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Results for “Integrin alphaVbeta3”

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PubMed1999

alphavbeta3 and vitronectin expression by normal ovarian surface epithelial cells: role in cell adhesion and cell proliferation.

The alphavbeta3 integrin and its ligand vitronectin are expressed by differentiated epithelial ovarian carcinomas and carcinoma cell lines in culture. Moreover, alphavbeta3/vitronectin interaction influences adhesion and migration of ovarian carcinoma cells in culture. For a better understanding of the behavior of these carcinomas, it appeared necessary to study the characteristics of their normal counterpart, the ovarian surface epithelium (OSE). The present study showed that normal cultured human OSE cells, like the carcinoma cells, have the ability to synthesize vitronectin. The vitronectin receptor, alphavbeta3 integrin, is also expressed by OSE cells and is localized in focal contacts close to paxillin, a focal contact-specific protein, and p125(FAK), a cytoskeletal and signaling molecule. This localization suggested an active participation of the integrin in the adhesion and/or proliferation of OSE cells. Indeed, the use of a blocking antibody demonstrated that alphav integrins promote OSE cell adhesion on vitronectin but not on fibronectin and that these integrins are required for maximal proliferative activity. The results suggest a role of the alphavbeta3/vitronectin system in normal OSE physiology and demonstrate that the expression of this system by well-differentiated ovarian carcinomas reflects the retention of normal cell properties.

Adult↗

PubMed2006

Use of RNA interference to inhibit integrin subunit alphaV-mediated angiogenesis.

One of the central molecules in capillary formation during angiogenesis is the integrin alphaVbeta3. The aim of this study was to inhibit alphaV-mediated angiogenesis in vitro using RNAs (siRNA) as well as antisense oligodeoxyribonucleotides (asON). Five siRNAs, against the alphaV chain of alphaVbeta3, and three asON, which had the respective sequence of the antisense sequence of three of the siRNAs molecules, were examined. Two of the siRNAs and their respective asON were designed on the basis of computer-predicted secondary structure analysis of alphaV mRNA. The different molecules were transfected into human umbilical vein endothelials cells (HUVEC) using lipofection. Following stimulation by PMA, two siRNAs showed a dose-dependent inhibition of PMA-induced alphaV mRNA and protein upregulation, as assessed by real-time RT-PCR and flow cytometry. At a concentration of 25 nM a complete inhibition of protein upregulation was found using siRNAs while transfection of the respective asON sequences reduced the protein upregulation only by 44%. To evaluate the anti-angiogenic potential a cell culture model of human angiogenesis based on the co-cultivation of endothelial cells and dermal fibroblasts was used. Transfection of the siRNA sequence (50 nM) resulted in an inhibition of the total length of capillary-like tubules by 40.6% in comparison to 21.1% using the respective asON sequence. In conclusion, siRNA-based downregulation of alphaV expression showed a stronger inhibition of capillary tube formation in an angiogenesis in vitro assay, than asON having the same sequence as the antisense strand of the siRNAs. Therefore, siRNAs are useful tools for functional alphaV knock-down experiments and might be a therapeutic alternative for antagonists which bind directly to the integrins alphaVbeta3 or alphaVbeta5.

Apoptosis↗

PubMed2001

Angiogenesis in autosomal-dominant polycystic kidney disease.

BACKGROUND: Autosomal-dominant polycystic kidney disease (ADPKD) is a genetic disorder that is responsible for approximately 10% of all cases of end-stage renal disease (ESRD). It is characterized by the formation of epithelial cell cysts, an increase in the extracellullar matrix, and vascular alterations believed to be the result of compression by the cysts. Our recent observations demonstrated a rich vascular network on the surface of the cysts, and thus, we postulated that angiogenesis could be a factor in the progression of ADPKD. METHODS: Kidneys removed from patients with ADPKD were studied using (1) angiographs, (2) immunostaining [factor VIII-related antigen, vascular endothelial growth factor (VEGF), VEGF receptors 1 and 2 (VEGFR-1 and VEGFR-2), metalloproteinase-2 (MMP-2), and integrin alphavbeta3], and (3) Western blot analysis and enzyme-linked immunosorbent assay. The expression of VEGF165 in ADPKD cells in culture was determined. RESULTS: There was (1) an extensive capillary network in the cyst wall of ADPKD kidneys, (2) morphological evidence of vascular malformations, (3) expression of VEGF165 in cyst cells of VEGFR-2 in endothelial cells and an absence of VEGFR-1 in endothelial cells, (4) secretion of VEGF165 by ADPKD cyst cells in culture, and (5) coexpression of matrix MMP-2 and integrin alphavbeta3 in vessels from ADPKD. CONCLUSIONS: There is angiogenesis in ADPKD. This process may be necessary for cyst cells to grow and may be responsible for increased vascular permeability facilitating fluid secretion into the cysts. Neovascularization may result in the formation of aneurysms responsible for the renal bleeding in this disease.

Angiography↗

PubMed2004

Expression of developmental endothelial locus-1 in a subset of macrophages for engulfment of apoptotic cells.

A major function of macrophages is to engulf apoptotic cells to prevent them from releasing noxious materials as they die. Milk fat globule-EGF-factor 8 (MFG-E8) is a glycoprotein secreted by activated macrophages that works as a bridge between apoptotic cells and phagocytes by specifically recognizing phosphatidylserine exposed on apoptotic cells. In this study, we found that developmental endothelial locus-1 (Del-1), originally identified as an embryonic endothelial cell protein that binds alphavbeta3 integrin, is structurally and functionally homologous to MFG-E8. That is, both consist of a signal sequence, two epidermal growth factor domains and two factor VIII-homologous domains (C1 and C2). Del-1 bound to the apoptotic cells by recognizing phosphatidylserine via the factor VIII-homologous domains with an affinity similar to that of MFG-E8. The phagocytic activity of NIH 3T3 cells against apoptotic cells was enhanced by Del-1 through an interaction between the epidermal growth factor domain in Del-1 and alphavbeta3 integrin expressed in the NIH 3T3 cells. Screening of primary macrophages and macrophage cell lines for the expression of MFG-E8 and Del-1 indicated that MFG-E8 and Del-1 are expressed in different sets of macrophages. These results suggest the existence of macrophage subsets that use MFG-E8 or Del-1 differently to engulf apoptotic cells.

Adjuvants, Immunologic↗

PubMed2000

Mice lacking beta3 integrins are osteosclerotic because of dysfunctional osteoclasts.

Osteoclasts express the alphavbeta3 integrin, an adhesion receptor that has been implicated in bone resorption and that is therefore a potential therapeutic target. To assess the role of this heterodimer in skeletal development in vivo, we engineered mice in which the gene for the beta3 integrin subunit was deleted. Bone marrow macrophages derived from these mutants differentiate in vitro into numerous osteoclasts, thus establishing that alphavbeta3 is not necessary for osteoclast recruitment. Furthermore, the closely related integrin, alphavbeta5, does not substitute for alphavbeta3 during cytokine stimulation or authentic osteoclastogenesis. beta3 knockout mice, but not their heterozygous littermates, develop histologically and radiographically evident osteosclerosis with age. Despite their increased bone mass, beta3-null mice contain 3.5-fold more osteoclasts than do heterozygotes. These mutant osteoclasts are, however, dysfunctional, as evidenced by their reduced ability to resorb whale dentin in vitro and the significant hypocalcemia seen in the knockout mice. The resorptive defect in beta3-deficient osteoclasts may reflect absence of matrix-derived intracellular signals, since their cytoskeleton is distinctly abnormal and they fail to spread in vitro, to form actin rings ex vivo, or to form normal ruffled membranes in vivo. Thus, although it is not required for osteoclastogenesis, the integrin alphavbeta3 is essential for normal osteoclast function.

Animals↗

PubMed2002

Identification of critical residues for ligand binding in the integrin beta3 I-domain by site-directed mutagenesis.

To define the structural basis of ligand recognition by alphaIIb beta3, we conducted site-directed mutagenesis of residues located on the top surface of the beta3 I-domain that is homologous to the I-domain of several alpha subunits and contains a putative ligand binding site. Here we identify D158 and N215 in beta3 as novel residues critical for ligand binding. Alanine substitution of D158 or N215 abolished binding of a ligand-mimetic antibody and fibrinogen to alphaIIb beta3 induced by different types of integrin activation. CHO cells expressing recombinant alphaIIb beta3 bearing D158A or N215A mutation did not adhere to fibrinogen. These mutations had the same effect on ligand binding to another beta3 integrin, alphaVbeta3. Compared to the alphaI-domain structure, the betaB-betaC loop containing D158 in the beta3 I-domain is quite different in length and sequence. These results suggest that the structure for ligand recognition is different in the betaI- and alphaI-domains.

Animals↗

PubMed2002

Integrin structure: new twists and turns in dynamic cell adhesion.

The divalent-cation-dependent binding of alphabeta heterodimeric integrins to their ligands regulates most cellular processes. Integrin-ligand interactions are tightly controlled by inside-out activation signals. Ligand-bound integrins in turn transduce outside-in signals typical of other receptors. Precise information of how ligands bind to integrins is restricted to that of a small vWF A-type domain present in some alpha-subunits (alphaA). Both inside-out and outside-in signals elicit tertiary and quaternary changes in integrins, but the precise nature and scope and of these changes are unknown. The recently solved structures of the extracellular segment of integrin alphaVbeta3 in its unliganded and liganded states are generating exciting new insights into the design, wiring, function and regulation of this protein family. The structures reveal a surprising degree of flexibility at defined regions in the structure that is potentially controlled by cations. The quaternary structure of the ligand-binding region bears a striking resemblance to the nucleotide-binding pocket of G-proteins, implying analogous activation and signaling mechanisms. Structural links exist through which ligand-induced tertiary changes may be translated into quaternary changes and vice versa. The structures also raise the tantalizing hypothesis that alphaA is a regulated endogenous integrin ligand, so that no special regulatory features are needed in this integrin. These findings provide the framework for new investigations of structure-activity relationships in integrins, with important implications for targeting these receptors therapeutically [corrected].

Animals↗

PubMed2001

MMP-2 colocalizes with caveolae on the surface of endothelial cells.

We examined the spatial distribution of MMP-2 on the surface of human endothelial cells using immunofluorescence and confocal microscopy. Staining endothelial cells with MMP-2-specific antibodies revealed a punctate labeling at the basolateral side of the cell periphery, which colocalized with patches of caveolin-1, a major constituent of the caveolae. This colocalization was confirmed by immunogold electron microscopy. MT1-MMP, TIMP-2, and the alphavbeta3 integrin exhibited a similar pattern of staining, with pericellular patches that colocalized with either MMP-2 or caveolin-1. The presence of MT1-MMP and TIMP-2 in caveolae patches could be seen only after treatment with concanavalin A, which induced MMP-2 activation but had no noticeable effect on the pattern or intensity of MMP-2 immunostaining. In contrast, MMP-9 and TIMP-1 staining showed a pattern completely different from that of MMP-2 and TIMP-2, with positive spots uniformly distributed throughout the cell body. Our data show that MMP-2, its activator the MT1-MMP, and its proposed receptor, the alphavbeta3 integrin, are all targeted to the same membrane microdomains on the endothelial cell, thereby restricting matrix proteolysis to a limited microenvironment at the cell surface.

Adult↗

PubMed2005

Retargeting of adenoviral vector using basic fibroblast growth factor ligand for malignant glioma gene therapy.

OBJECT: Adenovirus vector (AdV)-mediated gene delivery has been recently demonstrated in clinical trials as a novel potential treatment for malignant gliomas. Combined coxsackievirus B and adenovirus receptor (CAR) has been shown to function as an attachment receptor for multiple adenovirus serotypes, whereas the vitronectin integrins (alphavbeta3 and alphavbeta5) are involved in AdV internalization. In resected glioma specimens, the authors demonstrated that malignant gliomas have varying levels of CAR, alphavbeta3, and alphavbeta5 expression. METHODS: A correlation between CAR expression and the transduction efficiency of AdV carrying the green fluorescent protein in various human glioblastoma multiforme (GBM) cell lines and GBM primary cell lines was observed. To increase transgene activity in in vitro glioma cells with low or deficient levels of CAR, the authors used basic fibroblast growth factor (FGF2) as a targeting ligand to redirect adenoviral infection through its cognate receptor, FGF receptor 1 (FGFR1), which was expressed at high levels by all glioma cells. These findings were confirmed by in vivo study data demonstrating enhanced transduction efficiency of FGF2-retargeted AdV in CAR-negative intracranial gliomas compared with AdV alone, without evidence of increased angiogenesis. CONCLUSIONS: Altogether, the results demonstrated that AdV-mediated gene transfer using the FGF2/FGFR system is effective in gliomas with low or deficient levels of CAR and suggested that FGF2-retargeting of AdV may be a promising approach in glioma gene therapy.

Adenoviridae↗

PubMed2003

Centrifugation facilitates transduction of green fluorescent protein in human monocytes and macrophages by adenovirus at low multiplicity of infection.

Due to their phagocytic and poorly proliferative nature, it has been difficult to transfect human monocytes and macrophages. Adenoviral vectors have recently allowed transduction of a high percentage of human macrophages, but only after CSF upregulation of the integrins, alphavbeta3 or alphavbeta5, during culture for 48 h, a time allowing significant monocyte to macrophage differentiation. In our hands, after 24-h incubation with M-CSF (20 ng/ml) and a further 24-h incubation with an adenoviral vector encoding green fluorescent protein (AdV-GFP) [multiplicity of infection (MOI)=50:1], only 35% of CD14-positive cells express GFP. We report that centrifugation of these cells with AdV-GFP at 2000 x g for 1 h at 37 degrees C significantly enhanced the number of cells expressing GFP (to 65%) and the level of GFP expression per transduced cell (fivefold). The viability of the cells was not compromised (<5 % CD14-positive cells were 7-aminoactinomycin D (7AAD)-positive after 24 h AdV-GFP exposure at MOI=50:1). Centrifugation allowed efficient transduction of monocytes and macrophages with an MOI at least tenfold lower than otherwise required and did not activate the transduced cells or affect their ability to produce TNFalpha or IL-1beta in response to lipopolysaccharide (LPS). This methodology was also suitable for transducing large numbers of in vitro monocyte-derived macrophages (MDMac) and macrophages isolated from synovial fluids with up to 75-80% of CD14-positive cells transduced after 24-h exposure to AdV-GFP (50:1) and centrifugation (2000 x g). This methodology should provide significant expression of transgenes in human monocytes and macrophages.

Adenoviridae↗

PubMed2001

Cell-surface transglutaminase promotes fibronectin assembly via interaction with the gelatin-binding domain of fibronectin: a role in TGFbeta-dependent matrix deposition.

Assembly of fibronectin into a fibrillar matrix is critical for regulation of cell growth and migration, embryogenesis and wound healing. We have previously shown that cell-surface tissue transglutaminase serves as an integrin-binding adhesion coreceptor for fibronectin. Here we report that transglutaminase strongly promotes fibronectin assembly mediated by alpha5beta1 integrin. This effect is independent from transglutaminase-mediated enzymatic crosslinking of fibronectin and separate from the ability of transglutaminase to stimulate cell spreading. Surface transglutaminase increases the binding of fibronectin to cells via interaction with its gelatin-binding domain that contains modules I6II1,2I7-9 and lacks integrin-binding motifs. The gelatin-binding fragment of fibronectin binds to surface transglutaminase on cells in suspension but does not interact with cell monolayers where surface transglutaminase is occupied by fibronectin. Surface transglutaminase colocalizes with growing fibronectin fibrils at early timepoints of matrix formation and remains codistributed with fibronectin matrices thereafter. The observed stimulation of matrix assembly by transglutaminase is blocked by the gelatin-binding fragment of fibronectin, but is not strongly perturbed by its N-terminal fragment consisting of modules I1-5. These results implicate an interaction between transglutaminase and the gelatin-binding domain of fibronectin in matrix assembly and suggest its role in initiation of fibrillogenesis. However, blocking antibodies against alpha5beta1 integrin or the cell-binding fragment of fibronectin that contains modules III2-11 most strongly suppress matrix formation and abolish the effects of transglutaminase. Hence, transglutaminase cooperates with but can not substitute for alpha5beta1 integrin in fibronectin assembly. Treatment of fibroblasts with transforming growth factor beta (TGFbeta) significantly increases surface expression of transglutaminase and its association with beta1 integrins, but not with alphaVbeta3 integrin. TGFbeta enhances the binding of fibronectin to the cell surface and elevates matrix formation, whereas antibody against transglutaminase or the gelatin-binding fragment of fibronectin suppresses these effects, indicating an involvement of transglutaminase in TGFbeta-dependent fibronectin assembly. Therefore, TGFbeta-induced fibronectin matrix deposition during normal wound healing or fibrotic disorders may depend on upregulation of integrin-associated surface transglutaminase.

Animals↗

PubMed2006

Expression pattern of alphavbeta3 and alphavbeta5 integrin mRNA in mouse fetal gonads.

The alphavbeta3 and alphavbeta5 integrins are known as transmembrane receptors capable of binding to the RGD amino acid peptide sequence. In mouse early gonadogenesis, some proteins containing the RGD sequence are deposited into extracellular space and participate in morphogenesis. We analyzed the expression patterns of the alphavbeta3 and alphavbeta5 integrins in mouse developing gonads (10.5-13.5 days post coitum) using whole-mount in situ hybridization. The alphav integrin mRNA was homogenously expressed in developing gonadal regions. On the other hand, the beta3 integrin mRNA was found only in large and round cells (presumptive germ cells), whereas beta5 integrin was localized in gonadal somatic cells, with the exception of coelomic epithelial cells. The beta3 integrin-expressed cells were determined to be primordial germ cells because the number of these cells was drastically reduced in busulfan-treated gonads. In this study, we demonstrated that the alphavbeta3 and alphavbeta5 integrins are widely localized in the mouse developing gonads and discussed their presumptive functions on mouse gonadogenesis.

Animals↗

PubMed2005

alphavbeta3- and alpha5beta1-integrin blockade inhibits myogenic constriction of skeletal muscle resistance arterioles.

In isolated resistance arterioles with spontaneous tone, ligation of alpha4beta1- and alpha5beta1-integrins induces vasoconstriction whereas ligation of alphavbeta3-integrin induces vasodilation. However, whether integrins directly participate in myogenic constriction to pressure elevation is not known. To answer this question, isolated rat skeletal muscle arterioles were exposed to step increments in pressure in the absence or presence of peptides and function-blocking antibodies known to bind alpha4beta1-, alpha5beta1-, or alphavbeta3-integrins while vessel diameter was continually monitored. Myogenic constriction, as assessed by the ability of isolated arterioles to reduce their diameter in response to two consecutive increments in intraluminal pressure (90-110 and 110-130 cmH2O), was not affected by treatment with any of the control peptides (RAD, LEV), a control antibody (anti-rat major histocompatibility complex), an alpha4beta1-integrin-binding peptide (LDV), or an anti-alpha4-integrin antibody. In contrast, alpha5beta1-integrin blockade with either anti-alpha5- or anti-beta1-integrin antibody caused a significant inhibition of myogenic constriction. Also, both RGD peptide and anti-beta3-integrin antibody inhibited myogenic constriction. These results indicate that alpha5beta1- and alphavbeta3-integrins are necessary for myogenic constriction and further suggest that integrins are part of the mechanosensory apparatus responsible for the ability of vascular smooth muscle cells to detect and/or respond to changes in intraluminal pressure.

Animals↗

PubMed2000

Regulation of alphaVbeta3 and alphaVbeta5 integrins by dexamethasone in normal human osteoblastic cells.

Long-term administration of pharmacological doses of glucocorticoids inhibits bone formation and results in osteoporosis. Since integrin-mediated cell-matrix interactions are essential for osteoblast function, we hypothesized that the detrimental effect of glucocorticoids on bone derived, at least in part, from decreased integrin-matrix interactions. Because alphavbeta3 and alphavbeta5 integrins can interact with several bone matrix proteins, we analyzed the effects of dexamethasone (Dex) on the expression of these integrins in normal human osteoblastic cells. We found adhesion of these cells to osteopontin and vitronectin to be dependent on alphavbeta3 and alphavbeta5, respectively; this ligand specificity was not altered by Dex. The effects of Dex on the adhesion of human osteoblastic cells to osteopontin and vitronectin were biphasic with an increase after 2 days, followed by a decrease after 8 days of treatment. Consistently, surface alphavbeta3 and alphavbeta5 integrins, which were increased after 2 days of Dex treatment, were decreased after 8 days. Similarly, total cellular alphav, beta3, and beta5 proteins, which were increased by Dex early in the culture, were diminished after 8 days. Metabolic labeling studies indicated that Dex exhibited biphasic regulation on the biosynthesis of alphavbeta5, with stimulation observed during the second day of treatment, followed by inhibition during the 8th day of exposure. By contrast, the biosynthesis of alphavbeta3 was inhibited by Dex on day 1 and remained inhibited on day 8. Analysis of the mRNA indicated that alphav and beta5 levels were increased by Dex during early exposure (1-3 days), followed by inhibition after prolonged exposure (>/=7 days). By contrast, Dex decreased beta3 mRNA level at all the time points analyzed. Consistently, Dex decreased beta3 promoter activity after 1 day and persisted over 8-day period. By contrast, Dex stimulated beta5 promoter activity after 1 or 2 days but had no effect after 8 days. To further evaluate mechanism(s) leading to the decreased integrin expression after prolonged Dex treatment, mRNA stability was analyzed. Dex was found to accelerate the degradation of alphav, beta3 and beta5 mRNA after an 8-day treatment. Thus, the regulation of alphavbeta3 was dependent on transcription and posttranscriptional events whereas the expression of alphavbeta5 was dependent mainly on posttranscriptional events after prolonged Dex treatment. In conclusion, Dex exhibited time-dependent regulation on the expression of alphavbeta3 and alphavbeta5 integrins in normal human osteoblastic cells. Short-term exposure to Dex increased the levels of alphavbeta3 and alphavbeta5 on the surface and cell adhesion to osteopontin and vitronectin whereas long-term exposure to Dex decreased the expression of both integrins and inhibited the cell adhesion to matrix proteins.

Antigens, CD↗

PubMed2004

Enhancement of the efficacy of an antagonist of an extracellular receptor by attachment to the surface of a biocompatible carrier.

PURPOSE: In order to improve the in vitro and in vivo efficacy of an integrin antagonist (IA) of the extracellular domain of the alphavbeta3 integrin, a receptor upregulated on tumor neovasculature, the IA was attached to the surface of a dextran-coated liposome (DCL). IA-DCLs were characterized in vitro, and the pharmacokinetic and antitumor properties were assessed in vivo. METHODS: The in vitro binding properties were measured with purified integrin, endothelial cells, and melanoma cells. The pharmacokinetic parameters were measured in healthy mice with 14C-labeled IA-DCLs and anti-tumor efficacy was assessed with the M21 human melanoma xenograft mouse model. RESULTS: In vitro, IC50 values for IA-DCLs and IA are similar, and IA-DCLs inhibit cell proliferation relative to controls. IA-DCLs are stable in serum, and the pharmacokinetic half-life in mice is 23 h. In the M21/mouse model, statistically significant inhibition of tumor growth was observed for mice treated with IA-DCLs, whereas controls including saline, DCLs lacking IA, and cyclo(RGDfV) were ineffective. Increased apoptosis and a reduction in vessel counts relative to controls were present in tumors from animals treated with IA-DCLs. CONCLUSIONS: These results demonstrate that IA-DCLs are potent anti-angiogenic therapeutic agents with superior in vivo activity and pharmacology compared to unmodified IA.

Angiogenesis Inhibitors↗

PubMed2005

Insulin-like growth factor-I signaling in smooth muscle cells is regulated by ligand binding to the 177CYDMKTTC184 sequence of the beta3-subunit of alphaVbeta3.

The response of smooth muscle cells to IGF-I requires ligand occupancy of the alphaVbeta3 integrin. We have shown that vitronectin (Vn) is required for IGF-I-stimulated migration or proliferation, whereas the anti-alphaVbeta3 monoclonal antibody, LM609, which inhibits ligand binding, blocks responsiveness of these cells to IGF-I. The amino acids 177-184 ((177)CYDMKTTC(184)) within the extracellular domain of beta3 have been proposed to confer the ligand specificity of alphaVbeta3; therefore, we hypothesized that ligand binding to the 177-184 cysteine loop of beta3 may be an important regulator of the cross talk between alphaVbeta3 and IGF-I in SMCs. Here we demonstrate that blocking ligand binding to a specific amino acid sequence within the beta3 subunit of alphaVbeta3 (i.e. amino acids 177-184) blocked Vn binding to the beta3 subunit of alphaVbeta3 and correspondingly beta3 phosphorylation was decreased. In the presence of this antibody, IGF-I-stimulated Shc phosphorylation and ERK 1/2 activation were impaired, and this was associated with an inhibition in the ability of IGF-I to stimulate an increase in migration or proliferation. Furthermore, in cells expressing a mutated form of beta3 in which three critical residues within the 177-184 sequence were altered beta3 phosphorylation was decreased. This was associated with a loss of IGF-I-stimulated Shc phosphorylation and impaired smooth muscle cell proliferation in response to IGF-I. In conclusion, we have demonstrated that the 177-184 sequence of beta3 is necessary for Vn binding to alphaVbeta3 and that ligand occupancy of this site is necessary for an optimal response of smooth muscle cells to IGF-I.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Intracranial microenvironment reveals independent opposing functions of host alphaVbeta3 expression on glioma growth and angiogenesis.

alphaVbeta3 integrins are overexpressed in the host-derived vasculature of glioblastoma multiform (GBM) and are believed to contribute to angiogenesis and tumor growth. To directly address the role of host alphaVbeta3 expression in GBM growth and behavior, we intracranially implanted integrin beta3-expressing GBM cells into beta3 wild type (WT) or beta3 knock out (KO) mice and monitored angiogenesis and growth. GBM in beta3 WT animals had a vessel density greater than that in beta3 KO animals, consistent with a pro-angiogenic, pro-tumorigenic view of host integrin function. GBM in beta3 WT animals, however, were no larger than those in beta3 KO animals, because GBM in beta3WT animals were infiltrated with a higher number of tumor necrosis factor alpha-secreting, apoptosis-inducing macrophages than the tumors in the corresponding beta3 KO animals. The tumor-suppressive effects of host beta3 expression could be reversed by macrophage depletion or by transplantation of bone marrow from beta3 KO animals into beta3 WT animals, both of which significantly increased tumor growth independently of tumor vessel density. Taken together, these results show that host alphaVbeta3 integrin expression has opposing actions in the intracranial setting, enhancing tumor vascularization and growth while independently enhancing macrophage-mediated tumor elimination. Appropriate management of these functions could lead to enhanced efficacy of anti-integrin based therapies for glioma.

Animals↗

PubMed2004

Interactions of foot-and-mouth disease virus with soluble bovine alphaVbeta3 and alphaVbeta6 integrins.

At least four members of the integrin family of receptors, alphaVbeta1, alphaVbeta3, alphaVbeta6, and alphaVbeta8, have been identified as receptors for foot-and-mouth disease virus (FMDV) in vitro. Our investigators have recently shown that the efficiency of receptor usage appears to be related to the viral serotype and may be influenced by structural differences on the viral surface (H. Duque and B. Baxt, J. Virol. 77:2500-2511, 2003). To further examine these differences, we generated soluble alphaVbeta3 and alphaVbeta6 integrins. cDNA plasmids encoding the individual complete integrin alphaV, beta3, and beta6 subunits were used to amplify sequences encoding the subunits' signal peptide and ectodomain, resulting in subunits lacking transmembrane and cytoplasmic domains. COS-1 cells were transfected with plasmids encoding the soluble alphaV subunit and either the soluble beta3 or beta6 subunit and labeled with [35S]methionine-cysteine. Complete subunit heterodimeric integrins were secreted into the medium, as determined by radioimmunoprecipitation with specific monoclonal and polyclonal antibodies. For the examination of the integrins' biological activities, stable cell lines producing the soluble integrins were generated in HEK 293A cells. In the presence of divalent cations, soluble alphaVbeta6 bound to representatives of type A or O viruses, immobilized on plastic dishes, and significantly inhibited viral replication, as determined by plaque reduction assays. In contrast, soluble alphaVbeta3 was unable to bind to immobilized virus of either serotype; however, virus bound to the immobilized integrin, suggesting that FMDV binding to alphaVbeta3 is a low-affinity interaction. In addition, soluble alphaVbeta3 did not neutralize virus infectivity. Incubation of soluble alphaVbeta6 with labeled type A12 or O1 resulted in a significant inhibition of virus adsorption to BHK cells, while soluble alphaVbeta3 caused a low (20 to 30%), but consistent, inhibition of virus adsorption. Virus incubated with soluble alphaVbeta6 had a lower sedimentation rate than native virus on sucrose density gradients, but the particles retained all of their structural proteins and still contained bound integrin and, therefore, were not exhibiting characteristics of a picornavirus A particle.

Animals↗