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PubMed2005

Effect of ganglioside and tetraspanins in microdomains on interaction of integrins with fibroblast growth factor receptor.

The functional interaction ("cross-talk") of integrins with growth factor receptors has become increasingly clear as a basic mechanism in cell biology, defining cell growth, adhesion, and motility. However, no studies have addressed the microdomains in which such interaction takes place nor the effect of gangliosides and tetraspanins (TSPs) on such interaction. Growth of human embryonal WI38 fibroblasts is highly dependent on fibroblast growth factor (FGF) and its receptor (FGFR), stably associated with ganglioside GM3 and TSPs CD9 and CD81 in the ganglioside-enriched microdomain. Adhesion and motility of these cells are mediated by laminin-5 ((LN5) and fibronectin (FN) through alpha3beta1 and alpha5beta1 integrin receptors, respectively. When WI38 cells or its transformant VA13 cells were adhered to LN5 or FN, alpha3beta1 or alpha5beta1 were stimulated, giving rise to signaling to activate FGFR through tyrosine phosphorylation and inducing cell proliferation under serum-free conditions without FGF addition. Types and intensity of signaling during the time course differed significantly depending on the type of integrin stimulated (alpha3beta1 versus alpha5beta1), and on cell type (WI38 versus VA13). Such effect of cross-talk between integrins and FGFR was influenced strongly by the change of GM3 and TSPs. (i) GM3 depletion by P4 caused enhanced tyrosine phosphorylation of FGFR and Akt followed by MAPK activation, without significant change of ceramide level. GM3 depletion also caused enhanced co-immunoprecipitation of FGFR with alpha3/alpha5/beta1 and of these integrins with CD9/CD81. (ii) LN5- or FN-dependent proliferation of both WI38 and VA13 was strongly enhanced by GM3 depletion and by CD9/CD81 knockdown by siRNA. Thus, integrin-FGFR cross-talk is strongly influenced by GM3 and/or TSPs within the ganglioside-enriched microdomain.

Antigens, CD↗
PubMed1999

Fine mapping of inhibitory anti-alpha5 monoclonal antibody epitopes that differentially affect integrin-ligand binding.

The high-affinity interaction of integrin alpha5beta1 with the central cell-binding domain of fibronectin requires both the Arg-Gly-Asp (RGD) sequence (in the tenth type III repeat) and a second site Pro-His-Ser-Arg-Asn (PHSRN) in the adjacent ninth type III repeat, which synergizes with RGD. Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA) is a novel peptidic ligand for alpha5beta1, identified by phage display, which blocks alpha5beta1-mediated cell adhesion to fibronectin. A key question is the location of the binding sites for these ligand sequences within the integrin. In this study we have identified residues that form part of the epitopes of three inhibitory anti-alpha5 monoclonal antibodies (mAbs): 16, P1D6 and SNAKA52. These mAbs have distinct functional properties. mAb 16 blocks the recognition of RGD and RRETAWA, whereas P1D6 blocks binding to the synergy sequence. The binding of SNAKA52 is inhibited by anti-beta1 mAbs, indicating that its epitope is close to the interface between the alpha and beta subunits. Residues in human alpha5 were replaced with the corresponding residues in mouse alpha5 by site-directed mutagenesis; wild-type or mutant human alpha5 was expressed on the surface of alpha5-deficient Chinese hamster ovary cells. mAb binding was assessed by flow cytometry and by adhesion to the central cell-binding domain of fibronectin or RRETAWA by cell attachment assay. All three epitopes were located to different putative loops in the N-terminal domain of alpha5. As expected, disruption of these epitopes had no effect on ligand recognition by alpha5beta1. The locations of these epitopes are consistent with the beta-propeller model for integrin alpha-subunit structure and allow us to propose a topological image of the integrin-ligand complex.

Amino Acid Sequence↗
PubMed2002

Proximal tubular epithelial cell integrins respond to high glucose by altered cell-matrix interactions and differentially regulate matrixin expression.

Thickening of the tubular basement membrane (TBM) occurs in diabetic nephropathy, but the effects of high glucose on the functional aspects of proximal tubular epithelial cells are not clearly understood. In the present study, we examined the effects of elevated glucose concentrations on (a) integrin expression by human proximal tubular epithelial cells (HK-2) and integrin-mediated interactions with type IV collagen (colIV) and laminin, major components of TBM; (b) the expression of matrixins/matrix metalloproteinases (MMPs), which is regulated by integrins; and (c) the expression of tissue inhibitors of metalloproteinases (TIMPs). HK-2 cells cultured in 25 mM glucose underwent a reduction of the expression of alpha3, beta1, alpha(v)beta3, and alpha5 integrin subunits, with a concomitant increase of the alpha2 subunit, compared with cells grown in 5 mM glucose. Adhesion experiments demonstrated that high glucose led to increased cell adhesion on either colIV or laminin. Experiments of competition of adhesion using anti-integrin antibodies indicated that HK-2 cells in 5 mM glucose used mainly alpha(v)beta3 and alpha5beta1 integrins to adhere to colIV, whereas in 25 mM glucose they additionally used alpha2beta1. In the case of laminin, a beta1-mediated adhesion was observed when HK-2 cells were in 5 mM glucose, whereas in 25 mM glucose, alpha2beta1 and alpha(v)beta3 were also involved. Elevated glucose concentrations resulted in decreased expression of MMP-9 and MMP-2, whereas an increase in TIMP-1 and a decrease in TIMP-2 expression were observed. We also examined which integrins mediated the expression and secretion of matrixins MMP-2 and MMP-9. Ligation of alpha3beta1 with mAbs resulted in induction of MMP-2 expression and secretion, whereas antibody ligation of alpha(v)beta3 led to down-regulation of MMP-9. The above data implicate integrins of proximal tubular epithelial cells in the regulation of MMPs and in the development of TBM thickening in diabetic nephropathy.

Cell Line↗
PubMed2006

Adherence and internalization of Streptococcus gordonii by epithelial cells involves beta1 integrin recognition by SspA and SspB (antigen I/II family) polypeptides.

Streptococcus gordonii is a commensal bacterium that colonizes the hard and soft tissues present in the human mouth and nasopharynx. The cell wall-anchored polypeptides SspA and SspB expressed by S. gordonii mediate a wide range of interactions with host proteins and other bacteria. In this article we have determined the role of SspA and SspB proteins, which are members of the streptococcal antigen I/II (AgI/II) adhesin family, in S. gordonii adherence and internalization by epithelial cells. Wild-type S. gordonii DL1 expressing AgI/II polypeptides attached to and was internalized by HEp-2 cells, whereas an isogenic AgI/II- mutant was reduced in adherence and was not internalized. Association of S. gordonii DL1 with HEp-2 cells triggered protein tyrosine phosphorylation but no significant actin rearrangement. By contrast, Streptococcus pyogenes A40 showed 50-fold higher levels of internalization and this was associated with actin polymerization and interleukin-8 upregulation. Adherence and internalization of S. gordonii by HEp-2 cells involved beta1 integrin recognition but was not fibronectin-dependent. Recombinant SspA and SspB polypeptides bound to purified human alpha5beta1 integrin through sequences present within the NAV (N-terminal) region of AgI/II polypeptide. AgI/II polypeptides blocked interactions of S. gordonii and S. pyogenes with HEp-2 cells, and S. gordonii DL1 cells expressing AgI/II proteins inhibited adherence and internalization of S. pyogenes by HEp-2 cells. Conversely, S. gordonii AgI/II- mutant cells did not inhibit internalization of S. pyogenes. The results suggest that AgI/II proteins not only promote integrin-mediated internalization of oral commensal streptococci by host cells, but also potentially influence susceptibility of host tissues to more pathogenic bacteria.

Adhesins, Bacterial↗
PubMed1997

Fibroblast growth factor-2 potentiates vascular smooth muscle cell migration to platelet-derived growth factor: upregulation of alpha2beta1 integrin and disassembly of actin filaments.

Fibroblast growth factor-2 (FGF-2) has been implicated in vascular smooth muscle cell (SMC) migration, a key process in vascular disease. We demonstrate here that FGF-2 promotes SMC motility by altering beta1 integrin-mediated interactions with the extracellular matrix (ECM). FGF-2 significantly increased surface expression of alpha2beta1, alpha3beta1, and alpha5beta1 integrins on human SMCs, as assessed by flow cytometry. The greatest increase was for the collagen-binding alpha2beta1 integrin. Despite this, FGF-2 did not increase SMC adhesion to type I collagen but instead promoted SMC elongation and SMC motility. The latter was evaluated by using a microchemotaxis chamber and by digital time-lapse video microscopy. Although FGF-2 was not chemotactic for human SMCs, cells preincubated with FGF-2 displayed a 3.1-fold increase in migration to the undersurface of porous type I collagen-coated membranes and a 2.1-fold increase in migration speed on collagen. Furthermore, chemotaxis to platelet-derived growth factor-BB on collagen was significantly greater in SMCs exposed to FGF-2. FGF-2-induced elongation and migration on collagen were inhibited by a blocking anti-alpha2beta1 antibody; however, SMC adhesion to collagen was unaffected. SMC migration on fibronectin was also enhanced by FGF-2, although less prominently: migration through porous membranes increased 1.8-fold, and migration speed increased 1.3-fold. Also, FGF-2 completely disassembled the smooth muscle alpha-actin-containing stress fiber network contemporaneously with the change in integrin expression and cell shape. We conclude that (1) exogenous FGF-2 promotes SMC migration and potentiates chemotaxis to PDGF-BB; (2) the promigratory effect of FGF-2 is especially prominent on type I collagen and is mediated by upregulation of alpha2beta1 integrin; and (3) FGF-2 disassembles actin stress fibers, which may promote differential utilization of alpha2beta1 integrin for motility but not adhesion. This dynamic SMC-ECM interplay may be an important mechanism by which FGF-2 facilitates SMC motility in vivo.

Actin Cytoskeleton↗
PubMed2003

Developmental and regional differences in the consolidation of long-term potentiation.

The alpha5beta1 integrin is present in high concentrations in the apical dendrites of pyramidal neurons in adult rats but is virtually absent in the basal dendrites. Moreover, alpha5beta1 does not appear in apical dendritic branches until the third post-natal week. Given that integrins contribute to the consolidation of synaptic plasticity, these results raise the possibility of developmental and regional differences in the stability of long-term potentiation (LTP). The present study tested this point using a LTP reversal paradigm in field CA1 of hippocampal slices. In accord with earlier reports, low-frequency afferent stimulation (5 Hz) introduced 30 s after theta burst stimulation (TBS) completely reversed LTP but was ineffective 30 min and 60 min later in slices from adult rats. The same low-frequency trains caused a partial reversal of LTP when applied 30 and 60 min post-TBS in slices from 21-day-old rats and a complete reversal at all time points in slices from 10-day-old rats. LTP in the basal dendrites of adult rats did not fully consolidate; i.e. potentiation was partially reversed by low-frequency stimulation even after delays of 30 or 60 min. Moreover, spaced (10 min) applications of 5- Hz pulses beginning at 30 min post-TBS completely erased LTP. The reversal effect in both apical and basal dendrites was blocked by N-methyl-D-aspartic acid receptor antagonists but an integrin antagonist had differential effects across the two dendritic domains. These results constitute evidence that the stability of LTP increases with age in the apical dendrites but remains incomplete even in adulthood in the basal dendrites. The possibilities that the developmental and regional variations in LTP consolidation are correlated with integrin expression and linked to different types of memory processing are discussed.

Animals↗
PubMed2005

ZO-1: lamellipodial localization in a corneal fibroblast wound model.

PURPOSE: To explore the roles of ZO-1 in corneal fibroblasts and myofibroblasts in a model of wounding. METHODS: Antibodies were used to identify ZO-1 in cultured rabbit corneal fibroblasts by immunocytochemistry, Western blot analysis, and immunoprecipitation. For colocalization studies, antibodies to beta-catenin, cadherins, connexins, integrins, alpha-actinin, and cortactin were used. G- and F-actin were identified by DNase and rhodamine phalloidin, respectively. To study ZO-1 localization during cell migration, confluent corneal fibroblasts were subjected to scrape-wounding and evaluated by immunocytochemistry. RESULTS: As predicted from previous studies, ZO-1 colocalized with cadherins and connexin 43 in intercellular junctions. The study revealed a new finding: ZO-1 was also detected at the leading edge of lamellipodia, especially in motile wounded fibroblasts and in freshly plated fibroblasts, before the formation of cell-cell contacts. In fibroblast lysates, ZO-1 largely partitioned to the detergent-soluble fraction compared with myofibroblast lysates, indicating that much of the fibroblast ZO-1 is not associated with insoluble structural components. Lamellipodial ZO-1 colocalized with G-actin, alpha-actinin, and cortactin, which are proteins involved with actin remodeling and cell migration. Integrins alpha5beta1 and alphavbeta3 also localized to the leading edge of migrating fibroblasts, and the association of ZO-1 with integrin was confirmed by immunoprecipitation. Finally, alkaline phosphatase treatment of fibroblast lysate decreased the molecular mass of ZO-1 in lysates of cells grown in serum, demonstrating that, in activated fibroblasts, ZO-1 is phosphorylated. CONCLUSIONS: ZO-1's appearance at the leading edge of migrating fibroblasts makes it a candidate for a role in the initiation and organization of integrin-dependent fibroblast adhesion complexes formed during migration and adhesion. Further, phosphorylation of ZO-1 may regulate its cellular localization.

Animals↗
PubMed1998

Inhibition of vascular smooth muscle cell growth by inhibition of fibronectin matrix assembly.

The regulation of vascular smooth muscle cell (VSMC) proliferation by the fibronectin matrix was tested by treating human umbilical artery smooth muscle cells (HUASMCs) with a recombinant fragment of fibronectin (protein III1-C) that has previously been shown to modulate fibronectin matrix assembly. III1-C inhibited HUASMC proliferation by 75% to 90%. The inhibition of growth was time dependent; III1-C had no effect on DNA synthesis after 0 to 5 hours of treatment but did have an effect at 24 hours and beyond. III1-C did not stimulate apoptosis in these cells, indicating that the inhibition of proliferation was not due to an induction of programmed cell death. The effects of III1-C on cell growth were only specific for normal diploid smooth muscle cells. III1-C had no effect on the proliferation of IMR-90 fibroblasts, endothelial cells, NIH 3T3 cells, or the rat aortic smooth muscle cell line A7r5. However, III1-C did inhibit proliferation by primary rat aortic smooth muscle cells. An analysis of HUASMC fibronectin receptor (integrin alpha5beta1) distribution revealed that III1-C did not inhibit alpha5beta1 localization to focal contacts. Moreover, III1-C had no effect on the relative expression levels of seven different integrin subunits on HUASMCs. However, III1-C did inhibit fibronectin matrix assembly by rat aortic smooth muscle cells, HUASMCs, A7r5 cells, IMR-90 cells, and endothelial cells. An analysis of fibronectin synthesis indicated that the inhibition of fibronectin matrix assembly by III1-C was not due solely to a decrease in fibronectin synthesis. Finally, treatment of HUASMCs with anti-fibronectin monoclonal antibody L8 (which is known to inhibit fibronectin matrix assembly) also decreased the rate of HUASMC DNA synthesis. These results demonstrate that III1-C inhibits VSMC proliferation and suggest that this effect may be mediated by the inhibition of fibronectin matrix assembly.

Animals↗
PubMed1999

Restricted and coordinated expression of beta3-integrin and bone sialoprotein during cultured osteoblast differentiation.

In this study, the expression of beta3-integrin was examined in relationship to the restricted expression of bone sialoprotein (BSP). Immunohistochemical analysis indicated that the alpha(v)beta3-integrin was coincident and proximal to BSP expression in the fetal mandible bovine osteoblast culture model. Alpha(v)beta3-integrin expression was expressed predominantly in a region proximal to, but not including, the substrate adherent cells. In comparison, the alpha5beta1-integrin was expressed in a generalized pattern throughout the culture layers in a coordinated fashion to fibronectin. The temporal expression of beta1- and beta3-integrin was evaluated using RT-PCR and southern blot analysis. Unlike the generalized expression of beta1-integrin, beta3-integrin was restricted to days 3 and 5 of the culture period. The previous demonstration of similar restriction of BSP expression and the present colocalization of BSP suggests the potential coordinated expression of a specific extracellular matrix ligand with a select integrin. Beta3-integrin/BSP adhesion-mediated signaling may play a significant role in the process of osteoblast morphodifferentiation.

Animals↗
PubMed2003

Generation of a monoclonal antibody to a cryptic site common to both integrin beta1 as well as gelatinase MMP9.

Integrins are one class of cell surface receptors that have been implicated in the regulation of a diverse set of cellular processes, including cell adhesion, migration, and invasion as well as gene expression, differentiation, and signal transduction. Cellular invasion not only requires the adhesive properties of integrins but also the proteolytic properties of matrix-degrading enzymes, such as the metalloproteinases (MMPs). Previous studies have shown that integrin alphavbeta3 is a receptor for MMP2, localizing its proteinase activity to the cell surface, ultimately leading to site-specific extracellular matrix (ECM) degradation. Here we develop reagents to investigate the possibility of an interplay between MMP9 and integrin alpha5beta1. With the use of EV22 viral studies, the tetrapeptide sequence, LRSG, was shown to be a dimerizing sequence mediating beta1 integrin binding to EV22. The same study also showed that cellular infection could be halted with the use of LRSG-containing peptides. In a later study, in an effort to isolate inhibitors of the MMP family, LRSG sequence was identified as one capable of binding MMP9. Interestingly, MMP9 contains an LRSG sequence, raising the possibility that MMP9 binds the cell surface via beta1 integrins through the dimerizing LRSG motif. We used the LRSG-containing sequence from beta1 integrins as an antigen to generate the monoclonal antibody (MAB) FM155 in the mouse model. MAB FM155 will help identify a cryptic epitope, LRSG, and its role in matrix remodeling as well as tumor growth, cancer cell migration, and angiogenesis.

Amino Acid Sequence↗
PubMed2000

EC3, a heterodimeric disintegrin from Echis carinatus, inhibits human and murine alpha4 integrin and attenuates lymphocyte infiltration of Langerhans islets in pancreas and salivary glands in nonobese diabetic mice.

The venom of Echis carinatus suchoreki contains a monomeric disintegrin echistatin (Mr 5,500 Da) that strongly inhibits alphaIIbbeta3, alphavbeta3, and alpha5beta1 integrins and a heterodimeric disintegrin called EC3 (M(r) 14,762 Da). At nanomolar concentration, EC3 inhibits adhesion of human cell lines expressing alpha4beta1 and alpha4beta7 to immobilized VCAM-1; it has a lower inhibitory effect on alpha5beta1-mediated cell adhesion. In this study, we demonstrated that EC3, in contrast to echistatin, inhibited binding of monoclonal anti-alpha4 and anti-alpha5 antibodies to cells expressing alpha4beta7. In a dose-dependent manner and to the same extent, EC3 inhibited adhesion of Jurkat cells and murine splenic lymphocytes to immobilized VCAM-1, whereas echistatin was not active. EC3 injected intraperitoneally into nonobese diabetic (NOD mice) suppressed development of insulitis and sialoadenitis, whereas echistatin had no significant effect. We propose that the effect of EC3 is mediated, at least, in part, by blocking alpha4beta1 and alpha4beta7 on murine lymphocytes.

Amino Acid Sequence↗
PubMed1999

Integrin family of cell adhesion molecules in the injured brain: regulation and cellular localization in the normal and regenerating mouse facial motor nucleus.

Integrins are a large family of heterodimeric glycoproteins that play a crucial role in cell adhesion during development, inflammation, and tissue repair. In the current study, we investigated the localization of different integrin subunits in the mouse facial motor nucleus and their regulation after transection of the facial nerve. In the normal mouse brain, there was clear immunoreactivity for alpha5-, alpha6-, and beta1-integrin subunits on blood vessel endothelia and for alphaM- and beta2-subunits on resting parenchymal microglia. Facial nerve transection led to an up-regulation of the beta1-subunit on the axotomized neurons and an increase in the alpha4-, alpha5-, alpha6-, beta1-, alphaM-, alphaX-, and beta2-subunits on the adjacent, activated microglia. Quantification of the microglial integrins revealed two different expression patterns. The subunits alpha5 and alpha6 showed a monophasic increase with a maximum at day 4, the alphaM-subunit a biphasic regulation, with an early peak at day 1 and an elevated plateau between day 14 and 42. At day 14, there was also an influx of lymphocytes immunoreactive for the alpha4beta1- and alphaLbeta2-integrins, which aggregated at sites of neural debris and phagocytotic microglia. This finding was accompanied by a significant increase of the alpha5beta1-integrin on blood vessel endothelia. In summary, facial axotomy is followed by a strong and cell-type-specific expression of integrins on the affected neurons and on surrounding microglia, lymphocytes, and vascular endothelia. The presence of several, strikingly different temporal patterns suggests a selective involvement of these molecules in the different adhesive events during regeneration in the central nervous system.

Animals↗
PubMed2001

Hydroxylapatite binds more serum proteins, purified integrins, and osteoblast precursor cells than titanium or steel.

The implant material hydroxylapatite (HA) has been shown in numerous studies to be highly biocompatible and to osseointegrate well with existing bone; however, the molecular mechanisms at work behind this osseointegration remain largely unexplored. One possibility is that the implant, exposed to the patient's blood during surgery, adsorbs known cell adhesive proteins such as fibronectin and vitronectin from the serum. Osteoblast precursors could then adhere to these proteins through integrin-mediated mechanisms. In the present study, we have used a quantitative ELISA assay to test the hypothesis that hydroxylapatite will adsorb more fibronectin and vitronectin from serum than two commonly used hard-tissue materials, commercially pure titanium, and 316L stainless steel. We further used the ELISA, as well as a standard cell adhesion assay, to test the hypothesis that increased protein adsorption will lead to better binding of purified integrins alpha5beta1 and alpha(v)beta3 and osteoblast precursor cells to the HA than to the metals. Our results show that fibronectin, vitronectin, alpha5beta1, alpha(v)beta3, and osteoblast precursor cells do indeed bind better to HA than to the metals, suggesting that improved integrin-mediated cell binding may be one of the mechanisms leading to better clinical bone integration with HA-coated implants.

Adsorption↗
PubMed1999

Beta1-integrins control spontaneous adhesion and motility of human progenitor thymocytes and regulate differentiation-dependent expression of the receptor for hyaluronan-mediated motility.

The functions of the receptor for hyaluronan-mediated motility (RHAMM) and beta1-integrin in adhesion and motility were analysed for human progenitor multinegative (CD3- 4- 8- 19-) thymocytes (MN Thy). Both alpha4beta1- and alpha5beta1-integrins are expressed by MN Thy, but only alpha4beta1 mediates fibronectin (FN)-dependent adhesion and motility. Freshly isolated MN Thy lack expression of RHAMM and their motility is RHAMM independent. Prolonged surface expression of RHAMM on MN Thy is dependent upon FN. RHAMM expression, which occurs prior to surface expression of CD3/T-cell receptor (TCR), was found to be inhibited by cross-linking of alpha4-, alpha5- and beta1-integrins, as was the prolonged FN-dependent phase of RHAMM expression. To confirm that RHAMM expression had been down-regulated rather than rendered cryptic by treatment with immobilized anti-integrin monoclonal antibody (MoAb), RHAMM mRNA levels were analysed. Transcription of RHAMM was decreased 7-12-fold by treatment with immobilized anti-alpha4 or anti-alpha5, and twofold by anti-beta1. Prior to expression of CD3/TCR and RHAMM, alpha4beta1 regulates migratory behaviour. After MN Thy differentiate to acquire CD3/TCR in vitro or in vivo, their motility becomes dependent upon both RHAMM and beta1-integrins. Integrins play a direct role in FN-dependent, RHAMM-independent motility of MN Thy, and an indirect role in RHAMM-dependent motility. This work shows that beta1-integrins are primary mediators and regulators of fundamental cell behaviours required during migratory phases of T-cell differentiation that occur prior to the expression of CD3/TCR.

Antibodies, Monoclonal↗
PubMed2006

The role of beta1 integrin subfamily in anchorage-dependent apoptosis of breast carcinoma cells differing in multidrug resistance.

Integrin expression was investigated in MCF-7 human breast adenocarcinoma line and in the MCF-7Dox line, which was selected from MCF-7 by a resistance to multiple antitumor drugs (MDR). We have shown that acquisition of MDR was accompanied by a drastically reduced expression of some integrins of the beta1-subfamily (alpha2beta1, alpha3beta1, alpha6beta1) and of alpha vbeta5 intergin in the adenocarcinoma cells. In contrast, expression of alpha5beta1 integrin was markedly increased in the MDR cells. Along with multiple antitumor drug resistance, MCF-7Dox cells demonstrate elevated resistance to anchorage-dependent apoptosis (anoikis) and enhanced in vitro invasive activity. To elucidate the implication of beta1-integrins in the above phenotypic modifications, the effect of beta1-integrin signaling was assayed. Stimulation of beta1-mediated signaling was accomplished by treating of the cells with antibodies to the beta1-subunit common for members of the beta1-subfamily. These data show that activation of beta1-integrin signaling markedly upregulated anoikis of the adenocarcinoma cells.

Anoikis↗
PubMed2005

Inhibition of pathologic retinal neovascularization by alpha-defensins.

Proliferative retinopathies, such as those complicating prematurity and diabetes, are major causes of blindness. A prominent feature of these retinopathies is excessive neovascularization, which is orchestrated by the hypoxia-induced vascular endothelial growth factor (VEGF) stimulating endothelial cells and the integrin-mediated adhesive interactions of endothelial cells with extracellular matrix components such as fibronectin (FN). Recently, we demonstrated that alpha-defensins interfere with alpha5beta1-FN interactions and dependent endothelial cell functions. Here, alpha-defensins were studied in hypoxia-induced proliferative retinopathy. In vitro, alpha-defensins specifically inhibited alpha5beta1-integrin-dependent migration of bovine retinal endothelial cells (BRECs) to FN, attenuated the VEGF-stimulated increase in endothelial permeability, and blocked BREC proliferation and capillary sprout formation in 3-dimensional fibrin-matrices. An up-regulation of beta1-integrin and FN was observed in the retinal vessels in the mouse model of hypoxia-induced retinal angiogenesis. Systemic and local administration of alpha-defensins reduced retinal neovascularization by 45% and 60%, respectively, and this effect was comparable to the inhibitory effect of alpha5beta1-blocking antibody. alpha-Defensins were detected in human diabetic retinas associated with normal retinal vessels but were absent from proliferative lesions. Together, these data show that alpha-defensins inhibit pathologic retinal neovascularization in vivo and may provide a clinically efficient strategy against proliferative retinopathies.

Animals↗
PubMed2000

Defining fibronectin's cell adhesion synergy site by site-directed mutagenesis.

Fibronectin's RGD-mediated binding to the alpha5beta1 integrin is dramatically enhanced by a synergy site within fibronectin III domain 9 (FN9). Guided by the crystal structure of the cell-binding domain, we selected amino acids in FN9 that project in the same direction as the RGD, presumably toward the integrin, and mutated them to alanine. R1379 in the peptide PHSRN, and the nearby R1374 have been shown previously to be important for alpha5beta1-mediated adhesion (Aota, S., M. Nomizu, and K.M. Yamada. 1994. J. Biol. Chem. 269:24756-24761). Our more extensive set of mutants showed that R1379 is the key residue in the synergistic effect, but other residues contribute substantially. R1374A decreased adhesion slightly by itself, but the double mutant R1374A-R1379A was significantly less adhesive than R1379A alone. Single mutations of R1369A, R1371A, T1385A, and N1386A had negligible effects on cell adhesion, but combining these substitutions either with R1379A or each other gave a more dramatic reduction of cell adhesion. The triple mutant R1374A/P1376A/R1379A had no detectable adhesion activity. We conclude that, in addition to the R of the PHRSN peptide, other residues on the same face of FN9 are required for the full synergistic effect. The integrin-binding synergy site is a much more extensive surface than the small linear peptide sequence.

Amino Acid Sequence↗
PubMed2004

Integrin regulation of cell signalling and motility.

Integrins clearly play a key role in regulating both mitogenic signalling and cell migration. Thus integrins modulate the efficiency of the Erk (extracellular-signal-regulated kinase)/MAP kinase (mitogen-activated protein kinase) pathway, acting at several distinct levels. We have shown that both cAMP-dependent protein kinase and PAKs (p21-activated kinases) play a role in integrin regulation of the Erk pathway, acting primarily at the level of Raf-1. Integrins and PAKs also play a role in the control of cell migration. Thus we have discovered a novel protein that links the alpha5beta1 integrin to migration controlled by Rho-family GTPases. This protein, termed Nischarin, is a large cytosolic macromolecule that is not related to well-known protein families. The N-terminus of Nischarin interacts with a short segment of the cytoplasmic domain of the alpha5 integrin subunit. Overexpression of Nischarin alters actin organization and inhibits Rac-driven cell migration and tumour cell invasion. Use of effector domain mutants of Rac suggest that Nischarin acts downstream of Rac, probably at the level of PAK-family kinases. These studies emphasize the intricate connection between integrins and Rho-family GTPases and their effectors in controlling both mitogenesis and migration.

Animals↗