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PubMed2003

Physiological levels of tumstatin, a fragment of collagen IV alpha3 chain, are generated by MMP-9 proteolysis and suppress angiogenesis via alphaV beta3 integrin.

We demonstrate a physiological role for tumstatin, a cleavage fragment of the alpha3 chain of type IV collagen (Col IValpha3), which is present in the circulation. Mice with a genetic deletion of Col IValpha3 show accelerated tumor growth associated with enhanced pathological angiogenesis, while angiogenesis associated with development and tissue repair are unaffected. Supplementing Col IValpha3-deficient mice with recombinant tumstatin to a normal physiological concentration abolishes the increased rate of tumor growth. The suppressive effects of tumstatin require alphaVbeta3 integrin expressed on pathological, but not on physiological, angiogenic blood vessels. Mice deficient in matrix metalloproteinase-9, which cleaves tumstatin efficiently from Col IValpha3, have decreased circulating tumstatin and accelerated growth of tumor. These results indicate that MMP-generated fragments of basement membrane collagen can have endogenous function as integrin-mediated suppressors of pathologic angiogenesis and tumor growth.

Animals↗
PubMed2000

Contraction of collagen matrices mediated by alpha2beta1A and alpha(v)beta3 integrins.

The (beta)1-null fibroblastic cell line GD25 and its derivatives were studied to gain an understanding of the roles of (beta)1 and (beta)3 integrins in the initial (1-hour) contraction of collagen gels. Stable transfectants of GD25 cells expressing the (beta)1A splice variant of (beta)1 ((beta)1A-GD25) did not express (alpha)2(beta)1A and did not adhere to collagen. After transfection of (alpha)2 into (beta)1A-GD25 cells, the (alpha)2(beta)1A-GD25 transfectants contracted collagen gels in the presence of serum, whereas (beta)1A-GD25 cells did not. The GD25 parental cells, however, also contracted collagen gels. Collagen gel contraction by GD25 cells was blocked by antibodies to (alpha)v(beta)3 or a RGD-containing peptide, indicating that (alpha)v(beta)3 is the integrin responsible for mediation of contraction by GD25 cells. Collagen gel contraction by (alpha)2(beta)1A-GD25 cells was not inhibited by antibodies to (alpha)v(beta)3 or RGD-containing peptide, but was inhibited by anti-(alpha)2 antibody. Flow cytometry demonstrated negligible expression of (alpha)v(beta)3 by (beta)1A-GD25 and (alpha)2(beta)1A-GD25 cells when compared to GD25 cells. Platelet derived growth factor (PDGF) and sphingosine-1-phosphate (S1P) enabled gel contraction by (alpha)2(beta)1A-GD25 and GD25 cells, respectively, in the absence of serum. PDGF-stimulated contraction by (alpha)2(beta)1A-GD25 cells was attenuated in the presence of inhibitors of phosphatidylinositol-3-kinase whereas such inhibitors had no effect on S1P-stimulated contraction by GD25 cells. These experiments using the (beta)1-null GD25 cells and (beta)1A and (alpha)2(beta)1A transfectants demonstrate that (alpha)2(beta)1A and (alpha)v(beta)3 independently mediate collagen gel contraction and are regulated by different serum factors and signaling pathways.

Cell Line↗
PubMed2006

Bone sialoprotein enhances migration of bone marrow stromal cells through matrices by bridging MMP-2 to alpha(v)beta3-integrin.

UNLABELLED: BMSCs migrate through matrix barriers and differentiate into osteoblasts. BSP enhances osteogenic cell migration through basement membrane and collagen matrices in vitro by localizing MMP-2 on the cell surface through alpha(v)beta(3)-integrin. INTRODUCTION: The specific mechanisms by which bone marrow stromal cells (BMSCs) leave their primary sites, move through matrices encountered during homing to their site of final differentiation, and remove preexisting matrices in preparation for bone matrix production are not well understood. MATERIALS AND METHODS: The enhanced migration of human osteoblast precursor cells through matrix barriers by bone sialoprotein (BSP) was studied by a modified Boyden-chamber assay. The bridging of normally soluble matrix metalloproteinase 2 (MMP-2) to the cell surface receptor, alpha(v)beta(3)-integrin, by BSP was analyzed by flow cytometry. RESULTS: BSP enhanced the in vitro passage of BMSCs and pre-osteoblasts through matrix barriers (Matrigel and denatured type I collagen) in a dose-dependent manner. An intact ArgGlyAsp (RGD) was required in the BSP for enhanced migration through the barriers but was not sufficient, as shown by the inactivity of two other SIBLING (Small Integrin-Binding LIgand, N-linked Glycoprotein) family members, osteopontin and dentin matrix protein-1. The specificity of the BSP enhancement activity was apparently caused by this molecule's ability to bridge MMP-2 to the cell surfaces. CONCLUSIONS: Pre-osteoblasts and their BMSC precursors may use MMP-2/BSP/integrin complexes to disrupt matrix barriers during migration to their final destinations in vivo.

Bone Marrow Cells↗
PubMed2001

Podosomes in osteoclast-like cells: structural analysis and cooperative roles of paxillin, proline-rich tyrosine kinase 2 (Pyk2) and integrin alphaVbeta3.

Macrophages and osteoclasts develop unique contact sites with the extracellular matrix called podosomes. Podosomes have been associated with migratory and invasive cell characteristics, but a basic mechanism outlining their function is lacking. We have used chicken and human monocytes differentiating in vitro into osteoclast-like cells in the presence of RANKL-ODF to study these cytoskeletal structures. During the differentiation process, podosomes are redistributed from the cell body in early macrophages to the cell periphery in increasingly spread and multinucleated cells expressing high levels of integrin alphaVbeta3. Immunofluorescence with anti-phosphotyrosine antibodies revealed increased tyrosine-phosphorylation at the basal tips of these podosomes. RANKL-ODF treatment reinforced the peripheral location of podosomes and initiated their partial fusion to larger F-actin-containing structures that displayed reduced levels of tyrosine phosphorylation. Paxillin and the FAK-related kinase Pyk2 colocalized with integrin alphaVbeta3 in the juxtamembrane region surrounding individual podosomes. In lysates of macrophages and differentiated osteoclasts both paxillin and Pyk2 associated with synthetic and recombinant polypeptides containing the C-terminal region of the integrin beta3 cytoplasmic domain. These in vitro interactions were direct and they were abolished by substitutions in the beta3 integrin peptides known to disrupt integrin function in vivo. The marked adhesion-dependent tyrosinephosphorylation of Pyk2 and paxillin however did not detectably alter their interaction with beta3 tail peptides in cell lysates. Our results provide novel insight into the molecular architecture and the phosphorylation dynamics in podosomes. Moreover, they outline a novel potential mechanism for the recruitment of paxillin and Pyk2 to beta3 integrin-dependent cell contacts.

Actins↗
PubMed1996

Intracellular calcium and calcineurin regulate neutrophil motility on vitronectin through a receptor identified by antibodies to integrins alphav and beta3.

Buffering of intracellular calcium ([Ca2+]i) or inhibition of the calcium/calmodulin-dependent phosphatase, calcineurin, results in neutrophils being unable to detach from vitronectin with a consequent loss of motility. Treatment of [Ca2+]i-buffered or calcineurin-inhibited neutrophils with monoclonal antibodies (MoAbs) to beta3 or alphav beta3 integrins allowed neutrophils to detach and restored motility. Quantitative immunofluorescence and flow cytometry showed that MoAbs specific for beta3, alphav, or alphav beta3 integrins bind to neutrophils. Immunolocalization studies using antibodies to the highly conserved cytoplasmic domains of alphav and beta3 also identified the receptor on neutrophils. Whereas antibodies to alphav, alphav beta3, and beta3 recognized the receptor in intact cells, only the beta3 MoAb immunoprecipitated the receptor from a neutrophil cell lysate. The alpha subunit co-immunoprecipitated by the beta3 antibody reacted with an antibody to alphav by Western blot. Peptide maps of V8 protease digests showed a strong similarity in alpha and beta chains precipitated by antibodies to beta3 from neutrophils and endothelial cells. These results indicate that [Ca2+]i and calcineurin regulate neutrophil motility on vitronectin through an alphav beta3-like receptor. Although we cannot rule out the possibility that neutrophils have an isoform of alphav, such an isoform would have to be similar enough to react with alphav- and alphav beta3-specific MoAbs in intact cells.

Amino Acid Sequence↗
PubMed1999

Integrin alpha(v)beta3 (vitronectin receptor) is a candidate receptor for the virulent echovirus 9 strain Barty.

The enterovirus echovirus 9 strain Barty (E9/Barty) is pathogenic for newborn mice as well as for humans. In contrast to the apathogenic prototype strain Hill, strain Barty encodes an RGD motif in the C-terminal part of the structural protein VP1. Data are presented that show that E9/Barty binds its target cells via contact of the RGD motif to the alpha(v)beta3 integrin (vitronectin receptor), whereas prototype Hill uses a different, still unidentified receptor site. Furthermore, virus titres of murine muscle tissue were compared after infection of newborn and 1-, 2-, 3- and 12-week-old mice. The replication capacity of the virus decreased dramatically with age of the infected mice. Since E9/Barty does not replicate or replicates only poorly in mice older than about 5 days, and expression of the vitronectin receptor is reported to be down-regulated in striated muscle tissue during development, it is suggested that susceptibility of mice to this echovirus infection is controlled by the availability of alpha(v)beta3 integrin.

Animals↗
PubMed2001

Green fluorescent protein (GFP) tagged to the cytoplasmic tail of alphaIIb or beta3 allows the expression of a fully functional integrin alphaIIb(beta3): effect of beta3GFP on alphaIIb(beta3) ligand binding.

Using green fluorescent protein (GFP) as an autofluorescent tag, we report the first successful visualization of a beta3 integrin in a living cell. GFP fused in frame to the cytoplasmic tail of either alphaIIb or beta3 allowed normal expression, heterodimerization, processing and surface exposure of alphaIIbGFPbeta3 and alphaIIb(beta3)GFP receptors in Chinese hamster ovary (CHO) cells. Direct microscopic observation of the autofluorescent cells in suspension following antibody-induced alphaIIb(beta3) capping revealed an intense autofluorescent cap corresponding to unlabelled immunoclustered GFP-tagged alphaIIb(beta3). GFP-tagged alphaIIbbeta3 receptors mediated fibrinogen-dependent cell adhesion, were readily detectable in focal adhesions of unstained living cells and triggered p125(FAK) tyrosine phosphorylation similar to wild-type alphaIIb(beta3) (where FAK corresponds to focal adhesion kinase). However, GFP tagged to beta3, but not to alphaIIb, induced spontaneous CHO cell aggregation in the presence of soluble fibrinogen, as well as binding of the fibrinogen mimetic monoclonal antibody PAC1 in the absence of alphaIIb(beta3) receptor activation. Time-lapse imaging of living transfectants revealed a characteristic redistribution of GFP-tagged alphaIIb(beta3) during the early stages of cell attachment and spreading, starting with alphaIIb(beta3) clustering at the rim of the cell contact area, that gradually overlapped with the boundary of the attached cell, and, with the onset of cell spreading, to a reorganization of alphaIIb(beta3) in focal adhesions. Taken together, our results demonstrate that (1) fusion of GFP to the cytoplasmic tail of either alphaIIb or beta3 integrin subunits allows normal cell surface expression of a functional receptor, and (2) structural modification of the beta3 integrin cytoplasmic tail, rather than the alphaIIb subunit, plays a major role in alphaIIb(beta3) affinity modulation. With the successful direct visualization of functional alphaIIb(beta3) receptors in living cells, the generation of autofluorescent integrins in transgenic animals will become possible, allowing new approaches to study the dynamics of integrin functions.

Animals↗
PubMed1999

The human platelet alphaIIb gene is not closely linked to its integrin partner beta3.

alphaIIbb3 integrin is a heterodimeric receptor facilitating platelet aggregation. Both genes are on chromosome 17q21.32. Intergenic distance between them has been reported to be 125 to 260 kilobasepairs (kb) by pulsed-field gel electrophoresis (PFGE) genomic analysis, suggesting that they may be regulated coordinately during megakaryopoiesis. In contrast, other studies suggest these genes are greater than 2.0 megabasepairs (mb) apart. Because of the potential biological implications of having these two megakaryocytic-specific genes contiguous, we attempted to resolve this discrepancy. Taking advantage of large kindreds with mutations in either alphaIIb or beta3, we have developed a genetic linkage map between the thyroid receptor hormone-1 gene (THRA1) and beta3 as follows: cen-THRA1-BRCA1-D17S579/alphaIIb-beta3-qte r, with a distance of 1.3 centiMorgans (cM) between alphaIIb and beta3 and the two genes being oriented in the same direction. PFGE genomic and YAC clone analysis showed that the beta3 gene is distal and >/=365 kb upstream of alphaIIb. Additional restriction mapping shows alphaIIb is linked to the erythrocyte band 3 (EPB3) gene, and beta3 to the homeobox HOX2b gene. Analysis of alphaIIb(+)-BAC and P1 clones confirm that the EPB3 gene is approximately 110 kb downstream of the alphaIIb gene. Sequencing the region surrounding the human alphaIIb locus showed the Granulin gene approximately 18 kb downstream to alphaIIb, and the KIAA0553 gene approximately 5.7 kb upstream. This organization is conserved in the murine sequence. These studies show that alphaIIb and beta3 are not closely linked, with alphaIIb flanked by nonmegakaryocytic genes, and imply that they are unlikely to share common regulatory domains during megakaryopoiesis.

Anion Exchange Protein 1, Erythrocyte↗
PubMed2000

Von Willebrand's factor mediates the adherence of human tumoral cells to human endothelial cells and ticlopidine interferes with this effect.

The aim of this study was to explore whether von Willebrand's factor (vWF) plays a role in the adhesion of human colon tumor cells to human endothelial cells in our coculture system. Cell colony density was evaluated basally (endothelial plus colon tumor cells) and following the addition of: purified vWF, vWF plus vWF-blocking antibodies, antibodies against various integrins and adhesion molecules (alpha2 b integrin, beta1 integrin, beta3 integrin, intercellular adhesion molecule-I, intercellular adhesion molecule-II, vitronectin receptor CD61 CD51, laminin alpha6/beta4 receptor), and various drugs inhibiting the hemostatic system (ticlopidine, heparin, acetyl salicylic acid [ASA], defibrotide, indobuphen, dipyridamole, sulfinpyrazone). Furthermore, vWF concentration was measured in the supernatant fluid of the coculture system basally and following the addition of the above-listed drugs. Cell colony density (as measured by light absorption) increased by 33% following the addition of vWF and returned to a value similar to the basal level with antibodies against vWF, while it did not change significantly following the addition of antibodies against the other integrins or adhesion molecules tested. The same parameter was reduced by 35% following the addition of ticlopidine, while it showed a smaller or no change with the other drugs tested. Similarly, vWF concentration in the cell coculture supernatant showed the greatest reduction (from 0.22 to 0.11 mg/mL) following the addition of ticlopidine. These data suggest that vWF mediates the adherence of human tumor cells to human endothelial cells and that ticlopidine interferes with this effect.

Cell Adhesion↗
PubMed2003

Phosphatidylinositol phosphate kinase type 1gamma and beta1-integrin cytoplasmic domain bind to the same region in the talin FERM domain.

Talin is an essential component of focal adhesions that couples beta-integrin cytodomains to F-actin and provides a scaffold for signaling proteins. Recently, the integrin beta3 cytodomain and phosphatidylinositol phosphate (PIP) kinase type 1gamma (a phosphatidylinositol 4,5-bisphosphate-synthesizing enzyme) were shown to bind to the talin FERM domain (subdomain F3). We have characterized the PIP kinase-binding site by NMR using a 15N-labeled talin F2F3 polypeptide. A PIP kinase peptide containing the minimal talin-binding site formed a 1:1 complex with F2F3, causing a substantial number of chemical shift changes. In particular, two of the three Arg residues (Arg339 and Arg358), four of eight Ile residues, and one of seven Val residues in F3 were affected. Although a R339A mutation did not affect the exchange kinetics, R358A or R358K mutations markedly weakened binding. The Kd for the interaction determined by Trp fluorescence was 6 microm, and the R358A mutation increased the Kd to 35 microm. Comparison of these results with those of the crystal structure of a beta3-integrin cytodomain talin F2F3 chimera shows that both PIP kinase and integrins bind to the same surface of the talin F3 subdomain. Indeed, binding of talin present in rat brain extracts to a glutathione S-transferase integrin beta1-cytodomain polypeptide was inhibited by the PIP kinase peptide. The results suggest that ternary complex formation with a single talin FERM domain is unlikely, although both integrins and PIP kinase may bind simultaneously to the talin anti-parallel dimer.

Amino Acid Sequence↗
PubMed2005

Antimetastatic effect of salvicine on human breast cancer MDA-MB-435 orthotopic xenograft is closely related to Rho-dependent pathway.

PURPOSE: Salvicine is a novel DNA topoisomerase II inhibitor with potent anticancer activity. In present study, the effect of salvicine against metastasis is evaluated using human breast carcinoma orthotopic metastasis model and its mechanism is further investigated both in animal and cellular levels. EXPERIMENTAL DESIGN: The MDA-MB-435 orthotopic xenograft model was applied to detect the antimetastatic effect of salvicine. Potential target candidates were detected and analyzed by microarray technology. Candidates were verified and explored by reverse transcription-PCR and Western blot. Salvicine activities on stress fiber formation, invasion, and membrane translocation were further investigated by immunofluorescence, invasion, and ultracentrifugal assays. RESULTS: Salvicine significantly reduced the lung metastatic foci of MDA-MB-435 orthotopic xenograft, without affecting primary tumor growth obviously. A comparison of gene expression profiles of primary tumors and lung metastatic focus between salvicine-treated and untreated groups using the CLOTECH Atlas human Cancer 1.2 cDNA microarray revealed that genes involved in tumor metastasis, particularly those closely related to cell adhesion and motility, were obviously down-regulated, including fibronectin, integrin alpha3, integrin beta3, integrin beta5, FAK, paxillin, and RhoC. Furthermore, salvicine significantly down-regulated RhoC at both mRNA and protein levels, greatly inhibited stress fiber formation and invasiveness of MDA-MB-435 cells, and markedly blocked translocation of both RhoA and RhoC from cytosol to membrane. CONCLUSION: The unique antimetastatic action of salvicine, particularly its specific modulation of cell motility in vivo and in vitro, is closely related to Rho-dependent signaling pathway.

Animals↗
PubMed1999

Molecular prognostic markers in intermediate-thickness cutaneous malignant melanoma.

BACKGROUND: The limitations of morphologic criteria alone in determining the prognosis for a patient with a particular intermediate-thickness primary melanoma have prompted efforts to identify other markers. METHODS: In this study, the authors analyzed expression of p53, beta1 integrin, and beta3 integrin in primary tumors from 111 patients with intermediate-thickness malignant melanoma. RESULTS: Eighty-nine (80%) had detectable p53 protein, 58 (52%) expressed beta1 integrin, and 71 (64%) expressed beta3 integrin. Patients with beta3 positive melanomas were more likely to die of their disease (32 of 71 patients, 45%) than those with beta3 negative tumors (3 of 40 patients, 8%) (P < 0.0001). The number of involved lymph nodes, Clark's level, beta1 integrin expression, thickness, and mitotic rate also had prognostic significance. beta3 integrin was associated with subsequent lung metastases and beta1 integrin with lymph node involvement. CONCLUSIONS: Integrin expression, along with histopathologic criteria, is a prognostic marker for intermediate-thickness malignant melanoma and may indicate the site of subsequent metastasis. These observations may have clinical utility and suggest areas for future investigation.

Antigens, CD↗
PubMed2001

Activation of alpha(v)beta3-vitronectin binding is a multistage process in which increases in bond strength are dependent on Y747 and Y759 in the cytoplasmic domain of beta3.

Integrin receptors serve as mechanical links between the cell and its structural environment. Using alpha(v)beta3 integrin expressed in K562 cells as a model system, the process by which the mechanical connection between alpha(v)beta3 and vitronectin develops was analyzed by measuring the resistance of these bonds to mechanical separation. Three distinct stages of activation, as defined by increases in the alpha(v)beta3-vitronectin binding strength, were defined by mutational, biochemical, and biomechanical analyses. Activation to the low binding strength stage 1 occurs through interaction with the vitronectin ligand and leads to the phosphorylation of Y747 in the beta3 subunit. Stage 2 is characterized by a 4-fold increase in binding strength and is dependent on stage1 and the phosphorylation of Y747. Stage 3 is characterized by a further 2.5-fold increase in binding strength and is dependent on stage 2 events and the availability of Y759 for interaction with cellular proteins. The Y747F mutant blocked the transition from stage 1 to stage 2, and the Y759F blocked the transition from stage 2 to stage 3. The data suggest a model for tension-induced activation of alpha(v)beta3 integrin.

Antigens, CD↗
PubMed2005

Increased expression of integrin alpha(v)beta3 contributes to the establishment of autocrine TGF-beta signaling in scleroderma fibroblasts.

The constitutive secretion of latent TGF-beta by many cell types in culture suggests that extracellular mechanisms to control the activity of this potent cytokine are important in the pathogenesis of the diseases in which this cytokine may be involved, including fibrotic disorders. In this study, we focused on the alpha(v)beta3 integrin, which is recently demonstrated to function as an active receptor for latent TGF-beta1 through its interaction with latency-associated peptide-beta1, and investigated the involvement of this integrin in the pathogenesis of scleroderma. Scleroderma fibroblasts exhibited increased alpha(v)beta3 expression compared with normal fibroblasts in vivo and in vitro. In scleroderma fibroblasts, ERK pathway was constitutively activated and such abnormality induced the up-regulation of alpha(v)beta3. Transient overexpression of alpha(v)beta3 in normal fibroblasts induced the increase in the promoter activity of human alpha2(I) collagen gene and the decrease in that of human MMP-1 gene. These effects of alpha(v)beta3 were almost completely abolished by the treatment with anti-TGF-beta Ab or TGF-beta1 antisense oligonucleotide. Furthermore, the addition of anti-alpha(v)beta3) Ab reversed the expression of type I procollagen protein and MMP-1 protein, the promoter activity of human alpha2(I) collagen gene, and the myofibroblastic phenotype in scleroderma fibroblasts. These results suggest that the up-regulated expression of alpha(v)beta3 contributes to the establishment of autocrine TGF-beta loop in scleroderma fibroblasts, and this integrin is a potent target for the treatment of scleroderma.

Autocrine Communication↗
PubMed2002

Evidence that the platelet integrin alphaIIb beta3 is regulated by the integrin-linked kinase, ILK, in a PI3-kinase dependent pathway.

Platelet aggregation is mediated by the integrin alphaIIb beta3 which is activated by intracellular signals during platelet activation. We have attempted to determine if ILK ("Integrin-Linked Kinase") is involved in the regulation of alphaIIb beta3 function. ILK co-immunoprecipitated with beta3 in stimulated platelets. Using confocal microscopy, ILK was detected in the cytoplasm of resting platelets. ADP or PMA stimulation led to its translocation to the plasma membrane. In parallel, there was a transient increase in ILK kinase activity, association with and phosphorylation of beta3. Inhibition of PI3-kinase by two unrelated inhibitors (wortmannin and LY294002) prevented ILK-related functions. However, it did not prevent the conformational change in alphaIIb beta3 (shown by PAC-1 binding), although integrin affinity for fibrinogen was decreased as measured using FITC-fibrinogen. Furthermore, aggregate formation was reduced. Thus ILK transiently associates with and phosphorylates beta3 in a PI3-kinase dependent manner suggesting that it participates at an intermediate stage in a critical mechanism for assuring large stable aggregates.

Active Transport, Cell Nucleus↗
PubMed2002

PlA polymorphism of integrin beta 3 differentially modulates cellular migration on extracellular matrix proteins.

OBJECTIVE: Cell migration is central to multiple physiological and pathologic processes and involves interactions between integrins on the cell surface and the extracellular matrix. The Leu33Pro (PlA) polymorphism of integrin beta3 has been reported to be associated with a greater rate of restenosis after angioplasty, a process involving endothelial and smooth muscle cell migration. We have addressed the possibility that the Leu33Pro polymorphism could modify the migratory behavior of Chinese hamster ovary (CHO) cells expressing the beta3-containing integrin complexes. METHODS AND RESULTS: Haptotactic migratory responses of CHO alpha(IIb)beta3 cells to fibronectin and vitronectin were not statistically different between the Leu33 and Pro33 cells. However, CHO cells with the Pro33 (PlA2) polymorphism had an enhanced haptotactic migratory response to fibrinogen and von Willebrand Factor. This enhanced migration (1) could be blocked by the alpha(IIb)beta3-complex-specific neutralizing mAb 10E5, by 7E3, a neutralizing mAb specific for the beta3 integrin, and by the alpha(IIb)beta3-blocking peptide Integrilin; (2) was not observed with a CHO cell line expressing an activating beta3 Cys435 to Ala mutation; and (3) was attributable to increased activity of mitogen-activated protein kinase and cyclooxygenase. CHO cell lines expressing the Pro33 isoform of alpha(v)beta3 had an enhanced haptotactic migratory response to vitronectin and osteopontin but not fibrinogen. CONCLUSIONS: The Leu33Pro polymorphism alters the migratory behavior of cells on extracellular matrix substrates, and the alpha subunit influences the substrate specificity of this genetic effect.

Animals↗
PubMed2006

Integrin beta3-mediated Src activation regulates apoptosis in IEC-6 cells via Akt and STAT3.

Intestinal epithelial (IEC-6) cells are resistant to apoptosis following the inhibition of ODC (ornithine decarboxylase) and subsequent polyamine depletion. The depletion of polyamines rapidly activates NF-kappaB (nuclear factor kappaB) and STAT3 (signal transducer and activator of transcription 3), which is responsible for the observed decrease in apoptosis. Since both NF-kappaB and STAT3 signalling pathways can be activated by Src kinase, we examined its role in the antiapoptotic response. Inhibition of ODC by DFMO (alpha-difluoromethylornithine) increased the activity of Src and ERK1/2 (extracellular-signal-regulated kinase 1/2) within 30 min, which was prevented by exogenous polyamines added to the DFMO-containing medium. Conversely, epidermal growth factor-mediated Src and ERK1/2 activation was not prevented by the addition of polyamines. Inhibition of Src with PP2 {4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine} and a DN-Src (dominant-negative Src) construct prevented the activation of Akt, JAK (Janus kinase) and STAT3. Spontaneous apoptosis was increased in DN-Src-expressing cells and the protective effect of polyamine depletion was lost. Polyamine depletion by DFMO increased integrin beta3 Tyr785 phosphorylation. Cells plated on fibronectin had significantly higher beta3 phosphorylation and Src activation compared with plastic. Exogenous polyamines added to the fibronectin matrix prevented Src activation. Arg-Gly-Asp-Ser inhibited beta3, Src and Akt phosphorylation and sensitized polyamine-depleted cells to tumour necrosis factor alpha/cycloheximide-mediated apoptosis. Fibronectin activated Src and subsequently protected cells from apoptosis. Together, these results suggest that the inhibition of ODC rapidly removes a small pool of available polyamines triggering the activation of beta3 integrin, which in turn activates Src. The subsequent Akt and JAK activation is accompanied by translocation of NF-kappaB and STAT3 to the nucleus and the synthesis of antiapoptotic proteins.

Animals↗
PubMed1999

Induction of vascular smooth muscle cell tenascin-C gene expression by denatured type I collagen is dependent upon a beta3 integrin-mediated mitogen-activated protein kinase pathway and a 122-base pair promoter element.

Tenascin-C is an extracellular matrix glycoprotein, the expression of which is upregulated in remodeling arteries. In previous studies we showed that the presence of tenascin-C alters vascular smooth muscle cell shape and amplifies their proliferative response by promoting growth factor receptor clustering and phosphorylation. Moreover, we demonstrated that denatured type I collagen induces smooth muscle cell tenascin-C protein production via beta3 integrins. In the present study, we examine the pathway by which beta3 integrins stimulate expression of tenascin-C, and define a promoter sequence that is critical for its induction. On native collagen, A10 smooth muscle cells adopt a stellate morphology and produce low levels of tenascin-C mRNA and protein, whereas on denatured collagen they spread extensively and produce high levels of tenascin-C mRNA and protein, which is incorporated into an elaborate extracellular matrix. Increased tenascin-C synthesis on denatured collagen is associated with elevated protein tyrosine phosphorylation, including activation of extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2). beta3 integrin function-blocking antibodies attenuate ERK1/2 activation and tenascin-C protein synthesis. Consistent with these findings, treatment with the specific MEK inhibitor, PD 98059, results in suppression of tenascin-C protein synthesis. To investigate whether beta3 integrin-dependent activation of ERK1/2 regulates the tenascin-C promoter, we transfected A10 cells with a full-length (approx. 4 kb) mouse tenascin-C gene promoter-chloramphenicol acetyltransferse reporter construct and showed that, relative to native collagen, its activity is increased on denatured collagen. Next, to identify regions of the promoter involved, we examined a series of tenascin-C promoter constructs with 5' deletions and showed that denatured collagen-dependent promoter activity was retained by a 122-base pair element, located -43 to -165 bp upstream of the RNA start site. Activation of this element was suppressed either by blocking beta3 integrins, or by preventing ERK1/2 activation. These observations demonstrate that smooth muscle cell binding to beta3 integrins activates the mitogen activated protein kinase pathway, which is required for the induction of tenascin-C gene expression via a potential extracellular matrix response element in the tenascin-C gene promoter. Our data suggest a mechanism by which remodeling of type I collagen modulates tenascin-C gene expression via a beta3 integrin-mediated signaling pathway, and as such represents a paradigm for vascular development and disease whereby smooth muscle cells respond to perturbations in extracellular matrix composition by altering their phenotype and patterns of gene expression.

Animals↗