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PubMed2004

The bi-directional translocation of MARCKS between membrane and cytosol regulates integrin-mediated muscle cell spreading.

The regulation of the cytoskeleton is critical to normal cell function during tissue morphogenesis. Cell-matrix interactions mediated by integrins regulate cytoskeletal dynamics, but the signaling cascades that control these processes remain largely unknown. Here we show that myristoylated alanine-rich C-kinase substrate (MARCKS) a specific substrate of protein kinase C (PKC), is regulated by alpha5beta1 integrin-mediated activation of PKC and is critical to the regulation of actin stress fiber formation during muscle cell spreading. Using MARCKS mutants that are defective in membrane association or responsiveness to PKC-dependent phosphorylation, we demonstrate that the translocation of MARCKS from the membrane to the cytosol in a PKC-dependent manner permits the initial phases of cell adhesion. The dephosphorylation of MARCKS and its translocation back to the membrane permits the later stages of cell spreading during the polymerization and cross-linking of actin and the maturation of the cytoskeleton. All of these processes are directly dependent on the binding of alpha5beta1 integrin to its extracellular matrix receptor, fibronectin. These results demonstrate a direct biochemical pathway linking alpha5beta1 integrin signaling to cytoskeletal dynamics and involving bi-directional translocation of MARCKS during the dramatic changes in cellular morphology that occur during cell migration and tissue morphogenesis.

Actins↗
PubMed2005

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

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

Amino Acid Sequence↗
PubMed2006

N-glycosylation of the beta-propeller domain of the integrin alpha5 subunit is essential for alpha5beta1 heterodimerization, expression on the cell surface, and its biological function.

The N-glycosylation of integrin alpha5beta1 is thought to play crucial roles in cell spreading, cell migration, ligand binding, and dimer formation, but the underlying mechanism remains unclear. To investigate the importance of the N-glycans of this integrin in detail, sequential site-directed mutagenesis was carried out to remove single or combined putative N-glycosylation sites on the alpha5 integrin. Removal of the putative N-glycosylation sites on the beta-propeller, Thigh, Calf-1, or Calf-2 domains of the alpha5 subunit resulted in a decrease in molecular weight compared with the wild type, suggesting that all of these domains contain attached N-glycans. Importantly, the absence of N-glycosylation sites (sites 1-5) on the beta-propeller resulted in the persistent association of integrin subunit with calnexin in the endoplasmic reticulum, which subsequently blocked heterodimerization and its expression on the cell surface. Interestingly, the activities for cell spreading and migration for the alpha5 subunit carrying only three potential N-glycosylation sites (3-5 sites) on the beta-propeller were comparable with those of the wild type. In contrast, mutation of these three sites resulted in a significant decrease in cell spreading as well as functional expression, although the total expression level of the Delta3-5 mutant on the cell surface was comparable with that of wild type. Furthermore, we found that site 5 is a most important site for its expression on the cell surface, whereas the S5 mutant did not show any biological functions. Taken together, this study reveals for the first time that the N-glycosylation on the beta-propeller domain of the alpha5 subunit is essential for heterodimerization and biological functions of alpha5beta1 integrin and might also be useful for studies of the molecular structure.

Animals↗
PubMed1997

The alphavbeta3 integrin regulates alpha5beta1-mediated cell migration toward fibronectin.

This study examines the interactions of alphavbeta3 and alpha5beta1 in the regulation of cell migration. Human embryonic kidney (HEK) 293 cells that express alpha5beta1 endogenously were transfected with alphavbeta3 and beta3 mutants, and their attachment and migration to fibronectin (Fn) and vitronectin (Vn) were measured. An alphavbeta3 blocking antibody and the alphavbeta3 ligand cyclic G-Pen-GRGDSPC-A inhibited alpha5beta1-mediated migration toward Fn, but not attachment to Fn. This function was alphavbeta3-specific since alphavbeta5 transfection and alphavbeta5 blocking antibody did not produce this effect. Mutations introduced into the beta3 integrin subunit to dissect this phenomenon revealed the following. Disruption of the ligand binding domain by the Glanzmann thrombasthenia mutation beta3-D119Y constitutively abolished migration toward both Vn and Fn, and attachment to Vn but not to Fn. Insertion of the Glanzmann mutation beta3-S752P into the cytoplasmic domain or its truncation (beta3-Delta717) abolished binding to Vn but not to Fn. Inhibition of migration toward Fn was inhibited in these cells by alphavbeta3 blocking antibody. alphavbeta3-mediated inhibition was, however, abolished by truncation of the transmembrane domain (beta3-Delta693). These findings demonstrate alphavbeta3 regulation of alpha5beta1-mediated cell migration and suggest that the beta3 transmembrane domain is essential for this function.

Cell Line↗
PubMed2001

Antibodies to alpha5beta1 and alpha(v)beta3 integrins react with Candida albicans alcohol dehydrogenase.

It has been hypothesized that Candida albicans possesses integrin-like receptors on its cell surface. This is because C. albicans binds numerous fluid-phase extracellular matrix (ECM) proteins on its cell surface and adheres to the same ECM proteins when immobilized. In addition, numerous antibodies to human integrins (receptors for ECM proteins) bind to the fungal cell surface and in so doing inhibit the binding of the respective proteins. To demonstrate the presence of such a cell surface integrin, a cDNA library of C. albicans yeast cells was screened with polyclonal antiserum to the human fibronectin receptor (alpha5beta1 integrin). Clones isolated by this screening technique also reacted specifically to antiserum against the human vitronectin receptor (alpha(v)beta3 integrin). DNA sequence analysis of the cloned insert predicted a 350 aa protein (37 kDa). This predicted protein showed 75% homology at the nucleotide sequence level to alcohol dehydrogenase (ADH) of Saccharomyces cerevisiae. In vitro transcription/translation of the cloned inserts yielded a 37 kDa protein that was immunoprecipitated with antibodies to the alpha5beta1 and alpha(v)beta3 integrins and an antibody to a C. albicans fibronectin receptor. These antibodies and an mAb to the human vitronectin receptor demonstrated an antigen of -37 kDa present in the cell-wall preparations of C. albicans and in spent growth medium. All four antibodies reacted with authentic ADH. The possible significance of these results in relation to C. albicans adherence is discussed.

Alcohol Dehydrogenase↗
PubMed2004

Fibronectin matrix assembly regulates alpha5beta1-mediated cell cohesion.

Integrin-extracellular matrix (ECM) interactions in two-dimensional (2D) culture systems are widely studied (Goldstein and DiMilla, 2002. J Biomed. Mater. Res. 59, 665-675; Koo et al., 2002. J. Cell Sci. 115, 1423-1433). Less understood is the role of the ECM in promoting intercellular cohesion in three-dimensional (3D) environments. We have demonstrated that the alpha5beta1-integrin mediates strong intercellular cohesion of 3D cellular aggregates (Robinson et al., 2003. J. Cell Sci. 116, 377-386). To further investigate the mechanism of alpha5beta1-mediated cohesivity, we used a series of chimeric alpha5beta1-integrin-expressing cells cultured as multilayer cellular aggregates. In these cell lines, the alpha5 subunit cytoplasmic domain distal to the GFFKR sequence was truncated, replaced with that of the integrin alpha4, the integrin alpha2, or maintained intact. Using these cells, alpha5beta1-integrin-mediated cell aggregation, compaction and cohesion were determined and correlated with FN matrix assembly. The data presented demonstrate that cells cultured in the absence of external mechanical support can assemble a FN matrix that promotes integrin-mediated aggregate compaction and cohesion. Further, inhibition of FN matrix assembly blocks the intercellular associations required for compaction, resulting in cell dispersal. These results demonstrate that FN matrix assembly contributes significantly to tissue cohesion and represents an alternative mechanism for regulating tissue architecture.

Animals↗
PubMed2004

Characterization of plasminogen as an adhesive ligand for integrins alphaMbeta2 (Mac-1) and alpha5beta1 (VLA-5).

Plasminogen (Pg) has been implicated in many biologic processes involving extracellular proteolysis. We investigated whether Pg, by virtue of its capacity to be deposited within the extracellular matrix, can serve as a ligand for cell surface integrins. We report here that Pg supports cell adhesion by engaging integrins alphaMbeta2 and alpha5beta1. The immobilized Glu-Pg, but not its derivatives with the N-terminal peptide lacking, plasmin and Lys-Pg, supported efficient adhesion that was abolished by anti-alphaMbeta2 and anti-alpha5beta1 integrin-specific monoclonal antibodies (mAbs). In addition, lysine binding sites of Glu-Pg contributed to cell adhesion inasmuch as tranexamic acid and epsilon-aminocaproic acid inhibited cell adhesion. The involvement of alphaMbeta2 and alpha5)beta1 in adhesion to Glu-Pg was demonstrable with blood neutrophils, U937 monocytoid cells, and genetically engineered alphaMbeta2-transfected human embryonic kidney (HEK) 293 cells. In alphaMbeta2, the alphaMI-domain is the binding site for Glu-Pg because the "I-less" form of alphaMbeta2 did not support cell adhesion and the recombinant alphaMI-domain bound Glu-Pg directly. In comparison with cell adhesion, the binding of soluble Glu-Pg to cells and the concomitant generation of plasmin activity was inhibited by anti-alpha5beta1 but not by anti-alphaMbeta2. These findings identify Glu-Pg as an adhesive ligand for integrins alphaMbeta2 and alpha5beta1 and suggest that alpha5beta1 may participate in the binding of soluble Glu-Pg and assist in its activation.

Amino Acid Sequence↗
PubMed2001

A signaling pathway from the alpha5beta1 and alpha(v)beta3 integrins that elevates bcl-2 transcription.

Integrin-mediated cell adhesion is necessary for the survival of many cell types, and loss of adhesion causes apoptosis. We have previously shown that the alpha5beta1 integrin supports cell survival on fibronectin and increases Bcl-2 protein expression. Here we show that bcl-2 transcription is elevated in cells that attach to fibronectin through alpha(v)beta1 or to vitronectin through alpha(v)beta3 but is not elevated in cells attaching through the alpha(v)beta1 integrin. Bcl-2 protein expression and protection from apoptosis under serum-free conditions correlated with bcl-2 transcription. This integrin-mediated regulation of bcl-2 is Shc- and FAK-dependent, and activation of Ras by FAK is required. Furthermore, Ras mediates this up-regulation of bcl-2 by activating the phosphatidylinositol 3-kinase-AKT pathway. Mitogen-activated protein kinase did not appear to be necessary for the activation of bcl-2 transcription. Therefore, our work characterizes the pathway that mediates the effect of integrins on bcl-2 transcription and cell survival.

Animals↗
PubMed2006

The effect of heparin-functionalized PEG hydrogels on three-dimensional human mesenchymal stem cell osteogenic differentiation.

Poly(ethylene glycol) (PEG) hydrogels functionalized with heparin were utilized as a three-dimensional culture system for human mesenchymal stem cells (hMSCs). Heparin-functionalized hydrogels supported hMSC viability, as quantified through live/dead imaging, and induced osteogenic differentiation, as measured by increased alkaline phosphatase (ALP) production and osteopontin (OPN) and collagen I (COL I) gene expression over the 5-week study. Further exploration of the potential mechanism of heparin-induced osteogenic differentiation was performed. Specifically, the availability of bone morphogenetic protein 2 (BMP2) and fibronectin (FN) in the culture system was controlled and hMSC osteogenic differentiation was evaluated as a function of the microenvironment. BMP2 availability increased both ALP production and OPN gene expression, while FN increased ALP production, but not OPN gene expression. Furthermore, immunostaining of integrin expression revealed that viability and differentiation were differentially affected by integrin production, where both alpha5beta1 and alphavbeta3 integrin-ligand interactions supported viability, while only the alpha5beta1 integrin played a role in hMSC osteogenic differentiation.

Alkaline Phosphatase↗
PubMed2003

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

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

Animals↗
PubMed2003

Membrane type-1 matrix metalloproteinase (MT1-MMP) processing of pro-alphav integrin regulates cross-talk between alphavbeta3 and alpha2beta1 integrins in breast carcinoma cells.

We have recently demonstrated that in breast carcinoma MCF7 cells MT1-MMP processes the alphav, alpha3, and alpha5 integrin precursors generating the respective mature S-S-linked heavy and light alpha-chains. The precursor of alpha2 integrin subunit was found resistant to MT1-MMP proteolysis. The processing of the alphav subunit by MT1-MMP facilitated alphavbeta3-dependent adhesion, activation of FAK signaling pathway, and migration of MCF7 cells on vitronectin. To elucidate further the effects of MT1-MMP on cellular integrins, we examined the functional activity of alpha5beta1 and alpha2beta1 integrins in MCF7 cells expressing MT1-MMP. Either expression of MT1-MMP alone or its coexpression with alphavbeta3 failed to affect the functionality of alpha5beta1 integrin, and adhesion of cells to fibronectin. MT1-MMP, however, profoundly affected the cross-talk involving alphavbeta3 and alpha2beta1 integrins. In MT1-MMP-deficient cells, integrin alphavbeta3 suppressed the functional activity of the collagen-binding alpha2beta1 integrin receptor and diminished cell adhesion to type I collagen. Coexpression of MT1-MMP with integrin alphavbeta3 restored the functionality of alpha2beta1 integrin and, consequently, the ability of MCF7 cells to adhere efficiently to collagen. We conclude that the MT1-MMP-controlled cross-talk between alphavbeta3 and alpha2beta1 integrins supports binding of aggressive, MT1-MMP-, and alphavbeta3 integrin-expressing malignant cells on type I collagen, the most common substratum of the extracellular matrix.

Breast Neoplasms↗
PubMed2003

The tissue engineeting puzzle: a molecular perspective.

The inability of biomaterial scaffolds to functionally integrate into surrounding tissue is one of the major roadblocks to developing new biomaterials and tissue-engineering scaffolds. Despite considerable advances, current approaches to engineering cell-surface interactions fall short in mimicking the complexity of signals through which surrounding tissue regulates cell behavior. Cells adhere and interact with their extracellular environment via integrins, and their ability to activate associated downstream signaling pathways depends on the character of adhesion complexes formed between cells and their extracellular matrix. In particular, alpha5beta1 and alphavbeta3 integrins are central to regulating downstream events, including cell survival and cell-cycle progression. In contrast to previous findings that alphavbeta3 integrins promote angiogenesis, recent evidence argues that alphavbeta3 integrins may act as negative regulators of proangiogenic integrins such as alpha5beta1. This suggests that fibronectin is critical for scaffold vascularization because it is the only mammalian adhesion protein that binds and activates alpha5beta1 integrins. Cells are furthermore capable of stretching fibronectin matrices such that the protein partially unfolds, and recent computational simulations provide structural models of how mechanical stretching affects fibronectin function. We propose a model whereby excessive tension generated by cells in contact to biomaterials may in fact render fibronectin fibrils nonangiogenic and potentially inhibit vascularization. The model could explain why current biomaterials independent of their surface chemistries and textures fail to vascularize.

Animals↗
PubMed1999

Attachment and chemotaxis of melanocytes after ultraviolet irradiation in vitro.

Because ultraviolet (UV) radiation is able to influence the spatial distribution of melanocytes in melanocytic naevi in vivo, we investigated the influence of UV radiation on the ability of melanocytes to adhere to the extracellular matrix proteins fibronectin, laminin and collagen type IV in vitro. In addition, chemotaxis of melanocytes was studied using both fibronectin and the supernatants from irradiated, as well as non-irradiated, keratinocytes and fibroblasts as attractants. Melanocyte attachment to fibronectin was significantly increased 48 h after a single UV irradiation at 30 mJ/cm2 in comparison with that of non-irradiated melanocytes, whereas attachment to laminin and collagen type IV showed only minor changes after UV exposure. The UV-induced increase in attachment to fibronectin was suppressed by preincubation with antibodies against alpha5beta1 or alphavbeta3 integrin. Both immunohistochemistry and flow cytometric analysis showed an increase in alpha5beta1 integrin expression on melanocytes after UV exposure. The chemotaxis of melanocytes to fibronectin was not influenced by UV exposure. A decreasing migration rate of melanocytes towards the supernatants of UVA-irradiated fibroblasts was observed with increasing UVA doses. The chemotactic effects of conditioned medium of keratinocytes towards melanocytes was not influenced either by UVB or by UVA. The results indicate that UV radiation may alter the ability of melanocytes to adhere to certain substrates by modification of integrin expression. Because fibronectin, as the major target protein of UV-altered attachment, is located in the dermis, the UV-induced morphological changes in melanocytic lesions, with an increase in suprabasally located melanocytes within the epidermis, may be due to other changes in the adhesive properties of melanocytes.

Cell Adhesion↗
PubMed1999

Integrin-fibronectin interactions at the cell-material interface: initial integrin binding and signaling.

Integrin receptors mediate cell adhesion to extracellular matrices and provide signals that direct proliferation and differentiation. Integrin binding involves receptor-ligand interactions at the cell-substrate interface and assembly and reorganization of structural and signaling elements at the cytoplasmic face. Using a cross-linking/extraction/reversal method to quantify bound integrins, we demonstrate that the density of alpha5beta1 integrin-fibronectin bonds increases linearly with ligand density, as predicted by simple receptor-ligand equilibrium. This linear relationship is consistent with linear increases in cell adhesion strength with receptor and ligand surface densities. Furthermore, we show that phosphorylation of FAK, a tyrosine kinase involved in early integrin-mediated signaling, increases linearly with the number of integrin-Fn bonds. These linear relationships suggest the absence of cooperative effects in the initial stages of mechanical coupling and adhesion-mediated signaling.

Cell Adhesion↗
PubMed2003

Alpha4beta1 integrin/ligand interaction inhibits alpha5beta1-induced stress fibers and focal adhesions via down-regulation of RhoA and induces melanoma cell migration.

We have studied the function of the Hep III fibronectin domain in the cytoskeletal response initiated by alpha5beta1 integrin-mediated adhesion. Melanoma cells formed stress fibers and focal adhesions on the RGD-containing FNIII7-10 fragment. Coimmobilization of FNIII4-5, a fragment spanning Hep III and containing the alpha4beta1 ligand H2 with FNIII7-10, or addition of soluble FNIII4-5 to cells preattached to FNIII7-10, inhibited stress fibers and induced cytoplasmic protrusions. This effect involved alpha4beta1 since: 1) mutations in H2 reverted the inhibition; 2) other alpha4beta1 ligands (CS-1, VCAM-1), an anti-alpha4 mAb, or alpha4 expression in HeLa cells inhibited stress fibers. This activity was apparently cryptic in fibronectin or large fibronectin fragments, but exposed upon proteolytic degradation. Indeed purified peptic fragments containing H2, inhibited stress fibers when mixed with FNIII7-10 or fibronectin. RhoA activation with LPA or transfection with V14RhoA reverted the inhibitory effect and induced stress fibers on FNIII7-10+FNIII4-5. Furthermore, addition of alpha4beta1 ligands to FNIII7-10, down-regulated RhoA and activated p190RhoGAP, which localized to cytoplasmic protrusions. alpha4beta1/ligand interaction induced cell migration, monitored by video microscopy and wound healing assays. These data indicate that alpha4beta1 provides an antagonistic signal to alpha5beta1 by interfering with the RhoA activation pathway and this leads to melanoma cell migration.

Cell Adhesion↗
PubMed1998

TNF-alpha-induced matrix Fn disruption and decreased endothelial integrity are independent of Fn proteolysis.

Exposure of confluent pulmonary arterial endothelial monolayers to tumor necrosis factor (TNF)-alpha causes both a reorganization and/or disruption of fibronectin (Fn) in the extracellular matrix and an increase in transendothelial protein permeability. However, the factors initiating this response to TNF-alpha have not been defined. Because TNF-alpha can induce proteinase expression in endothelial cells, we determined whether proteinases cause both the alteration of the Fn matrix and the permeability increase as is often speculated. Incubation of calf pulmonary arterial endothelial monolayers with TNF-alpha (200 U/ml) for 18 h caused a disruption of the Fn matrix and an increase in transendothelial protein permeability. A reduced colocalization of cell-surface alpha5beta1-Fn integrins with the Fn fibers in focal contacts was also observed. TNF-alpha treatment of endothelial monolayers with matrices prelabeled with 125I-human Fn (hFn) did not cause the release of Fn fragments or alter the content of Fn antigen in the medium as analyzed by SDS-PAGE coupled with autoradiography. Both the content and fragmentation pattern of Fn within the cell layer and the insoluble Fn matrix also appeared unchanged after TNF-alpha exposure as confirmed by Western immunoblot. Fn-substrate zymography revealed that TNF-alpha increased the expression of two proteinases within the conditioned medium in which activity could be blocked by aprotinin but not by EDTA, 1,10-phenanthroline, leupeptin, or pepstatin. However, inhibition of the Fn proteolytic activity of these two serine proteinases did not prevent either the TNF-alpha-induced disruption of the Fn matrix or the increase in permeability. Thus the reorganization and/or disruption of the Fn matrix and the temporally associated increase in endothelial permeability caused by TNF-alpha appear not to be due to proteolytic degradation of Fn within the extracellular matrix. In contrast, decreased alpha5beta1-Fn integrin interaction with Fn fibers in the matrix may be important in the response to TNF-alpha exposure.

Animals↗
PubMed2004

Role of alphavbeta5 integrins and vitronectin in Pseudomonas aeruginosa PAK interaction with A549 respiratory cells.

Bacterial adherence to mammalian cells and their internalization are thought to participate in Pseudomonas aeruginosa pathogenicity. In this study, we explored the role of alpha5beta1 and alphavbeta5 integrins and their natural ligands, fibronectin (Fn) and vitronectin (Vn), in P. aeruginosa interaction with epithelial cells by using the PAK reference bacterial strain, A549 respiratory, and SKOV-3 human ovarian cell lines. The host cell cytoskeleton and cellular tyrosine kinases seem to be solicited during the PAK-respiratory cell interaction: cytochalasin D and genistein decreased the bacterial adherence and internalization. Blocking antibodies to alphavbeta5 integrins were the only antibodies tested to have inhibitory activity against PAK adherence to A549 cells. PAK internalization by A549 and SKOV-3 cells was markedly decreased in the presence of blocking antibodies to Vn and alphavbeta5 integrins. Addition of Vn in excess restored PAK invasion of both A549 and SKOV-3 cells in the presence of anti-Vn antibodies. Immunofluorescence experiments revealed that, in the presence of bacteria, the Vn fibrillar network disappeared, and alphavbeta5 staining was concentrated in sites where adherent bacteria were present. Taken together, these findings suggest that alphavbeta5 integrins, and their natural ligand Vn, are involved in PAK entry into human epithelial cells.

Antibodies, Blocking↗
PubMed2001

Transformation of chicken embryo fibroblasts by v-src uncouples beta1 integrin-mediated outside-in but not inside-out signaling.

Adhesion of cells to extracellular matrix is mediated by integrin family receptors. The process of receptor-ligand binding is dependent on metabolic energy and is regulated by intracellular signals, termed inside-out signals. The strength of the initial alpha5beta1-mediated adhesion of v-src-transformed chicken embryo fibroblasts (v-srcCEF) was similar to that of normal CEF. A chemically cross-linked fibronectin substrate was able to restore cell spreading and the ability of v-srcCEF to assemble a fibronectin matrix. Over time, v-srcCEF showed decreased adhesion due to the reduction of alpha5beta1-fibronectin bonds consequent on the reduction of substrate-bound fibronectin due to the secretion of proteases by v-srcCEF. Excess synthesis of hyaluronic acid by v-srcCEF also reduced the alpha5beta1-fibronectin bonds and contributed to cell detachment at later times in culture. Thus, the adhesion defects were not due to a failure of alpha5beta1 function and adhesion of the v-srcCEF was alpha5beta1 dependent. Integrin-mediated adhesion also produces signals that affect cell proliferation and cell differentiation. An early consequence of these "outside-in" signals was the phosphorylation of FAK Y397 in direct proportion to the number of alpha5beta1-fibronectin bonds formed. In contrast, v-srcCEF had an increased level of phosphorylation on five different tyrosines in FAK, and none of these phosphorylation levels were sensitive to the number of alpha5beta1-fibronectin bonds. In the absence of serum, CEF proliferation was sensitive to changes in alpha5beta1-mediated adhesion levels. Transformation by v-src increased the serum-free proliferation rate and made it insensitive to alpha5beta1-mediated adhesion. Thus, the v-srcCEF were insensitive to the normal outside-in signals from alpha5beta1 integrin.

Animals↗