaen, Zoé I; Spencer-Dene, Bradley; Stone, Richard K; Boeing, Stefan; Wculek, Stefanie K; Cordero, Julia; Tan, Ee H; Ridgway, Rachel; Brunton, Val G; Sahai, Erik; Gerhardt, Holger; Behrens, Axel; Malanchi, Ilaria; Sansom, Owen J; Thompson, Barry J

    2016-05-15

    The skin is a squamous epithelium that is continuously renewed by a population of basal layer stem/progenitor cells and can heal wounds. Here, we show that the transcription regulators YAP and TAZ localise to the nucleus in the basal layer of skin and are elevated upon wound healing. Skin-specific deletion of both YAP and TAZ in adult mice slows proliferation of basal layer cells, leads to hair loss and impairs regeneration after wounding. Contact with the basal extracellular matrix and consequent integrin-Src signalling is a key determinant of the nuclear localisation of YAP/TAZ in basal layer cells and in skin tumours. Contact with the basement membrane is lost in differentiating daughter cells, where YAP and TAZ become mostly cytoplasmic. In other types of squamous epithelia and squamous cell carcinomas, a similar control mechanism is present. By contrast, columnar epithelia differentiate an apical domain that recruits CRB3, Merlin (also known as NF2), KIBRA (also known as WWC1) and SAV1 to induce Hippo signalling and retain YAP/TAZ in the cytoplasm despite contact with the basal layer extracellular matrix. When columnar epithelial tumours lose their apical domain and become invasive, YAP/TAZ becomes nuclear and tumour growth becomes sensitive to the Src inhibitor Dasatinib. PMID:26989177

  9. Integrin signalling regulates YAP and TAZ to control skin homeostasis

    PubMed Central

    Elbediwy, Ahmed; Vincent-Mistiaen, Zoé I.; Spencer-Dene, Bradley; Stone, Richard K.; Boeing, Stefan; Wculek, Stefanie K.; Cordero, Julia; Tan, Ee H.; Ridgway, Rachel; Brunton, Val G.; Sahai, Erik; Gerhardt, Holger; Behrens, Axel; Malanchi, Ilaria; Sansom, Owen J.; Thompson, Barry J.

    2016-01-01

    ABSTRACT The skin is a squamous epithelium that is continuously renewed by a population of basal layer stem/progenitor cells and can heal wounds. Here, we show that the transcription regulators YAP and TAZ localise to the nucleus in the basal layer of skin and are elevated upon wound healing. Skin-specific deletion of both YAP and TAZ in adult mice slows proliferation of basal layer cells, leads to hair loss and impairs regeneration after wounding. Contact with the basal extracellular matrix and consequent integrin-Src signalling is a key determinant of the nuclear localisation of YAP/TAZ in basal layer cells and in skin tumours. Contact with the basement membrane is lost in differentiating daughter cells, where YAP and TAZ become mostly cytoplasmic. In other types of squamous epithelia and squamous cell carcinomas, a similar control mechanism is present. By contrast, columnar epithelia differentiate an apical domain that recruits CRB3, Merlin (also known as NF2), KIBRA (also known as WWC1) and SAV1 to induce Hippo signalling and retain YAP/TAZ in the cytoplasm despite contact with the basal layer extracellular matrix. When columnar epithelial tumours lose their apical domain and become invasive, YAP/TAZ becomes nuclear and tumour growth becomes sensitive to the Src inhibitor Dasatinib. PMID:26989177

  10. Mechanism of action of cyclic beta-1,2-glucan synthetase from Agrobacterium tumefaciens: competition between cyclization and elongation reactions.

    PubMed Central

    Williamson, G; Damani, K; Devenney, P; Faulds, C B; Morris, V J; Stevens, B J

    1992-01-01

    We have examined some aspects of the mechanism of cyclic beta-1,2-glucan synthetase from Agrobacterium tumefaciens (235-kDa protein, gene product of the chvB region). The enzyme produces cyclic beta-1,2-glucans containing 17 to 23 glucose residues from UDP-glucose. In the presence of added cyclic beta-1,2-glucans (> 0.5 mg/ml) (containing 17 to 23 glucose residues), the enzyme instead synthesizes larger cyclic beta-1,2-glucans containing 24 to 30 glucose residues. This is achieved by de novo synthesis and not by disproportion reactions with the added product. This is interpreted as inhibition of the specific cyclization reaction for the synthesis of cyclic beta-1,2-glucans containing 17 to 23 glucose residues but with no concomitant effect on the elongation (polymerization) reaction. Temperature and detergents both affect the distribution of sizes of cyclic beta-1,2-glucans, but glucans containing 24 to 30 glucose residues are not produced. We suggest that the size distribution of cyclic beta-1,2-glucan products depends on competing elongation and cyclization reactions. PMID:1459942

  11. Urokinase Receptors Promote β1 Integrin Function through Interactions with Integrin α3β1

    PubMed Central

    Wei, Ying; Eble, Johannes A.; Wang, Zemin; Kreidberg, Jordan A.; Chapman, Harold A.

    2001-01-01

    The urokinase receptor (uPAR) is linked to cellular migration through its capacity to promote pericellular proteolysis, regulate integrin function, and mediate cell signaling in response to urokinase (uPA) binding. The mechanisms for these activities remain incompletely defined, although uPAR was recently identified as a cis-acting ligand for the β2 integrin CD11b/CD18 (Mac-1). Here we show that a major β1 integrin partner for uPAR/uPA signaling is α3. In uPAR-transfected 293 cells uPAR complexed (>90%) with α3β1 and antibodies to α3 blocked uPAR-dependent vitronectin (Vn) adhesion. Soluble uPAR bound to recombinant α3β1 in a uPA-dependent manner (Kd < 20 nM) and binding was blocked by a 17-mer α3β1 integrin peptide (α325) homologous to the CD11b uPAR-binding site. uPAR colocalized with α3β1 in MDA-MB-231 cells and uPA (1 nM) enhanced spreading and focal adhesion kinase phosphorylation on fibronectin (Fn) or collagen type I (Col) in a pertussis toxin- and α325-sensitive manner. A critical role of α3β1 in uPA signaling was verified by studies of epithelial cells from α3-deficient mice. Thus, uPAR preferentially complexes with α3β1, promoting direct (Vn) and indirect (Fn, Col) pathways of cell adhesion, the latter a heterotrimeric G protein-dependent mechanism of signaling between α3β1 and other β1 integrins. PMID:11598185

  12. Magnetic field-responsive release of transforming growth factor beta 1 from heparin-modified alginate ferrogels.

    PubMed

    Kim, Hwi; Park, Honghyun; Lee, Jae Won; Lee, Kuen Yong

    2016-10-20

    Stimuli-responsive polymeric systems have been widely used for various drug delivery and tissue engineering applications. Magnetic stimulation can be also exploited to regulate the release of pharmaceutical drugs, growth factors, and cells from hydrogels in a controlled manner, on-demand. In the present study, alginate ferrogels containing iron oxide nanoparticles were fabricated via ionic cross-linking, and their various characteristics were investigated. The deformation of the ferrogels was dependent on the polymer concentration, calcium concentration, iron oxide concentration, and strength of magnetic field. To modulate the release of transforming growth factor beta 1 (TGF-β1) under magnetic stimulation, alginate was chemically modified with heparin, as TGF-β1 has a heparin-binding domain. Alginate was first modified with ethylenediamine, and heparin was then conjugated to the ethylenediamine-modified alginate via carbodiimide chemistry. Conjugation of heparin to alginate was confirmed by infrared spectroscopy and proton nuclear magnetic resonance spectroscopy. Sustained release of TGF-β1 from alginate-g-heparin ferrogels was achieved, and application of a magnetic field to the ferrogels regulated TGF-β1 release, resultantly enhancing chondrogenic differentiation of ATDC5 cells, which were used as a model chondrogenic cell line. Alginate-based ferrogels that release drugs in a controlled manner may therefore be useful in many biomedical applications. PMID:27474590

  13. Minimal Synthetic Cells to Study Integrin-Mediated Adhesion

    PubMed Central

    Frohnmayer, Johannes P; Brüggemann, Dorothea; Eberhard, Christian; Neubauer, Stefanie; Mollenhauer, Christine; Boehm, Heike; Kessler, Horst; Geiger, Benjamin; Spatz, Joachim P

    2015-01-01

    To shed light on cell-adhesion-related molecular pathways, synthetic cells offer the unique advantage of a well-controlled model system with reduced molecular complexity. Herein, we show that liposomes with the reconstituted platelet integrin αIIbβ3 as the adhesion-mediating transmembrane protein are a functional minimal cell model for studying cellular adhesion mechanisms in a defined environment. The interaction of these synthetic cells with various extracellular matrix proteins was analyzed using a quartz crystal microbalance with dissipation monitoring. The data indicated that integrin was functionally incorporated into the lipid vesicles, thus enabling integrin-specific adhesion of the engineered liposomes to fibrinogen- and fibronectin-functionalized surfaces. Then, we were able to initiate the detachment of integrin liposomes from these surfaces in the presence of the peptide GRGDSP, a process that is even faster with our newly synthesized peptide mimetic SN529, which specifically inhibits the integrin αIIbβ3. PMID:26257266

  14. Talins and kindlins; partners in integrin-mediated adhesion

    PubMed Central

    Calderwood, David A; Campbell, Iain D; Critchley, David R

    2014-01-01

    Integrin receptors provide a dynamic tightly-regulated link between the extracellular matrix (or cellular counter-receptors) and intracellular cytoskeletal and signalling networks, enabling cells to sense and respond to their chemical and physical environment. Talins and kindlins, two families of FERM–domain proteins, bind the cytoplasmic tail of integrins, recruit cytoskeletal and signalling proteins involved in mechano-transduction, and synergise to activate integrin binding to extracellular ligands. New data reveal the domain structure of full-length talin, provide insights into talin-mediated integrin activation, and show that RIAM recruits talin to the plasma membrane while vinculin stabilises talin in cell–matrix junctions. How Kindlins’ act is less well defined, but disease-causing mutations show that kindlins are also essential for integrin activation, adhesion, cell spreading and signalling. PMID:23860236

  15. Maintenance of Stem Cell Niche Integrity by a Novel Activator of Integrin Signaling

    PubMed Central

    Lee, Joo Yeun; Chang, Karen T.

    2016-01-01

    Stem cells depend critically on the surrounding microenvironment, or niche, for their maintenance and self-renewal. While much is known about how the niche regulates stem cell self-renewal and differentiation, mechanisms for how the niche is maintained over time are not well understood. At the apical tip of the Drosophila testes, germline stem cells (GSCs) and somatic stem cells share a common niche formed by hub cells. Here we demonstrate that a novel protein named Shriveled (Shv) is necessary for the maintenance of hub/niche integrity. Depletion of Shv protein results in age-dependent deterioration of the hub structure and loss of GSCs, whereas upregulation of Shv preserves the niche during aging. We find Shv is a secreted protein that modulates DE-cadherin levels through extracellular activation of integrin signaling. Our work identifies Shv as a novel activator of integrin signaling and suggests a new integration model in which crosstalk between integrin and DE-cadherin in niche cells promote their own preservation by maintaining the niche architecture. PMID:27191715

  16. Conformational coupling of integrin and Thy-1 regulates Fyn priming and fibroblast mechanotransduction

    PubMed Central

    Fiore, Vincent F.; Strane, Patrick W.; Bryksin, Anton V.; White, Eric S.; Hagood, James S.

    2015-01-01

    Progressive fibrosis is characterized by excessive deposition of extracellular matrix (ECM), resulting in gross alterations in tissue mechanics. Changes in tissue mechanics can further augment scar deposition through fibroblast mechanotransduction. In idiopathic pulmonary fibrosis, a fatal form of progressive lung fibrosis, previous work has shown that loss of Thy-1 (CD90) expression in fibroblasts correlates with regions of active fibrogenesis, thus representing a pathologically relevant fibroblast subpopulation. We now show that Thy-1 is a regulator of fibroblast rigidity sensing. Thy-1 physically couples to inactive αvβ3 integrins via its RGD-like motif, altering baseline integrin avidity to ECM ligands and also facilitating preadhesion clustering of integrin and membrane rafts via Thy-1’s glycophosphatidylinositol tether. Disruption of Thy-1–αvβ3 coupling altered recruitment of Src family kinases to adhesion complexes and impaired mechanosensitive, force-induced Rho signaling, and rigidity sensing. Loss of Thy-1 was sufficient to induce myofibroblast differentiation in soft ECMs and may represent a physiological mechanism important in wound healing and fibrosis. PMID:26459603

  17. Selective Detection of RGD-Integrin Binding in Cancer Cells Using Tip Enhanced Raman Scattering Microscopy.

    PubMed

    Xiao, Lifu; Wang, Hao; Schultz, Zachary D

    2016-06-21

    Ligand-receptor interactions play important roles in many biological processes. Cyclic arginine-glycine-aspartic acid (RGD) containing peptides are known to mimic the binding domain of extracellular matrix protein fibronectin and selectively bind to a subset of integrin receptors. Here we report the tip enhanced Raman scattering (TERS) detection of RGD-functionalized nanoparticles bound to integrins produces a Raman scattering signal specific to the bound protein. These results demonstrate that this method can detect and differentiate between two different integrins (α5β1 and αvβ3) bound to RGD-conjugated gold nanoparticles both on surfaces and in a cancer cell membrane. In situ measurements of RGD nanoparticles bound to purified α5β1 and αvβ3 receptors attached to a glass surface provide reference spectra for a multivariate regression model. The TERS spectra observed from nanoparticles bound to cell membranes are analyzed using this regression model and the identity of the receptor can be determined. The ability to distinguish between receptors in the cell membrane provides a new tool to chemically characterize ligand-receptor recognition at molecular level and provide chemical perspective on the molecular recognition of membrane receptors. PMID:27189228

  18. Transforming growth factor-beta 1 stimulates glomerular mesangial cell synthesis of the 72-kd type IV collagenase.

    PubMed Central

    Marti, H. P.; Lee, L.; Kashgarian, M.; Lovett, D. H.

    1994-01-01

    Transforming growth factor-beta 1 (TGF-beta 1) is generally considered to exert positive effects on the accumulation of extracellular matrices. These occur as the net result of enhanced matrix protein synthesis, diminished matrix metalloproteinase (MMP) synthesis, and augmented production of specific inhibitors, including the tissue inhibitor of metalloproteinases (TIMP-1). Given that glomerular TGF-beta 1 synthesis is induced by inflammation, the effects of this cytokine on synthesis of the 72-kd type IV collagenase and TIMP-1 by cultured human mesangial cells were evaluated. Concentrations of TGF-beta 1 of 5 ng/ml and above specifically stimulated the synthesis of the 72-kd type IV collagenase. This effect was independent of the stimulatory effect of TGF-beta 1 on TIMP-1 synthesis, which was maximal in a lower concentration range (0.1 to 1 ng/ml). Most significantly, the net effect at the higher concentrations of TGF-beta 1 was an excess of enzyme over the TIMP-1 inhibitor. Northern blot analysis of TGF-beta 1-stimulated human mesangial cells demonstrated a specific increase in the abundance of the 3.1 kb mRNA transcript encoding the 72-kd type IV collagenase, presumably mediated by a direct stimulation of 72-kd type IV collagenase mRNA transcription observed as early as 3 hours after exposure to TGF-beta 1. These studies were extended to an analysis of the expression of TGF-beta 1 and 72-kd type IV collagenase mRNAs in normal and nephritic rats. In normal animals, basal TGF-beta 1 and 72-kd type IV collagenase mRNA expression was observed in a strictly mesangial distribution. After induction of acute immune complex-mediated glomerulonephritis, there was a major increase in TGF-beta 1 and 72-kd type IV collagenase mRNA expression, which was strictly limited to the expanded, hypercellular mesangial compartment. Enhanced synthesis of the mesangial type IV collagenase in response to TGF-beta 1 released during glomerular inflammatory processes could have an important

  19. Targeted inactivation of β1 integrin induces β3 integrin switching, which drives breast cancer metastasis by TGF-β

    PubMed Central

    Parvani, Jenny G.; Galliher-Beckley, Amy J.; Schiemann, Barbara J.; Schiemann, William P.

    2013-01-01

    Mammary tumorigenesis and epithelial–mesenchymal transition (EMT) programs cooperate in converting transforming growth factor-β (TGF-β) from a suppressor to a promoter of breast cancer metastasis. Although previous reports associated β1 and β3 integrins with TGF-β stimulation of EMT and metastasis, the functional interplay and plasticity exhibited by these adhesion molecules in shaping the oncogenic activities of TGF-β remain unknown. We demonstrate that inactivation of β1 integrin impairs TGF-β from stimulating the motility of normal and malignant mammary epithelial cells (MECs) and elicits robust compensatory expression of β3 integrin solely in malignant MECs, but not in their normal counterparts. Compensatory β3 integrin expression also 1) enhances the growth of malignant MECs in rigid and compliant three-dimensional organotypic cultures and 2) restores the induction of the EMT phenotypes by TGF-β. Of importance, compensatory expression of β3 integrin rescues the growth and pulmonary metastasis of β1 integrin–deficient 4T1 tumors in mice, a process that is prevented by genetic depletion or functional inactivation of β3 integrin. Collectively our findings demonstrate that inactivation of β1 integrin elicits metastatic progression via a β3 integrin–specific mechanism, indicating that dual β1 and β3 integrin targeting is necessary to alleviate metastatic disease in breast cancer patients. PMID:24006485

  20. Lamellipodial tension, not integrin/ligand binding, is the crucial factor to realise integrin activation and cell migration.

    PubMed

    Schulte, Carsten; Ferraris, Gian Maria Sarra; Oldani, Amanda; Galluzzi, Massimiliano; Podestà, Alessandro; Puricelli, Luca; de Lorenzi, Valentina; Lenardi, Cristina; Milani, Paolo; Sidenius, Nicolai

    2016-01-01

    The molecular clutch (MC) model proposes that actomyosin-driven force transmission permits integrin-dependent cell migration. To investigate the MC, we introduced diverse talin (TLN) and integrin variants into Flp-In™ T-Rex™ HEK293 cells stably expressing uPAR. Vitronectin variants served as substrate providing uPAR-mediated cell adhesion and optionally integrin binding. This particular system allowed us to selectively analyse key MC proteins and interactions, effectively from the extracellular matrix substrate to intracellular f-actin, and to therewith study mechanobiological aspects of MC engagement also uncoupled from integrin/ligand binding. With this experimental approach, we found that for the initial PIP2-dependent membrane/TLN/f-actin linkage and persistent lamellipodia formation the C-terminal TLN actin binding site (ABS) is dispensable. The establishment of an adequate MC-mediated lamellipodial tension instead depends predominantly on the coupling of this C-terminal TLN ABS to the actomyosin-driven retrograde actin flow force. This lamellipodial tension is crucial for full integrin activation eventually determining integrin-dependent cell migration. In the integrin/ligand-independent condition the frictional membrane resistance participates to these processes. Integrin/ligand binding can also contribute but is not necessarily required. PMID:26616200

  1. Cross-talk between TGF-beta/SMAD and integrin signaling pathways in regulating hypertrophy of mesenchymal stem cell chondrogenesis under deferral dynamic compression.

    PubMed

    Zhang, Tianting; Wen, Feng; Wu, Yingnan; Goh, Graham Seow Hng; Ge, Zigang; Tan, Lay Poh; Hui, James Hoi Po; Yang, Zheng

    2015-01-01

    The molecular mechanisms of mechanotransduction in regulating mesenchymal stem cell (MSC) chondrogenesis are not fully understood and represent an area of growing investigation. In this study, human MSC was subjected to chondrogenic differentiation in chitosan-coated poly L-lactide-co-ɛ-caprolactone scaffolds under free swelling or deferral dynamic compression conditions. The effect of deferral dynamic compression to MSC chondrogenesis and late stage hypertrophy development was investigated, and the involvement of TGF-β/SMAD pathway and integrin β1 signaling was analyzed. Deferral dynamic compression enhanced cartilage formation and suppressed chondrocyte hypertrophy. Differential cell morphology and cytoskeletal organization were induced under dynamic compression, together with the activation of TGF-β/Activin/Nodal and suppression of the BMP/GDP signaling. This was accompanied by the repression of integrin/FAK/ERK signaling in the non-hypertrophic cells when compared to the free swelling samples. Inhibition studies blocking TGF-β/Activin/Nodal signaling heightened hypertrophy, activate BMP/SMAD1/5/8 and integrin signaling, while inhibition of integrin-ECM interaction suppressed hypertrophy and activate TGF-β/SMAD2/3 in the free-swelling samples. This study demonstrates the roles of TGF-β/SMAD and integrin signaling, and suggests cross-talk between these two signaling pathways, in regulating the compression-driven hypertrophy development. PMID:25453975

  2. Complete integrin headpiece opening in eight steps

    PubMed Central

    Zhu, Jieqing; Zhu, Jianghai

    2013-01-01

    Carefully soaking crystals with Arg-Gly-Asp (RGD) peptides, we captured eight distinct RGD-bound conformations of the αIIbβ3 integrin headpiece. Starting from the closed βI domain conformation, we saw six intermediate βI conformations and finally the fully open βI with the hybrid domain swung out in the crystal lattice. The β1-α1 backbone that hydrogen bonds to the Asp side chain of RGD was the first element to move followed by adjacent to metal ion-dependent adhesion site Ca2+, α1 helix, α1’ helix, β6-α7 loop, α7 helix, and hybrid domain. We define in atomic detail how conformational change was transmitted over long distances in integrins, 40 Å from the ligand binding site to the opposite end of the βI domain and 80 Å to the far end of the hybrid domain. During these movements, RGD slid in its binding groove toward αIIb, and its Arg side chain became ordered. RGD concentration requirements in soaking suggested a >200-fold higher affinity after opening. The thermodynamic cycle shows how higher affinity pays the energetic cost of opening. PMID:23798730

  3. Exposure to particulates, microorganisms, beta(1-3)-glucans, and endotoxins during soybean harvesting.

    PubMed

    Roy, Chad J; Thorne, Peter S

    2003-01-01

    Sclerotinia sclerotiorum is an emerging fungal pathogen affecting soybeans in the United States. In response to its emergence, exposures to particulates, bioaerosols, endotoxins, S. sclerotiorum, and beta(1-3)-glucans were characterized during soybean harvesting. Air sampling was performed on soybean harvesters (combines) and on the farmers in closed cabs as personal samples during harvesting at 17 farms in 1997 and repeated at 15 in 1998. S. sclerotiorum infestation was evident in the fields at 8 of the sites (44%). The geometric mean concentrations (and geometric standard deviations) measured on the combines in 1998 were as follows: total dust, 11.9 (2.8) mg/m(3); inhalable dust 11.7 (6.4) mg/m(3); and beta(1-3)-glucans, 5027 (7) ng/m(3). Values for the personal samples in 1998 were as follows: total dust, 1.2 (6.7) mg/m(3); inhalable dust, 1.1 (5.3); and beta(1-3)-glucans, 674 (9) ng/m(3). These concentrations were two- to threefold higher than in the previous year. Ambient endotoxin concentrations were 4438 EU/m(3) in Year I and 459 EU/m(3) in Year II. Particle size distribution measurements on the combines yielded mass median aerodynamic diameters of 6.6 microm on the combine and 4.0 microm inside the combine cab. Closed combine cabs provided an average workplace protection factor of 11.7 for total dust. Nevertheless, personal exposures to organisms inside combine cabins ranged from 3.6 x 10(4) to 4.0 x 10(8) organisms/m(3). These data indicate the potential exists for high exposures to organic dust and bioaerosols during soybean harvesting. PMID:12908864

  4. IFN beta 1a as Glucocorticoids-Sparing Therapy in a Patient with CLIPPERS

    PubMed Central

    Rico, María; Villafani, Javier; Tuñón, Alberto; Mateos, Valentín; Oliva-Nacarino, Pedro

    2016-01-01

    Patient: Male, 31 Final Diagnosis: CLIPPERS Symptoms: Ataxia • diplopia Medication: IFNbeta 1a Clinical Procedure: — Specialty: Neurology Objective: Rare disease Background: Chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids (CLIPPERS) is a recently described inflammatory disease of the central nervous system, distinguished by brainstem- and spinal cord-centered lesions with a characteristic contrast enhancement on MRI, a lymphocytic perivascular infiltrate on pathological exam, and a dramatic response to and dependence on steroids therapy. Since its initial description in 2010, different glucocorticoid-sparing agents, mostly immunosuppressant drugs, have been used to minimize the dosage, but these therapies also carry the risk of important secondary effects. We present the first reported case of CLIPPERS treated with interferon beta 1a as add-on therapy. Case Report: A previously healthy 31-year-old man presented with gait ataxia and dysarthria. MRI showed pons-centered hyperintense patchy lesions on T2-weighted images. Additional tests ruled out other possible diagnoses and symptoms reversed with intravenous methylprednisolone. Over the years the patient presented with several episodes of deterioration each year, which were partly reversed with glucocorticoid therapy, but leaving him with growing sequelae. Four years after the initial event, treatment with interferon-beta-1a was initiated, achieving reduced frequency of the relapses to 1 every 4 years, which were no longer associated to increasing disability. This allowed reducing glucocorticoids to 30 mg of Deflazacort every other day. Conclusions: Interferon beta-1a could be an alternative to corticosteroid-combined therapy in CLIPPERS and its more benign profile of secondary effects compared to immunosuppressants could make it an attractive choice. PMID:26813773

  5. Nuclear-factor-{kappa}B (NF-{kappa}B) and radical oxygen species play contrary roles in transforming growth factor-{beta}1 (TGF-{beta}1)-induced apoptosis in hepatocellular carcinoma (HCC) cells

    SciTech Connect

    Wang Fang Kaur, Swayamjot; Cavin, Lakita G.; Arsura, Marcello

    2008-12-26

    Nuclear-Factor-{kappa}B (NF-{kappa}{beta} can counteract transforming growth factor-{beta}1 (TGF-{beta}1)-induced apoptosis in malignant hepatocytes through up-regulation of its downstream genes, such as X-linked inhibitor of apoptosis protein (XIAP). Reports have demonstrated that TGF-{beta}1 can induce oxidative stress, and c-Jun N-terminal Kinase1 (JNK1) is indispensable for TGF-{beta}1-induced apoptosis pathway, but the relationship between radical oxygen species (ROS) and the activation of JNKs is still unclear. In the present study, we found that ROS can induce JNK activation in TGF-{beta}1 mediated apoptosis in hepatocytes. The inhibitors of hydrogen peroxide and superoxide, which were produced by mitochondria under stress, could inhibit the phosphorylation of c-Jun in XIAP knockdown cells. In conclusion, it is the first time to show that both NF-{kappa}B and antioxidants can counteract TGF-{beta}1-induced apoptosis in hepatic cell death through JNK1 pathway.

  6. Polymorphism of HF (beta 1H-globulin) in three Asian populations (Bangladeshis, Tibetans and Indonesians).

    PubMed

    Kido, Akira; Susukida, Rie; Oya, Masakazu; Fujitani, Noboru; Kimura, Hiroshi; Hara, Masaaki

    2003-03-01

    The polymorphism of HF (beta 1H-globulin) was investigated in three Asian populations (Bangladeshis, Tibetans and Indonesians) by means of isoelectric focusing and immunoblotting. Phenotypes associated with three common alleles (HF*A, HF*B and HF*Q0) and a rare allele HF*A1 were identified. The observed numbers of phenotypes were in accordance with the numbers expected under the Hardy-Weinberg equilibrium. HF*A1 seems to be a unique allele of the East-Asian Mongoloids including Tibetans and Indonesians. PMID:12712773

  7. The selectivity of beta-adrenoceptor antagonists at the human beta1, beta2 and beta3 adrenoceptors.

    PubMed

    Baker, Jillian G

    2005-02-01

    Beta-adrenoceptor antagonists ("beta-blockers") are one of the most widely used classes of drugs in cardiovascular medicine (hypertension, ischaemic heart disease and increasingly in heart failure) as well as in the management of anxiety, migraine and glaucoma. Where known, the mode of action in cardiovascular disease is from antagonism of endogenous catecholamine responses in the heart (mainly at beta1-adrenoceptors), while the worrisome side effects of bronchospasm result from airway beta2-adrenoceptor blockade. The aim of this study was to determine the selectivity of beta-antagonists for the human beta-adrenoceptor subtypes. (3)H-CGP 12177 whole cell-binding studies were undertaken in CHO cell lines stably expressing either the human beta1-, beta2- or the beta3-adrenoceptor in order to determine the affinity of ligands for each receptor subtype in the same cell background. In this study, the selectivity of well-known subtype-selective ligands was clearly demonstrated: thus, the selective beta1 antagonist CGP 20712A was 501-fold selective over beta2 and 4169-fold selective over beta3; the beta2-selective antagonist ICI 118551 was 550- and 661-fold selective over beta1 and beta3, respectively, and the selective beta3 compound CL 316243 was 10-fold selective over beta2 and more than 129-fold selective over beta1. Those beta2-adrenoceptor agonists used clinically for the treatment of asthma and COPD were beta2 selective: 29-, 61- and 2818-fold for salbutamol, terbutaline and salmeterol over beta1, respectively. There was little difference in the affinity of these ligands between beta1 and beta3 adrenoceptors. The clinically used beta-antagonists studied ranged from bisoprolol (14-fold beta1-selective) to timolol (26-fold beta2-selective). However, the majority showed little selectivity for the beta1- over the beta2-adrenoceptor, with many actually being more beta2-selective. This study shows that the beta1/beta2 selectivity of most clinically used beta-blockers is

  8. Glucocorticoids down-regulate beta 1-adrenergic-receptor expression by suppressing transcription of the receptor gene.

    PubMed Central

    Kiely, J; Hadcock, J R; Bahouth, S W; Malbon, C C

    1994-01-01

    The expression of beta 2-adrenergic receptors is up-regulated by glucocorticoids. In contrast, beta 1-adrenergic receptors display glucocorticoid-induced down-regulation. In rat C6 glioma cells, which express both of these subtypes of beta-adrenergic receptors, the synthetic glucocorticoid dexamethasone stimulates no change in the total beta-adrenergic receptor content, but rather shifts the beta 1:beta 2 ratio from 80:20 to 50:50. Radioligand binding and immunoblotting demonstrate a sharp decline in beta 1-adrenergic receptor expression. Metabolic labelling of cells with [35S]-methionine in tandem with immunoprecipitation by beta 1-adrenergic-receptor-specific antibodies reveals a sharp decline in the synthesis of the receptor within 48 h for cells challenged with glucocorticoid. Steady-state levels of beta 1-adrenergic-receptor mRNA declined from 0.47 to 0.26 amol/microgram of total cellular RNA within 2 h of dexamethasone challenge, as measured by DNA-excess solution hybridization. The stability of receptor mRNA was not influenced by glucocorticoid; the half-lives of the beta 1- and beta 2-subtype mRNAs were 1.7 and 1.5 h respectively. Nuclear run-on assays revealed the basis for the down-regulation of receptor expression, i.e. a sharp decline in the relative rate of transcription for the beta 1-adrenergic-receptor gene in nuclei from dexamethasone-treated as compared with vehicle-treated cells. These data demonstrate transcriptional suppression as a molecular explanation for glucocorticoid-induced down-regulation of beta 1-adrenergic receptors. Images Figure 1 Figure 2 Figure 6 PMID:8092990

  9. Functional Implications of the Subcellular Localization of Ethylene-Induced Chitinase and [beta]-1,3-Glucanase in Bean Leaves.

    PubMed Central

    Mauch, F.; Staehelin, L. A.

    1989-01-01

    Plants respond to an attack by potentially pathogenic organisms and to the plant stress hormone ethylene with an increased synthesis of hydrolases such as chitinase and [beta]-1,3-glucanase. We have studied the subcellular localization of these two enzymes in ethylene-treated bean leaves by immunogold cytochemistry and by biochemical fractionation techniques. Our micrographs indicate that chitinase and [beta]-1,3-glucanase accumulate in the vacuole of ethylene-treated leaf cells. Within the vacuole label was found predominantly over ethylene-induced electron dense protein aggregates. A second, minor site of accumulation of [beta]-1,3-glucanase was the cell wall, where label was present nearly exclusively over the middle lamella surrounding intercellular air spaces. Both kinds of antibodies labeled Golgi cisternae of ethylene-treated tissue, suggesting that the newly synthesized chitinase and [beta]-1,3-glucanase are processed in the Golgi apparatus. Biochemical fractionation studies confirmed the accumulation in high concentrations of both chitinase and [beta]-1,3-glucanase in isolated vacuoles, and demonstrated that only [beta]-1,3-glucanase, but not chitinase, was present in intercellular washing fluids collected from ethylene-treated leaves. Based on these results and earlier studies, we propose a model in which the vacuole-localized chitinase and [beta]-1,3-glucanase are used as a last line of defense to be released when the attacked host cells lyse. The cell wall-localized [beta]-1,3-glucanase, on the other hand, would be involved in recognition processes, releasing defense activating signaling molecules from the walls of invading pathogens. PMID:12359894

  10. FGF-1 reverts epithelial-mesenchymal transition induced by TGF-{beta}1 through MAPK/ERK kinase pathway.

    PubMed

    Ramos, Carlos; Becerril, Carina; Montaño, Martha; García-De-Alba, Carolina; Ramírez, Remedios; Checa, Marco; Pardo, Annie; Selman, Moisés

    2010-08-01

    Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal lung disease characterized by the expansion of the fibroblast/myofibroblast population and aberrant remodeling. However, the origin of mesenchymal cells in this disorder is still under debate. Recent evidence indicates that epithelial-mesenchymal transition (EMT) induced primarily by TGF-beta1 plays an important role; however, studies regarding the opposite process, mesenchymal-epithelial transition, are scanty. We have previously shown that fibroblast growth factor-1 (FGF-1) inhibits several profibrogenic effects of TGF-beta1. In this study, we examined the effects of FGF-1 on TGF-beta1-induced EMT. A549 and RLE-6TN (human and rat) alveolar epithelial-like cell lines were stimulated with TGF-beta1 for 72 h, and then, in the presence of TGF-beta1, were cultured with FGF-1 plus heparin for an additional 48 h. After TGF-beta1 treatment, epithelial cells acquired a spindle-like mesenchymal phenotype with a substantial reduction of E-cadherin and cytokeratins and concurrent induction of alpha-smooth muscle actin measured by real-time PCR, Western blotting, and immunocytochemistry. FGF-1 plus heparin reversed these morphological changes and returned the epithelial and mesenchymal markers to control levels. Signaling pathways analyzed by selective pharmacological inhibitors showed that TGF-beta1 induces EMT through Smad pathway, while reversion by FGF-1 occurs through MAPK/ERK kinase pathway, resulting in ERK-1 phosphorylation and Smad2 dephosphorylation. These findings indicate that TGF-beta1-induced EMT is reversed by FGF-1 and suggest therapeutic approaches to target this process in IPF. PMID:20495078

  11. Inhibiting and stimulating effects of TGF-. beta. 1 on osteoclastic bone resorption in fetal mouse bone organ cultures

    SciTech Connect

    Dieudonne, S.C.; Foo, P.; van Zoelen, E.J.; Burger, E.H. )

    1991-05-01

    The effects of TGF-{beta} 1 on osteoclastic resorption of fetal mouse calvaria and long bones at various stages of development was studied in organ culture. In resorbing calvariae and long bones with an established marrow cavity TGF-beta 1 (4-10 ng/ml) had a stimulating effect on 45Ca release that was partially inhibited by indomethacin. In primitive long bones, however, which were explanted before osteoclast invasion and excavation of a marrow cavity had started, TGF-beta 1 (1-4 ng/ml) inhibited 45Ca release by an indomethacin-insensitive mechanism. Histomorphometry of long bones after staining for tartrate-resistant acid phosphatase (TRAP) revealed that TGF-beta 1 treatment inhibited the migration of TRAP-positive cells from periosteum to developing marrow cavity and inhibited cell fusion. However, the formation of (mononuclear) TRAP-positive cells in the periosteum-perichondrium was strongly enhanced. These data suggest that TGF-beta 1 modulates various steps in the cascade of osteoclast development, recruitment, and activation in different ways, involving both prostaglandin-mediated and prostaglandin-independent pathways. Therefore the net effect of exogenous TGF-beta 1 on osteoclastic resorption in bone organ cultures depends on the relative prevalence of osteoclast progenitors, precursors, and mature osteoclasts in the tissue under study.

  12. Alternative statistical methods for estimating efficacy of interferon beta-1b for multiple sclerosis clinical trials

    PubMed Central

    2011-01-01

    Background In the randomized study of interferon beta-1b (IFN beta-1b) for multiple sclerosis (MS), it has usually been evaluated the simple annual relapse rate as the study endpoint. This study aimed to investigate the performance of various regression models using information regarding the time to each recurrent event and considering the MS specific data generation process, and to estimate the treatment effect of a MS clinical trial data. Methods We conducted a simulation study with consideration of the pathological characteristics of MS, and applied alternative efficacy estimation methods to real clinical trial data, including 5 extended Cox regression models for time-to-event analysis, a Poisson regression model and a Poisson regression model with Generalized Estimating Equations (GEE). We adjusted for other important covariates that may have affected the outcome. Results We compared the simulation results for each model. The hazard ratios of real data were estimated for each model including the effects of other covariates. The results (hazard ratios of high-dose to low-dose) of all models were approximately 0.7 (range, 0.613 - 0.769), whereas the annual relapse rate ratio was 0.714. Conclusions The precision of the treatment estimation was increased by application of the alternative models. This suggests that the use of alternative models that include recurrence event data may provide better analyses. PMID:21612661

  13. Localization of beta-(1,3)-glucanase in the mycelium of Sclerotium rolfsii.

    PubMed Central

    Kritzman, G; Chet, I; Henis, Y

    1978-01-01

    The role of the lytic enzyme beta-(1,3)-glucanase in cell wall synthesis and its distribution in the mycelium of the fungus Sclerotium rolfsii were studied. Enzyme activity was determined after enzyme extraction with Triton X-100 from a cell wall preparation. Specific zones of immunofluorescence appeared in the hyphal tips, clamp connections, new septa, and lateral branching when a specific antiserum was used with the indirect method of the fluorescent antibody staining. Enzymatic activity in the cell wall preparation was inactivated by diethylpyrocarbonate. However, 69% of the total enzymatic activity was present in a latent form which was not affected by the ester. This result suggests that most of the beta-(1,3)-glucanase was present along the hyphal cell walls in a "masked" form. An active enzyme appeared only in those regions which showed immunofluorescence. The activity of glucan synthetase, an enzyme essential for wall formation, was higher in the branching funus grown on L-threonine-supplemented synthetic medium than in the synthetic medium-grown fungus. Images PMID:350824

  14. Integrin Activation States and Eosinophil Recruitment in Asthma

    PubMed Central

    Johansson, Mats W.; Mosher, Deane F.

    2013-01-01

    Eosinophil arrest and recruitment to the airway in asthma are mediated, at least in part, by integrins. Eosinophils express α4β1, α6β1, αLβ2, αMβ2, αXβ2, αDβ2, and α4β7 integrins, which interact with counter-receptors on other cells or ligands in the extracellular matrix. Whether a given integrin-ligand pair mediates cell adhesion and migration depends on the activation state of the integrin. Integrins exist in an inactive bent, an intermediate-activity extended closed, and a high-activity extended open conformation. Integrin activation states can be monitored by conformation-specific monoclonal antibodies (mAbs). Studies in mice indicate that both β1 and β2 integrins mediate eosinophil recruitment to the lung. In vitro studies indicate that α4β1 and αMβ2 are the principal integrins mediating eosinophil adhesion, including to vascular cell adhesion molecule-1 and the novel αMβ2 ligand periostin. In vivo, blood eosinophils have intermediate-activity β1 integrins, as judged by mAb N29, apparently resulting from eosinophil binding of P-selectin on the surface of activated platelets, and have a proportion of their β2 integrins in the intermediate conformation, as judged by mAb KIM-127, apparently due to exposure to low concentrations of interleukin-5 (IL-5). Airway eosinophils recovered by bronchoalveolar lavage (BAL) after segmental antigen challenge have high-activity β1 integrins and high-activity αMβ2 that does not require IL-5. Here we review information on how the activation states of eosinophil β1 and β2 integrins correlate with measurements of eosinophil recruitment and pulmonary function in asthma. Blood eosinophil N29 reactivity is associated with decreased lung function under various circumstances in non-severe asthma and KIM-127 with BAL eosinophil numbers, indicating that intermediate-activity α4β1 and αMβ2 of blood eosinophils are important for eosinophil arrest and consequently for recruitment and aspects of asthma. PMID

  15. Integrin α3 Mutations with Kidney, Lung, and Skin Disease

    PubMed Central

    Has, Cristina; Spartà, Giuseppina; Kiritsi, Dimitra; Weibel, Lisa; Moeller, Alexander; Vega-Warner, Virginia; Waters, Aoife; He, Yinghong; Anikster, Yair; Esser, Philipp; Straub, Beate K.; Hausser, Ingrid; Bockenhauer, Detlef; Dekel, Benjamin; Hildebrandt, Friedhelm; Bruckner-Tuderman, Leena; Laube, Guido F.

    2012-01-01

    SUMMARY Integrin α3 is a transmembrane integrin receptor subunit that mediates signals between the cells and their microenvironment. We identified three patients with homozygous mutations in the integrin α3 gene that were associated with disrupted basement-membrane structures and compromised barrier functions in kidney, lung, and skin. The patients had a multiorgan disorder that included congenital nephrotic syndrome, interstitial lung disease, and epidermolysis bullosa. The renal and respiratory features predominated, and the lung involvement accounted for the lethal course of the disease. Although skin fragility was mild, it provided clues to the diagnosis. PMID:22512483

  16. Thyroid hormones and tetrac: new regulators of tumour stroma formation via integrin αvβ3.

    PubMed

    Schmohl, Kathrin A; Müller, Andrea M; Wechselberger, Alexandra; Rühland, Svenja; Salb, Nicole; Schwenk, Nathalie; Heuer, Heike; Carlsen, Janette; Göke, Burkhard; Nelson, Peter J; Spitzweg, Christine

    2015-12-01

    To improve our understanding of non-genomic, integrin αvβ3-mediated thyroid hormone action in tumour stroma formation, we examined the effects of triiodo-l-thyronine (T3), l-thyroxine (T4) and integrin-specific inhibitor tetrac on differentiation, migration and invasion of mesenchymal stem cells (MSCs) that are an integral part of the tumour's fibrovascular network. Primary human bone marrow-derived MSCs were treated with T3 or T4 in the presence of hepatocellular carcinoma (HCC) cell-conditioned medium (CM), which resulted in stimulation of the expression of genes associated with cancer-associated fibroblast-like differentiation as determined by qPCR and ELISA. In addition, T3 and T4 increased migration of MSCs towards HCC cell-CM and invasion into the centre of three-dimensional HCC cell spheroids. All these effects were tetrac-dependent and therefore integrin αvβ3-mediated. In a subcutaneous HCC xenograft model, MSCs showed significantly increased recruitment and invasion into tumours of hyperthyroid mice compared to euthyroid and, in particular, hypothyroid mice, while treatment with tetrac almost completely eliminated MSC recruitment. These studies significantly improve our understanding of the anti-tumour activity of tetrac, as well as the mechanisms that regulate MSC differentiation and recruitment in the context of tumour stroma formation, as an important prerequisite for the utilisation of MSCs as gene delivery vehicles. PMID:26307023

  17. Epstein-Barr virus latent membrane protein-2A induces ITAM/Syk- and Akt-dependent epithelial migration through αv-integrin membrane translocation.

    PubMed

    Fotheringham, Julie A; Coalson, Nicole E; Raab-Traub, Nancy

    2012-10-01

    Epstein-Barr virus (EBV) is a highly prevalent herpesvirus associated with epithelial cancers, including nasopharyngeal carcinoma (NPC). The EBV protein latent membrane protein 2 (LMP2) is expressed in NPC tumor tissue and has been shown to induce transformation, inhibit differentiation, and promote migration of epithelial cells. In this study, the effect of LMP2A on migration of human epithelial cells was further analyzed. LMP2A expression induced migration in human foreskin keratinocytes (HFK) and HaCaT keratinocytes measured by wound healing scratch assay and chemoattractant-induced Transwell migration assay. The induction of migration by LMP2A required the ITAM signaling domain of LMP2A and activation of the Syk tyrosine kinase. LMP2A-induced Transwell migration required the Akt signaling pathway, and activation of Akt by LMP2A required the ITAM signaling domain of LMP2A. LMP2A also induced phosphorylation of the Akt target GSK3β, a Wnt signaling mediator that has been shown to regulate the activity of focal adhesion kinase (FAK), a tyrosine kinase activated by clustering and ligand interaction of integrins. Inhibition of either FAK or its signaling mediator Src kinase inhibited LMP2A-induced migration. Interestingly, αV-integrin was greatly increased in membrane-enriched fractions by LMP2A, and a neutralizing antibody to αV-integrin blocked migration, suggesting that the effects of LMP2A on membrane-localized αV-integrin promoted migration. The results of this study indicate that LMP2A expression in human epithelial cells induces αV-integrin-dependent migration through a mechanism requiring ITAM-mediated Syk and Akt activation and inducing membrane translocation or stabilization of αV-integrin and FAK activation. The specific effects of LMP2A on an integrin with a diverse repertoire of ligand specificities could promote migration of different cell types and be initiated by multiple chemoattractants. PMID:22837212

  18. Suppression of integrin activation by the membrane-distal sequence of the integrin alphaIIb cytoplasmic tail.

    PubMed Central

    Yamanouchi, Jun; Hato, Takaaki; Tamura, Tatsushiro; Fujita, Shigeru

    2004-01-01

    Integrin cytoplasmic tails regulate integrin activation including an increase in integrin affinity for ligands. Although there is ample evidence that the membrane-proximal regions of the alpha and beta tails interact with each other to maintain integrins in a low-affinity state, little is known about the role of the membrane-distal region of the alpha tail in regulation of integrin activation. We report a critical sequence for regulation of integrin activation in the membrane-distal region of the alphaIIb tail. Alanine substitution of the RPP residues in the alphaIIb tail rendered alphaIIbbeta3 constitutively active in a metabolic energy-dependent manner. Although an alphaIIb/alpha6Abeta3 chimaeric integrin, in which the alphaIIb tail was replaced by the alpha6A tail, was in an energy-dependent active state to bind soluble ligands, introduction of the RPP sequence into the alpha6A tail inhibited binding of an activation-dependent antibody PAC1. In alphaIIb/alpha6Abeta3, deleting the TSDA sequence from the alpha6A tail or single amino acid substitutions of the TSDA residues inhibited alphaIIb/alpha6Abeta3 activation and replacing the membrane-distal region of the alphaIIb tail with TSDA rendered alphaIIbbeta3 active, suggesting a stimulatory role of TSDA in energy-dependent integrin activation. However, adding TSDA to the alphaIIb tail containing the RPP sequence of the membrane-distal region failed to activate alphaIIbbeta3. These results suggest that the RPP sequence after the GFFKR motif of the alphaIIb tail suppresses energy-dependent alphaIIbbeta3 activation. These findings provide a molecular basis for the regulation of energy-dependent integrin activation by alpha subunit tails. PMID:14723599

  19. Angiotensin II increases CTGF expression via MAPKs/TGF-{beta}1/TRAF6 pathway in atrial fibroblasts

    SciTech Connect

    Gu, Jun; Liu, Xu; Wang, Quan-xing; Tan, Hong-wei; Guo, Meng; Jiang, Wei-feng; Zhou, Li

    2012-10-01

    The activation of transforming growth factor-{beta}1(TGF-{beta}1)/Smad signaling pathway and increased expression of connective tissue growth factor (CTGF) induced by angiotensin II (AngII) have been proposed as a mechanism for atrial fibrosis. However, whether TGF{beta}1/non-Smad signaling pathways involved in AngII-induced fibrogenetic factor expression remained unknown. Recently tumor necrosis factor receptor associated factor 6 (TRAF6)/TGF{beta}-associated kinase 1 (TAK1) has been shown to be crucial for the activation of TGF-{beta}1/non-Smad signaling pathways. In the present study, we explored the role of TGF-{beta}1/TRAF6 pathway in AngII-induced CTGF expression in cultured adult atrial fibroblasts. AngII (1 {mu}M) provoked the activation of P38 mitogen activated protein kinase (P38 MAPK), extracellular signal-regulated kinase 1/2(ERK1/2) and c-Jun NH(2)-terminal kinase (JNK). AngII (1 {mu}M) also promoted TGF{beta}1, TRAF6, CTGF expression and TAK1 phosphorylation, which were suppressed by angiotensin type I receptor antagonist (Losartan) as well as p38 MAPK inhibitor (SB202190), ERK1/2 inhibitor (PD98059) and JNK inhibitor (SP600125). Meanwhile, both TGF{beta}1 antibody and TRAF6 siRNA decreased the stimulatory effect of AngII on TRAF6, CTGF expression and TAK1 phosphorylation, which also attenuated AngII-induced atrial fibroblasts proliferation. In summary, the MAPKs/TGF{beta}1/TRAF6 pathway is an important signaling pathway in AngII-induced CTGF expression, and inhibition of TRAF6 may therefore represent a new target for reversing Ang II-induced atrial fibrosis. -- Highlights: Black-Right-Pointing-Pointer MAPKs/TGF{beta}1/TRAF6 participates in AngII-induced CTGF expression in atrial fibroblasts. Black-Right-Pointing-Pointer TGF{beta}1/TRAF6 participates in AngII-induced atrial fibroblasts proliferation. Black-Right-Pointing-Pointer TRAF6 may represent a new target for reversing Ang II-induced atrial fibrosis.

  20. Astrocytic αVβ3 Integrin Inhibits Neurite Outgrowth and Promotes Retraction of Neuronal Processes by Clustering Thy-1