Expression of soluble, recombinant alphavbeta3 integrin fragments in Escherichia coli

No prokaryotic expression of integrin alphavbeta3 has been reported so far. We report here the expression of C-terminally truncated alphavbeta3 receptors in E. coli considering the known features required for dimerization and ligand binding. The expressed protein was insoluble despite of the addition of 'solubilizers' to the culture medium. Osmotic stress conditions combined with added exogenous solutes resulted in a small part of soluble receptor. The alphavbeta3 variants were purified from inclusion bodies or from soluble cytoplasmic maltose binding protein fusions. Heterodimerization of the subunits was proved by immunoprecipitation assays. Receptor-ligand binding was found to depend on the concentration. A competition assay with RGD peptides referred to unspecific receptor-ligand interaction. The latter fact was consistent with the finding that soluble receptors did not bind on RGD peptide-coupled sepharose (GRGDSPK sepharose).     

N-Aryl-gamma-lactams as integrin alphavbeta3 antagonists

Novel alphavbeta3 antagonists based on the N-aryl-gamma-lactam scaffold were prepared. SAR studies led to the identification of potent antagonists for alphavbeta3 receptor with excellent selectivity against the structurally related alpha(IIb)beta3 receptor. Additional interactions of N-aryl-gamma-lactam derivatives with alphavbeta3 were found when compared to c(-RGDf[NMe]V-) peptide antagonist. The effects of the conformation and configuration of the gamma-lactam core on the binding were also assessed.     

alphavbeta3 integrin and angiogenesis: a moody integrin in a changing environment

In the adult, angiogenesis, the formation of new blood vessels from pre-existing vasculature contributes to the pathogenesis of many disorders including cancer. The role of adhesion molecules, especially integrins, in pathological angiogenesis has long been the subject of investigation, mostly because of their potential as anti-angiogenic targets. Recent studies have highlighted the complexities connected with understanding the roles of one particular integrin, alphavbeta3, in neovascularization. This integrin is notoriously promiscuous and its precise functions in angiogenesis are unclear. Here, I have firstly summarized some of the salient features of the roles played by alphavbeta3 during angiogenesis; secondly attempted to address the apparently conflicting issues surrounding this topic; and finally raised some questions that appear to be unanswered.     

Expression of alphaV and beta3 integrin subunits during implantation in pig

Integrin are adhesion molecules involved in uterine-conceptus interactions during the perimplantation period. In this study, the expression of alphaV and beta3 integrin subunits in endometrium during implantation in pigs was investigated. The immunohistochemical location was performed on paraformaldehyde-fixed, paraffin-embedded tissue sections, and the mRNA expression of alphaV was detected in endometrium. In addition, serum levels of estradiol, progesterone, follicle-stimulating hormone, and luteinizing hormone were measured on Days 0, 12, 18, and 25 of pregnancy. The results indicate that endometrium expressed integrin alphaV and beta3 in all stages examined. The most intensive staining for integrin alphaV and beta3 was observed in endometrial stroma in porcine pregnancy on Day 18. The mRNA of alphaV integrin strongly expressed on Day 18, and moderately expressed on Days 12 and 25. The correlation between serum hormone level and the mRNA expression of alphaV integrin was not significant. The expression patterns of integrin alphaV and beta3 during implantation provide insights into the important physiological function of alphaVbeta3 integrin in pig, and the strong expression of integrin alphaV and beta3 in mid-implantation may indicate its crucial role in successful implantation and embryo survival.     

Distribution of alphavbeta3, alphavbeta5 integrins and the integrin associated protein--IAP (CD47) in human glomerular diseases

The alphav integrins present on the membrane of numerous cells, mediate attachment to matrix proteins, cell proliferation, migration and survival. We studied the expression of alphav integrinis and CD47 (a beta3 chain integrin associated protein) in various forms of glomerulonephritis (GN) characterized by mesangial proliferation and/or increased mesangial matrix. In normal glomeruli, epithelial cells expressed alphavbeta3, alphavbeta5 and CD47; endothelial cells expressed alpha5beta1 and CD47; mesangial cells expressed alphavbeta5, CD47, and to a less extent alphavbeta3. In acute post infectious GN (APIGN), membrano-proliferative GN (MPGN) and diabetic nephropathy(DN), we observed that the beta3 chain, normally expressed by mesangial cells, was not detectable in the mesangium while its expression by epithelial cells was not modified. Parallel to the disappearance of alphavbeta3, the CD47 expression was decreased on the mesangial cells in MPGN, APIGN and DN. The expression of alphavbeta5 was clearly increased on podocytes and on proliferating mesangial cells in APIGN. By contrast, the mesangial expression of alphavbeta was normal or decreased in DN. The alpha5 chain of integrin, absent on normal mesangial cell, was expressed on proliferating mesangial cells in MPGN and APIGN. Thus, we observed modifications of alphavbeta3 and alphavbeta5 expression during human GN. The modulations of alphavbeta3 and alphavbeta5 expression differed according to the different glomerular cell types and were not parallel in glomerular cells: alphavbeta3 was decreased (and alphavbeta5 unchanged) on proliferating mesangial cells and alphavbeta5 was increased (and alphavbeta3 unchanged) in podocytes. This may reflect the existence of two distinct regulatory pathways.     

Near-infrared optical imaging of integrin alphavbeta3 in human tumor xenografts

In vivo optical imaging is potentially useful for evaluating the presence of tumor markers that are targets of molecular medicine. Here we report the synthesis and characterization of integrin alphavbeta3-targeted peptide cyclo(Lys-Arg-Gly-Asp-Phe) [c(KRGDf )] labeled with fluorescence dyes with wavelength spanning from the visible/near infrared (Cy5.5) to the true near infrared (IRDye800) for optical imaging. In vitro, the peptide-dye conjugates bound specifically to tumor cells expressing alphavbeta3. When administered intravenously into mice at a dose of 6 nmol /mouse, the conjugates accumulated in tumors expressing alphavbeta3. The tumor-to-background ratios for human KS1767 Kaposi's sarcoma in mice injected with Cy5.5-c(KRGDf ) and Cy5.5 were 5.5 and 1.5, respectively. Preinjection of c(KRGDf ) blocked the uptake of Cy5.5-c(KRGDf ) in tumors by 89%. In alphavbeta3-positive M21 and alphavbeta3-negative M21-L human melanoma, fluorescence intensity in the tumor of mice injected with IRDye800 - c(KRGDf ) was 2.3 and 1.3 times that in normal tissue, respectively. Dynamic imaging revealed that Cy5.5- c(KRGDf ) was rapidly taken up by KS1767 tumor immediately after bolus injection. The rate of its uptake in the tumor was reduced by preinjection of c(KRGDf ) in an interval time-dependent manner. Our data suggest that near-infrared fluorescence imaging may be applied to the detection of tumors expressing integrin alphavbeta3 and to the assessment of the optimal biological dose and schedule of targeted therapies.     

Regulation of podosomes by integrin alphavbeta3 and Rho GTPase-facilitated phosphoinositide signaling

In osteoclasts, polyphosphoinositides such as phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5 trisphosphate (PI(3,4,5)P3) are produced in response to integrin alphavbeta3 signaling and they have a critical role in actin cytoskeleton remodeling. The levels of PI(4,5)P2 and PI(3,4,5)P3 are regulated by Rho GTPase through the activation of phosphatidylinositol 4-phosphate 5-kinase (PI4P-5 kinase) and phospatidylinositol 3-kinase (PI3 kinase), respectively. Interaction of PI(4,5)P2 with gelsolin and Wiscott-Aldrich syndrome protein (WASP) is critical for podosome assembly/disassembly and actin ring formation in osteoclasts. Interaction of PI(3,4,5)P3 with gelsolin functions in orchestrating the podosome signaling complex consisting of several key signaling molecules. Gelsolin deficiency has been shown to block podosome assembly and motility in mouse osteoclasts. However, these osteoclasts are able to form a WASP-containing actin ring and retain their resorptive function. The TAT-mediated delivery of gelsolin phosphoinositide-binding domains into osteoclasts resulted in production of podosome clusters and disruption of actin ring formation. Hence, these osteoclasts were hypomotile and less resorptive. Our observations suggest that both PI(4,5)P2 and PI(3,4,5)P3 are involved in regulating osteoclast functions through modulation of severing, capping, and nucleating functions of actin-binding proteins.     

Rac recruits high-affinity integrin alphavbeta3 to lamellipodia in endothelial cell migration

Integrin alphavbeta3 has an important role in the proliferation, survival, invasion and migration of vascular endothelial cells. Like other integrins, alphavbeta3 can exist in different functional states with respect to ligand binding. These changes involve both affinity modulation, by which conformational changes in the integrin heterodimer govern affinity for individual extracellular matrix proteins, and avidity modulation, by which changes in lateral mobility and integrin clustering affect the binding of cells to multivalent matrices. Here we have used an engineered monoclonal antibody Fab (antigen-binding fragment) named WOW-1, which binds to activated integrins alphavbeta3 and alphavbeta5 from several species, to investigate the role of alphavbeta3 activation in endothelial cell behaviour. Because WOW-1 is monovalent, it is insensitive to changes in integrin clustering and therefore reports only changes in affinity. WOW-1 contains an RGD tract in its variable region and binds only to unoccupied, high-affinity integrins. By using WOW-1, we have identified the selective recruitment of high-affinity integrins as a mechanism by which lamellipodia promote formation of new adhesions at the leading edge in cell migration.     

Phenylbutyrates as potent,orally bioavailable vitronectin receptor (integrin alphavbeta3) antagonists

In our continuing efforts to identify small molecule vitronectin receptor antagonists, we have discovered a series of phenylbutyrate derivatives, exemplified by 16, which have good potency and excellent oral bioavailability (approximately 100% in rats). This new series is derived conceptually from opening of the seven-membered ring of SB-265123.     

Engagement of alphavbeta3 integrin regulates proliferation and apoptosis of hepatic stellate cells

Hepatic stellate cells are the major source of the extracellular matrix that accumulates in fibrotic liver. During progressive liver fibrosis, hepatic stellate cells proliferate, but during resolution of fibrosis there is extensive stellate cell apoptosis that coincides with degradation of the liver scar. We have examined the possibility that the fate of stellate cells is influenced by the extracellular matrix through the intermediary of alpha(v)beta(3) integrin. alpha(v)beta(3) integrin was expressed by activated, myofibroblastic rat and human stellate cells in culture. Antagonism of this integrin using neutralizing antibodies, echistatin, or small inhibitory RNA to silence alpha(v) subunit expression inhibited stellate cell proliferation and their expression of proliferating cell nuclear antigen and activated forms of p44 and p42 MAPK. These alpha(v)beta(3) antagonists also increased apoptosis of cultured stellate cells, and this was associated with an increase in the BAX/BCL-2 protein ratio, induction of nuclear DNA fragmentation, and activation of intracellular caspase-3. Expression of tissue inhibitor of metalloproteinases-1 by activated stellate cells was reduced by the alpha(v)beta(3) antagonists, while matrix metalloproteinase-9 synthesis was enhanced. Stellate cells incubated with active recombinant matrix metalloproteinase-9 showed enhanced apoptosis, while cells treated with a synthetic inhibitor of this protease showed increased survival. Our studies suggest that alpha(v)beta(3) integrin regulates the fate of hepatic stellate cells. Degradation of alpha(v)beta(3) ligands surrounding activated stellate cells during resolution of liver fibrosis might decrease alpha(v)beta(3) integrin ligation, suppressing stellate cell proliferation and inducing a fibrolytic, matrix metalloproteinase-secreting phenotype that may prime stellate cells for apoptosis.     

Synthesis, SAR and in vitro evaluation of new cyclic Arg-Gly-Asp pseudopentapeptides containing a s-cis peptide bond as integrin alphavbeta3 and alphavbeta5 ligands

The solid-phase synthesis of two diastereomeric cyclic pseudopeptides containing the Arg-Gly-Asp sequence and the dipeptide isostere 2-amino-3-oxotetrahydro-1H-pyrrolizine-7a(5H)-carboxylic acid (GPTM) is described. Competition binding assays to purified alphavbeta3 and alphavbeta5 integrins with respect to [125I]echistatin showed a high inhibitory activity for the (2S,7aS)-GPTM derivative. Effects of the structural constraint induced by the two enantiomeric scaffolds (2R,7aR)-GPTM and (2S,7aS)-GPTM on the conformation of Arg-Gly-Asp sequence have been computationally investigated using as a reference the recently solved X-ray structure of cyclo(Arg-Gly-Asp-d-Phe-[N-Me]Val) in complex with the extracellular fragment of the alphavbeta3 receptor. The computational method disclosed the key role played by a bridging water molecule on differentiating the two ligands by a diverse stabilization of the ligand-protein complex.     

Syk, c-Src, the alphavbeta3 integrin, and ITAM immunoreceptors, in concert, regulate osteoclastic bone resorption

In this study, we establish that the tyrosine kinase Syk is essential for osteoclast function in vitro and in vivo. Syk^−/− osteoclasts fail to organize their cytoskeleton, and, as such, their bone-resorptive capacity is arrested. This defect results in increased skeletal mass in Syk^−/− embryos and dampened basal and stimulated bone resorption in chimeric mice whose osteoclasts lack the kinase. The skeletal impact of Syk deficiency reflects diminished activity of the mature osteoclast and not impaired differentiation. Syk regulates bone resorption by its inclusion with the αvβ3 integrin and c-Src in a signaling complex, which is generated only when αvβ3 is activated. Upon integrin occupancy, c-Src phosphorylates Syk. αvβ3-induced phosphorylation of Syk and the latter's capacity to associate with c-Src is mediated by the immunoreceptor tyrosine-based activation motif (ITAM) proteins Dap12 and FcRγ. Thus, in conjunction with ITAM-bearing proteins, Syk, c-Src, and αvβ3 represent an essential signaling complex in the bone-resorbing osteoclast, and, therefore, each is a candidate therapeutic target.     

Contortrostatin, a homodimeric disintegrin, binds to integrin alphavbeta5

Contortrostatin is a homodimeric disintegrin from snake venom. We have shown that contortrostatin binds to integrins alphaIIbbeta3, alpha5beta1, and alphavbeta3. We now use several criteria to demonstrate the binding of contortrostatin to alphavbeta5. First, incubation of T24 cells, which express alphavbeta3 and alphavbeta5, with antibody against alphavbeta3 failed to completely inhibit adhesion of cells to vitronectin. However, pretreatment of the cells with contortrostatin or the combination of antibodies against alphavbeta3 and alphavbeta5 completely blocked adhesion to vitronectin. By contrast, either anti-alphavbeta5 alone or contortrostatin blocked adhesion of an alphavbeta3-negative T24 subline. Second, contortrostatin as well as anti-alphavbeta5 inhibits invasion of OVCAR-5, which express only alphavbeta5. Third, contortrostatin binds to purified alphavbeta5 in a saturable manner. Finally, radioligand binding assays yielded a K(d) value of 24 nM for [(125)I]contortrostatin binding to alphavbeta5. This investigation identifies alphavbeta5 as a binding site for contortrostatin. Blockage of alphavbeta5 by contortrostatin inhibits alphavbeta5-mediated adhesion and invasion.     

RGD-dependent binding of procathepsin X to integrin alphavbeta3 mediates cell-adhesive properties

Secreted lysosomal cysteine proteases (cathepsins) are involved in degradation and remodeling of the extracellular matrix, thus contributing to cell adhesion and migration. Among the eleven human lysosomal cysteine proteases, only procathepsin X contains an RGD motif located in a highly exposed region of the propeptide, which may allow binding of the proenzyme to RGD-recognizing integrins. Here, we have tested procathepsin X for cell-adhesive properties and found that it supports integrin alpha(v)beta(3)-dependent attachment and spreading of human umbilical vein endothelial cells. Using site-directed mutants of procathepsin X, we proved that this effect is mediated by the RGD sequence within the proregion of the protease. Endogenous procathepsin X is transported to the plasma membrane, accumulates in vesicles at lamellipodia of the human umbilical vein endothelial cell, and is partly associated with the cell surface, as shown by immunofluorescence. In addition, procathepsin X is partly co-localized with integrin beta(3), as detected by immunogold electron microscopy. A direct interaction between endogenous procathepsin X and alpha(v)beta(3) was demonstrated by co-immunoprecipitation. Moreover, surface plasmon resonance analysis revealed significant and RGD-dependent binding of procathepsin X to integrin alpha(v)beta(3). Our results provide for the first time evidence that the extracellular function of cathepsin X may include binding to integrins thereby modulating the attachment of migrating cells to ECM components.     

Discovery of small molecule inhibitors of integrin alphavbeta3 through structure-based virtual screening

Inhibitors of integrin alphavbeta3 have been implicated in the treatment of a variety of diseases, including tumor metastasis, neovascularization, osteoporosis, and rheumatoid arthritis. It is therefore desirable to develop new types of small molecule inhibitors of integrin alphavbeta3. Here we describe the discovery of novel classes of small molecule inhibitors, via structure-based virtual screening, that target the ligand binding site of integrin alphavbeta3. Application of the docking procedure for screening of a commercially available compound database resulted in a 1774-fold reduction in the size of the screening set (88695 to 50 compounds) and gave a hit-rate of 14% upon biological evaluation (IC50 value ranging from 30 to 200 microM). The best hit, compound 37, 3,4-dichloro-phenylbiguanide, showed inhibitory activity, in a time- and dose-dependent manner, in both cell motility and angiogenesis assays. Based on the best hit, compound 37, a more effective derivative compound 62 has been identified. Furthermore, molecular graphics analyses of a series of substituted phenylbiguanides were carried out to predict the binding mode between the active compounds and integrin alphavbeta3. Our results indicate that the substituted phenylbiguanides might be involved in the inhibition of bivalent cation-mediated ligand binding of integrin alphavbeta3.     

Tricyclic pharmacophore-based molecules as novel integrin alphavbeta3 antagonists. Part IV: preliminary control of alphavbeta3 selectivity by meta-oriented substitution

To establish the in vivo efficacy of alphavbeta3/alphaIIbbeta3 dual antagonists possessing a tricyclic pharmacophore, a corresponding alphavbeta3-selective antagonist was required as a control. We initially took two synthetic approaches to obtain alphavbeta3-selective antagonists based on the RGD recognition pattern or on modification of the dihedral angle between the central benzene ring and the adjacent heterocycle, but both proved unsuccessful. However, synthesis of novel antagonists with meta-substitution of the central benzene ring generated weak selectivity for alphavbeta3 over alphaIIbbeta3 for the first time in the family of compounds with the tricyclic pharmacophore. Optimization of meta-oriented antagonists furnished an alphavbeta3-selective antagonist exhibiting inhibitory activity not only in a receptor-binding assay, but also in a cell adhesion assay.     

Epithelial membrane protein-2 regulates surface expression of alphavbeta3 integrin in the endometrium

The four-transmembrane protein epithelial membrane protein-2 (EMP2) was recently identified as an endometrial protein necessary for blastocyst implantation, but the mechanism of this role is uncertain. In other cell types, EMP2 controls delivery of certain classes of proteins to the cell surface, including various integrin isoforms (a class of receptors implicated in endometrial-blastocyst interaction). Since alphavbeta3 integrin is an important endometrial molecule involved in blastocyst interaction, we evaluated the role of EMP2 in modulating integrin expression in the HEC1A endometrial cell line and endometrial epithelium in vivo. Elevation of EMP2 expression in HEC1A cells selectively increased the expression of alphavbeta3 integrin on the plasma membrane and was functional as judged by increased cell binding to an alphavbeta3 ligand, fibronectin. Conversely, reduction in EMP2 expression using an EMP2 specific ribozyme decreased the cell alphavbeta3 surface expression. The influence of EMP2 on alphavbeta3 integrin was also observed in vivo as reduction of EMP2 using ribozymes or short hairpin RNA diminished alphavbeta3 integrin expression in glandular and luminal uterine epithelium. Colocalization and coimmunoprecipitation studies suggested that EMP2 and alphavbeta3 integrin predominantly exist in a physically associated state. This study demonstrates for the first time the influence of EMP2 on alphavbeta3 surface expression and suggests that surface trafficking of integrin alphavbeta3 by EMP2 during the window of implantation may be a mechanism for its requirement in endometrial-blastocyst interaction.