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PubMed2001

Extracellular matrix-derived peptide binds to alpha(v)beta(3) integrin and inhibits angiogenesis.

Angiogenesis is associated with several pathological disorders as well as with normal physiological maintenance. Components of vascular basement membrane are speculated to regulate angiogenesis in both positive and negative manner. Recently, we reported that tumstatin (the NC1 domain of alpha 3 chain of type IV collagen) and its deletion mutant tum-5 possess anti-angiogenic activity. In the present study, we confirm that the anti-angiogenic activity of tumstatin and tum-5 is independent of disulfide bond requirement. This property of tum-5 allowed us to use overlapping synthetic peptide strategy to identify peptide sequence(s) which possess anti-angiogenic activity. Among these peptides, only the T3 peptide (69-88 amino acids) and T7 peptide (74-98 amino acids) inhibited proliferation and induced apoptosis specifically in endothelial cells. The peptides, similar to tumstatin and the tum-5 domain, bind and function via alpha(v)beta(3) in an RGD-independent manner. Restoration of a disulfide bond between two cysteines within the peptide did not alter the anti-angiogenic activity. Additionally, these studies show that tumstatin peptides can inhibit proliferation of endothelial cells in the presence of vitronectin, fibronectin, and collagen I. Anti-angiogenic effect of the peptides was further confirmed in vivo using a Matrigel plug assay in C57BL/6 mice. Collectively, these experiments suggest that the anti-angiogenic activity of tumstatin is localized to a 25-amino acid region of tumstatin and it is independent of disulfide bond linkage. Structural features and potency of the tumstatin peptide make it highly feasible as a potential anti-cancer drug.

Alkylation↗
PubMed2001

Insulin-like growth factor I increases alpha Vbeta 3 affinity by increasing the amount of integrin-associated protein that is associated with non-raft domains of the cellular membrane.

Insulin-like growth factor I (IGF-I) stimulates an increase in alpha(V)beta(3) ligand binding. Stimulation of smooth muscle cells by IGF-I requires alpha(V)beta(3) ligand occupancy, and enhanced alpha(V)beta(3) ligand occupancy augments IGF-I actions. Therefore, IGF-I-induced changes in alpha(V)beta(3) ligand binding may act to further enhance IGF-I actions. Integrin-associated protein (IAP) has been shown to be associated with alpha(V)beta(3) and is required for the binding of alpha(V)beta(3) to vitronectin-coated beads. We therefore investigated whether IGF-I could stimulate IAP-alpha(V)beta(3) association resulting in enhanced ligand binding. IGF-I stimulated an increase in IAP-alpha(V)beta(3) association. This was due, at least in part, to an IGF-I-stimulated redistribution of IAP from the Triton-insoluble fraction of the cell to the Triton-soluble fraction of the cell, where most of the alpha(V)beta(3) was located. Inhibition of the phosphatidylinositol 3-kinase pathway blocked both the redistribution of IAP and the increase in IAP-alpha(V)beta(3) association, providing further evidence that the redistribution of IAP is essential for the increase in association. An anti-IAP monoclonal antibody, blocked both the IGF-I-stimulated increase in IAP-alpha(V)beta(3) complex formation and cell migration. IGF-I-stimulated translocation of IAP and increase in IAP-alpha(V)beta(3) association represent an important process by which IGF-I modulates alpha(V)beta(3) ligand binding and cellular responses.

Animals↗
PubMed1996

Protein kinase C regulates alpha v beta 5-dependent cytoskeletal associations and focal adhesion kinase phosphorylation.

Integrins alpha v beta 3 and alpha v beta 5 both mediate cell adhesion to vitronectin yet trigger different postligand binding events. Integrin alpha v beta 3 is able to induce cell spreading, migration, angiogenesis, and tumor metastasis without additional stimulators, whereas alpha v beta 5 requires exogenous activation of protein kinase C (PKC) to mediate these processes. To investigate this difference, the ability of beta 3 or beta 5 to induce colocalization of intracellular proteins was assessed by immunofluorescence in hamster CS-1 melanoma cells. We found that alpha v beta 5 induced colocalization of talin, alpha-actinin, tensin, and actin very weakly relative to alpha v beta 3. alpha v beta 5 was able to efficiently induce colocalization of focal adhesion kinase (FAK); however, it was unable to increase phosphorylation of FAK on tyrosine. Activation of PKC by adding phorbol ester to alpha v beta 5-expressing cells induced spreading, increased colocalization of alpha-actinin, tensin, vinculin, p130cas and actin, and triggered tyrosine phosphorylation of FAK. Unexpectedly, talin colocalization remained low even after activation of PKC. Treatment of cells with the PKC inhibitor calphostin C inhibited spreading and the colocalization of talin, alpha-actinin, tensin, and actin for both alpha v beta 3 and alpha v beta 5. We conclude that PKC regulates localization of cytoskeletal proteins and phosphorylation of FAK induced by alpha v beta 5. Our results also show that FAK can be localized independent of its phosphorylation and that cells can spread and induce localization of other focal adhesion proteins in the absence of detectable talin.

Animals↗
PubMed1996

Thrombospondin modulates alpha v beta 3 function through integrin-associated protein.

Integrin-associated protein (IAP) is a receptor for the carboxyl-terminal "cell-binding domain" (CBD) of thrombospondin 1 (TS1). IAP associates with alpha v beta 3 integrin and mAbs against IAP inhibit certain integrin functions. Here we examine the effects of the TS1 CBD and 4N1K (KRFYVVMWKK), a cell-binding peptide derived from it, on the adhesion and spreading on vitronectin (VN) of C32 human melanoma cells which express IAP, alpha v beta 3, and alpha v beta 5. Cells adhere to VN at low surface densities via alpha v beta 5 and spread very slowly while adhesion to higher density VN involves both alpha v beta 5 and alpha v beta 3 and results in rapid spreading. Spreading of the cells, but not adhesion, on sparse VN coatings is markedly enhanced by the presence of soluble TS1, the recombinant CBD and 4N1K, but not the "mutant" peptide 4NGG, KRFYGGMWKK, which fails to bind IAP. This enhanced spreading is completely blocked by mAb LM609 against alpha v beta 3 and the anti-IAP mAb B6H12. Correlated with this enhanced spreading is increased tyrosine phosphorylation of focal adhesion kinase (FAK), paxillin, and a protein of ca. 90 kD. The enhanced spreading induced by TS1 and 4N1K and the constitutive spreading on higher density VN are both blocked by calphostin C (100 nM), wortmannin (10 nM), and tyrosine kinase inhibitors. In contrast, pertussis toxin specifically blocks only the TS1 stimulated spreading on low density VN, indicating that IAP exerts its effects on signal transduction via a heterotrimeric Gi protein acting upstream of a common cell spreading pathway which includes PI-3 kinase, PKC, and tyrosine kinases.

Amino Acid Sequence↗
PubMed1998

Modulation of calcium current in arteriolar smooth muscle by alphav beta3 and alpha5 beta1 integrin ligands.

Vasoactive effects of soluble matrix proteins and integrin-binding peptides on arterioles are mediated by alphav beta3 and alpha5 beta1 integrins. To examine the underlying mechanisms, we measured L-type Ca2+ channel current in arteriolar smooth muscle cells in response to integrin ligands. Whole-cell, inward Ba2+ currents were inhibited after application of soluble cyclic RGD peptide, vitronectin (VN), fibronectin (FN), either of two anti-beta3 integrin antibodies, or monovalent beta3 antibody. With VN or beta3 antibody coated onto microbeads and presented as an insoluble ligand, current was also inhibited. In contrast, beads coated with FN or alpha5 antibody produced significant enhancement of current after bead attachment. Soluble alpha5 antibody had no effect on current but blocked the increase in current evoked by FN-coated beads and enhanced current when applied in combination with an appropriate IgG. The data suggest that alphavbeta3 and alpha5 beta1 integrins are differentially linked through intracellular signaling pathways to the L-type Ca2+ channel and thereby alter control of Ca2+ influx in vascular smooth muscle. This would account for the vasoactive effects of integrin ligands on arterioles and provide a potential mechanism for wound recognition during tissue injury.

Animals↗
PubMed2001

Convergence of alpha(v)beta(3) integrin- and macrophage colony stimulating factor-mediated signals on phospholipase Cgamma in prefusion osteoclasts.

The macrophage colony stimulating factor (M-CSF) and alpha(v)beta(3) integrins play critical roles in osteoclast function. This study examines M-CSF- and adhesion-induced signaling in prefusion osteoclasts (pOCs) derived from Src-deficient and wild-type mice. Src-deficient cells attach to but do not spread on vitronectin (Vn)-coated surfaces and, contrary to wild-type cells, their adhesion does not lead to tyrosine phosphorylation of molecules activated by adhesion, including PYK2, p130(Cas), paxillin, and PLC-gamma. However, in response to M-CSF, Src(-/-) pOCs spread and migrate on Vn in an alpha(v)beta(3)-dependent manner. Involvement of PLC-gamma activation is suggested by using a PLC inhibitor, U73122, which blocks both adhesion- and M-CSF-mediated cell spreading. Furthermore, in Src(-/-) pOCs M-CSF, together with filamentous actin, causes recruitment of beta(3) integrin and PLC-gamma to adhesion contacts and induces stable association of beta(3) integrin with PLC-gamma, phosphatidylinositol 3-kinase, and PYK2. Moreover, direct interaction of PYK2 and PLC-gamma can be induced by either adhesion or M-CSF, suggesting that this interaction may enable the formation of integrin-associated complexes. Furthermore, this study suggests that in pOCs PLC-gamma is a common downstream mediator for adhesion and growth factor signals. M-CSF-initiated signaling modulates the alpha(v)beta(3) integrin-mediated cytoskeletal reorganization in prefusion osteoclasts in the absence of c-Src, possibly via PLC-gamma.

Androstadienes↗
PubMed2002

P21-activated kinase 4 interacts with integrin alpha v beta 5 and regulates alpha v beta 5-mediated cell migration.

p21-activated kinase 1 (PAK1) can affect cell migration (Price et al., 1998; del Pozo et al., 2000) and modulate myosin light chain kinase and LIM kinase, which are components of the cellular motility machinery (Edwards, D.C., L.C. Sanders, G.M. Bokoch, and G.N. Gill. 1999. Nature Cell Biol. 1:253-259; Sanders, L.C., F. Matsumura, G.M. Bokoch, and P. de Lanerolle. 1999. SCIENCE: 283:2083-2085). We here present a novel cell motility pathway by demonstrating that PAK4 directly interacts with an integrin intracellular domain and regulates carcinoma cell motility in an integrin-specific manner. Yeast two-hybrid screening identified PAK4 binding to the cytoplasmic domain of the integrin beta 5 subunit, an association that was also found in mammalian cells between endogenous PAK4 and integrin alpha v beta 5. Furthermore, we mapped the PAK4 binding to the membrane-proximal region of integrin beta 5, and identified an integrin-binding domain at aa 505-530 in the COOH terminus of PAK4. Importantly, engagement of integrin alpha v beta 5 by cell attachment to vitronectin led to a redistribution of PAK4 from the cytosol to dynamic lamellipodial structures where PAK4 colocalized with integrin alpha v beta 5. Functionally, PAK4 induced integrin alpha v beta 5-mediated, but not beta1-mediated, human breast carcinoma cell migration, while no changes in integrin cell surface expression levels were observed. In conclusion, our results demonstrate that PAK4 interacts with integrin alpha v beta 5 and selectively promotes integrin alpha v beta 5-mediated cell migration.

Amino Acid Sequence↗
PubMed1997

alphavbeta3 integrin mediates the cell-adhesive capacity and biological activity of basic fibroblast growth factor (FGF-2) in cultured endothelial cells.

Fibroblast growth factor-2 (FGF-2) immobilized on non-tissue culture plastic promotes adhesion and spreading of bovine and human endothelial cells that are inhibited by anti-FGF-2 antibody. Heat-inactivated FGF-2 retains its cell-adhesive activity despite its incapacity to bind to tyrosine-kinase FGF receptors or to cell-surface heparan sulfate proteoglycans. Recombinant glutathione-S-transferase-FGF-2 chimeras and synthetic FGF-2 fragments identify two cell-adhesive domains in FGF-2 corresponding to amino acid sequences 38-61 and 82-101. Both regions are distinct from the FGF-receptor-binding domain of FGF-2 and contain a DGR sequence that is the inverse of the RGD cell-recognition sequence. Calcium deprivation, RGD-containing eptapeptides, soluble vitronectin (VN), but not fibronectin (FN), inhibit cell adhesion to FGF-2. Conversely, soluble FGF-2 prevents cell adhesion to VN but not FN, thus implicating VN receptor in the cell-adhesive activity of FGF-2. Accordingly, monoclonal and polyclonal anti-alphavbeta3 antibodies prevent cell adhesion to FGF-2. Also, purified human alphavbeta3 binds to immobilized FGF-2 in a cation-dependent manner, and this interaction is competed by soluble VN but not by soluble FN. Finally, anti-alphavbeta3 monoclonal and polyclonal antibodies specifically inhibit mitogenesis and urokinase-type plasminogen activator (uPA) up-regulation induced by free FGF-2 in endothelial cells adherent to tissue culture plastic. These data demonstrate that FGF-2 interacts with alphavbeta3 integrin and that this interaction mediates the capacity of the angiogenic growth factor to induce cell adhesion, mitogenesis, and uPA up-regulation in endothelial cells.

Amino Acid Motifs↗
PubMed1999

Alphavbeta3 integrin binding affinity and specificity of SM256 in various species.

This study was undertaken to define the alphavbeta3 binding affinity and specificity of the low-molecular-weight nonpeptide integrin antagonist, SM256. SM256 demonstrated high potency (IC50, 0.057+/-0.030 nM) in inhibiting vitronectin binding to purified human alphavbeta3 receptors. Additionally, SM256 inhibited alphavbeta3-mediated human umbilical vein endothelial cell (HUVEC) or 293/beta3 (beta3-transfected cell line) adhesion to fibrinogen with IC50 values of 0.0054+/-0.0058 and 0.0023+/-0.0012 microM, respectively. SM256 demonstrated a relatively high degree of specificity for human alphavbeta3-mediated functions as compared with other human integrins including alphavbeta5 (IC50, 0.92+/-0.69 microM), alphaIIbbeta3 (IC50, 0.72+/-0.07 microM), alpha4/beta1 (IC50, >100 microM) and alpha5/beta1 (IC50, 2.3+/-2.1 microM). SM256 demonstrated different degree of species specificity in blocking alphavbeta3-mediated cellular adhesion with relatively higher affinity to dog (IC50, 0.005+/-0.002 microM), rabbit (IC50, 0.021+/-0.01 microM), mouse (IC50, 0.035+/-0.01 microM), and pig (IC50, 0.41+/-0.24 microM) endothelial or smooth-muscle cell alphavbeta3-mediated adhesion. Additionally, SM256 demonstrated high degree of alphavbeta3 specificity as compared with alphavbeta5, alpha5beta1, or alphaIIbbeta3-mediated binding in these species. SM256 is a potent alphavbeta3, antagonist with high affinity and specificity for alphavbeta3-mediated functions. Additionally, a comparable alphavbeta3 affinity for SM256 was demonstrated with endothelial cells obtained from various species (dog, mouse, rabbit, and pig) as compared with that from human.

Animals↗
PubMed1996

Evidence of the involvement of phosphatidylinositol 3-kinase in the migration, actin stress fiber formation, and alpha v beta 3-integrin-mediated adherence of human melanoma cells.

Tumor invasion and formation of metastases are major obstacles for a successful therapy of melanomas. Metastasis is thought to require multiple steps such as alpha v beta 3-integrin-mediated adhesion, proteolytic digestion of extracellular matrix by metalloproteinase-2, and reorganization of the actin cytoskeleton. To analyze the functional role of phosphatidylinositol 3-kinase in these processes, melanoma cells were treated with the fungal metabolite wortmannin. Wortmannin inhibited phosphatidylinositol 3-kinase activity in melanoma cells and migration in an equally concentration-dependent fashion. Flow cytometric analysis of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phallacidin-stained actin network indicated reduction of actin filaments by wortmannin. Fluorescence laser confocal microscopy experiments revealed breakdown of actin stress fibers. In addition, wortmannin inhibited alpha v beta 3-integrin-mediated adhesion of melanoma cells to vitronectin. Since flow cytometric measurements did not show altered expression of the alpha v beta 3-integrin at the cell surface, avidity changes of the alpha v beta 3-integrin by wortmannin are suggested. In contrast to the actin analysis and adhesion assays, wortmannin had no influence on mRNA expression or on protein secretion of metalloproteinase-2. These data provide evidence that phosphatidylinositol 3-kinase is an essential signal transduction protein required for migration of melanoma cells, regulating formation of the actin stress fiber as well as alpha v beta 3-integrin-mediated adhesion.

Actins↗
PubMed2000

Normal development, wound healing, and adenovirus susceptibility in beta5-deficient mice.

Integrins have been shown to play important roles in embryonic development, wound healing, metastasis, and other biological processes. alphavbeta5 is a receptor for RGD-containing extracellular matrix proteins that has been suggested to be important in cutaneous wound healing and adenovirus infection. To examine the in vivo function of this receptor, we have generated mice lacking beta5 expression, using homologous recombination in embryonic stem cells. Mice homozygous for a null mutation of the beta5 subunit gene develop, grow, and reproduce normally. Keratinocytes harvested from beta5(-/-) mice demonstrate impaired migration on and adhesion to the alphavbeta5 ligand, vitronectin. However, the rate of healing of cutaneous wounds is not different in beta5(-/-) and beta5(+/+) mice. Furthermore, keratinocytes and airway epithelial cells obtained from null mice show adenovirus infection efficiency equal to that from wild-type mice. These data suggest that alphavbeta5 is not essential for normal development, reproduction, adenovirus infection, or the healing of cutaneous wounds.

Adenoviridae↗
PubMed1999

Exposure of human vascular endothelial cells to sustained hydrostatic pressure stimulates proliferation. Involvement of the alphaV integrins.

The present study investigated the effects of sustained hydrostatic pressure (SHP; up to 4 cm H2O) on human umbilical vein endothelial cell (HUVEC) proliferation, focal adhesion plaque (FAP) organization, and integrin expression. Exposure of HUVECs to SHP stimulated cell proliferation and a selective increase in the expression of integrin subunit alphaV. The increase in alphaV was observed as early as 4 hours after exposure to pressure and preceded detectable increases in the bromodeoxyuridine labeling index. Laser confocal microscopy studies demonstrated colocalization of the alphaV integrin to FAPs. The individual FAPs in pressure-treated cells demonstrated a reduced area and increased aspect ratio and were localized to both peripheral and more central regions of the cells, in contrast to the predilection for the cell periphery in cells maintained under control pressure conditions. The pressure-induced changes in alphaV distribution had functional consequences on the cells: adhesivity of the cells to vitronectin was increased, and alphaV antagonists blocked the pressure-induced proliferative response. Thus, the present study suggests a role for alphaV integrins in the mechanotransduction of pressure by endothelial cells.

Antigens, CD↗
PubMed2001

Eptifibatide and 7E3, but not tirofiban, inhibit alpha(v)beta(3) integrin-mediated binding of smooth muscle cells to thrombospondin and prothrombin.

BACKGROUND: Our objective was to determine whether abciximab, eptifibatide, or tirofiban inhibited ligand binding to alpha(v)beta(3) integrins on human aortic smooth muscle cells (HASMCs) or human umbilical vein endothelial cells (HUVECs). Abciximab binds alpha(IIb)beta(3) on platelets and alpha(v)beta(3) on HUVECs with similar affinity, whereas eptifibatide and tirofiban are thought to be highly specific for alpha(IIb)beta(3). The conclusion that eptifibatide does not bind vascular alpha(v)beta(3) integrins may be premature, however, because recent studies have demonstrated that the affinity of alpha(v)beta(3) for various ligands, including antagonists, is subject to modulation. METHODS AND RESULTS: Abciximab and 7E3, the anti-beta(3) integrin monoclonal antibody from which abciximab was derived, bound alpha(v)beta(3) on HASMCs in a specific and saturable manner and with an affinity similar to binding to alpha(IIb)beta(3) on platelets. 7E3 and eptifibatide inhibited alpha(v)beta(3)-mediated attachment of HASMCs to thrombospondin (TSP) and prothrombin but had no effect on alpha(v)beta(5)- or beta(1)-mediated HASMC attachment to vitronectin-, collagen-, or fibronectin-coated or noncoated tissue culture plates. The inhibitory effect of eptifibatide was similar in magnitude and not additive to that of 7E3. Eptifibatide and 7E3 inhibited alpha(v)beta(3)-mediated attachment of HUVECs. Tirofiban had only nonspecific effects on HASMC attachment to extracellular matrix proteins. In cell proliferation assays, eptifibatide inhibited alpha(v)beta(3)-mediated responses to soluble TSP by HASMCs and beta(3) integrin-expressing HEK cells. CONCLUSIONS: Eptifibatide and 7E3, but not tirofiban, specifically inhibit alpha(v)beta(3)-mediated binding of human smooth muscle and endothelial cells.

Abciximab↗
PubMed2006

Blockade of alpha v beta3 and alpha v beta5 integrins by RGD mimetics induces anoikis and not integrin-mediated death in human endothelial cells.

Alpha v integrins are thought to play an important role in tumor angiogenesis. However, discrepancies between findings with Arg-Gly-Asp (RGD) mimetics, which block angiogenesis in animal models, and knockout mice, in which loss of some alpha v integrins enhances tumor angiogenesis, raise questions concerning the function of these integrins and the precise role of alpha v substrate mimetics in antiangiogenic therapies. We have examined the effects of a novel non-peptide RGD mimetic, S 36578-2, on human endothelial cells to elucidate its antagonist activity and to identify possible agonist functions. S 36578-2 is highly selective for alpha v beta3 and alpha v beta5 integrins and induces detachment, caspase-8 activation, and apoptosis in human umbilical endothelial cells (HUVECs) plated on vitronectin. Importantly, the compound has no effect on the morphology or survival of cells plated on interstitial matrix components such as fibronectin, and it does not potentiate the apoptotic process in suspended cells. Identical results were obtained with a cyclic RGD peptide with similar target specificity. In microvascular endothelial cells, S 36578-2-induced death was also linked to its antiadhesive effect, with established lines markedly more resistant than primary cultures to the antiadhesive and proapoptotic effects. Altogether, these findings have important implications for the development of this class of antiangiogenics.

Anoikis↗
PubMed2000

Preliminary communication. MRNA expression of MT1-MMP, MMP-9, cathepsin K, and TRAP in highly enriched osteoclasts cultured on several matrix proteins and ivory surfaces.

We demonstrated that the transcriptional expression of MT1-MMP, MMP-9, cathepsin K, and TRAP in highly enriched osteoclasts were regulated by different matrix proteins that bind to integrin on osteoclast, such as collagen type I (CoI), fibronectin (FN), vitronectin (VN), osteopontin (OPN), and ivory. Results suggested that the OPN-integrin alphavbeta3 binding plays a more important role than CoI-alpha2beta1 binding in the regulation of osteoclast activity.

Animals↗
PubMed2004

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

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

Alkaline Phosphatase↗
PubMed1998

Contortrostatin, a homodimeric snake venom disintegrin, is a potent inhibitor of osteoclast attachment.

Disintegrins are small disulfide-rich proteins containing an Arg-Gly-Asp (RGD) sequence near their carboxyl terminus. These polypeptides inhibit binding of adhesion molecules to their receptors (integrins) on the surface of cells. Osteoclasts express integrins, heterodimeric cell surface adhesion receptors, that have been shown to be involved in interactions with the extracellular matrix (ECM), including attachment to bone and bone resorption. It has recently been shown that disintegrins effectively inhibit attachment of osteoclasts to components of the ECM and also disrupt osteoclast-mediated bone resorption. Here we characterize the effects of contortrostatin (CTS), a novel homodimeric snake venom disintegrin, on osteoclast attachment. Plastic dishes coated with CTS were able to support osteoclast attachment with a high affinity (EC50,CTS = 86 +/- 6.7 nM) similar to that of vitronectin (VTN; EC50,VTN = 80 +/- 20 nM). Further, CTS was observed to inhibit completely osteoclast attachment to fetal bovine serum (FBS; IC50,FBS = 0.36 +/- 0.04 nM) and VTN (IC50,VTN = 0.85 +/- 0.13 nM). We used monoclonal antibodies directed against the beta1 (monoclonal antibody [MAb] CD29) and beta3 (MAb F11) integrin subunits to explore the mechanism of osteoclast attachment to immobilized CTS. Only MAb F11 inhibited attachment to immobilized CTS (IC50 = 0.41 +/- 0.12 microg/ml), suggesting that binding to CTS is mediated in part by a beta3 integrin, presumably the alpha(v)beta3 VTN receptor. In further support of an integrin-mediated mechanism, binding of osteoclasts to CTS is inhibited by the RGD peptide, GRGDSP. CTS was also more potent (IC50,FBS = 0.36 +/- 0.04 nM) at inhibiting osteoclast attachment to FBS-coated wells than the monomeric snake venom disintegrin echistatin (IC50,FBS = 8.9 +/- 1.5 nM) or VTN (IC50FBS = 97.5 +/- 25.5 nM). Taken together, these data suggest that the snake venom disintegrin CTS is a potent inhibitor of beta3 integrin-mediated osteoclast attachment, presumably involving the VTN receptor (an alpha[v]beta3 integrin). Further studies of the mechanism of CTS-osteoclast interactions may aid in the design of peptide mimetics to act as antiresorptive agents for the treatment of osteoporosis and other skeletal pathology.

Animals↗
PubMed1994

Integrin expression in middle ear cholesteatoma.

Cholesteatoma is lined by a squamous keratinizing epithelium exhibiting most of the features of normal epidermis. In this study, we investigated by immunohistochemistry the expression of integrin adhesion molecules in primary acquired and recurrent cholesteatomas, and compared it with common epidermal cysts and normal human skin. The results showed that cholesteatoma epithelium exhibited a markedly augmented expression of alpha v integrin subunit and a corresponding increased deposition of vitronectin (alpha v ligand) in the surrounding stroma as compared to epidermal cyst and normal human skin. In contrast, the expression pattern of alpha 2 beta 1, alpha 3 beta 1, and alpha 6 beta 4 integrins as well as the distribution of laminin, collagen IV and fibronectin were similar in cholesteatomas, epidermal cysts and normal human skin. Similar staining pattern was observed in primary acquired and recurrent cholesteatoma.

Antigens, CD↗