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Results for “Integrin alphaVbeta3”

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PubMed2005

Canstatin acts on endothelial and tumor cells via mitochondrial damage initiated through interaction with alphavbeta3 and alphavbeta5 integrins.

Canstatin, the noncollagenous domain of collagen type IV alpha-chains, belongs to a series of collagen-derived angiogenic inhibitors. We have elucidated the functional receptors and intracellular signaling induced by canstatin that explain its strong antitumor efficacy in vivo. For this purpose, we generated a canstatin-human serum albumin (CanHSA) fusion protein, employing the HSA moiety as an expression tag. We show that CanHSA triggers a crucial mitochondrial apoptotic mechanism through procaspase-9 cleavage in both endothelial and tumor cells, which is mediated through cross-talk between alphavbeta3- and alphavbeta5-integrin receptors. As a point of reference, we employed the first three kringle domains of angiostatin (K1-3), fused with HSA, which, in contrast to CanHSA, act only on endothelial cells through alphavbeta3-integrin receptor-mediated activation of caspase-8 alone, without ensuing mitochondrial damage. Taken together, these results provide insights into how canstatin might exert its strong anticancer effect.

Angiogenesis Inhibitors↗

PubMed2006

Molecular mediators of alphavbeta3-induced endothelial cell survival.

The alphavbeta3 integrin interaction with the extracellular matrix (ECM) plays an essential role in inhibiting apoptosis in endothelial cells. We have recently shown that alphavbeta3 ligation on rat aortic endothelial cells (RAECs) specifically activates the transcription factor nuclear factor kappaB (NF-kappaB) and promotes cell survival. Inhibiting NF-kappaB nuclear translocation abolished the protective effect of alphavbeta3 ligands. Here, we report that ligation of alphavbeta3 by its ligand, osteopontin (OPN), induces the phosphorylation and activation of inhibitory kappa B kinase beta IKKbeta and promotes the specific degradation of inhibitory kappa Balpha (IkappaBalpha) in RAECs. Overexpression of a dominant negative (DN) IKKbeta protein prevents IkappaBalpha phosphorylation, NF-kappaB activation, and inhibits the protective effects of OPN. The NF-kappaB-inducing kinase (NIK) has been shown to be one of the upstream kinases involved in IKK activation. OPN-mediated NF-kappaB activity is increased upon NIK wild-type (WT) overexpression and blocked following NIK DN overexpression. In addition, NIK-/-mouse embryonic fibroblasts (MEFs) plated on OPN display reduced NF-kappaB activity and decreased IkappaBalpha phosphorylation compared to NIK+/+MEFs. Finally, functional inhibition of integrin beta3-dependent NF-kappaB signaling decreases OPN-induced IkappaBalpha, IKKbeta and NIK phosphorylation. These studies for the first time show that the alphavbeta3-NF-kappaB-dependent endothelial survival pathway is dependent on IkappaBalpha, IKKbeta, and NIK.

Animals↗

PubMed1996

Beta3 integrin expression in melanoma predicts subsequent metastasis.

Previous studies have suggested that differential expression and/or activation of integrins facilitates metastatic progression in murine and human melanoma. While recent data show that the integrin alphavbeta3 is involved in tumor angiogenesis and that tumor growth may be abrogated by alphavbeta3 inhibitors in vitro, the clinical significance of beta3 integrin expression in human malignant melanoma is not known. To assess the prognostic value of beta3 integrin expression, we examined primary cutaneous melanomas from 160 patients followed for a mean of 98 months or until death. We quantified the percentage of tumor area stained with beta3 integrin Ab CD-61 using an image analyzer. beta3 integrin expression was detected in 107/160 primary melanomas (69%). beta3-integrin-positive (beta3+) tumors were thicker (mean 2.98 +/- 0.3 mm) than beta3-integrin-negative (beta3-) melanomas (mean 1.64 +/- 0.2 mm) (P = 0.002). Patients with beta3+ melanomas were more likely to relapse (57/107, 53%) and to die from disease (45/107, 42%) than those with beta3- tumors (6/53, 11%; and 4/53, 8%, respectively) (P < 0.001). Overall survival was greater for beta3- than for beta3+ patients (mean 102 +/- 9 vs 69 +/- 6 months) (P = 0.001). These data show that beta3 integrin expression in primary cutaneous melanoma predicts subsequent metastatic progression. Further study of beta3 integrins in the development of melanoma metastases may yield new therapeutic strategies, as well as prognostic information, for the treatment of this cancer.

Antigens, CD↗

PubMed2006

CT domain of CCN2/CTGF directly interacts with fibronectin and enhances cell adhesion of chondrocytes through integrin alpha5beta1.

Searching for CCN family protein 2/connective tissue growth factor (CCN2/CTGF) interactive proteins by yeast-two-hybrid screening, we identified fibronectin 1 gene product as a major binding partner of CCN2/CTGF in the chondrosarcoma-derived chondrocytic cell line HCS-2/8. Only the CT domain of CCN2/CTGF bound directly to fibronectin (FN). CCN2/CTGF and its CT domain enhanced the adhesion of HCS-2/8 cells to FN in a dose-dependent manner. The CCN2/CTGF-enhancing effect on cell adhesion to FN was abolished by a blocking antibody against alpha5beta1 integrin (alpha5beta1), but not by one against anti-alphavbeta3 integrin. These findings suggest for the first time that CCN2/CTGF enhances chondrocyte adhesion to FN through direct interaction of its C-terminal CT domain with FN, and that alpha5beta1 is involved in this adhesion.

Binding Sites↗

PubMed1999

Progression-related expression of beta3 integrin in melanomas and nevi.

The expression of the beta3 integrin subunit was investigated in 130 fixed, paraffin-embedded specimens of human melanomas and nevi using two different monoclonal antibodies. Expression was not observed in melanocytes and was absent or low in most nevi. In primary melanomas, expression was absent or low in the nontumorigenic radial growth phase, which includes the classes of in situ and microinvasive melanomas. In contrast, expression was high in the tumorigenic or vertical growth phase compartment of many primary melanomas and in most metastatic melanomas. Expression patterns were similar with the two antibodies, SSA6 and SAP, and was membrane-related as well as cytoplasmically expressed. In those nevi that reacted focally, the reactivity tended to occur in the dermal component of neurotized nevi, and in Spitz nevi, where the reactivity was stronger and more diffuse. A few dysplastic nevi showed focal reactivity of the junctional component. These results are consistent with tumor progression-related expression of the beta3 integrin, which is expressed in melanocytic tumors as the alphavbeta3 integrin, having affinity for matrix molecules, including vitronectin and fibronectin. In all melanomas, and in the subset of tumorigenic vertical growth phase melanomas, expression increased with thickness (P < .01). For this reason, and because ligation of this integrin has been shown in vitro to have several properties that may be related to the malignant phenotype, it is likely that expression of this marker may have prognostic value. However, because of its consistent and strong expression in Spitz nevi, the diagnostic utility of this marker will likely be limited.

Aged↗

PubMed2006

Effect of RGD secondary structure and the synergy site PHSRN on cell adhesion, spreading and specific integrin engagement.

The relationship between the form of cell adhesion, ligand presentation, and cell receptor function was characterized using model Langmuir-Blodgett supported films, containing lipid-conjugated peptide ligands, in which isolated variables of the ligand presentation were systematically altered. First, the conformation of an adhesive Arginine-Glycine-Aspartic acid (RGD) peptide was varied by synthesizing linear and looped RGD peptide-containing amphiphiles and subsequently measuring the impact on the function of human umbilical vein endothelial cells. Secondly, the contribution of non-contiguous ligands to cellular engagement was assessed using multi-component biomimetic films. The peptide amphiphiles were composed of fibronectin-derived headgroups--GRGDSP, and its synergy site Pro-His-Ser-Arg-Asn (PHSRN)--attached to hydrocarbon tails. The peptide amphiphiles were diluted using polyethylene glycol (PEG) amphiphiles, where PEG inhibited non-specific cell adhesion. Cells adhered and spread on GRGDSP/PEG systems in a dose-dependent manner. The presentation of GRGDSP influenced integrin cell surface receptor specificity. Results demonstrated that beta1-containing integrins mediated adhesion to the linear GRGDSP presentation to a greater extent than did the alphavbeta3 integrin, and looped GRGDSP preferentially engaged alphavbeta3. GRGDSP/PHSRN/PEG mixtures that closely mimicked the RGD-PHSRN distance in fibronectin, enhanced cell spreading over their two-component analogues. This study demonstrated that controlling the microenvironment of the cell was essential for biomimetics to modulate specific binding and subsequent signaling events.

Amino Acid Sequence↗

PubMed2005

Critical role of beta3 integrin in experimental postmenopausal osteoporosis.

UNLABELLED: We show that mice lacking beta3 integrin are protected from OVX-induced bone loss. Using a lentiviral-based strategy to express beta3 mutants in beta3(-/-) mice, we also show that beta3(S752), but not beta3(Y747/Y759), is important for osteoclastic bone resorption in vivo. INTRODUCTION: Mice lacking the beta3 integrin have dysfunctional osteoclasts and therefore accumulate bone mass with age. Thus, the alphavbeta3 integrin is a potential anti-osteoporosis target. Identifying components of the beta3 integrin that determine its function in vivo is essential for therapeutically exploiting the antiresorptive properties of alphavbeta3. MATERIALS AND METHODS: We used DXA and histomorphometry to assess bone loss after ovariectomy in wildtype and beta3 integrin null mice. We used lentiviral vectors carrying various human beta3 (hbeta3) integrin constructs to transduce beta3(-/-) bone marrow and reconstituted lethally irradiated beta3(-/-) mice with the transduced marrow. The expressed constructs include the intact integrin and two mutants, namely hbeta3(Y747F/Y759F) and hbeta3(S752P), each of which induces the bleeding dyscrasia, Glanzmann's thrombasthenia, in humans. Two months after transplantation, the expression of hbeta3 was measured by flow cytometry of marrow-derived macrophages. Osteoclast differentiation and function were assessed ex vivo by TRACP and actin-ring staining, respectively. Reconstituted mice were ovariectomized, and bone loss was assessed by DXA, histomorphometry, and serum TRACP5b assay. RESULTS: beta3(-/-) mice are protected from ovariectomy-induced bone loss, showing no difference in BMD compared with sham-operated controls. We successfully expressed hbeta3 integrins in beta3(-/-) hosts using lentiviral transduction of bone marrow. Two months after transplantation, 25-35% of marrow-derived macrophages expressed the hbeta3 constructs. Similar to its effect in vitro, hbeta3(WT) completely rescued the osteoclast and platelet phenotype of beta3(-/-) mice. Whereas platelet function remained deranged in beta3(-/-) mice overexpressing hbeta3(Y747F/Y759F), osteoclast function was fully restored. In contrast, beta3(-/-) mice expressing hbeta3(S752P) continued to exhibit prolonged bleeding times and dysfunctional osteoclasts in vitro and ex vivo. Most importantly, hbeta3(WT) and hbeta3(Y747F/Y759F) transplanted mice underwent equivalent ovariectomy-induced bone loss, whereas, like those bearing the control vector, hbeta3(S752P) transplanted mice were protected. CONCLUSIONS: Functional beta3 integrin is required for ovariectomy-induced bone loss. beta3(S752), but not beta3(Y747/Y759), is critical for osteoclast function in vivo.

Acid Phosphatase↗

PubMed2001

Overexpression of homeobox gene HOXD3 induces coordinate expression of metastasis-related genes in human lung cancer cells.

Homeobox-containing genes are expressed in spatiotemporal fashion during embryogenesis and act as master transcription-regulating factors which control the expression of a variety of genes involved in morphogenesis. They are also expressed in a tissue-specific manner in normal adult tissues and appear to give cells spatial information in the maintenance of their architectural integrity. We transfected a HOXD3 class I homeobox-containing gene into human lung cancer A549 cells and investigated alterations in gene expressions and phenotypes related to the maintenance of tissue architecture in HOXD3-overexpressing A549 cells. In the HOXD3-overexpressing cell lines, expression of E-cadherin was lost and plakoglobin was strongly repressed, whereas integrin alpha3 and beta3 were up-regulated and N-cadherin and integrin alpha4 were newly expressed. Compared with parental and control transfectant lines, the HOXD3-overexpressing cell lines showed highly motile and invasive activity. Blocking experiments using anti-integrin beta1 and beta3 suggested that the increased haptotaxis of the HOXD3-overexpressing cells to vitronectin resulted from increased expression and activation of integrin alphavbeta3, and that overexpression of the HOXD3 gene converted the integrin beta1-dependent haptotaxis to fibronectin into both integrin beta1- and beta3-dependent one. HOXD3 overexpression increased production of matrix-degrative enzymes including matrix metalloproteinase-2 and urokinase-plasminogen activator. When the tumor cells were intravenously injected into the tail veins of nude mice, HOXD3 transfectants formed a significantly large number of metastatic foci in lungs compared with the control transfectants. These findings suggest that HOXD3 can act as a metastasis-promoting gene in human lung cancer A549 cells.

Antigens, CD↗

PubMedDate not supplied

Adhesion molecules and implantation.

Endometrial receptivity towards embryo implantation is a complex process that involves the ovary, endometrium and embryo. The dialog between the ovary and the endometrium provides the hormonal stimulus for establishment of a successful pregnancy. The hormones estrogen and progesterone act in concert to stimulate the expression of key molecules necessary for embryos to attach and invade. It is thought that initial attachment of the embryo involves cell adhesion events. The best characterized cell adhesion molecule on the luminal surface of the endometrium is the alphavbeta3 integrin. Its ligand osteopontin (OPN) is co-localized with alphavbeta3 and may play a role in endometrial or embryo signaling or facilitate embryo attachment to the apical surface prior to invasion. Surprising new evidence suggests that these two proteins are differentially regulated. Acting directly on endometrial epithelium, progesterone stimulates OPN expression. Using a stromal-mediated paracrine mechanism, HB-EGF or other EGF molecules appear to stimulate epithelial alphavbeta3 expression. In this article, we review what is known about these two pathways.

Animals↗

PubMed2002

Involvement of alphavbeta 3 integrin and disruption of endothelial fibronectin network during the adhesion of the human ovarian adenocarcinoma cell line IGROV1 on the human umbilical vein cell extracellular matrix.

Like the majority of tumor cells, ovarian cancer cell growth is critically dependent on their neovascularization. Adhesion molecules and cellular events that lead to ovarian tumor cell interactions with endothelial extracellular matrix surrounding the vasculature are poorly identified. To understand the role of alphavbeta3 integrin and its ligand fibronectin in this process, we used in vitro coculture models with IGROV1 human ovarian adenocarcinoma cell line and human umbilical vein endothelial cells (HUVEC). Adhesion assays revealed a strong ability of IGROV1 cells to adhere to HUVEC-ECM. alphavbeta3 is mainly implicated and seems to cooperate with alpha5beta1 integrin in this event. Immunofluorescence staining revealed the presence of alphavbeta3 and alpha5beta1 in IGROV1 cells adhering on HUVEC-ECM at regions of cell sub-stratum contacts. Furthermore, our data showed the absence of fibronectin staining in IGROV1 cells and the disruption of the HUVEC-ECM fibrillar fibronectin network under IGROV1 cell influence. In situ experiments in ovarian neoplastic tissue corroborated the absence of fibronectin in the tumor and its strong detection in vasculature. These findings suggest the active participation of alphavbeta3 and alpha5beta1 integrins and the reorganization of endothelial fibronectin during the adhesion of IGROV1 cells to HUVEC-ECM whereas IGROV1 cells seem to be unable to synthesize fibronectin. Thus, fibronectin integrin receptors expressed by ovarian tumor cells and endothelial fibronectin may be of importance in ovarian carcinoma neovascularization and during tumor-vasculature interactions.

Adenocarcinoma↗

PubMed2001

Developmental regulation of alphav integrins produces functional changes in astrocyte behavior.

To examine the role of the extracellular matrix in regulating astrocyte behavior we previously characterized alphav integrin expression on postnatal astrocytes in vitro and found that they express alphavbeta5 and alphavbeta8. Here we show that differentiation of immature cells into astrocytes is accompanied by developmental regulation of alphav integrins, downregulation of alphavbeta1 and alphavbeta8, and upregulation of alphavbeta5. In addition, using two previously described astrocyte cell lines, we found that the neurite-permissive A7 cell line expressed high levels of alphavbeta1 in addition to alphavbeta5 and alphavbeta8, while the neurite-inhibitory Neu7 cell line expressed only alphavbeta5. To examine integrin function we generated clones of the Neu7 cell line expressing alphavbeta1 or alphavbeta3 in addition to alphavbeta5. This showed that the parent Neu7 cells migrated more slowly than the A7 cells on fibronectin and vitronectin, but that Neu7 cells expressing alphavbeta1 or alphavbeta3 integrins showed enhanced migration on fibronectin and vitronectin, respectively. These results show that alphav integrin expression is regulated during astrocyte development and confirm an instructive role in cell migration for alphavbeta1 in embryonic cells and alphavbeta3 in astroglial tumors.

Animals↗

PubMed2003

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

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

Animals↗

PubMed1996

Role of beta3 integrins in melanoma cell adhesion to activated platelets under flow.

Mechanisms mediating tumor cell attachment to the vessel wall under flow conditions are largely unknown. Therefore we analyzed the ability of human melanoma cells to adhere to an immobilized matrix during blood flow and determined the role of platelets in this process. In a parallel plate flow chamber, M21 melanoma cells were suspended in human blood and perfused over a collagen I matrix at a wall shear rate of 50 s-1 (2 dynes/cm2) to simulate venous flow over a thrombogenic surface. Melanoma cell interaction with the matrix or blood cells and platelets was monitored and quantified by fluorescence and confocal laser microscopy. Despite their ability to adhere to collagen I under static conditions, M21 cells failed to attach directly to this matrix during blood flow. However, they associated with adherent thrombi, and this resulted in stable melanoma cell arrest. Inhibition of platelet activation or platelet integrin alphaIIbbeta3 function abolished M21 cell attachment. Melanoma cell interaction with thrombi was specific and required beta3 integrin expression. M21-L cells which lack integrin alphavbeta3 failed to associate with thrombi and to arrest during blood flow. Transfection of these cells with the integrin subunits alphav or alphaIIb resulted in variants expressing alphavbeta3, as in the wild type, or alphaIIbbeta3. Both variants were able to associate with thrombi and to arrest during blood flow. Therefore, beta3 integrin-mediated binding to activated platelets represents an efficient mechanism for melanoma cell arrest under flow, and this may contribute to the role of platelets in hematogenous metastasis.

Antigens, CD↗

PubMed1998

Integrin alphavbeta5 participates in the binding of photoreceptor rod outer segments during phagocytosis by cultured human retinal pigment epithelium.

PURPOSE: To examine the expression of integrin vitronectin receptors (integrins alphavbeta3 and alphavbeta5) in native human fetal retinal pigment epithelium (RPE) and cultured hunman fetal RPE and to examine the role of RPE VnRs in the phagocytosis of photoreceptor rod outer segments (ROS). METHODS: Monoclonal antibodies against human integrin subunit alphav and heterodimers alphavbeta3 (LM609) and alphavbeta5 (P1F6) were used to label freshly isolated human fetal RPE explant and cultured human fetal RPE and to immunoprecipitate membrane proteins from cultured human RPE. Effects of antibodies and peptides that inhibit integrin vitronectin receptors on phagocytosis of ROS by RPE were determined using cultured human fetal RPE and fluorescein-labeled bovine outer segments. RESULTS: Antibodies against integrin subunit alphav and against alphavbeta5 (P1F6) labeled the RPE apical membrane in both native tissue and cultured RPE, while anti-alphavbeta3 antibody (LM609) labeled less than 1% of cultured RPE and did not label native RPE. Antibodies against alphav and alphavbeta5 P1F6 also immunoprecipitated 2 protein bands corresponding to integrin subunits alphav and beta5 from a membrane extract of cultured human RPE. The peptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP, 1 mM) inhibited the total ROS uptake (externally bound and ingested) by 48% (P < 0.001) and ingestion of ROS by 37% (P < 0.001). Antibody PIF6 (50 microg/ml) inhibited the total ROS uptake and the ingestion of ROS by 63% (P < 0.001) and 43% (P < 0.01), respectively. CONCLUSIONS: The integrin alphavbeta5 vitronectin receptor is expressed on the apical membrane of human RPE and participates in the binding of photoreceptor ROS during phagocytosis by cultured human RPE.

Animals↗

PubMed2000

Cell growth and matrix invasion of EBV-immortalized human B lymphocytes is regulated by expression of alpha(v) integrins.

alpha(v) integrins have been shown to play an important role in epithelial-derived cell migration, cell growth and tumor invasion/metastasis, however their role on cells of hematopoietic origin is less clear. Epstein-Barr virus (EBV), a human herpesvirus associated with several lymphoproliferative disorders in man, induces expression of alpha(v) integrins on transformed B lymphocytes. In the studies reported here, we show that EBV infection increases alpha(v), beta3 and beta5 integrin subunit mRNAs as well as upregulates the expression of the alphavbeta3 integrin protein on human B cells. Among the nine different EBV proteins expressed in latently infected B cells (nuclear and plasma membrane-associated), only LMP1, LMP2A and EBNA2 were shown to selectively transactivate the alpha(v) integrin promoter. Treatment of EBV-transformed B cells with alpha(v) antisense oligonucleotides specifically reduced cell surface expression of alpha(v) integrins, inhibited cell growth in low serum, reduced cell invasion in matrigels and decreased expression of metalloprotease, MMP9. These studies indicate that alpha(v) integrins play a significant role in EBV-induced B-lymphocyte proliferation and invasion. Strategies to interfere with alphav integrin expression and/or function may therefore be of potential value in the treatment of EBV-associated lymphoproliferative disorders.

Antigens, CD↗

PubMed1999

Targeted disruption of fibronectin-integrin interactions in human gingival fibroblasts by the RI protease of Porphyromonas gingivalis W50.

Cell surface integrins mediate interactions between cells and their extracellular matrix and are frequently exploited by a range of bacterial pathogens to facilitate adherence and/or invasion. In this study we examined the effects of Porphyromonas gingivalis proteases on human gingival fibroblast (HGF) integrins and their fibronectin matrix. Culture supernatant from the virulent strain W50 caused considerably greater loss of the beta1 integrin subunit from HGF in vitro than did that of the beige-pigmented strain W50/BE1. Prior treatment of the W50 culture supernatant with the protease inhibitor Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) blocked its effects on cultured cells, indicating that this process is proteolytically mediated. Purified arginine-specific proteases from P. gingivalis W50 were able to mimic the effects of the whole-culture supernatant on loss of beta1 integrin expression. However purified RI, an alpha/beta heterodimer in which the catalytic chain is associated with an adhesin chain, was 12 times more active than RIA, the catalytic monomer, in causing loss of the alpha5beta1 integrin (fibronectin receptor) from HGF. No effect was observed on the alphaVbeta3 integrin (vitronectin receptor). The sites of action of RI and RIA were investigated in cells exposed to proteases pretreated with TLCK to inactivate the catalytic component. Use of both monoclonal antibody 1A1, which recognizes only the adhesin chain of RI, and a rabbit antibody against P. gingivalis whole cells indicated localization of RI on the fibroblasts in a clear, linear pattern typical of that seen with fibronectin and alpha5beta1 integrin. Exact colocalization of RI with fibronectin and its alpha5beta1 receptor was confirmed by double labeling and multiple-exposure photomicroscopy. In contrast, RIA bound to fibroblasts in a weak, patchy manner, showing only fine linear or granular staining. It is concluded that the adhesin component of RI targets the P. gingivalis arginine-protease to sites of fibronectin deposition on HGF, contributing to the rapid loss of both fibronectin and its main alpha5beta1 integrin receptor. Given the importance of integrin-ligand interactions in fibroblast function, their targeted disruption by RI may represent a novel mechanism of damage in periodontal disease.

Animals↗

PubMed1999

Interaction of metargidin (ADAM-15) with alphavbeta3 and alpha5beta1 integrins on different haemopoietic cells.

Metargidin (ADAM-15) is a type I transmembrane glycoprotein belonging to the ADAM (A Disintegrin and Metalloprotease Domain) family of proteins and is widely expressed in different tissues and cell types. Members of this family contain an amino-terminal metalloprotease domain followed by a disintegrin domain, a cysteine-rich region and a membrane proximal EGF-like domain. The disintegrin domain of metargidin contains an RGD tripeptide sequence, suggesting that it may potentially interact with the integrin family of proteins. Here we identify integrin ligands for metargidin on haemopoietic cells, by using a chimeric protein containing the extracellular domain of metargidin fused to the Fc portion of human IgG. Binding activity to a panel of human cell lines was analysed by solid-phase cell-adhesion assays. Metargidin bound to a monocytic cell line, U937, and a T cell line, MOLT-4, in a specific manner. Adhesion was divalent cation- and temperature- dependent and strongly enhanced by Mn2+, all features of integrin-mediated binding. Using a panel of anti-integrin antibodies we show that alphavbeta3 is a ligand for metargidin on U937 cells. In contrast, for MOLT-4 cells, the integrin alpha5beta1 contributes to cell binding. Adhesion was mediated by the disintegrin domain of metargidin as RGD-based peptides inhibited cell binding to both cell lines. The specificity of the interaction between both alphavbeta3 and alpha5beta1 and metargidin was further confirmed by solid-phase adhesion assays using purified recombinant integrins. These results together indicate that metargidin can function as a cell adhesion molecule via interactions with alphavbeta3 and alpha5beta1 integrins.

ADAM Proteins↗

PubMed2002

The fibronectin-binding integrins alpha5beta1 and alphavbeta3 differentially modulate RhoA-GTP loading, organization of cell matrix adhesions, and fibronectin fibrillogenesis.

We have studied the formation of different types of cell matrix adhesions in cells that bind to fibronectin via either alpha5beta1 or alphavbeta3. In both cases, cell adhesion to fibronectin leads to a rapid decrease in RhoA activity. However, alpha5beta1 but not alphavbeta3 supports high levels of RhoA activity at later stages of cell spreading, which are associated with a translocation of focal contacts to peripheral cell protrusions, recruitment of tensin into fibrillar adhesions, and fibronectin fibrillogenesis. Expression of an activated mutant of RhoA stimulates alphavbeta3-mediated fibrillogenesis. Despite the fact that alpha5beta1-mediated adhesion to the central cell-binding domain of fibronectin supports activation of RhoA, other regions of fibronectin are required for the development of alpha5beta1-mediated but not alphavbeta3-mediated focal contacts. Using chimeras of beta1 and beta3 subunits, we find that the extracellular domain of beta1 controls RhoA activity. By expressing both beta1 and beta3 at high levels, we show that beta1-mediated control of the levels of beta3 is important for the distribution of focal contacts. Our findings demonstrate that the pattern of fibronectin receptors expressed on a cell dictates the ability of fibronectin to stimulate RhoA-mediated organization of cell matrix adhesions.

Actins↗