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Results for “Integrin alphaVbeta3”

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PubMed1998

Adhesion of human umbilical vein endothelial cells to the immediate-early gene product Cyr61 is mediated through integrin alphavbeta3.

Cyr61 is a member of a family of growth factor-inducible immediate-early gene products thought to act cooperatively with the activities of growth factors. Upon synthesis, Cyr61 is secreted and is predominantly incorporated into the extracellular matrix. Recently, we demonstrated that Cyr61 promotes cell adhesion and migration and augments growth factor-induced DNA synthesis (Kireeva, M. L., Mo, F.-E., Yang, G. P., and Lau, L. F. (1996) Mol. Cell. Biol. 16, 1326-1334). In the present study, we investigated possible candidate receptor(s) on human umbilical vein endothelial cells (HUVECs) mediating adhesion to Cyr61. Under both serum-containing and serum-free conditions, adhesion of HUVECs to Cyr61 was dose-dependent, saturable, and abolished by affinity-purified anti-Cyr61 antibodies. Cell adhesion to Cyr61 was divalent cation-dependent and specifically inhibited by the peptide RGDS and LM609, a monoclonal antibody against integrin alphavbeta3. Furthermore, purified alphavbeta3 bound directly to an affinity matrix of Cyr61-coupled Sepharose 4B, and this interaction was specifically blocked by anti-Cyr61 antibodies. Additionally, in a solid phase binding assay, soluble Cyr61 bound to immobilized alphavbeta3 in a dose-dependent manner, and half-saturation binding occurred at approximately 5 nM Cyr61. As expected, the interaction of Cyr61 with immobilized alphavbeta3 was blocked by RGDS and LM609. In sum, these results identified Cyr61 as a novel ligand for alphavbeta3 and indicate that the adhesion of HUVECs to Cyr61 is mediated through interaction with this integrin. The possibility that integrin alphavbeta3 functions as a signaling receptor for Cyr61 accounts for most if not all activities that can be ascribed to Cyr61 to date and suggests a mechanism of action discussed herein.

Antibodies, Monoclonal↗

PubMed2001

Podosomes in osteoclast-like cells: structural analysis and cooperative roles of paxillin, proline-rich tyrosine kinase 2 (Pyk2) and integrin alphaVbeta3.

Macrophages and osteoclasts develop unique contact sites with the extracellular matrix called podosomes. Podosomes have been associated with migratory and invasive cell characteristics, but a basic mechanism outlining their function is lacking. We have used chicken and human monocytes differentiating in vitro into osteoclast-like cells in the presence of RANKL-ODF to study these cytoskeletal structures. During the differentiation process, podosomes are redistributed from the cell body in early macrophages to the cell periphery in increasingly spread and multinucleated cells expressing high levels of integrin alphaVbeta3. Immunofluorescence with anti-phosphotyrosine antibodies revealed increased tyrosine-phosphorylation at the basal tips of these podosomes. RANKL-ODF treatment reinforced the peripheral location of podosomes and initiated their partial fusion to larger F-actin-containing structures that displayed reduced levels of tyrosine phosphorylation. Paxillin and the FAK-related kinase Pyk2 colocalized with integrin alphaVbeta3 in the juxtamembrane region surrounding individual podosomes. In lysates of macrophages and differentiated osteoclasts both paxillin and Pyk2 associated with synthetic and recombinant polypeptides containing the C-terminal region of the integrin beta3 cytoplasmic domain. These in vitro interactions were direct and they were abolished by substitutions in the beta3 integrin peptides known to disrupt integrin function in vivo. The marked adhesion-dependent tyrosinephosphorylation of Pyk2 and paxillin however did not detectably alter their interaction with beta3 tail peptides in cell lysates. Our results provide novel insight into the molecular architecture and the phosphorylation dynamics in podosomes. Moreover, they outline a novel potential mechanism for the recruitment of paxillin and Pyk2 to beta3 integrin-dependent cell contacts.

Actins↗

PubMed2005

Fibronectin-induced proliferation in thyroid cells is mediated by alphavbeta3 integrin through Ras/Raf-1/MEK/ERK and calcium/CaMKII signals.

We recently demonstrated in an immortalized thyroid cell line that integrin stimulation by fibronectin (FN) simultaneously activates two signaling pathways: Ras/Raf/MAPK kinase (Mek)/Erk and calcium Ca2+/calcium calmodulin-dependent kinase II (CaMKII). Both signals are necessary to stimulate Erk phosphorylation because CaMKII modulates Ras-induced Raf-1 activity. In this study we present evidence that extends these findings to normal human thyroid cells in primary culture, demonstrating its biological significance in a more physiological cell model. In normal thyroid cells, immobilized FN-induced activation of p21Ras and Erk phosphorylation. This pathway was responsible for FN-induced cell proliferation. Concurrent increase of intracellular Ca2+ concentration and CaMKII activation was observed. Both induction of p21Ras activity and increase of intracellular Ca2+ concentration were mediated by FN binding to alphavbeta3 integrin. Inhibition of the Ca2+/CaMKII signal pathway by calmodulin or CaMKII inhibitors completely abolished the FN-induced Erk phosphorylation. Binding to FN induced Raf-1 and CaMKII to form a protein complex, indicating that intersection between Ras/Raf/Mek/Erk and Ca2+/CaMKII signaling pathways occurred at Raf-1 level. Interruption of the Ca2+/CaMKII signal pathway arrested cell proliferation induced by FN. We also analyzed thyroid tumor cell lines that displayed concomitant aberrant integrin expression and signal transduction. These data confirm that integrin activation by FN in normal thyroid cells generates Ras/Raf/Mek/Erk and Ca2+/CaMKII signaling pathways and that both are necessary to stimulate cell proliferation, whereas in thyroid tumors integrin signaling is altered.

Calcium Signaling↗

PubMed1998

Cloning and characterization of developmental endothelial locus-1: an embryonic endothelial cell protein that binds the alphavbeta3 integrin receptor.

We have taken advantage of an enhancer trap event in a line of transgenic mice to identify a unique developmentally regulated endothelial cell locus (Del1). The protein encoded in this locus contains three EGF-like repeats homologous to those in Notch and related proteins, including an EGF-like repeat that contains an RGD motif, and two discoidin I-like domains. Del1 is shown to be a matrix protein and to promote adhesion of endothelial cells through interaction with the alphavbeta3 integrin receptor. Embryonic endothelial-like yolk sac cells expressing recombinant Del1 protein, or grown on an extracellular matrix containing Del1 protein, are inhibited from forming vascular-like structures. Expression of Del1 protein in the chick chorioallantoic membrane leads to loss of vascular integrity and promotes vessel remodeling. Del1 is thus a new ligand for the alphavbeta3 integrin receptor and may function to regulate vascular morphogenesis or remodeling in embryonic development.

Amino Acid Sequence↗

PubMed1996

Involvement of integrin alphavbeta3 in cell adhesion, motility, and liver metastasis of murine RAW117 large cell lymphoma.

The molecules that mediate metastatic cell homing to specific organ sites remain largely unidentified. As a target organ for metastasis, the liver is a unique environment characterized by fenestrated sinusoidal endothelium, lack of a complete basement membrane, and production of serum components, including fibronectin and vitronectin. We examined a series of marine RAW117 large cell lymphoma variants selected in vivo for liver-colonizing properties (H10 >> L17 > P). Compared with L17 or P cells, the highly liver-colonizing H1O cells expressed much higher levels of surface integrin alphavbeta3, as shown by affinity chromatography, immunoprecipitation, and flow cytometry. H10 cells adhered at higher rates to vitronectin and fibronectin than to fibrinogen, fibrin, laminin, and type I collagen. Among the RGD peptides, H10 cells adhered at significantly higher rates to the polymeric RGD peptide (glycyl-arginyl-glycyl-aspartyl-serine)4 than to monomeric RGD peptides. H10 cells were able to spread on immobilized vitronectin with highly polarized morphology but not on fibronectin. In contrast, the poorly liver-metastatic P and L17 cells did not adhere or spread well on vitronectin or fibronectin. H10 cells also migrated toward vitronectin concentration gradients. Blocking cell surface alphavbeta3 molecules with specific anti-beta3 monoclonal antibodies resulted in significant decreases in the adhesion of H10 cells to vitronectin and (glycyl-arginyl-glycyl-aspartyl-serine)4 and significant inhibition of the formation of experimental liver metastases. These data suggest an important role of integrin alphavbeta3 in the metastasis of RAW117 cells to the liver.

Amino Acid Sequence↗

PubMed2005

Rhodostomin inhibits thrombin-enhanced adhesion of ROS 17/2.8 cells through the blockade of alphavbeta3 integrin.

Osteosarcoma is a very malignant bone tumor which has a high metastatic potential and usually lead to poor prognosis. The adhesion of tumor cells to the endothelium or extracellular matrix (ECM) is an essential step in the metastatic cascade. We investigated the effect of thrombin on the adhesion activity of the osteosarcoma cell line, ROS 17/2.8. Incubation with the low concentrations of thrombin (0.01-5 U/ml, 5 min to 24 h) elevated the adhesion activity of ROS 17/2.8 to both human umbilical vein endothelial cells (HUVEC) and extracellular matrix, with the peak effect at the concentration of 0.5 U/ml for 30 min at 37 degrees C. The ROS 17/2.8 cells responded to thrombin by a peak effect of increased adhesion to HUVEC (5.5 folds vs. control) and fibronectin (4.8 folds) after thrombin pretreatment (0.5 U/ml, 30 min, 37 degrees C). Pretreatment with monoclonal antibodies against beta3 integrins, including anti-alphavbeta3, 10E5 and 7E3, effectively antagonized the thrombin-enhanced cell adhesion activity, whereas anti-alpha3beta1 and anti-alpha5beta1 did not antagonize the enhanced cell adhesion. Rhodostomin, an Arg-Gly-Asp (RGD)-containing snake venom peptide, and synthetic peptide RGDS also blocked the thrombin-enhanced ROS 17/2.8 cell adhesion. This study demonstrated that thrombin enhanced the cell adhesion of ROS 17/2.8 cells to HUVEC or ECM through an upregulation of beta3 integrins, and rhodostomin was a strong inhibitor on thrombin-enhanced cell adhesion, either to HUVEC or fibronectin substratum.

Animals↗

PubMed2001

Vascular smooth muscle cell growth-promoting factor/F-spondin inhibits angiogenesis via the blockade of integrin alphavbeta3 on vascular endothelial cells.

Vascular smooth muscle cell growth-promoting factor (VSGP) was originally isolated from bovine ovarian follicular fluid as a stimulator of vascular smooth muscle cell proliferation. Homology searches indicate that bovine and human VSGPs are orthologs of rat F-spondin. Here, we examined whether recombinant human VSGP/F-spondin affected the biological activities of endothelial cells. VSGP/F-spondin did not affect the proliferation of human umbilical vein endothelial cells (HUVECs); however, it did inhibit VEGF- or bFGF-stimulated HUVEC migration. To clarify the mechanism of this inhibitory effect, we examined the adhesion of HUVECs to extracellular matrix proteins. VSGP/F-spondin specifically inhibited the spreading of HUVECs on vitronectin via the functional blockade of integrin alphavbeta3. As a result, VSGP/F-spondin inhibited the tyrosine phosphorylation of focal adhesion kinase (FAK) when HUVECs were plated on vitronectin. Moreover, VSGP/F-spondin inhibited the activation of Akt when HUVECs on vitronectin were stimulated with VEGF. VSGP/F-spondin inhibited tube formation by HUVECs in vitro and neovascularization in the rat cornea in vivo. These results indicate that VSGP/F-spondin inhibits angiogenesis at least in part by the blockade of endothelial integrin alphavbeta3.

Animals↗

PubMed2000

PECAM-1/CD31 trans-homophilic binding at the intercellular junctions is independent of its cytoplasmic domain; evidence for heterophilic interaction with integrin alphavbeta3 in Cis.

PECAM-1/CD31 is a cell adhesion and signaling molecule that is enriched at the endothelial cell junctions. Alternative splicing generates multiple PECAM-1 splice variants, which differ in their cytoplasmic domains. It has been suggested that the extracellular ligand-binding property, homophilic versus heterophilic, of these isoforms is controlled by their cytoplasmic tails. To determine whether the cytoplasmic domains also regulate the cell surface distribution of PECAM-1 splice variants, we examined the distribution of CD31-EGFPs (PECAM-1 isoforms tagged with the enhanced green fluorescent protein) in living Chinese hamster ovary cells and in PECAM-1-deficient endothelial cells. Our results indicate that the extracellular, rather than the cytoplasmic domain, directs PECAM-1 to the cell-cell borders. Furthermore, coculturing PECAM-1 expressing and deficient cells along with transfection of CD31-EGFP cDNAs into PECAM-1 deficient cells reveal that this PECAM-1 localization is mediated by homophilic interactions. Although the integrin alphavbeta3 has been shown to interact with PECAM-1, this trans-heterophilic interaction was not detected at the borders of endothelial cells. However, based on cocapping experiments performed on proT cells, we provide evidence that the integrin alphavbeta3 associates with PECAM-1 on the same cell surface as in a cis manner.

Alternative Splicing↗

PubMed2000

Role of alpha5beta1 and alphavbeta3 integrins on smooth muscle cell spreading and migration in fibrin gels.

Fibrin is found at sites of vascular injury and is one of the major matrix ligands for beta3 integrins. Blocking the beta3 integrin on smooth muscle cell is hypothesized as a potential target to prevent restenosis because it could inhibit cell attachment and migration into fibrin provisional matrix. Human aortic smooth muscle cells (HNB18E6E7) spread stably in plasma gels within 24 h. Cell spreading was dramatically blocked by simultaneous use of alpha5beta1 and alphavbeta3 integrin antibodies (P <0.0001), however, blocking of either integrin alone failed to inhibit spreading. GPenGRGDSPCA, which has been considered a specific alphavbeta3 antagonist, inhibited spreading at 500 microM, suggesting that the peptide blocked both alpha5beta1 and alphavbeta3. Similarly, invasive migration into fibrin gels was blocked by simultaneous use of both alpha5beta1 and alphavbeta3 antibodies, however, blocking of either integrin alone failed to effect cell migration. Another migration assay using transwell indicated similar results. In conclusion, both alpha5beta1 and alphavbeta3 integrins are responsible for smooth muscle cell spreading and migration into fibrin gels. These data suggest that blocking beta3 integrin alone would not affect smooth muscle cell interaction with fibrin.

Cell Adhesion↗

PubMed1998

Adhesion of activated platelets to endothelial cells: evidence for a GPIIbIIIa-dependent bridging mechanism and novel roles for endothelial intercellular adhesion molecule 1 (ICAM-1), alphavbeta3 integrin, and GPIbalpha.

Although it has been reported that activated platelets can adhere to intact endothelium, the receptors involved have not been fully characterized. Also, it is not clear whether activated platelets bind primarily to matrix proteins at sites of endothelial cell denudation or directly to endothelial cells. Thus, this study was designed to further clarify the mechanisms of activated platelet adhesion to endothelium. Unstimulated human umbilical vein endothelial cell (HUVEC) monolayers were incubated with washed, stained, and thrombin-activated human platelets. To exclude matrix involvement, HUVEC were harvested mechanically and platelet binding was measured by flow cytometry. Before the adhesion assay, platelets or HUVEC were treated with different receptor antagonists. Whereas blockade of platelet beta1 integrins, GPIbalpha, GPIV, P-selectin, and platelet-endothelial cell adhesion molecule (PECAM)-1 did not reduce platelet adhesion to HUVEC, blockade of platelet GPIIbIIIa by antibodies or Arg-Gly-Asp (RGD) peptides markedly decreased adhesion. Moreover, when platelets were treated with blocking antibodies to GPIIbIIIa-binding adhesive proteins, including fibrinogen and fibronectin, and von Willebrand factor (vWF), platelet binding was also reduced markedly. Addition of fibrinogen, fibronectin, or vWF further increased platelet adhesion, indicating that both endogenous platelet-exposed and exogenous adhesive proteins can participate in the binding process. Evaluation of the HUVEC receptors revealed predominant involvement of intercellular adhesion molecule (ICAM)-1 and alphavbeta3 integrin. Blockade of these two receptors by antibodies decreased platelet binding significantly. Also, there was evidence that a component of platelet adhesion was mediated by endothelial GPIbalpha. Blockade of beta1 integrins, E-selectin, P-selectin, PECAM-1, vascular cell adhesion molecule (VCAM)-1 and different matrix proteins on HUVEC did not affect platelet adhesion. In conclusion, we show that activated platelet binding to HUVEC monolayers is mediated by a GPIIbIIIa-dependent bridging mechanism involving platelet-bound adhesive proteins and the endothelial cell receptors ICAM-1, alphavbeta3 integrin, and, to a lesser extent, GPIbalpha.

Anti-Bacterial Agents↗

PubMed2007

Activated alphavbeta3 integrin targeting in injury-induced vascular remodeling.

There is currently no imaging modality to track the remodeling process, a common feature of a broad spectrum of vasculopathies, in vivo. alphavbeta3 Integrin is up-regulated in proliferating vascular cells. RP748, a novel peptidomimetic tracer, binds specifically to the activated alphavbeta3 conformer and exhibits favorable binding characteristics for in vivo imaging. In a model of injury-induced vascular remodeling in apoE null mice, RP748 localization to the injured carotid arteries parallels vascular cell proliferation, providing an opportunity to image the remodeling process in vivo.

Animals↗

PubMed2006

Discovery of diphenylmethanepropionic and dihydrostilbeneacetic acids as antagonists of the integrin alphavbeta3.

A peptidomimetic inhibitor of the integrin alpha(v)beta(3) has been substantially modified to produce several new nonpeptidic antagonists. These inhibitors are simpler to synthesize and belong to new classes of scaffolds. Some of the compounds served as the initial lead for further optimization, which led to the discovery of potent and selective inhibitors of the integrin alpha(v)beta(3).

Acetates↗

PubMed2002

The alphaVbeta3 integrin regulates insulin-like growth factor I (IGF-I) receptor phosphorylation by altering the rate of recruitment of the Src-homology 2-containing phosphotyrosine phosphatase-2 to the activated IGF-I receptor.

The alphaVbeta3 integrin is an important determinant of IGF-I-stimulated receptor phosphorylation and biological actions. Blocking ligand occupancy of alphaVbeta3 with the distintegrin echistatin reduces IGF-I-stimulated receptor phosphorylation, and it inhibits cellular migration and DNA synthesis responses to IGF-I. We have shown that recruitment of the tyrosine phosphatase Src-homology 2-containing phosphotyrosine phosphatase-2 (SHP-2) to the IGF-I receptor (IGF-IR) is an important determinant of the duration of IGF-IR phosphorylation. These studies were undertaken to determine whether an alteration in the recruitment of SHP-2 to the receptor in the presence of echistatin could account for the decrease in receptor phosphorylation. Following an overnight exposure of smooth muscle cell cultures to echistatin, the addition of IGF-I was accompanied by rapid dephosphorylation of IGF-IR compared with cells exposed to media alone. This was associated with an increase in the rate of SHP-2 recruitment to the IGF-IR. In cells expressing a catalytically inactive form of SHP-2, prior exposure to echistatin had no effect on the rate of receptor dephosphorylation. In contrast to the usual physiologic situation in which following IGF-I exposure SHP-2 is recruited to IGF-IR via SHP-2 substrate-1 (SHPS-1) in the presence of echistatin, SHPS-1 was not used for SHP-2 recruitment. Our findings show that IRS-1 may substitute for SHPS-1 under these conditions. These results demonstrate that the activation state of alphaVbeta3 is an important regulator of the duration of IGF-IR phosphorylation and subsequent downstream signaling and that this regulation is mediated through changes in the subcellular localization of SHP-2.

Antigens, Differentiation↗

PubMed2005

Alphavbeta3 integrin and cofilin modulate K1735 melanoma cell invasion.

Cytoskeletal reorganization is partially mediated through cofilin, an actin assembly regulatory protein. Cofilin activity is modulated by reversible phosphorylation at Ser3. In this study, using K1735 murine melanoma cells, we examined the relationship between beta3-integrin expression, phosphorylation of cofilin, and metalloproteinase production. The levels of phosphorylated cofilin were 10-fold higher in cells expressing alphavbeta3 than in alphavbeta3-negative cells when plated on vitronectin for 30 min. However, by 60 min, phosphorylation of cofilin was greater in the beta3-negative cells. Expression of the wild type (WT) or non-phosphorylatable cofilin (A3 mutant) increased melanoma cell migration on vitronectin and invasion through a reconstituted basement membrane. Expression of a pseudophosphorylated, poorly active cofilin (E3 mutant) reduced cell motility. Expression of active cofilin accelerated the phosphorylation of FAK at Y397 and at Y576, strongly implicating cofilin as a mediator of cell signaling. The expression of MT1-MMP and MMP2 was also increased by expression of wild type or A3 cofilin. A 50% reduction of both enzymes was observed by the expression of the E3 cofilin. Overexpression of non-phosphorylatable cofilin was sufficient to induce the expression of MT1-MMP and MMP2 in the beta3-negative M2Tbeta3 cells. Interestingly, the invasion of M2Tbeta3 cells could be sustained by overexpression of cofilin A3. These results suggest that the integrin alphavbeta3 and cofilin together regulate K1735 melanoma cell invasion.

Actin Depolymerizing Factors↗

PubMed2006

Analysis of the interaction between RGD-expressing adenovirus type 5 fiber knob domains and alphavbeta3 integrin reveals distinct binding profiles and intracellular trafficking.

Adenovirus (Ad) vectors are used widely for experimental and therapeutic gene transfer. Ad-mediated gene delivery is often inefficient and, thus, there is considerable interest in developing Ad vectors that overcome biological barriers to efficient virus uptake. For this strategy to succeed, it is imperative that the interaction between such Ad vectors and their novel receptors is well understood. In this study, three surface-exposed loops (HI, CD and IJ loops) on the Ad5 fiber knob domain were selected as sites for insertion of an alphavbeta3 integrin-binding RGD sequence. Three RGD-containing Ad5 fiber knob-domain mutants were produced as recombinant proteins and all were shown to interact with soluble alphavbeta3 integrin by using biomolecular cell-free assays. Cell adsorption and subsequent internalization and intracellular trafficking of each of these proteins were assessed by confocal microscopy. Whilst the Ad5 fiber knob domain expressing the RGD sequence in the HI and CD loops bound with similar association and dissociation profiles, the fiber knob domain expressing the RGD sequence in the IJ loop bound with slower association and faster dissociation rates. By using molecular modelling, it was shown that the Ad5 fiber knob domain in which the RGD peptide was expressed in the IJ loop was only capable of binding to one alphavbeta3 integrin molecule per trimer. In contrast, fiber knob domains in which the RGD peptide was expressed in the HI and CD loops were capable of binding to one integrin molecule per monomer. These differences in the interactions between each mutant and alphavbeta3 may explain our observation that the three RGD-bearing Ad5 fiber knob domains demonstrated similar internalization rates, but distinct patterns of endosomal transport and escape.

Adenoviruses, Human↗

PubMed2005

Rigidity sensing at the leading edge through alphavbeta3 integrins and RPTPalpha.

Cells require optimal substrate stiffness for normal function and differentiation. The mechanisms for sensing matrix rigidity and durotaxis, however, are not clear. Here we showed that control, Shp2-/-, integrin beta1-/-, and talin1-/- cell lines all spread to a threefold greater area on fibronectin (FN)-coated rigid polyacrylamide surfaces than soft. In contrast, RPTPalpha-/- cells spread to the same area irrespective of rigidity on FN surfaces but spread 3x greater on rigid collagen IV-coated surfaces than soft. RPTPalpha and alphavbeta3 integrins were shown previously to be colocalized at leading edges and antibodies to alphavbeta3 blocked FN rigidity sensing. When FN beads were held with a rigid laser trap at the leading edge, stronger bonds to the cytoskeleton formed than when held with a soft trap; whereas back from the leading edge and in RPTPalpha-/- cells, weaker bonds were formed with both rigid and soft laser traps. From the rigidity of the trap, we calculate that a force of 10 pN generated in 1 s is sufficient to activate the rigidity response. We suggest that RPTPalpha and alphavbeta3 at the leading edge are critical elements for sensing FN matrix rigidity possibly through SFK activation at the edge and downstream signaling.

Animals↗

PubMed2006

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.

Actin Cytoskeleton↗

PubMed2003

Integrin alphavbeta3 binding to human alpha5-laminins facilitates FGF-2- and VEGF-induced proliferation of human ECV304 carcinoma cells.

Human ECV304 cells respond reproducibly by tube formation to complex basement membrane matrices. Laminins are major glycoproteins of basement membranes. We therefore studied the ability of ECV304 cells to attach to defined laminin isoforms and to fibronectin, and identified the involved laminin receptors. The cells bound poorly to fibronectin, to some extent to laminin-1, whereas laminin-2/4 and -10/11 were strong adhesive substrates. Antibody perturbation assays showed that adhesion to laminin-1 was mediated by integrin alpha6beta1, and adhesion to laminin-2/4 by cooperative activity of integrins alpha3beta1 and alpha6beta1. Adhesion of ECV 304 cells to laminin-10/11 was mainly mediated by integrins alpha3beta1, with minor involvement of alpha6beta1/4 and alphavbeta3. Solid-phase binding assays confirmed that integrin alphavbeta3 binds human laminin-10/11 and -10, in an RGD-dependent fashion. Although integrin alphavbeta3 played a very minor role in cell adhesion to laminin-10/11, this interaction facilitated growth factor-induced proliferation of ECV304 cells. In response to FGF-2 or VEGF, the cells proliferated better when attached on laminin-10/11 than on laminin-1, -2/4, or gelatin. The proliferation induced by the joint application of laminin-10/11 and either one of the growth factors could be blocked by antibodies against integrin alphavbeta3. Fragments of several other basement membrane components are known to interact with alphavbeta3. The current data show that that integrin alphavbeta3 can bind intact alpha5-containing laminin trimers. Since the laminin alpha5 chain is broadly expressed in adult basement membranes, this interaction could be physiologically important. Our data suggest that this interaction is involved in the regulation of cellular responses to growth factors known to be involved in epithelial and endothelial development.

Cell Division↗