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Results for “Integrin alpha2”

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PubMed1998

Expression of the VLA family of integrins in coeliac intestinal mucosa.

The integrins, a family of cell surface proteins, mediate cell adhesion and may influence within the intestinal mucosa processes such as migration and/or proliferation and differentiation of enterocytes and lymphocytes. The aim of this study was to examine the distribution pattern of integrin subunits (VLA alpha1, alpha2, alpha3, alpha4, alpha5, alpha6, beta1 chains) in normal intestinal mucosa and in that of patients with active coeliac disease (CD) and CD in remission. Immunohistochemical techniques and double immunostainings with monoclonal antibodies were used for investigation of the VLA alpha family of integrins and beta1 chain distribution. While the majority of the findings are consistent with the few data previously reported in the literature, surprising is the finding of a lack of expression of VLAalpha1 on the intraepithelial lymphocytes in the coeliac mucosa. The deficient VLA alpha1 expression on IEL in coeliac but not in normal mucosa may imply a genetic variation or a specific deficiency of gene expression during T cell differentiation and activation.

Antibodies, Monoclonal↗

PubMed2001

Extracellular matrix molecules improve periodontal ligament cell adhesion to anorganic bone matrix.

Bone replacement graft (BRG) materials are used in periodontal therapy to encourage new bone formation. Extracellular matrix proteins may improve periodontal ligament fibroblast (PDLF) attachment to these materials. We demonstrate that PDLFs adhere well to the extracellular matrix (ECM) proteins fibronectin, vitronectin, laminin, and collagen types I and IV. PDLFs express numerous ECM-receptor integrin subunit transcripts (alpha1, alpha2, alpha3, alpha4, alpha5, alpha11, beta1, beta5, and beta8) at high levels, while others (alpha6, alpha9, alphaV, beta3, beta6, and beta7) are expressed at reduced levels. Despite the fact that PDLFs adhere well to fibronectin and collagen type IV bound to plastic, and express integrins that recognize these ECM proteins, they do not attach well to anorganic bovine bone matrix (ABM) coated with these same proteins. However, the addition of vitronectin, laminin, or collagen type I to these same ABMs substantially increased PDL cell attachment. Thus, selective use of ECM proteins may be clinically useful in promoting cell attachment to ABM and bone regrowth.

Adsorption↗

PubMed2006

Localization of extracellular matrix receptors on the chondrocyte primary cilium.

A single primary cilium is found in chondrocytes and other connective tissue cells. We have previously shown that extracellular matrix (ECM) macromolecules such as collagen fibers closely associate with chondrocyte primary cilia, and their points of contact are characterized by electron-opaque plaques suggesting a direct link between the ECM and the cilium. This study examines the expression of receptors for ECM molecules on chondrocyte primary cilia. Embryonic chick sterna were fluorescently labeled with antibodies against alpha and beta integrins, NG2, CD44, and annexin V. Primary cilia were labeled using acetylated alpha-tubulin antibody. Expression of ECM receptors was examined on chondrocyte plasma membranes and their primary cilia using immunofluorescence and confocal microscopy. All receptors examined showed a punctate distribution on the plasma membrane. alpha2, alpha3, and beta1 integrins and NG2 were also present on primary cilia, whereas annexin V and CD44 were excluded. The number of receptor-positive cilia varied from 8/50 for NG2 to 43/50 for beta1 integrin. This is the first study to demonstrate the expression of integrins and NG2 on chondrocyte primary cilia. The data strongly suggest that chondrocyte primary cilia have the necessary machinery to act as mechanosensors, linking the ECM to cytoplasmic organelles responsible for matrix production and secretion.

Animals↗

PubMed1999

Expression and function of cell adhesion molecules on fetal liver, cord blood and bone marrow hematopoietic progenitors: implications for anatomical localization and developmental stage specific regulation of hematopoiesis.

The mechanism of localization, migration, and regulation of hematopoiesis at different stages of ontogeny is not well understood, but may relate to the specific adhesive interactions between hematopoietic stem cells and their microenvironment at different ontogenic stages. We studied the expression of cell adhesion molecules (CAM) on fetal liver (FL), umbilical cord blood (UCB) and adult bone marrow (ABM) CD34+ cells, and the adhesion of committed progenitors (CFC) from all three sources to ABM stromal layers and purified extracellular matrix proteins. Compared to ABM CFC, significantly more UCB CFC and fewer FL CFC adhered to ABM stroma. Adhesion of FL CFC to fibronectin (FN), the 75 kD RGD containing FN fragment and the 33-66 kD COOH-terminal heparin binding FN fragment was also significantly less than that of ABM CFC. Like ABM CFC, the adhesion of FL CFC was mediated through alpha4beta1 and alpha5beta1 integrins. Of note, more FL CD34+ cells expressed alpha5 integrins and the number of alpha4, alpha5 and beta1 integins per cell (mean channel frequency) was similar or higher for FL CD34+ cells than ABM CD34+ cells. Further, treatment of FL CFC with a beta1 integrin activating antibody (8A2), increased adhesion of FL CFC to FN to the same level as that of 8A2 treated ABM CFC. This suggests that the alpha4beta1 and alpha5beta1 integrins on FL CD34+ cells may be present in a low avidity/affinity state. We also show that unlike ABM, FL CD34+ cells expressed alpha2 and that approximately 20% FL CFC adhered to collagen IV. Further, alpha2beta1 integrin on FL CFC was functional since their engagement, either by adhesion to collagen IV or through blocking alpha2 antibodies, transmitted proliferation inhibitory signals. In contrast to alpha4b and alpha5beta1 integrin dependent adhesion, alpha2beta1 dependent adhesion of FL CFC to collagen IV was not enhanced after treatment with 8A2. The reason for this is not clear but suggests that alpha2 integrins on FL CFC are maximally activated. This novel adhesive interaction with collagen IV, reminiscent of that described for CML progenitors, may have a role in the extramedullary localization of FL hematopoiesis or its developmental stage-specific regulation by its microenvironment. Studies to evaluate these possibilities are underway.

Adult↗

PubMed2004

Adhesion of HT-29 colon carcinoma cells to endothelial cells requires sequential events involving E-selectin and integrin beta4.

HT-29 colon carcinoma cells attach to TNFalpha-activated human umbilical vein endothelial cells (HUVECs) by their specific binding to E-selectin. This interaction activates, in the cancer cells, the MAPK SAPK2/p38, which leads to their transendothelial migration (Laferrière et al., J Biol Chem 2001; 276: 33762). In this study, we investigated the role of E-selectin in activating integrins to modulate adhesion and regulate integrin-mediated events. Blocking the integrins from HT-29 cells (alpha2, alpha3, alpha6, alphav/beta5, beta1 and beta4) with specific antibodies revealed a role for beta4 integrin in their adhesion to TNFalpha-treated HUVEC. The beta4 integrin-dependent adhesion was maximal after 30 min, whereas the-E-selectin-dependent adhesion was maximal after 15 min. Integrin beta4 became quickly phosphorylated upon addition of HT-29 cells to endothelial cells and the effect was independent of the expression of E-selectin. Moreover, a recombinant E-selectin/Fc chimera did not induce the phosphorylation of beta4. The phosphorylation of beta4 is not required for adhesion since adhesion was not affected in HT-29 cells that express a truncated form of beta4 that is deleted from its cytoplasmic phosphorylatable domain. However, the expression of the non-phosphorylatable deletant of beta4 was associated with decreased transendothelial cell migration underscoring the key role for the cytoplasmic domain of beta4 in cell migration. We suggest: 1) that the adhesion of HT-29 cells to activated endothelial cells follows at least two essential sequential steps involving the binding of E-selectin to its receptor on carcinoma cells and then the binding of beta4 to its own receptor on endothelial cells; 2) that the phosphorylation of integrin beta4 contributes to enhance the motile potential of cancer cells and increase their trans-endothelial migration. Overall, our results indicate that the interaction of metastatic cancer cells with endothelial cells implies a specific sequence of signaling events that ultimately leads to an increase in their efficient transendothelial migration.

Cell Adhesion↗

PubMed1998

Tetraspanin CD9 is associated with very late-acting integrins in human vascular smooth muscle cells and modulates collagen matrix reorganization.

CD9, a member of the tetraspanin family, and very late-acting (VLA) integrins are known to associate and form functional units on the surface of several cell types. We studied the changes in expression of CD9 and beta1-integrins (CD29, VLA) in human vascular smooth muscle cells (VSMCs) under in vitro culture conditions mimicking proliferative vascular diseases. We also investigated possible interactions between CD9 and VLA integrins in VSMCs. We found that CD9 is highly expressed in VSMCs and is subject to modulation, depending on the proliferative/contractile state of the cells. In the contractile phenotype, the levels of CD9, CD81, another tetraspanin, and CD29 are approximately 50% of those found in the proliferative phenotype. Coimmunoprecipitation experiments showed physical association between CD9 and CD29. CD9 was mainly associated with alpha2 and alpha3-integrins (CD49b and c) and also with alpha5-integrin to a weaker extent. Functionally, the addition of anti-CD9 monoclonal antibodies (MoAbs) doubled the extent of collagen gel contraction mediated by VSMCs, a model for the reorganization of the extracellular collagen matrix occurring in the vessel wall. Anti-CD29 MoAbs inhibited gel contraction, but anti-CD9 MoAbs counteracted this inhibitory effect of anti-CD29 MoAbs. Transfection of human CD9 into Chinese hamster ovary cells more than doubled the extent of Chinese hamster ovary cell-mediated collagen gel contraction (130% stimulation), confirming a role for CD9 in extracellular matrix reorganization. Thus, CD9 seems to be involved in the modulation of VLA integrin-mediated collagen matrix reorganization by VSMCs. These findings suggest that high CD9 expression is associated with a proliferative state of VSMCs. The role of CD9 could be to modulate the function of VLA integrins on the surface of VSMCs.

Animals↗

PubMed1998

The effect of dexamethasone on integrin and laminin expression in cultured human trabecular meshwork cells.

Glucocorticoid treatment in vivo can produce a glaucoma similar in many ways to POAG. Treatment of trabecular meshwork cells in culture with dexamethasone allows the study of biochemical aspects of this disease process. The effects of dexamethasone on the expression of integrins and laminin in both normal and glaucomatous cultured human trabecular meshwork cells were evaluated. Human trabecular meshwork cell lines were cultured for 18 days in the presence or absence of 10(-7) m dexamethasone. Radioimmunoprecipitation was used to determine the relative expression of five alphaintegrin subunits. Laminin expression was evaluated with Western blots. Laminin was increased in all cell lines following dexamethasone treatment. alpha2, alpha5 and alphaV integrin chains showed consistent dexamethasone-induced changes in expression, while alpha3 and alpha4 subunits did not. There were no differences in the expression patterns for any of these integrin subunits between normal and glaucomatous cell lines. Increased laminin deposition as seen in this study with dexamethasone treatment may be partially responsible for the decreased outflow facility seen in both steroid-induced glaucoma and in POAG.

Blotting, Western↗

PubMed2002

The alpha(2)beta(1) and alpha(3)beta(1) integrins do not mediate attachment of endometrial cells to peritoneal mesothelium.

OBJECTIVE: To evaluate the possible role of mesothelial alpha(2)beta(1) and alpha(3)beta(1) integrins in the attachment of endometrial stromal cells (ESCs) and endometrial epithelial cells (EECs). DESIGN: In vitro study. SETTING: University medical center. PATIENT(S): Women of reproductive age (n = 26). MAIN OUTCOME MEASURE(S): Mesothelial cells were grown on collagen IV. Endometrial stromal cells and EECs were plated on mesothelial cells for 1 hour. Before plating, mesothelial cells or endometrial cells were incubated with antibodies to alpha2, alpha3, and beta1 integrin subunits. The effect of these antibodies on ESC and EEC binding to collagen IV and collagen I was also examined. The expression of collagen I, collagen IV, fibronectin, and laminin by cultured ESCs and EECs was examined. RESULT(S): The anti-integrin antibodies had no effect on endometrial binding to mesothelium. The beta1 integrin antibody decreased binding of ESCs and EECs to the collagen matrices. In culture, ESCs and EECs expressed collagen I, collagen IV, fibronectin, and laminin to varying degrees. CONCLUSION(S): The initial adhesion of ESCs and EECs to mesothelium is not mediated by beta1 integrins. In contrast, ESC and EEC attachment to collagen IV and collagen I, which are present in the submesothelial extracellular matrix, is mediated by beta1 integrins.

Adult↗

PubMed2005

Regulation by ultrasound treatment on the integrin expression and differentiation of osteoblasts.

It has been shown that ultrasound (US) stimulation accelerates fracture healing in the animal models and in clinical studies. However, the mechanism by which US achieves these outcomes is not clear. Here we investigated the effect of US stimulation on the differentiation of osteoblasts and osteoclastogenesis. The effect of different intensities of US stimulation (1 MHz, continuous wave) on the osteoblastic cell line MC3T3-E1 or primary cultured osteoblasts was examined. Flow cytometry showed that US stimulation at 125 mW/cm2 for 10 min transiently increased the surface expression of alpha2, alpha5, and beta1 integrins in both MC3T3-E1 and primary osteoblasts. Fluorocytochemistry showed that the actin cytoskeleton also reorganized in response to US stimulation. When the MC3T3-E1 cells were cultured in differentiation medium containing vitamin C and beta-glycerophosphate, long-term US stimulation (10 min/day for 11 days) increased mineralized nodule formation, collagen content, and alkaline phosphatase activity. The intensity at 125 mW/cm2 exerts the most prominent action. Effect of long-term US stimulation on the osteoclastogenesis was also examined. US stimulation at a power of 62.5 or 125 mW/cm2 markedly inhibited RANKL plus M-CSF-induced osteoclastic differentiation from bone marrow stromal cells. These findings suggest that US has a regulatory effect on the integrin expression and the differentiation of osteoblasts and osteoclastogenesis, which may contribute to the beneficial effects of US on the fracture repair.

3T3 Cells↗

PubMed2006

Activation of platelet alphaIIbbeta3 by an exogenous peptide corresponding to the transmembrane domain of alphaIIb.

A transmembrane domain heterodimer, acting in concert with a membrane-proximal cytoplasmic domain clasp, is thought to maintain integrins in a low affinity state. To test whether helix-helix interactions between the alphaIIb and beta3 transmembrane domains regulate the activity of integrin alphaIIbbeta3, we synthesized a soluble peptide corresponding to the alphaIIb transmembrane domain, designated alphaIIb-TM, and we studied its ability to affect alphaIIbbeta3 activity in human platelets. alphaIIb-TM was alpha-helical in detergent micelles and phospholipid vesicles, readily inserted into membrane bilayers, bound to intact purified alphaIIbbeta3, and specifically associated with the transmembrane domain of alphaIIb, rather than the transmembrane domains of beta3, alpha2, and beta1, other integrin subunits present in platelets. When added to suspensions of gel-filtered platelets, alphaIIb-TM rapidly induced platelet aggregation that was not inhibited by preincubating platelets with the prostaglandin E(1) or the ADP scavenger apyrase but was prevented by the divalent cation chelator EDTA. Furthermore, alphaIIb-TM induced fibrinogen binding to platelets but not the binding of osteopontin, a specific ligand for platelet alphavbeta3. The peptide also induced fibrinogen binding to recombinant alphaIIbbeta3 expressed by Chinese hamster ovary cells, confirming that its effect was independent of platelet signal transduction. Finally, transmission electron microscopy of purified alphaIIbbeta3 revealed that alphaIIb-TM shifted the integrin from a closed configuration with its stalks touching to an open configuration with separated stalks. These observations demonstrate that transmembrane domain interactions regulate integrin function in situ and that it is possible to target intra-membranous protein-protein interactions in a way that can have functional consequences.

Animals↗

PubMed2007

Three-dimensional collagen fiber remodeling by mesenchymal stem cells requires the integrin-matrix interaction.

Tissue engineering aiming to repair or regenerate damaged tissues necessitates fabricating three-dimensional biomaterial scaffolds with controlled porosity for delivering cells. To facilitate cell distribution, a strategy using stem cell-based fabrication of biomaterials was tested in type II collagen fibers. Human mesenchymal stem cells when delivered in type II collagen assembled and reorganized these matrices and differentiated into spherical chondrocytes with the synthesis of cartilage proteins. The cell-mediated assembly and reorganization of collagen fibers was not limitless and only restricted to an appropriate ratio of cell number and collagen amount. The blocking of alpha2 or beta1-integrin function with specific antibodies significantly impeded the collagen-assembly effects. In vitro chondrogenesis or in vivo cartilage formation of human mesenchymal stem cells was also dependent on the interactions between cells and surrounding matrices. This method for three-dimensional fabricating collagen fibers may generally be applied to other biomaterials, when combined with surface modification or ligand addition for cell adhesion.

Biocompatible Materials↗

PubMed2002

Transforming growth factor-beta1 modulates expression of adhesion and cytoskeletal proteins in human peritoneal fibroblasts.

OBJECTIVE: To determine the effects of TGF-beta1 on the expression of the alpha1, alpha2, alpha5, alpha(v), and alpha6 integrin subunits and on vinculin, and F-actin in human peritoneal fibroblasts. DESIGN: Descriptive study using cell culture, reverse transcriptase-polymerase chain reaction (RT-PCR), and immunofluorescent and confocal microscopy. SETTING: Academic medical center. PATIENT(S): Gynecological surgery patients. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Effects of TGF-beta1 on the steady state levels of alpha5, alpha(v), and alpha6 integrin transcripts were examined in the normal peritoneal fibroblasts using RT-PCR. Expression levels of the alpha1, alpha2, alpha5, alpha(v), and alpha6 integrin subunits and of F-actin were measured by immunofluorescent microscopy. The distribution pattern of the integrin subunits, vinculin, and F-actin were examined using confocal microscopy. RESULT(S): TGF-beta1 significantly up-regulated the expression levels of the alpha5, alpha(v), and alpha6 integrin subunits and modulated their expression pattern, resulting in relatively higher levels of these subunits in the focal contacts of peritoneal fibroblasts. It allocated vinculin expression primarily to the focal contacts of cells and caused distortion of F-actin structure. The transcript levels of the alpha5, alpha(v), and alpha6 integrin subunits were not altered by TGF-beta1 treatment. CONCLUSION(S): TGF-beta1 may promote postoperative adhesion formation by inducing the migration of peritoneal fibroblasts by altering the expression levels and patterns of specific integrin subunits, vinculin, and F-actin.

Actins↗

PubMed2006

Characterization of the bovine placenta by cytoskeleton, integrin receptors, and extracellular matrix.

The cytoskeleton together with integrin receptors and proteins of the extracellular matrix provide sensitive indices of the development and organization of the bovine placenta. The bovine placenta is classified as synepitheliochorial because migrating trophoblast giant cells fuse with single uterine epithelial cells. This phenomenon may be interpreted as a restricted trophoblast invasion. Bovine placentomes from early placentation until term can be characterized by indirect immunohistochemical methods. In order to do so, placental tissues are snap-frozen in liquid nitrogen or perfusion-fixed in formalin and embedded in paraffin. Depending on the antibodies used, the different cell types within the cow placenta are identified either on frozen sections or on paraffin sections according to the expression of different cytoskeletal filaments (alpha smooth muscle actin, different cytokeratins, desmin and vimentin). The specific expression of integrin receptors (subunits alpha1, alpha2, alpha3, alpha4, alpha5, alpha6, alpha(v), beta1, beta3, and alpha4) as well as proteins of the extracellular matrix (collagens type I and IV, fibronectin, and laminin) in the different cell populations is also examined.

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↗

PubMed2001

Alpha2,6-sialylation of cell-surface N-glycans inhibits glioma formation in vivo.

Human gliomas express very high levels of cell-surface alpha2,3-linked terminal sialic acids on glycoproteins bearing N-linked oligosaccharides, most notably on alpha3beta1 integrin, which is the predominant integrin found in these tumors. Alpha2,6-linked terminal sialic acids, however, are not expressed. Two stable transfectants were made using a tumorigenic human glioma cell line, U-373 MG. Galbeta1,4GlcNAc alpha2,6-sialyltransferase (ST6Gal I) transfectants were made to replace the endogenous alpha2,3-linked sialic acids with alpha2,6-linked sialic acids. And Galbeta1,3(4)GlcNAc alpha2,3-sialyltransferase (ST3Gal III) transfectants were made to increase further the expression of cell-surface, N-glycan, alpha2,3-linked sialic acids. Although ST3Gal III transfection resulted in increased invasivity when compared with parental U-373 MG and vector-transfected control cells in vitro, ST6Gal I transfection abolished invasion in vitro and induced alterations in both cell morphology, cell-spreading, and adhesion-mediated protein tyrosine phosphorylation. Furthermore, the ST6Gal I transfectants produced no intracranial tumors in severe combined immunodeficient mice, whereas parental U-373 MG cells, the vector-transfected control cells, and ST3Gal III-transfected U-373 MG cells did. These results suggest that both the linkage and expression levels of the terminal sialic acids of alpha3beta1 integrin N-glycans play an important role in glioma cell-extracellular matrix interactions. Thus, manipulating ST6Gal I gene expression may have therapeutic potential for the treatment of malignant gliomas.

Animals↗

PubMed2004

Effects on rotavirus cell binding and infection of monomeric and polymeric peptides containing alpha2beta1 and alphaxbeta2 integrin ligand sequences.

Integrin-using rotaviruses bind MA104 cell surface alpha2beta1 integrin via the Asp-Gly-Glu (DGE) sequence in virus spike protein VP4 and interact with alphaxbeta2 integrin during cell entry through outer capsid protein VP7. Infection is inhibited by the alpha2beta1 ligand Asp-Gly-Glu-Ala (DGEA) and the alphaxbeta2 ligand Gly-Pro-Arg-Pro (GPRP), and virus-alpha2beta1 binding is increased by alpha2beta1 activation. In this study, we analyzed the effects of monomers and polymers containing DGEA-, GPRP-, and DGEA-related peptides on rotavirus binding and infection in intestinal (Caco-2) and kidney (MA104) cells and virus binding to recombinant alpha2beta1. Blockade of rotavirus-cell binding and infection by peptides and anti-alpha2 antibody showed that Caco-2 cell entry is dependent on virus binding to alpha2beta1 and interaction with alphaxbeta2. At up to 0.5 mM, monomeric DGEA and DGAA inhibited binding to alpha2beta1 and infection. At higher concentrations, DGEA and DGAA showed a reduced ability to inhibit virus-cell binding and infection that depended on virus binding to alpha2beta1 but occurred without alteration in cell surface expression of alpha2, beta2, or alphavbeta3 integrin. This loss of DGEA activity was abolished by genistein treatment and so was dependent on tyrosine kinase signaling. It is proposed that this signaling activated existing cell surface alpha2beta1 to increase virus-cell attachment and entry. Polymeric peptides containing DGEA and GPRP or GPRP only were inhibitory to SA11 infection at approximately 10-fold lower concentrations than peptide monomers. As polymerization can improve peptide inhibition of virus-receptor interactions, this approach could be useful in the development of inhibitors of receptor recognition by other viruses.

Cell Line↗

PubMed2004

Integrins can directly mediate metastatic tumor cell adhesion within the liver sinusoids.

Tumor cells can show different malignant properties regarding their ability for organ-specific metastasis formation. Their adhesive and invasive characteristics mediated by various cell adhesion molecules appear to be crucial for this process. Using intravital fluorescence microscopy, we analyzed the adhesive and invasive interactions of circulating human colon carcinoma cells within the microvasculature of the liver in rats. The involvement of different cell adhesion molecules in specific tumor cell-host organ interactions was investigated. Single-cell suspensions of human colon carcinoma with low (HT-29P) and high (HT-29LMM) metastatic potential were fluorescence labeled with calcein-AM and intra-arterially injected into Sprague-Dawley rats. Initial interactions between different cell lines and the microvasculature of the liver were observed over 30 minutes and semiquantitatively analyzed. Different integrin subunits, carbohydrate ligands, and vascular cell adhesion molecule-1 were inhibited using function-blocking antibodies or by enzymatic removal. Inhibition of sialyl-Lewis(a) (sLe(a)) or enzymatic removal of selectin carbohydrate ligands significantly reduced metastatic cell adhesion. In addition, alpha6-, beta1-, and beta4-integrins can directly mediate cell adhesion within the hepatic microcirculation. Furthermore, alpha2-, alpha6-, beta1-, and beta4-integrins are involved in early tumor cell extravasation into the liver parenchyma. Organ-specific formation of colorectal metastases appears to be mainly mediated by specific interactions between circulating carcinoma cells and the vessel wall of target organs but not mechanical entrapment. Selectin-sLe(a) interactions with sinusoidal endothelial cells can play a key role in organ-specific targeting, but direct integrin-mediated cell adhesion to extracellular matrix components in the space of Disse appears to be required for the successful formation of liver metastases.

Animals↗

PubMed2004

Binding of alpha2 monoclonal antibody to human cervical tumor cell (SiHa) surface alpha2beta1 integrin modulates MMP-2 activity.

OBJECTIVE(S): The purpose was to study the interrelationship between cell surface integrin receptor (alpha2beta1) and matrixmetalloproteinases. METHODS: Immunoprecipitation and cell adhesion assay were done to assay alpha2beta1 and alpha3beta1 on SiHa cell surface. Zymogram was developed to assay secreted MMP activity of cells grown in presence of alpha2 monoclonal antibody. Immunoblot was developed to assay expression of MMP-2, FAK, and p-FAK. Plasma membrane-dependent activation of MMP2 was performed by incubating pure MMP-2 with membrane-enriched fraction isolated from SiHa cells. RESULTS: Immunoprecipitation and cell adhesion assay results confirmed the presence of alpha2beta1 receptor on SiHa cells. Zymographic analysis of serum-free media collected at different time points from SiHa cells grown on alpha2 monoclonal antibody-coated culture dishes showed the expression and activation of MMP-2 within 2-4 h, confirmed by immunoblot. Western blot of cells grown on alpha2-coated dishes for 30 min-4 h showed increased phosphorylation of FAK. Membrane-enriched fraction isolated from SiHa cells was found to specifically activate proMMP-2 to its activated forms within 30 min. CONCLUSION(S): The experimental findings strongly indicate that SiHa cell surface alpha2beta1 regulates MMP-2 expression. Increased phosphorylation of focal adhesion kinase (FAK) strongly indicates the possible role of FAK in signaling cascade. Incubation of SiHa cell membrane fraction with pure MMP-2 strongly confirms the cell membrane-dependent activation of proMMP2.

Antibodies, Monoclonal↗