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Results for “Integrin alphaVbeta3”

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PubMed2006

Peptides based on alphaV-binding domains of erythrocyte ICAM-4 inhibit sickle red cell-endothelial interactions and vaso-occlusion in the microcirculation.

Growing evidence shows that adhesion molecules on sickle erythrocytes interact with vascular endothelium leading to vaso-occlusion. Erythrocyte intercellular adhesion molecule-4 (ICAM-4) binds alphaV-integrins, including alphaVbeta3 on endothelial cells. To explore the contribution of ICAM-4 to vascular pathology of sickle cell disease, we tested the effects of synthetic peptides, V(16)PFWVRMS (FWV) and T(91)RWATSRI (ATSR), based on alphaV-binding domains of ICAM-4 and capable of inhibiting ICAM-4 and alphaV-binding in vitro. For these studies, we utilized an established ex vivo microvascular model system that enables intravital microscopy and quantitation of adhesion under shear flow. In this model, the use of platelet-activating factor, which causes endothelial oxidant generation and endothelial activation, mimicked physiological states known to occur in sickle cell disease. Infusion of sickle erythrocytes into platelet-activating factor-treated ex vivo rat mesocecum vasculature produced pronounced adhesion of erythrocytes; small-diameter venules were sites of maximal adhesion and frequent blockage. Both FWV and ATSR peptides markedly decreased adhesion, and no vessel blockage was observed with either of the peptides, resulting in improved hemodynamics. ATSR also inhibited adhesion in unactivated microvasculature. Although infused fluoresceinated ATSR colocalized with vascular endothelium, pretreatment with function-blocking antibody to alphaVbeta3-integrin markedly inhibited this interaction. Our data strengthen the thesis that ICAM-4 on sickle erythrocytes binds endothelium via alphaVbeta3 and that this interaction contributes to vaso-occlusion. Thus peptides or small molecule mimetics of ICAM-4 may have therapeutic potential.

Anemia, Sickle Cell↗

PubMed2003

CCN3 (NOV) is a novel angiogenic regulator of the CCN protein family.

CCN3 (NOV) is a matricellular protein of the CCN family, which also includes CCN1 (CYR61), CCN2 (CTGF), CCN4 (WISP-1), CCN5 (WISP-2), and CCN6 (WISP-3). During development, CCN3 is expressed widely in derivatives of all three germ layers, and high levels of expression are observed in smooth muscle cells of the arterial vessel wall. Altered expression of CCN3 has been observed in a variety of tumors, including hepatocellular carcinomas, Wilm's tumors, Ewing's sarcomas, gliomas, rhabdomyosarcomas, and adrenocortical carcinomas. To understand its biological functions, we have investigated the activities of purified recombinant CCN3. We show that in endothelial cells, CCN3 supports cell adhesion, induces directed cell migration (chemotaxis), and promotes cell survival. Mechanistically, CCN3 supports human umbilical vein endothelial cell adhesion through multiple cell surface receptors, including integrins alphavbeta3, alpha5beta1, alpha6beta1, and heparan sulfate proteoglycans. In contrast, CCN3-induced cell migration is dependent on integrins alphavbeta3 and alpha5beta1, whereas alpha6beta1 does not play a role in this process. Although CCN3 does not contain a RGD sequence, it binds directly to immobilized integrins alphavbeta3 and alpha5beta1, with half-maximal binding occurring at 10 nm and 50 nm CCN3, respectively. Furthermore, CCN3 induces neovascularization when implanted in rat cornea, demonstrating that it is a novel angiogenic inducer. Together, these findings show that CCN3 is a ligand of integrins alphavbeta3 and alpha5beta1, acts directly upon endothelial cells to stimulate pro-angiogenic activities, and induces angiogenesis in vivo.

Animals↗

PubMed2004

Inhibition of lipopolysaccharide-induced expression of inducible nitric oxide synthase by butein in RAW 264.7 cells.

Butein has been reported to exert anti-inflammatory effect but the possible mechanism involved is still unclear. Here, we report the inhibitory effect of butein on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) gene expression. Butein also inhibited the induction of tumor necrosis factor-alpha and cyclooxygenase 2 by LPS. To further investigate the mechanism responsible for the inhibition of iNOS gene expression by butein, we examined the effect of butein on LPS-induced nuclear factor-kappaB (NF-kappaB) activation. The LPS-induced DNA binding activity of NF-kappaB was significantly inhibited by butein, and this effect was mediated through inhibition of the degradation of inhibitory factor-kappaB and phosphorylation of Erk1/2 MAP kinase. Furthermore, increased binding of the osteopontin alphavbeta3 integrin receptor by butein may explain its inhibitory effect on LPS-mediated NO production. Taken together, these results suggest that butein inhibits iNOS gene expression, providing possible mechanisms for its anti-inflammatory action.

Animals↗

PubMed2006

Therapeutic effects of paclitaxel-containing ultrasound contrast agents.

Drug delivery vehicles that combine ultrasonic and molecular targeting are shown to locally concentrate a drug in a region-of-interest. The drug delivery vehicles, referred to as acoustically active lipospheres (AALs), are microbubbles surrounded by a shell of oil and lipid. In a region limited to the focal area of ultrasound application, circulating AALs are deflected by radiation force to a vessel wall and can subsequently be fragmented. Ligands targeting the alphavbeta3 integrin are conjugated to the AAL shell and increase in vitro binding by 26.5-fold over nontargeted agents. Toxicity assays demonstrate that paclitaxel-containing AALs exert a greater antiproliferative effect after insonation than free paclitaxel at an equivalent concentration. Lastly, ultrasound and molecular targeting are combined to deliver a model drug to the endothelium and interstitium of chorioallantoic membrane vasculature in vivo.

Animals↗

PubMed2000

Molecular analysis of the hematopoiesis supporting osteoblastic cell line U2-OS.

OBJECTIVE: Osteoblasts play an important role in regulating hematopoiesis in the bone marrow. Here we show that U2-OS, a widely used osteoblastic cell line derived from an osteosarcoma, has the capacity to support proliferation of human hematopoietic progenitor cells in vitro. In this study, U2-OS cells are characterized at the molecular level to unravel the molecular mechanisms underlying the support of hematopoiesis. MATERIALS AND METHODS: U2-OS was analyzed in great detail using RT-PCR and flow cytometry. In addition, a cDNA library was constructed and randomly sequenced to obtain insight in the repertoire of expressed molecules. RESULTS: A broad panel of growth factors and cytokines is expressed by U2-OS. TGF-beta, GM-CSF, c-kit ligand, and IL-7 are produced constitutively and IL-1beta, IL-6, IL-8, TNF-alpha, IFN-gamma, and MIP1-alpha are upregulated upon stimulation. In addition to those, mRNAs of the CC chemokine LARC and leukemia inhibitory factor were identified. U2-OS cells express high levels of beta1-integrins at the cell surface: VLA-2, VLA-3, VLA-4, VLA-5, VLA-6, and the integrin alphavbeta3. Besides integrins, ALCAM and NCAM are detected on the cell surface of U2-OS. Interestingly, we show that CD34(+) progenitor cells expressing ALCAM are highly proliferative when compared with CD34(+) ALCAM(low) cells, hinting at a role for ALCAM in anchoring progenitor cells to the bone marrow stroma. Interestingly, random sequencing of an U2-OS cDNA library yielded almost 10% of novel cDNAs with a potential role in hematopoiesis. The involvement of these novel molecules in hematopoiesis is an interesting target for future investigations. CONCLUSIONS: We conclude that U2-OS supports outgrowth of hematopoietic progenitor cells and accordingly expresses adhesion molecules and growth factors and a number of novel, as yet uncharacterized potentially interesting genes.

Activated-Leukocyte Cell Adhesion Molecule↗

PubMed2005

Function of endogenous inhibitors of angiogenesis as endothelium-specific tumor suppressors.

Disruption of the systemic angiogenesis balance to favor enhanced angiogenesis is speculated to represent a key step in the growth of tumors. Although a major emphasis has been placed on the increase of angiogenesis stimulators, such as VEGF, on the disruption of the angiogenic balance, the potential role of the physiological levels of endogenous inhibitors of angiogenesis on tumor growth is poorly understood. Here, we use three independent lines of mice deficient in tumstatin, endostatin, or thrombospondin-1 (TSP-1), to address the role that these endogenous angiogenesis inhibitors play in tumor growth. Our experiments demonstrate that normal physiological levels of these inhibitors serve to retard the growth of tumors, and that their absence leads to enhanced angiogenesis and a 2- to 3-fold increase in tumor growth. The tumor-suppressive action of TSP-1, endostatin, and tumstatin correlates with expression of CD36 receptor, alpha5beta1 integrin, and alphavbeta3 integrin on proliferating endothelial cells, respectively. Moreover, tumors grow 2-fold faster in the tumstatin/TSP-1 double-knockout mice, compared with either the tumstatin- or the TSP-1-deficient mice, strongly suggesting that ceiling rate of cancer growth is not completely dependent on the genetic defects of cancer cells but also depends on the host-derived tumor microenvironment. Additionally, tumor growth in transgenic mice overproducing endostatin specifically in the endothelial cells (a 1.6-fold increase in the circulating levels; mimicking Down's syndrome condition) is 3-fold slower than the tumor growth in wild-type mice. Collectively, our data suggest that physiological levels of endogenous inhibitors of angiogenesis can serve as endothelium-specific tumor suppressors.

Angiogenesis Inhibitors↗

PubMed2005

Integrin activation and matrix binding mediate cellular responses to mechanical stretch.

Mechanical tension is a critical determinant of cell growth, differentiation, apoptosis, migration, and development. Integrins have been implicated in sensing force but little is known about how forces are transduced to biochemical signals. We now show that mechanical strain stimulates conformational activation of integrin alphavbeta3 in NIH3T3 cells. Integrin activation is mediated by phosphoinositol 3-kinase and is followed by an increase in integrin binding to extracellular matrix proteins. Mechanical stretch stimulation of JNK was dependent on new integrin binding to extracellular matrix. These data define a molecular mechanism for the role of integrins in mechanotransduction.

Animals↗

PubMedDate not supplied

A new colloidal lipidic system for gene therapy.

A novel type of liposomal vector for gene therapy is described (Artificial Virus Particles; AVPs). This vector is based on the composition of retroviral envelopes, serum-resistant and non-toxic and smaller than 200 nm in size. The DNA is condensed using low molecular weight branched PEI. Equipment of these particles with a cyclic RGD peptide ligand as targeting device renders them selective for tumor endothelial and melanoma cells expressing high levels of alphavbeta3-integrins, and allows for an efficient delivery of the enclosed genetic material. The specificity of the vector system for melanoma cells could be further improved by using a melanocyte-specific tyrosinase promoter to drive transgene expression.

Cell Separation↗

PubMed2006

Regulation of osteoclast function and bone mass by RAGE.

The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily that has multiple ligands and is implicated in the pathogenesis of various diseases, including diabetic complications, neurodegenerative disorders, and inflammatory responses. However, the role of RAGE in normal physiology is largely undefined. Here, we present evidence for a role of RAGE in osteoclast maturation and function, which has consequences for bone remodeling. Mice lacking RAGE had increased bone mass and bone mineral density and decreased bone resorptive activity in vivo. In vitro-differentiated RAGE-deficient osteoclasts exhibited disrupted actin ring and sealing zone structures, impaired maturation, and reduced bone resorptive activity. Impaired signaling downstream of alphavbeta3 integrin was observed in RAGE(-/-) bone marrow macrophages and precursors of OCs. These results demonstrate a role for RAGE in osteoclast actin cytoskeletal reorganization, adhesion, and function, and suggest that the osteosclerotic-like phenotype observed in RAGE knockout mice is due to a defect in osteoclast function.

Actins↗

PubMed2004

Improved tumor targeting of radiolabeled RGD peptides using rapid dose fractionation.

Arginine-glycine-aspartic acid (RGD) peptides preferentially bind to alphavbeta3 integrin, an integrin expressed on newly formed endothelial cells and on various tumor cells. When labeled with beta-emitting radionuclides, these peptides can be used for peptide-receptor radionuclide therapy of malignant tumors. These studies aimed to investigate whether tumor targeting and tumor therapy could be optimized by dose fractionation. The RGD-peptide DOTA-E-[c(RGDfK)]2 was labeled with 111In for biodistribution experiments and with 90Y for therapy experiments. In mice with NIH:OVCAR-3 ovarian carcinoma xenografts, optimal tumor uptake was obtained at peptide doses up to 1.0 microg (4.8 %ID/g). A peptide dose of 5 microg, required to administer the maximum tolerable dose (MTD) 90Y-DOTA-E-[c(RGDfK)]2, was administered as 5 portions of 1.0 microg. Tumor uptake of the fifth portion was significantly higher than that of the single 5.0 microg portion (3.3 %ID/g versus 2.1 %ID/g). The therapeutic efficacy of 37 MBq 90Y-DOTA-E-[c(RGDfK)]2 (1 x 5.0 microg) was compared with that of 37 MBq administered in five equal portions (5 x 1.0 microg). No difference in tumor growth between the fractionated and the nonfractionated therapy was observed. In conclusion, dose fractionation resulted in higher radiation doses. However, therapeutic efficacy of the radiolabeled peptide was not significantly improved by dose fractionation.

Animals↗

PubMed2006

Electroconvulsive seizure increases adult hippocampal angiogenesis in rats.

Electroconvulsive seizure has a proven therapeutic application in the treatment of severe depression and treatment-resistant depression. Despite the efficacy of electroconvulsive seizure as a non-chemical antidepressant treatment, the mechanism of action is unclear. Elevation in hippocampal trophic factor expression and concomitant cellular proliferation are thought to play a role in its action. We examined whether the reported induction of angiogenic factors and endothelial cell proliferation leads to an increase in vascular density. Two hippocampal regions, the dentate gyrus and the stratum lacunosum moleculare (SLM), were examined employing a combination of vascular density quantification, angiogenic gene expression analysis and immunohistochemistry. A 6% increase in vascular density was observed in the dentate gyrus but this did not achieve statistical significance. The SLM of the hippocampus exhibited a robust 20-30% increase in vascular density and was accompanied by an increase in expression of inhibitor of differentiation-3. There was also an induction of the angiogenesis markers alphaVbeta3 integrin and Del1. Increases in the vascular density of the SLM could be in response to enhanced metabolic activity in this region. This is supported by the induction of glutamine synthetase and the glutamate transporter GLT1.

Animals↗

PubMed2001

Integrin alphavbeta3-mediated endocytosis of immobilized fibrinogen by A549 lung alveolar epithelial cells.

Fibrinogen (FBG), together with its polymerized form fibrin, modulates cellular responses during wound repair and tissue remodeling. Thus, we sought to determine whether A549 lung epithelial type II-like cells would endocytose insoluble, surface-bound FBG as a potential mechanism of alveolar matrix remodeling. Surface-bound FBG was endocytosed into either lysosomes or late endosomes by A549 cells through arg-gly-asp-dependent binding to alphavbeta3 but not alpha5beta1 integrin receptors. Soluble FBG added to confluent monolayers of A549 cells was not endocytosed. Unlike the uptake of the extracellular matrix glycoproteins vitronectin and thrombospondin by other cell types, endocytosis of FBG by A549 cells was neither inhibited by heparin nor dependent on binding to cell-surface heparan sulfate proteoglycans. FBG did not colocalize with endocytosed transferrin, whereas dextran showed partial colocalization with FBG in endocytic vesicles, suggesting nonclathrin-mediated endocytosis. Inhibition of actin filament polymerization blocked endocytosis of both dextran and FBG but not transferrin, providing further support that FBG is endocytosed via a nonclathrin pathway. Disruption of actin polymerization inhibited integrin-mediated cell spreading, which contributed to an overall reduction in FBG clearance that was most likely due to reduced cell migration and associated pericellular proteolysis. Trasylol inhibition of extracellular plasmin activity did not inhibit endocytosis of FBG. The endocytosed FBG was degraded to trichloroacetic acid-soluble fragments that showed an electrophoretic pattern distinctly different from plasmin-degraded FBG. Together, these results suggest that endocytosis of matrix-associated FBG by alveolar epithelial cells may be involved in the processes of alveolar tissue repair and matrix remodeling.

Cell Line↗

PubMed2004

Epinephrine acts through erythroid signaling pathways to activate sickle cell adhesion to endothelium via LW-alphavbeta3 interactions.

The possible role of physiologic stress hormones in enhancing adhesion of sickle erythrocytes (SS RBCs) to endothelial cells (ECs) in sickle cell disease (SCD) has not been previously explored. We have now found that up-regulation of intracellular cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) by epinephrine significantly increased sickle but not normal erythrocyte adhesion to both primary and immortalized ECs. Inhibition of serine/threonine phosphatases also enhanced sickle erythrocyte adhesion at least partially through a PKA-dependent mechanism. Adhesion was mediated through LW (intercellular adhesion molecule-4 [ICAM-4], CD242) blood group glycoprotein, and immunoprecipitation studies showed that LW on sickle but not on normal erythrocytes undergoes increased PKA-dependent serine phosphorylation as a result of activation. The major counter receptor for LW was identified as the alphavbeta3 integrin on ECs. These data suggest that adrenergic hormones such as epinephrine may initiate or exacerbate vaso-occlusion and thus contribute to the association of vaso-occlusive events with physiologic stress.

Anemia, Sickle Cell↗

PubMed2005

Limited rescue of osteoclast-poor osteopetrosis after successful engraftment by cord blood from an unrelated donor.

UNLABELLED: We report on a case of osteoclast-poor osteopetrosis who received a hematopoietic stem cell graft and, despite hematological engraftment, showed little signs of response in the skeletal defect. Clinical and laboratory studies supported the concept that the bone microenvironment remained abnormal, thus reducing the clinical response to transplantation. INTRODUCTION: Osteopetrosis is a rare genetic disorder characterized by severely reduced bone resorption resulting from a defect in either osteoclast development (osteoclast-poor osteopetrosis) or activation (osteoclast-rich osteopetrosis). Patients with osteoclast-rich osteopetrosis can be rescued by allogenic hematopoietic stem cell transplantation; however, little information exists concerning the success of transplantation as a treatment for osteoclast-poor osteopetrosis. We report on a child with osteoclast-poor osteopetrosis whose diagnosis was delayed, consequently receiving a cord blood transplant from an unrelated donor at the age of 8 years. Engraftment was deemed successful by peripheral blood genotyping, although >3 years after transplantation there was little rescue of the skeletal defect and anemia, and extramedullary hematopoiesis persisted. MATERIALS AND METHODS: Peripheral blood mononuclear cells from the osteopetrosis patient, before and after transplantation, were used to generate osteoclasts in vitro in the presence of macrophage colony-stimulating factor (M-CSF) and RANKL. RESULTS: Before transplantation few, small mononuclear osteoclasts formed (F-actin ring-positive cells, co-localizing with vitronectin receptor [alphavbeta3 integrin] and TRACP) associated with occasional, small resorption lacunae. Low levels of collagen C-terminal telopeptide (CTx) fragments were released from these cultures as assessed by ELISA (CrossLaps; patient, 12.85 nM; control, 448.6 nM). In contrast, osteoclasts formed in cultures after transplantation formed to a similar degree to control cultures from healthy individuals: large numbers of osteoclasts containing numerous nuclei were present, and approximately 50% of the surface of bone slices was resorbed, associated with intermediate levels of collagen fragment release (116.48 nM). The culture data reflect the histopathology and radiological findings and also support previous studies showing that neither M-CSF nor RANKL rescues osteoclast-poor osteopetrosis. CONCLUSIONS: This is the first case reported in which a successful hematopoietic engraftment failed to correct an osteopetrotic skeletal defect, and this finding may be credited to the age at which the child was transplanted.

Acid Phosphatase↗

PubMed2001

Potential role of a new anti-beta3 integrin antibody in the development of intimal hyperplasia after vascular surgery: an in vitro smooth muscle cell model.

The occurrence of intimal hyperplasia after vascular surgery is an ongoing concern in current clinical practice. Among the many factors involved in the development of this pathology, platelet adhesion and myointimal proliferation play a major role. Both these processes are mediated by integrins (mainly alphavbeta3 integrins). Over the past years, several substances have been designed to delay or inhibit the cell proliferation that leads to hyperplasia and mainly include monoclonal antibodies directed against integrins. The aim of the present study was to evaluate the effects of an antibody denoted P37 (anti beta3 integrin) on human smooth muscle cells (SMC) and its role in blocking the B3 subunit. To this end, SMC from human umbilical artery were cultured in the presence or absence of the cell substrate vitronectin (VN) and incubated with P37. After 4 days of treatment, determination was made of cell proliferation and migration. Smooth muscle cells grown on VN showed increased proliferation and migration compared to control VN-free cultures. However, the presence of P37 in the culture medium inhibited proliferation and reduced migration. Combined treatment with VN and P37 led to improved proliferation but VN was unable to reverse the effects on migration observed in the former cultures. Results suggest that in vitro, P37 is capable of blocking human SMC beta3 integrins and thus impedes cell proliferation and migration These findings may have clinical implications related to modulation of the development of hyperplasia.

Antibodies↗

PubMed2003

Heavy ion irradiation inhibits in vitro angiogenesis even at sublethal dose.

Angiogenesis is essential for tumor growth and metastasis. Because endothelial cells are genetically stable, they rarely acquire resistance to anticancer modalities, and could, thus, be a suitable target for radiation therapy. Heavy ion radiation therapy has attracted attention as an effective modality for cancer therapy because of its highly lethal effects, but the effects of heavy ion irradiation on in vitro cell function associated with angiogenesis have not been reported. Our study found that in vitro angiogenesis was inhibited by high linear energy transfer carbon ion irradiation even at sublethal dose (0.1 Gy). ECV304 and HUVEC human umbilical vascular endothelial cells were irradiated with 290 MeV carbon ion beams of approximately 110 keV/ micro m or 4 MV X-ray of approximately 1 keV/ micro m. Their adhesiveness and migration to vitronectin or osteopontin were inhibited, and capillary-like tube structures in three-dimensional culture were destroyed after carbon ion irradiation concomitant with the inhibition of matrix metalloproteinase-2 activity, down-regulation of alphaVbeta3 integrin, which is one of the adhesion molecules, slight up-regulation of membrane type1- matrix metalloproteinase, and significant up-regulation of tissue inhibitor of metalloproteinase-2. On the other hand, sublethal X-ray irradiation promoted migration of endothelial cells, and the capillary-like tube structure in three-dimensional culture progressed even after 16 Gy irradiation. These results provide an implication that heavy ion beam therapy could be superior to conventional photon beam therapy in preventive effects on in vitro angiogenesis even at sublethal dose, and might inhibit angiogenesis in vivo.

Capillaries↗

PubMed2006

Efficiency of recombinant human TNF in human cancer therapy.

Recombinant human tumour necrosis factor (TNF) has a selective effect on angiogenic vessels in tumours. Given that it induces vasoplegia, its clinical use has been limited to administration through isolated limb perfusion (ILP) for regionally advanced melanomas and soft tissue sarcomas of the limbs. When combined with the alkylating agent melphalan, a single ILP produces a very high objective response rate. In melanoma, the complete response (CR) rate is around 80% and the overall objective response rate greater than 90%. In soft tissue sarcomas that are inextirpable, ILP is a neoadjuvant treatment resulting in limb salvage in 80% of the cases. The CR rate averages 20% and the objective response rate is around 80%. The mode of action of TNF-based ILP involves two distinct and successive effects on the tumour-associated vasculature: first, an increase in endothelium permeability leading to improved chemotherapy penetration within the tumour tissue, and second, a selective killing of angiogenic endothelial cells resulting in tumour vessel destruction. The mechanism whereby these events occur involves rapid (of the order of minutes) perturbation of cell-cell adhesive junctions and inhibition of alphavbeta3 integrin signalling in tumour-associated vessels, followed by massive death of endothelial cells and tumour vascular collapse 24 hours later. New, promising approaches for the systemic use of TNF in cancer therapy include TNF targeting by means of single chain antibodies or endothelial cell ligands, or combined administration with drugs perturbing integrin-dependent signalling and sensitizing angiogenic endothelial cells to TNF-induced death.

Aged↗

PubMed2005

Distinct structural requirements for binding of the integrins alphavbeta6, alphavbeta3, alphavbeta5, alpha5beta1 and alpha9beta1 to osteopontin.

The extracellular matrix protein, osteopontin, is a ligand for several members of the integrin family, including alpha5beta1, alphavbeta3, alphavbeta5 and alpha9beta1. Osteopontin is a substrate for a number of extracellular proteases, including thrombin and the metalloproteases MMP-3 and MMP-7, which cleave osteopontin at sites close to or within the mapped integrin binding sites. Using affinity chromatography and cell adhesion assays, we now identify the integrin alphavbeta6 as an additional osteopontin receptor. Utilizing a series of recombinant forms of osteopontin, we compared the structural requirements for alphavbeta6 binding with those for the 4 other osteopontin-binding integrins. Like alpha5beta1, alphavbeta3 and alphavbeta5 (but not alpha9beta1), alphavbeta6 binds to the RGD site in osteopontin, since RGD peptide or mutation of this site to RAA completely inhibits alphavbeta6-mediated cell adhesion. For both alpha9beta1 and alpha5beta1, the N-terminal fragment generated by thrombin cleavage is a much better ligand than full length osteopontin, whereas thrombin-cleavage does not appear to be required for optimal adhesion to alphavbeta3, alphavbeta5 or alphavbeta6. A recombinant fragment predicted to be generated by MMP cleavage no longer supported alpha5beta1 or alpha9beta1-mediated adhesion, but adhesion mediated by alphavbeta5 or alphavbeta6 was unaffected. Finally, adhesion of alphavbeta5 or alphavbeta6 was inhibited by mutation of two aspartic acid residues upstream of the RGD site, whereas adhesion mediated by alphavbeta3, alpha5beta1 or alpha9beta1 was unaffected by these mutations. These results suggest that the hierarchy of integrin interactions with osteopontin can undergo complex regulation at least in part through the action of extracellular proteases.

Amino Acid Sequence↗