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Results for “Integrin alpha2beta1”

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PubMed1996

The alpha2beta1 integrin is a necessary co-receptor for collagen-induced activation of Syk and the subsequent phosphorylation of phospholipase Cgamma2 in platelets.

Although there are multiple potential collagen-binding proteins on platelets, the contribution of each to collagen-induced signaling events and platelet activation is unclear. We investigated which early platelet signaling events, if any, could be attributed specifically to the binding of collagen to one of its receptors, the alpha2beta1 integrin. Treatment of platelets with collagen induced a rapid activation of the non-receptor tyrosine kinase, Syk, as measured by an increase in phosphorylation and kinase activity. Collagen also induced the rapid phosphorylation of phospholipase Cgamma2 (PLCgamma2). The phosphorylation of both Syk and PLCgamma2, as well as platelet aggregation, was blocked by an anti-alpha2beta1 integrin monoclonal antibody (P1E6), demonstrating that collagen binding to alpha2beta1 is necessary for signaling. Cross-linking of the alpha2beta1 integrin with stimulatory monoclonal antibody against either the beta1 or alpha2 subunit stimulated the phosphorylation of both Syk and PLCgamma2. However, antibody stimulation was dependent on co-stimulation of the FcgammaII receptor (CD32) since specific F(ab')2 fragments did not induce Syk and PLCgamma2 phosphorylation. Thus, these results suggest that occupancy of alpha2beta1 by collagen is necessary, but that a co-receptor, in addition to alpha2beta1, is required for these collagen-induced signaling events. Moreover, the P1E6 antibody did not inhibit all collagen-induced tyrosine phosphorylation events, demonstrating that collagen also induces phosphorylation events that are independent of the alpha2beta1 integrin. In addition to Syk and PLCgamma2, we identified the FcgammaII receptor (FcgammaRII) as being rapidly phosphorylated in response to collagen stimulation, even in the absence of antibodies. Finally, to determine if Syk activation precedes and directly contributes to the phosphorylation of PLCgamma2, platelets were preincubated with the Syk-selective kinase inhibitor, piceatannol. A concentration of piceatannol that inhibited the phosphorylation and kinase activity of Syk, but had no effect on Src kinase activity, blocked the collagen-induced phosphorylation of PLCgamma2 and also inhibited collagen-induced platelet aggregation. Our results begin to delineate a signaling pathway whereby occupancy of the alpha2beta1 integrin is required, but not sufficient, for collagen-induced activation of Syk and the subsequent phosphorylation of PLCgamma2. These events are necessary for platelet activation and aggregation in response to collagen.

Antibodies↗

PubMed2006

Integrin alpha2beta1 regulates neutrophil recruitment and inflammatory activity in experimental colitis in mice.

BACKGROUND: Human inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis), is associated with leukocyte accumulation in the inflamed intestinal tissue. Recent studies strongly suggest a role of beta1 integrin receptors in regulating tissue damage and disease symptoms related to inflammatory bowel disease. The aim of this study was to investigate the role of the collagen-binding alpha2beta1 integrin (CD49b/CD29) in dextran sodium sulfate-induced colitis in mice. METHODS: Colitis was induced in mice through oral administration of 2% dextran sodium sulfate in drinking water. Rectal administration of anti-alpha2-monoclonal antibody (mAb) in 1 group was compared with oral treatment with betamethasone in another group and rectal administration of a control antibody in a third group. Clinical and histological signs of colitis, neutrophil infiltration into the colon mucosa, and gene expression of metalloproteinases were assessed. RESULTS: Rectal administration of anti-alpha2-mAb was found to significantly reduce weight loss from 13.5% +/- 6.5% to 2.2% +/- 0.2% (P = 0.013 versus control mAb) and mucosal neutrophil infiltration from 47.2 +/- 10.0 to 6.6 +/- 8.0 neutrophils per counted area (P < 0.05 versus control mAb). Metalloproteinase gene expression was suppressed through anti-alpha2-mAb treatment. The protective effect against colitis seen after anti-alpha2beta1 integrin treatment was found to be favorable to the effect seen after high-dose oral betamethasone. CONCLUSIONS: We demonstrate an alleviating action of the collagen-binding alpha2beta1 integrin in experimental colitis in mice and suggest that this effect is mediated by inhibition of neutrophil migration and activation. Local administration of function-blocking antibodies against integrin alpha2beta1 may provide novel avenues to treat inflammatory bowel disease.

Analysis of Variance↗

PubMed2004

Differential regulation of actin stress fiber assembly and proplatelet formation by alpha2beta1 integrin and GPVI in human megakaryocytes.

The actin cytoskeleton plays a major role in platelet function. In contrast, its precise role in the function of megakaryocytes (MKs) is less understood but may be important for a chemoattractive response and an efficient proplatelet formation. In the marrow microenvironment, mature MKs are in contact with the extracellular matrix, including fibrillar collagen type I. MKs express alpha2beta1 integrin and the immunoglobulin superfamily member glycoprotein VI (GPVI), the main receptors for collagen. Using function-blocking antibodies or specific ligands, we investigated in primary human MKs how alpha2beta1 integrin and GPVI regulate stress fiber formation, the primary actin structures needed for cell contraction. Stress fiber assembly requires synergistic activation of the MAPK/Erk1/2 pathway and the small guanosine triphosphatase Rho via its effector, Rho-associated coiled-coil kinase (ROCK). alpha2beta1 integrin is crucial for stress fiber formation, whereas GPVI triggers rapid and sustained activation of the Erk1/2 pathway. Strikingly, after a longer adhesion time, proplatelet formation was significantly inhibited by the engagement of alpha2beta1 integrin, not by GPVI, likely through the Rho/ROCK pathway. Thus, proplatelet formation in human MKs could be tightly regulated by differential interactions with their collagen receptors. We propose that this interaction with collagen prevents proplatelet formation within the marrow.

Blood Platelets↗

PubMed2006

Stable knockdown of polycystin-1 confers integrin-alpha2beta1-mediated anoikis resistance.

The mechanisms of action of polycystin-1 (PC1) have been difficult to dissect because of its interaction with multiple factors, the heterogeneity of the genetic mutations, and the complexity of the experimental animal models. Here, stable knockdown of PC1 in MDCK epithelial cells was achieved by lentiviral-mediated delivery of a specific small interfering RNA for PKD1. The reduction of PC1 expression prevented tubulogenesis in three-dimensional collagen type I culture in response to hepatocyte growth factor and induced formation of cysts. PC1 knockdown created a condition of haploinsufficiency that led to hyperproliferation, increased adhesion to collagen type I, and increased apoptosis. It was shown that the suppression of PC1 was associated with the increased expression of integrin-alpha2beta1 and reduced apoptosis in cells grown on collagen type I. The engagement of integrin-alpha2beta1 seemed to be essential for the survival because PC1 knockdown cells were significantly less susceptible to anoikis by a mechanism that was reversible by anti-integrin-alpha2beta1 blocking antibodies. Overall, these data link integrin-alpha2beta1 to some of the biologic functions that are ascribed to PC1 and establish the potential of this approach for the direct study of PC1 functions in a genetically defined background. Furthermore, these findings indicate that reduction of PC1 expression levels, rather than the loss of heterozygosity, may be sufficient to induce cystogenesis.

Animals↗

PubMed1997

Adhesion and activation of human platelets induced by convulxin involve glycoprotein VI and integrin alpha2beta1.

We analyzed the interaction of convulxin (Cvx), a 72-kDa protein isolated from the venom of Crotalus durissus terrificus, with human platelets. Cvx is a potent platelet agonist that induces an increase in the intracellular Ca2+ concentration ([Ca2+]i), granule exocytosis and aggregation. 125I-Labeled Cvx binds specifically and rapidly to platelets at binding sites of high and moderate affinity. Platelets adhere to immobilized Cvx in a time-dependent but cation-independent manner. Platelet exocytosis and aggregation induced by Cvx were inhibited by an anti-integrin alpha2beta1 monoclonal antibody (6F1) and by the Fab fragments of a polyclonal anti-glycoprotein VI (GPVI) antibody. Both the adhesion of platelets to Cvx and the Cvx-induced increase in [Ca2+]i were inhibited by anti-GPVI Fab fragments but not by 6F1. Ligand blotting assay showed that 125I-Cvx binds to a 57-kDa platelet protein with an electrophoretic mobility identical to that of GPVI. In addition, we observed the following: (i) 125I-Cvx binds to GPVI immunoprecipitated by the anti-GPVI antibody from a platelet lysate, and (ii) Cvx inhibits the binding of anti-GPVI IgG to GPVI. Taken together, these results demonstrate that GPVI behaves as a Cvx receptor and that the alpha2beta1 integrin appears to be involved in the later stages of Cvx-induced platelet activation, i.e. exocytosis and aggregation.

Antibodies, Monoclonal↗

PubMed2002

Production of soluble integrin alpha2beta1 heterodimer complex functionally active in vitro and in vivo.

Integrin alpha2beta1, which is a membrane protein consisting of noncovalently bound alpha2 and beta1 chains, mediates cell binding to collagen and plays a role in platelet functions. DNAs encoding the chimeric proteins in which the extracellular domains of each alpha2 and beta1 chain was fused to hinge and Fc regions of human IgG(1)gamma chain were cotransfected into CHO cells. Soluble integrin alpha2beta1 (salpha2beta1) in which alpha2 and beta1 chains were covalently bound by disulfide bonds was recovered from the culture supernatant. salpha2beta1 maintained functional characteristics of cell surface alpha2beta1 as indicated by cation-dependent binding to collagen and conformational changes induced by cations or ligand. Intravenously administered salpha2beta1 in rats colocalized with collagen in inflamed microvessels. Moreover, salpha2beta1-conjugated liposome administered intravenously reduced bleeding time of the thrombocytopenic mice. These results indicated that salpha2beta1 has pharmaceutical utilities as an agent for detecting injured vessels and a component of platelet substitute.

Animals↗

PubMed1999

Activation of protein kinase C by phorbol esters modulates alpha2beta1 integrin on MCF-7 breast cancer cells.

Cellular adhesions to other cells and to the extracellular matrix play crucial roles in the malignant progression of cancer. In this study, we investigated the role of protein kinase C (PKC) in the regulation of cell-substratum adhesion by the breast adenocarcinoma cell line MCF-7. A PKC activator, 12-O-tetradecanoylphorbol-l, 3-acetate (TPA), stimulated cell adhesion to laminin and collagen I in a dose-dependent manner over a 1- to 4-h interval. This enhanced adhesion was mediated by alpha2beta1 integrin, since both anti-alpha2 and anti-beta1 blocking antibodies each completely abrogated the TPA-induced adhesion. FACS analysis determined that TPA treatment does not change the cell surface expression of alpha2beta1 integrin over a 4-h time interval. However, alpha2beta1 levels were increased after 24 h of TPA treatment. Thus, the enhanced avidity of alpha2beta1-dependent cellular adhesion preceded the induction of alpha2beta1 cell surface expression. Northern blot analysis revealed that mRNA levels of both alpha2 and beta1 subunits were increased after exposure to TPA for 4 h, indicating that the induction of alpha2beta1 mRNA preceded that of its cell surface expression. This further suggested that the TPA-induced avidity of alpha2beta1 was independent of increased expression of alpha2beta1. Pretreatment of cells with the PKC inhibitor calphostin C partially antagonized the TPA-induced increase in expression of alpha2beta1 integrin expression and of alpha2beta1-mediated cellular adhesion. To identify a possible mechanism by which TPA could be acting to promote the rapid induction of alpha2beta1 adhesion, we treated the cells with the Rho-GTPase inhibitor Clostridium botulinumexotoxin C3. C3 inhibited TPA-induced adhesion to laminin and collagen I in a dose-dependant manner, suggesting a likely role for Rho in TPA-induced adhesion. Together, these results suggest that PKC can modulate the alpha2beta1-dependent adhesion of MCF-7 cells by two distinct mechanisms: altering the gene expression of integrins alpha2 and beta1 and altering the avidity of the alpha2beta1 integrin by a Rho-dependant mechanism.

ADP Ribose Transferases↗

PubMed2003

Alpha2beta1-integrin signaling by itself controls G1/S transition in a human adenocarcinoma cell line (Caco-2): implication of NADPH oxidase-dependent production of ROS.

In this work, we report that type IV collagen, mainly via alpha2beta1-integrin ligation, was able to induce cyclin expression and G1/S transition in a colic adenocarcinoma cell line (Caco-2) cultured without soluble growth factors or fetal bovine serum. This process involved Erk 1/2 activation and the production of reactive oxygen species (ROS) by a membrane-bound NADPH oxidase. Data presented here show that NADPH oxidase-dependent production of ROS increased following alpha2beta1-integrin ligation with type IV collagen or with a specific monoclonal antibody (Gi9 mAb). NADPH oxidase activation and, therefore, the production of ROS were shown to be involved in the increase of alpha2beta1-integrin plasma membrane expression, p38 MAPK phosphorylation, cyclin expression, and G1/S transition. We thus identified in this work a new integrin-signaling pathway in colon tumor cells involved in cell cycle regulation by the extracellular matrix.

Adenocarcinoma↗

PubMed2003

Butyrate downregulates alpha2beta1 integrin: a possible role in the induction of apoptosis in colorectal cancer cell lines.

BACKGROUND: Integrins mediate cell matrix adhesion and regulate cell growth and survival. In colonic epithelial cells, alpha(2)beta(1) integrin controls glandular differentiation and proliferation. Butyrate stimulates differentiation and induces apoptosis in vitro. AIMS: We investigated whether butyrate induction of apoptosis was associated with perturbation of integrin mediated cell matrix adhesion. METHODS: Three colonic cancer cell lines (SW1222, SW620, LS174T) were studied. Adhesion to extracellular matrix proteins, expression of alpha(2)beta(1) integrin, and apoptosis were studied in adherent cells after treatment with 4 mM butyrate. RESULTS: Butyrate decreased the attachment to type I collagen in SW620 cells and type I and IV collagen in LS174T cells. The decreased cell attachment was associated with downregulation of alpha(2)beta(1) integrin and increased apoptosis in adherent cells. No changes in alpha(2)beta(1) expression or matrix adhesion were seen in SW1222 cells, which were also found to be less sensitive to butyrate induction of apoptosis. Downregulation of alpha(2)beta(1) integrin preceded the detection of apoptosis. CONCLUSION: Apoptosis induced by butyrate is associated with downregulation of expression and functional activity of alpha(2)beta(1) integrin. Perturbation of cell matrix adhesion may be a novel mechanism by which butyrate induces apoptosis in colorectal cancer cells.

Apoptosis↗

PubMed2003

Collagen IV regulates Caco-2 migration and ERK activation via alpha1beta1- and alpha2beta1-integrin-dependent Src kinase activation.

Our previous work indicates intestinal epithelial cell ERK activation by collagen IV, a major component of the intestinal epithelial basement membrane, requires focal adhesion kinase (FAK) and suggests FAK and ERK may have important roles in regulating intestinal epithelial cell migration. We therefore sought to identify FAK downstream targets regulating intestinal epithelial cell spreading, migration, and ERK activation on collagen IV and the integrins involved. Both dominant-negative Src and Src inhibitor PP2 strongly inhibited collagen IV ERK activation in Caco-2 intestinal epithelial cells. Collagen IV stimulated Grb2 binding site FAK Y925 phosphorylation, which was inhibited by PP2 and required FAK Y397 autophosphorylation. Additionally, FAK Y925F expression blocked collagen IV ERK activation. alpha(1)beta(1)- Or alpha(2)beta(1)-integrin blockade with alpha(1)- or alpha(2)-integrin subunit antibodies indicated that either integrin can mediate adhesion, cell spreading, and FAK, Src, and ERK activation on collagen IV. Both dominant-negative Src and PP2 inhibited Caco-2 spreading on collagen IV. PP2 inhibited p130(Cas) tyrosine phosphorylation, but dominant-negative p130(Cas) did not inhibit cell spreading. PP2 inhibited Caco-2 migration on collagen IV much more strongly than the mitogen-activated protein kinase kinase inhibitor PD-98059, which completely inhibited collagen IV ERK activation. These results suggest a pathway for collagen IV ERK activation requiring Src phosphorylation of FAK Y925 not previously described for this matrix protein and suggest either alpha(1)beta(1)- or alpha(2)beta(1)-integrins can regulate Caco-2 spreading and ERK activation on collagen IV via Src. Additionally, these results suggest Src regulates Caco-2 migration on collagen IV primarily through ERK-independent pathways.

Antibodies, Blocking↗

PubMed2001

Platelet collagen receptor integrin alpha2beta1 activation involves differential participation of ADP-receptor subtypes P2Y1 and P2Y12 but not intracellular calcium change.

In agonist-induced platelet activation, the collagen platelet receptor integrin alpha2beta1 is activated to high-affinity states through ADP involvement [Jung, S.M. & Moroi, M. (2000) J. Biol. Chem. 275, 8016-8026]. Here we determined the ADP-receptor subtypes involved and their relative contributions to alpha2beta1 activation (assessed by soluble-collagen binding) using the P2Y12 antagonist AR-C69931MX and P2Y1 antagonists adenosine 3',5'-diphosphate (Ado(3,5)PP) and adenosine 3'-phosphate 5'-phosphosulfate (AdoPPS). All three inhibited alpha2beta1 activation induced by low or high ADP, low thrombin, or low collagen-related peptide (CRP) concentrations; however, AR-C69931MX was markedly more inhibitory than the P2Y1 antagonists, suggesting the greater contribution of P2Y12. Inhibition patterns by various combinations of AR-C69931MX, AdoPPS, and wortmannin suggested that P2Y1 and P2Y12 mediate alpha2beta1 activation through different pathways, with possible involvement of phosphoinositide 3-kinase in both. Low concentrations of the acetoxy-methyl derivative of 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetra-acetic acid (calcium chelator) markedly decreased alpha2beta1 activation by low thrombin or CRP, but did not affect that by low or high ADP. Measurements of intracellular Ca2+ level (fluorimetric method) and alpha2beta1 activation (soluble-collagen binding) in the same platelet preparation indicated that alpha2beta1 activation via ADP receptors was independent of intracellular Ca2+ release. Our data indicate that integrin alpha2beta1 activation by ADP occurs through an inside-out signaling mechanism involving differential contributions by P2Y1 and P2Y12 wherein each contributes to some portion of the activation, with the stronger contribution of P2Y12. Furthermore, intracellular Ca2+ increase is not directly related to integrin alpha2beta1 activation, meaning that it is separate from the calcium mobilization pathways that these two ADP receptors are involved in.

Adenosine Diphosphate↗

PubMed2004

Type I collagen synergistically enhances PDGF-induced smooth muscle cell proliferation through pp60src-dependent crosstalk between the alpha2beta1 integrin and PDGFbeta receptor.

Smooth muscle cells (SMCs) are exposed to both platelet-derived growth factor (PDGF) and type I collagen (CNI) at the time of arterial injury. In these studies we explore the individual and combined effects of these agonists on human saphenous vein SMC proliferation. PDGF-BB produced a 5.5-fold increase in SMC DNA synthesis whereas CNI stimulated DNA synthesis to a much lesser extent (1.6-fold increase). Alternatively, we observed an 8.3-fold increase in DNA synthesis when SMCs were co-incubated with CNI and PDGF-BB. Furthermore, stimulation of SMCs with PDGF-BB produced a significant increase in ERK-2 activity whereas CNI alone had no effect. Co-incubation of SMCs with PDGF-BB and CNI resulted in ERK-2 activity that was markedly greater than that produced by PDGF-BB alone. In a similar fashion, PDGF-BB induced phosphorylation of the PDGF receptor beta (PDGFRbeta) and CNI did not, whereas concurrent agonist stimulation produced a synergistic increase in receptor activity. Blocking antibodies to the alpha2 and beta1 subunits eliminated this synergistic interaction, implicating the alpha2beta1 integrin as the mediator of this effect. Immunoprecipitation of the alpha2beta1 integrin in unstimulated SMCs followed by immunoblotting for the PDGFRbeta as well as Src family members, pp60(src), Fyn, Lyn, and Yes demonstrated coassociation of alpha2beta1 and the PDGFRbeta as well as pp60(src). Incubation of cells with CNI and/or PDGF-BB did not change the degree of association. Finally, inhibition of Src activity with SU6656 eliminated the synergistic effect of CNI on PDGF-induced PDGFRbeta phosphorylation suggesting an important role for pp60(src) in the observed receptor crosstalk. Together, these data demonstrate that CNI synergistically enhances PDGF-induced SMC proliferation through Src-dependent crosstalk between the alpha2beta1 integrin and the PDGFRbeta.

Animals↗

PubMed2003

Statins regulate alpha2beta1-integrin expression and collagen I-dependent functions in human vascular smooth muscle cells.

HMG-CoA reductase inhibitors have direct vascular effects that contribute to plaque stability. In the current study, the authors demonstrate that the HMG-CoA reductase inhibitors atorvastatin and pravastatin augment the adhesion of human (HSMCs) and rat aortic smooth muscle cells (RASMCs) to collagen I via induction of alpha2beta1-integrin receptors. Atorvastatin (0.1 microM ) increased the adhesion of HSMCs to collagen I up to 2-fold (p < 0.01) and pravastatin (1.0 microM ) up to 1.8-fold (p < 0.01) after treatment of at least 24 h. This increase in adhesion was concentration dependent and was observed for treatment periods from 16 to 72 h. Inhibition of isoprenoid synthesis with mevalonate and geranyl-geraniol prevented the statin-induced effect on human and rat smooth muscle cells. Flow cytometry revealed an increased expression of alpha2- and beta1-integrins after treatment with atorvastatin (0.1 microM ) at 24 and 48 h. Atorvastatin increased levels of beta1-integrin mRNA after 12- and 24-h treatment in HSMCs, which was inhibited by mevalonate. Furthermore, atorvastatin (0.1 microM ) and pravastatin (1.0 microM ) inhibited chemotaxis of HSMCs on collagen I, which was also reversed by mevalonate treatment. In contrast, inhibition of beta1-integrins with a specific antibody nearly doubled (p < 0.01) the rate of chemotaxis. These data indicate that the chemotactic activity in HSMCs is inhibited in part by up-regulation of alpha2beta1-integrin receptors. The current study indicates that HMG-CoA reductase inhibitors increase cell-matrix interaction with collagen I via induction of alpha2beta1-integrins and increased adhesion to collagen I.

Animals↗

PubMed2003

Epidermal growth factor increased the expression of alpha2beta1-integrin and modulated integrin-mediated signaling in human cervical adenocarcinoma cells.

Epidermal growth factor (EGF) receptor (EGFR) is involved in various basic biochemical pathways and is thus thought to play an important role in cell migration. We examined the effect of EGF on motility, migration, and morphology of a human adenocarcinoma cell line CAC-1. EGF treatment increased the motility of cervical adenocarcinoma cells and promoted migration of the cells on fibronectin and type IV collagen. EGF induced morphological changes with lamellipodia during EGFR-mediated motility. The results of an immunoprecipitation study showed that EGF up-regulated the expression of alpha2beta1-integrin in a dose-dependent manner. EGF-induced cell migration was blocked by alpha2beta1-integrin antibody. Our results also showed that EGF treatment stimulated the level of tyrosine dephosphorylation of FAK, which is required for EGF-induced changes in motility, migration, and cell morphology. A tyrosine kinase inhibitor (ZD1839) blocked EGF-induced changes in cervical adenocarcinoma cells. The results suggest that EGF promotes cell motility and migration and increases the expression of alpha2beta1-integrin, possibly by decreasing FAK phosphorylation.

Adenocarcinoma↗

PubMed2000

Integrins alpha2beta1 and alpha4beta1 can mediate SA11 rotavirus attachment and entry into cells.

Most mammalian rotaviruses contain tripeptide amino acid sequences in outer capsid proteins VP4 and VP7 which have been shown to act as ligands for integrins alpha2beta1 and alpha4beta1. Peptides containing these sequences and monoclonal antibodies directed to these integrins block rotavirus infection of cells. Here we report that SA11 rotavirus binding to and infection of K562 cells expressing alpha2beta1 or alpha4beta1 integrins via transfection is increased over virus binding to and infection of cells transfected with alpha3 integrin or parent cells. The increased binding and growth were specifically blocked by a monoclonal antibody to the transfected integrin subunit but not by irrelevant antibodies. In our experiments, integrin activation with phorbol ester did not affect virus binding to cells. However, phorbol ester treatment of K562 parent and transfected cells induced endogenous gene expression of alpha2beta1 integrin, which was detectable by flow cytometry 16 h after treatment and quantitatively correlated with the increased level of SA11 virus growth observed after this time. Virus binding to K562 cells treated with phorbol ester 24 h previously and expressing alpha2beta1 was elevated over binding to control cells and was specifically blocked by the anti-alpha2 monoclonal antibody AK7. Virus growth in alpha4-transfected K562 cells which had also been induced to express alpha2beta1 integrin with phorbol ester occurred at a level approaching that in the permissive MA104 cell line. We therefore have demonstrated that two integrins, alpha2beta1 and alpha4beta1, are capable of acting as cellular receptors for SA11 rotavirus.

Antibodies, Monoclonal↗

PubMed2004

AP-1 regulates alpha2beta1 integrin expression by ERK-dependent signals during megakaryocytic differentiation of K562 cells.

Mitogen-activated protein kinases (MAPKs) have been implicated as regulators of cellular differentiation. The biological effect of MAPK signaling in the nucleus is achieved by signal-responsive transcription factors. Here, we have investigated the connection of MAPKs, transcription factor AP-1, and alpha2beta1 integrin expression in K562 cells undergoing differentiation along the megakaryocytic pathway. We report that three distinct MAPKs, ERK, JNK, and p38, are activated during the TPA-induced megakaryocytic differentiation. Activation of MAPK pathways is followed by acquisition of the AP-1 DNA-binding and transactivation capacities. AP-1 DNA-binding activity consists primarily of JunD, c-Fos, and Fra-1, and is accompanied with the increased expression and phosphorylation of these subunits. While inhibition of JNK mainly prevents expression and phosphorylation of JunD and c-Jun, inhibition of the ERK pathway suppresses both phosphorylation and expression of Jun proteins, and expression of c-Fos and Fra-1. Furthermore, only the activity of the ERK pathway is essential for the differentiation response, as determined by expression of alpha2beta1 (CD49b) integrin. The importance of AP-1 as a mediator ERK signaling during differentiation is demonstrated by the findings that expression of c-fos siRNA and dominant negative AP-1/c-Jun(bZIP) downregulate the TPA- and ERK-induced expression of alpha2beta1 integrin mRNAs and proteins. Conversely, coexpression of JunD or c-Jun and c-Fos can induce alpha2beta1 integrin expression independently of upstream signals. Taken together, the results show that AP-1 is a nuclear target of the ERK-pathway and mediates alpha2beta1 integrin expression during megakaryocytic differentiation of K562 cells.

Cell Differentiation↗

PubMed1999

Integrin alpha2beta1 recognizes laminin-2 and induces C-erb B2 tyrosine phosphorylation in metastatic human melanoma cells.

Previous studies have shown that tumor cells with metastatic propensity secrete more of the laminin alpha2 chain than non-metastatic tumor cells do, and that laminin-2, which contains the alpha2 chain, promotes cell adhesion better than laminin-1 (Jenq et al. (1994). Differentiation, 58, 29-36). The current studies were designed to determine whether a correlation exists between the expression of the laminin-2 isoform and the metastatic phenotype in melanoma cells. We found that expression of the laminin-2 isoform was upregulated in the metastatic melanoma cell lines tested. Cell attachment studies showed that metastatic melanoma cells attached more efficiently to laminin-2 substrates. Studies on integrin expression revealed that the presence of alpha2beta1 integrin correlated with expression of the laminin-2 isoform in metastatic melanoma cells; anti-integrin alpha2 antibody prevented cell attachment to laminin-2 substrates. The data suggest that the alpha2beta1 integrin is the receptor mediating cell attachment to the laminin-2 isoform. This interaction, mediated by the alpha2beta1 integrin, stimulates secretion of the 72 kD type IV collagenase and induces a specific 185 kD protein tyrosine phosphorylation. The 185 kD tyrosine-phosphorylated protein was identified as the p185/C-erb B2 oncoprotein by immunoprecipitation. These studies suggest that upregulation of expression of the laminin-2 chain correlates with the metastatic phenotype of melanoma cells and provides evidence that the specific p185/C-erb B2 tyrosine phosphorylation may be involved in integrin-mediated signaling during tumor cell invasion and metastasis.

Collagenases↗

PubMed2003

Leukotriene D4-induced adhesion of Caco-2 cells is mediated by prostaglandin E2 and upregulation of alpha2beta1-integrin.

Cell-cell and extracellular matrix adhesions play important roles in the progression of cancer. We investigated the involvement of the inflammatory mediator leukotriene D4 (LTD4) in the regulation of cell-matrix adhesion of colon cancer (Caco-2) cells. We observed that LTD4 acted via its CysLT1 receptor in these cells to induce increased adhesion to collagen I. LTD4 also enhanced the activation and expression of alpha2beta1-integrins on the cell surface, which we found to be responsible for mediating the increased adhesion to collagen I. LTD4 simultaneously augmented expression of the prostaglandin-generating enzyme cyclooxygenase-2 (COX-2) and increased prostaglandin E2 (PGE2) production in Caco-2 cells. The adhesive capacity of the Caco-2 cells was reduced by specific inhibition of COX-2 and was subsequently restored by PGE2, but not by LTD4. A selective PGE2 receptor antagonist abolished the increased adhesion and the augmented alpha2beta1-integrin expression induced by both PGE2 and LTD4. Summarizing, the inflammatory mediator LTD4 regulates the adhesive properties and migration of the Caco-2 cell line by upregulating COX-2 and stimulating PGE2-induced expression of alpha2beta1-integrins. This suggests that inflammatory mediators such as LTD4 can be involved in the dissemination and survival of colon cancer cells.

Caco-2 Cells↗