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PubMedDate not supplied

Cell adhesion molecule expression in the sheep thymus.

Cell adhesion molecules are potential regulating factors in both prethymic and intrathymic T cell development. An experimental challenge has been the development of a large animal model that facilitates in vivo studies of both intrathymic development and lymphocyte migration. To extend earlier studies of thymic development, we have developed a panel of monoclonal antibodies (mAb) to a variety of sheep cell adhesion molecules. Immunohistochemistry was used to define mAb reactivity and flow cytometry was used to quantify expression of cell adhesion molecules within the thymus. To facilitate flow cytometry definition of cortical thymocytes, mAbs were developed to the sheep CD1 antigen. Dual parameter flow cytometry provided a phenotypic characterization of cell adhesion molecule expression on both CD1(+) and CD1(-) sheep thymocyte populations. These studies demonstrated significantly enhanced cortical thymocyte expression of three cell adhesion molecules: beta1 integrin (CD29), ICAM-2 and LFA-3. The beta1 integrin cell adhesion molecule was also expressed at higher levels on CD1(+) thymocytes in post-natal lambs as compared to adult sheep. These studies of thymocyte membrane molecule expression should facilitate future investigations of sheep intrathymic development and T lymphocyte immigration.

Animals↗
PubMed2004

Loss of caveolin-1 polarity impedes endothelial cell polarization and directional movement.

The ability of a cell to move requires the asymmetrical organization of cellular activities. To investigate polarized cellular activity in moving endothelial cells, human endothelial cells were incubated in a Dunn chamber to allow migration toward vascular endothelial growth factor. Immunofluorescent staining with a specific antibody against caveolin-1 revealed that caveolin-1 was concentrated at the rear of moving cells. Similarly, monolayer scraping to induce random cell walk resulted in relocation of caveolin-1 to the cell rear. These results suggest that posterior polarization of caveolin-1 is a common feature both for chemotaxis and chemokinesis. Dual immunofluorescent labeling showed that, during cell spreading, caveolin-1 was compacted in the cell center and excluded from nascent focal contacts along the circular lamellipodium, as revealed by integrin beta1 and FAK staining. When cells were migrating, integrin beta1 and FAK appeared at polarized lamellipodia, whereas caveolin-1 was found at the posterior of moving cells. Notably, wherever caveolin-1 was polarized, there was a conspicuous absence of lamellipod protrusion. Transmission electron microscopy showed that caveolae, similar to their marker caveolin-1, were located at the cell center during cell spreading or at the cell rear during cell migration. In contrast to its unphosphorylated form, tyrosine-phosphorylated caveolin-1, upon fibronectin stimulation, was associated with the focal complex molecule phosphopaxillin along the lamellipodia of moving cells. Thus, unphosphorylated and phosphorylated caveolin-1 were located at opposite poles during cell migration. Importantly, loss of caveolin-1 polarity by targeted down-regulation of the protein prevented cell polarization and directional movement. Our present results suggest a potential role of caveolin polarity in lamellipod extension and cell migration.

Caveolae↗
PubMed2001

Leukotriene D(4) affects localisation of vinculin in intestinal epithelial cells via distinct tyrosine kinase and protein kinase C controlled events.

Local inflammatory reactions affect the integrity of intestinal epithelial cells, such as E-cadherin-mediated cell-cell interactions. To elucidate this event, we investigated the effects of an inflammatory mediator, leukotriene D(4 )(LTD(4)), on the phosphorylation status and properties of vinculin, a multi-binding protein known to interact with both the E-cadherin-catenin complex and the cytoskeleton. Treatment of an intestinal epithelial cell line with LTD(4 )induced rapid tyrosine phosphorylation of vinculin, which was blocked by the Src family tyrosine kinase inhibitor PP1. Simultaneously, LTD(4) caused an increased association between vinculin and actin, and that association was decreased by PP1. LTD(4) also induced dissociation of vinculin from alpha-catenin without affecting the catenin complex itself. This dissociation was not blocked by PP1 but was mimicked by the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA). Also, the PKC inhibitor GF109203X abolished both the LTD(4)- and the TPA-induced dissociation of vinculin from alpha-catenin. Furthermore, LTD(4) caused a colocalisation of vinculin with PKC-alpha in focal adhesions. This accumulation of vinculin was blocked by transfection with a dominant negative inhibitor of PKC (PKC regulatory domain) and also by preincubation with either GF109203X or PP1. Thus, various LTD(4)-induced phosphorylations of vinculin affect the release of this protein from catenin complexes and its association with actin, two events that are necessary for accumulation of vinculin in focal adhesions. Functionally this LTD(4)-induced redistribution of vinculin was accompanied by a PKC-dependent upregulation of active beta1 integrins on the cell surface and an enhanced beta1 integrin-dependent adhesion of the cells to collagen IV.

Actins↗
PubMed1999

Differences in phosphatase modulation of alpha4beta1 and alpha5beta1 integrin-mediated adhesion and migration of B16F1 cells.

It is well established that a biphasic relationship exists between the adhesive strength of beta1 integrins and their ability to mediate cell movement. Thus, cell movement increases progressively with adhesive strength, but beyond a certain point of optimal interaction, cell movement is reduced with further increases in adhesive function. The interplay between the various kinase and phosphatase activities provides the balance in beta1 integrin-mediated cell adhesion and migration. In the present study, the significance of protein tyrosine phosphatases (PTP) and ser/thr protein phosphatases (PP) in alpha4beta1 and alpha5beta1 integrin-mediated mouse melanoma B16F1 cell anchorage and migration on fibronectin was characterized using phosphatase inhibitors. At low fibronectin concentration, alpha5beta1 functioned as the predominant receptor for cell movement; a role for alpha4beta1 in B16F1 cell migration increased progressively with fibronectin concentration. Treatment of B16F1 cells with PTP inhibitors, sodium orthovanadate (Na3VO4) and phenylarsine oxide (PAO), or PP-1/2A inhibitor, okadaic acid (OA), abolished cell movement. Inhibition of cell movement by PAO and OA was associated by a reduction in the adhesive strength of alpha4beta1 and alpha5beta1. In contrast, treatment of B16F1 cells with Na3VO4 resulted in selective stimulation of the adhesive function of alpha5beta1, but not alpha4beta1. Therefore, our results demonstrate that (i) both PTP and PP-1/2A have roles in cell movement, (ii) modulation of cell movement by PTP and PP-1/2A may involve either a stimulation or reduction of beta1 integrin adhesive strength, and (iii) distinct phosphatase-mediated signaling pathways for differential regulation of the various beta1 integrins exist.

Animals↗
PubMed2006

A soluble truncated cadherin induces breast cancer cell apoptosis and growth inhibition.

PURPOSE: To identify the characteristics and function of the truncated cadherin cDNA which encodes a soluble molecule containing the sequence of VE-cadherin extracellular domain repeats from repeat 1 to 4 (designated as CED1-4) and a secreting signal peptide at N terminal. METHODS: A pMSCV/CED1-4 vector was constructed. Recombinant retrovirus ReCED1-4 and ReEmpty were produced by 293 package cells and transfected into MDA-MB435 human breast cancer cells. The expression of CED1-4 in transfectants and their supernatant was analyzed by RT-PCR and Western blot, respectively. MDA-MB435 cell proliferation assays were performed in vitro and in vivo. CED-14-induced apoptosis was demonstrated using Annexin V binding, TUNEL and caspase 3 assays. The expression of integrin beta1 and c-fos mRNA was detected by RT-PCR. RESULTS: The constructed soluble CED1-4 encoded 484 amino acids and a secreting signal peptide (27 amino acids). CED1-4 was expressed by MDA-MB435/CED1-4 cells, and detected in the supernatant of CED1-4 tranfectants. CED1-4 transfection significantly inhibited the growth of MDA-MB435 cells in vitro and in vivo. About 22-fold increase in the early apoptotic cells in MDA-MB435/CED1-4 cells was observed as compared with MDA-MB435/empty cells. Increased activity of caspase 3 in MDA-MB435/CED1-4 cells was more than two times as compared with that of the control cells. Interestingly, integrin beta1 transcriptional level in MDA-MB435/CED1-4 cells was down-regulated as compared with control cells. The resistance of fibronectin to CED1-4 apoptotic inducibility was confirmed by detection of caspase 3. The blockage of c-fos transcriptional expression was detected in MDA-MB435/CED1-4 cells. CONCLUSIONS: The soluble truncated cadherin may be considered an apoptotic inducer and growth inhibitor in the MDA-MB435 breast carcinoma cell line. Down-regulation of integrin beta1 and blockage of c-fos expression may be related to CED1-4-induced apoptosis and growth inhibition.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Real-time analysis of integrin-mediated chemotactic migration of T lymphocytes within 3-D extracellular matrix-like gels.

We have developed a novel 3-D gel reconstituted with major extracellular matrix (ECM) glycoproteins to follow the dynamics of migration of human T cells locomoting, in real-time, on gradients formed by representative chemoattractants: the C-C chemokine RANTES, and the pro-inflammatory cytokine IL-2. In the absence of chemoattractants, none of the T cells migrated directionally and the levels of random migration or cell polarization were low. However, major fractions of T cells placed in IL-2 and RANTES gradients in the gels polarized immediately after exposure to the chemoattractants. Shortly after polarization, 25% of the T cells migrated, in either a random or directional fashion, towards the sources of the chemoattractants; additional 5-10% of the cells remained polarized but stationary. The number of T cells migrating directionally towards RANTES or IL-2 peaked along with the formation of the chemotactic gradients. The directional migration of T cells was increased by a short pre-exposure to low doses of IL-2, which did not alter the level of expression of the beta1 integrins. The directional migration of T cells towards IL-2 and RANTES was mediated by IL-2R and pertussis toxin-sensitive receptors, respectively, and the directional, and to a lesser degree, the random locomotion of T cells induced by both chemoattractants required intact tyrosine kinase signaling and activities of the alpha4, alpha5, and, to a lesser degree, the alpha2 and alpha6 members the beta1 integrins. Our system enables the real-time tracking of individual locomoting lymphocytes and the analysis of their dynamic interactions with ECM components and cytokines.

Cell Movement↗
PubMed2006

The cleavage plane of corneal epithelial adhesion complex in traumatic recurrent corneal erosion.

PURPOSE: To determine the anatomic cleavage plane of the corneal epithelial adhesion complex in eyes with traumatic recurrent corneal erosion (RCE). METHODS: A loosened sheet of corneal epithelium was obtained from corneal epithelial wounds in eight patients with traumatic RCE, before each patient underwent phototherapeutic keratectomy. Three control groups were employed in the study, including normal corneal epithelial sheets obtained by mechanical separation, normal corneal tissues obtained by partial lamellar keratectomy during pterygial surgery, and corneal tissues from three residual corneoscleral rims of corneas donated for transplantation. Immunofluorescence staining was performed using monoclonal antibodies against integrins beta1 and beta4, laminin 5, and collagen VII to identify abnormalities in specific layers of the adhesion complex. RESULTS: In both experimental and control specimens, the suprabasal and basal cells stained positive for integrin beta1, and basal cells stained positive for integrin beta4. Similarly, a continuous line along the base of each epithelial sheet and each control specimen stained positive for laminin 5, a major basement membrane component. In contrast, in all controls there was a continuous linear staining pattern along the basement membrane of stain positive for collagen VII, a marker for the presence of fibrils that anchor corneal basement membrane to Bowman's layer, but epithelial sheets from eyes with RCE showed either a discontinuous line stained positive for collagen VII (three out of eight specimens) or no positively stained areas (five out of eight specimens). The results indicated the cleavage plane of RCE was located at collagen VII layer, between basement membrane and Bowman's layer. CONCLUSIONS: This study identified a defect in collagen fibrils that anchor the corneal epithelium basement membrane to Bowman's layer as the cause of corneal epithelial loss in cases of traumatic RCE. As hemidesmosomes do not seem to be impaired, a treatment specific to restore anchoring fibril function might prove helpful.

Adult↗
PubMed2001

Mechanism in the sequential control of cell morphology and S phase entry by epidermal growth factor involves distinct MEK/ERK activations.

Cell shape plays a role in cell growth, differentiation, and death. Herein, we used the hepatocyte, a normal, highly differentiated cell characterized by a long G1 phase, to understand the mechanisms that link cell shape to growth. First, evidence was provided that the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) cascade is a key transduction pathway controlling the hepatocyte morphology. MEK2/ERK2 activation in early G1 phase did not lead to cell proliferation but induced cell shape spreading and demonstration was provided that this MAPK-dependent spreading was required for reaching G1/S transition and DNA replication. Moreover, epidermal growth factor (EGF) was found to control this morphogenic signal in addition to its mitogenic effect. Thus, blockade of cell spreading by cytochalasin D or PD98059 treatment resulted in inhibition of EGF-dependent DNA replication. Our data led us to assess the first third of G1, is exclusively devoted to the growth factor-dependent morphogenic events, whereas the mitogenic signal occurred at only approximately mid-G1 phase. Moreover, these two growth factor-related sequential signaling events involved successively activation of MEK2-ERK2 and then MEK1/2-ERK1/2 isoforms. In addition, we demonstrated that inhibition of extracellular matrix receptor, such as integrin beta1 subunit, leads to cell arrest in G1, whereas EGF was found to up-regulated integrin beta1 and fibronectin in a MEK-ERK-dependent manner. This process in relation to cytoskeletal reorganization could induce hepatocyte spreading, making them permissive for DNA replication. Our results provide new insight into the mechanisms by which a growth factor can temporally control dual morphogenic and mitogenic signals during the G1 phase.

Animals↗
PubMed1998

Cross-talk between insulin receptor and integrin alpha5 beta1 signaling pathways.

The ligation and clustering of cell surface alphabeta heterodimeric integrins enhances cell adhesion and initiates signaling pathways that regulate such processes as cell spreading, migration, differentiation, proliferation and apoptosis. Here we show that insulin treatment of Chinese hamster ovary cells expressing insulin receptors (CHO-T) markedly promotes cell adhesion onto a fibronectin matrix, but not onto bovine serum albumin or poly-lysine. Incubation of cells with a GRGDSP peptide that specifically binds integrins (but not the nonspecific GRADSP peptide) abolishes this insulin effect, as does the potent phosphoinositide 3-kinase (PI 3-kinase) inhibitor wortmannin. Moreover, a specific blocking monoclonal anti-alpha5beta1 integrin antibody, PB-1, blocks insulin-stimulated cell adhesion onto fibronectin. Conversely, activating alpha5beta1 integrins on CHO-T cells by adherence onto fibronectin markedly potentiates the action of insulin to enhance insulin receptor and insulin receptor substrate (IRS)-1 tyrosine phosphorylation. Activation of alpha5beta1 integrin also markedly potentiates the recruitment of p85-associated PI 3-kinase activity to IRS-1 in response to submaximal levels of insulin in CHO-T cells. These data indicate that insulin potently activates integrin alpha5beta1 mediated CHO-T cell adhesion, while integrin alpha5beta1 signaling in turn enhances insulin receptor kinase activity and formation of complexes containing IRS-1 and PI 3-kinase. These findings raise the hypothesis that insulin receptor and alpha5beta1 integrin signaling act synergistically to enhance cell adhesion.

Androstadienes↗
PubMed1998

Integrin-mediated ras-extracellular regulated kinase (ERK) signaling regulates interferon gamma production in human natural killer cells.

Recent evidence indicates that integrin engagement results in the activation of biochemical signaling events important for regulating different cell functions, such as migration, adhesion, proliferation, differentiation, apoptosis, and specific gene expression. Here, we report that beta1 integrin ligation on human natural killer (NK) cells results in the activation of Ras/mitogen-activated protein kinase pathways. Formation of Shc-growth factor receptor-bound protein 2 (Grb2) and Shc-proline-rich tyrosine kinase 2-Grb2 complexes are the receptor-proximal events accompanying the beta1 integrin-mediated Ras activation. In addition, we demonstrate that ligation of beta1 integrins results in the stimulation of interferon gamma (IFN-gamma) production, which is under the control of extracellular signal-regulated kinase 2 activation. Overall, our data indicate that beta1 integrins, by delivering signals capable of triggering IFN-gamma production, may function as NK-activating receptors.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Regulation of alpha5beta1 integrin conformation and function by urokinase receptor binding.

Urokinase-type plasminogen activator receptors (uPARs), up-regulated during tumor progression, associate with beta1 integrins, localizing urokinase to sites of cell attachment. Binding of uPAR to the beta-propeller of alpha3beta1 empowers vitronectin adhesion by this integrin. How uPAR modifies other beta1 integrins remains unknown. Using recombinant proteins, we found uPAR directly binds alpha5beta1 and rather than blocking, renders fibronectin (Fn) binding by alpha5beta1 Arg-Gly-Asp (RGD) resistant. This resulted from RGD-independent binding of alpha5beta1-uPAR to Fn type III repeats 12-15 in addition to type III repeats 9-11 bound by alpha5beta1. Suppression of endogenous uPAR by small interfering RNA in tumor cells promoted weaker, RGD-sensitive Fn adhesion and altered overall alpha5beta1 conformation. A beta1 peptide (res 224NLDSPEGGF232) that models near the known alpha-chain uPAR-binding region, or a beta1-chain Ser227Ala point mutation, abrogated effects of uPAR on alpha5beta1. Direct binding and regulation of alpha5beta1 by uPAR implies a modified "bent" integrin conformation can function in an alternative activation state with this and possibly other cis-acting membrane ligands.

Amino Acid Sequence↗
PubMed2001

Integrin signaling via the PI3-kinase-Akt pathway increases neuronal resistance to glutamate-induced apoptosis.

Integrins are integral membrane proteins that mediate adhesive interactions of cells with the extracellular matrix and with other cells. Integrin engagement results in activation of intracellular signaling cascades that effect several different cellular responses including motility, proliferation and survival. Although integrins are known to provide cell survival signaling in various types of non-neuronal cells, the possibility that integrins modulate neuron survival has not been explored. We now report data demonstrating a neuroprotective function of integrins in embryonic hippocampal neurons. Neurons grown on laminin, an integrin ligand, exhibit increased resistance to glutamate-induced apoptosis compared with neurons grown on polylysine. Neurons expressed integrin beta1 and treatment of cultures with an antibody against integrin beta1 abolished the protective effect of laminin. Neurons maintained on laminin exhibited a sustained activation of the Akt signaling pathway demonstrated in immunoblot analyses using an antibody that selectively recognizes phosphorylated Akt. The neuroprotective effect of integrin engagement by laminin was mimicked by an IKLLI-containing integrin-binding peptide and was abolished by treatment of neurons with the PI3 kinase inhibitor wortmanin. Levels of the anti-apoptotic protein Bcl-2 were increased in neurons grown on laminin and decreased by wortmanin, suggesting a mechanism for the neuroprotective effect of integrin-mediated signaling. The ability of integrin-mediated signaling to prevent glutamate-induced apoptosis suggests a mechanism whereby neuron-substrate interactions can promote neuron survival under conditions of glutamate receptor overactivation.

Androstadienes↗
PubMed2001

Overexpression of the integrin-linked kinase mesenchymally transforms mammary epithelial cells.

Signals generated by the interaction of beta1 integrins with laminin in the basement membrane contribute to mammary epithelial cell morphogenesis and differentiation. The integrin-linked kinase (ILK) is one of the signaling moieties that associates with the cytoplasmic domain of beta1 integrin subunits with some specificity. Forced expression of a dominant negative, kinase-dead form of ILK subtly altered mouse mammary epithelial cell morphogenesis but it did not prevent differentiative milk protein expression. In contrast, forced overexpression of wild-type ILK strongly inhibited both morphogenesis and differentiation. Overexpression of wild-type ILK also caused the cells to lose the cell-cell adhesion molecule E-cadherin, become invasive, reorganize cortical actin into cytoplasmic stress fibers, and switch from an epithelial cytokeratin to a mesenchymal vimentin intermediate filament phenotype. Forced expression of E-cadherin in the latter mesenchymal cells rescued epithelial cytokeratin expression and it partially restored the ability of the cells to differentiate and undergo morphogenesis. These data demonstrate that ILK, which responds to interactions between cells and the extracellular matrix, induces a mesenchymal transformation in mammary epithelial cells, at least in part, by disrupting cell-cell junctions.

Adherens Junctions↗
PubMed2000

Antibody targeting of doxorubicin-loaded liposomes suppresses the growth and metastatic spread of established human lung tumor xenografts in severe combined immunodeficient mice.

Beta1 integrins, expressed on the cell surface of human non-small cell lung carcinomas, are used here as a target for the selective delivery of anti-cancer drug-loaded liposomes. Fab' fragments of a monoclonal antibody specific for human beta1 integrins were conjugated to sterically stabilized liposomes. Confocal microscopy of beta1 integrin-positive lung tumor cells incubated with fluorescently labeled anti-beta1 Fab immunoliposomes revealed a tumor-specific binding and efficient internalization of the liposomes into the tumor cells. The ability of these liposomes to deliver cytotoxic drugs to the tumor and kill these cells was demonstrated in vitro by incubating tumor cells with doxorubicin-loaded anti-beta1 Fab' immunoliposomes. The drug-loaded immunoliposomes were >30-fold more cytotoxic to the tumor cells than drug-loaded liposomes without antibody, nonspecific Fab' control immunoliposomes with drug or immunoliposomes without drug. The therapeutic efficacy of doxorubicin-loaded immunoliposomes was also evaluated in a metastatic human lung tumor xenograft/severe combined immunodeficient (SCID) mouse model. SCID mice that received i.v. injections of human lung tumor cells developed primary tumor nodules in the lung that subsequently metastasized to the liver and adrenal gland. Treatment of SCID mice bearing established lung tumor xenografts with doxorubicin-loaded anti-beta1 Fab immunoliposomes resulted in a significant suppression of tumor growth (monitored periodically by quantifying serum levels of a tumor marker), whereas tumors grew progressively in mice treated with control formulations. In addition to suppressing the growth of the primary lung tumor nodules, the immunoliposomes prevented the metastatic spread of the tumor to the liver and adrenal glands and increased the median survival time of the tumor-bearing mice. We conclude that Fab' immunoliposomes directed to tumor-associated integrins represent a potentially viable approach clinically for the selective delivery of drugs to solid tumors and may be useful in preventing the metastatic spread of lung cancer.

Adrenal Gland Neoplasms↗
PubMed2003

Integrin-mediated adhesion of human articular chondrocytes to cartilage.

OBJECTIVE: To determine 1) the kinetics and strength of adhesion of human articular chondrocytes to a cut cartilage surface, and 2) the role of specific integrins in mediating such adhesion, using an in vitro model. METHODS: Human articular chondrocytes isolated from cadaveric donors (mean +/- SD age 38 +/- 13 years) were cultured in high-density or low-density monolayer. Following release from culture with trypsin and a 2-2.5-hour recovery period, chondrocytes were analyzed either for adhesion to cartilage or for integrin expression by flow cytometry. RESULTS: Following culture in monolayer, adhesion of chondrocytes to cartilage increased with time, from 6-16% at 10 minutes to a maximum of 59-82% at 80-320 minutes. After 80 minutes of adhesion, the resistance of cells to flow-induced shear stress (50% detachment) was approximately 21 Pa. Chondrocyte adhesion to cartilage decreased with pretreatment of cells with monoclonal antibodies that bound to and blocked certain integrins. After an 80-minute incubation time, adhesion of chondrocytes cultured in high-density monolayer decreased from the value of IgG1-treated controls (55%) with blocking of the beta1 integrin subunit (to 23%) or with blocking of alpha 5 beta 1 (to 36%). Following expansion of chondrocytes in low-density monolayer, the mechanisms of adhesion to cartilage were generally similar. After an 80-minute incubation time, adhesion of chondrocytes cultured in low-density monolayer decreased from the value of IgG1-treated controls (62%) with blocking of the beta1 integrin subunit (to 30%) or with blocking of alpha 5 beta 1 (to 44%). Additionally, adhesion of these cells decreased to 46% by blocking of alpha v beta 5, with a similar trend in effect for chondrocytes cultured in high-density monolayer. Blocking of the alpha 1 or alpha 3 integrin subunits or alpha v beta 3 had no detectable effect on adhesion, even though these receptors were detected by flow cytometry. CONCLUSION: Under the culture and seeding conditions studied, beta1, alpha 5 beta 1, and alpha v beta 5 integrins mediate human chondrocyte adhesion to cartilage. These chondrocyte integrins have a potential role in the initial adhesion and retention of chondrocytes at a cartilage defect site following clinical procedures of chondrocyte transplantation.

Adult↗
PubMed1999

Targeted disruption of fibronectin-integrin interactions in human gingival fibroblasts by the RI protease of Porphyromonas gingivalis W50.

Cell surface integrins mediate interactions between cells and their extracellular matrix and are frequently exploited by a range of bacterial pathogens to facilitate adherence and/or invasion. In this study we examined the effects of Porphyromonas gingivalis proteases on human gingival fibroblast (HGF) integrins and their fibronectin matrix. Culture supernatant from the virulent strain W50 caused considerably greater loss of the beta1 integrin subunit from HGF in vitro than did that of the beige-pigmented strain W50/BE1. Prior treatment of the W50 culture supernatant with the protease inhibitor Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) blocked its effects on cultured cells, indicating that this process is proteolytically mediated. Purified arginine-specific proteases from P. gingivalis W50 were able to mimic the effects of the whole-culture supernatant on loss of beta1 integrin expression. However purified RI, an alpha/beta heterodimer in which the catalytic chain is associated with an adhesin chain, was 12 times more active than RIA, the catalytic monomer, in causing loss of the alpha5beta1 integrin (fibronectin receptor) from HGF. No effect was observed on the alphaVbeta3 integrin (vitronectin receptor). The sites of action of RI and RIA were investigated in cells exposed to proteases pretreated with TLCK to inactivate the catalytic component. Use of both monoclonal antibody 1A1, which recognizes only the adhesin chain of RI, and a rabbit antibody against P. gingivalis whole cells indicated localization of RI on the fibroblasts in a clear, linear pattern typical of that seen with fibronectin and alpha5beta1 integrin. Exact colocalization of RI with fibronectin and its alpha5beta1 receptor was confirmed by double labeling and multiple-exposure photomicroscopy. In contrast, RIA bound to fibroblasts in a weak, patchy manner, showing only fine linear or granular staining. It is concluded that the adhesin component of RI targets the P. gingivalis arginine-protease to sites of fibronectin deposition on HGF, contributing to the rapid loss of both fibronectin and its main alpha5beta1 integrin receptor. Given the importance of integrin-ligand interactions in fibroblast function, their targeted disruption by RI may represent a novel mechanism of damage in periodontal disease.

Animals↗
PubMed2006

Alpha-toxin interferes with integrin-mediated adhesion and internalization of Staphylococcus aureus by epithelial cells.

Staphylococcus aureus is an important human and animal pathogen. During infection, this bacterium is able to attach to and enter host cells by using its cell surface-associated factors to bind to the host's extracellular matrix (ECM) proteins. In this study, we determined that a protein exported by S. aureus, alpha-toxin, can interfere with the integrin-mediated adhesion and internalization of S. aureus by human lung epithelial cells (A549). The downregulation of alpha-toxin production significantly increased bacterial adhesion and invasion into the epithelial cells. In contrast, bacterial adhesion and invasion was inhibited by both overproduction of alpha-toxin and the addition of alpha-toxin to the culture medium. Moreover, our results showed that the quantitative effects on invasion closely parallel those of adherence. This suggests that the effect on invasion is probably secondary to, and a consequence of, the reduced adherence caused by alpha-toxin exposure. Specifically, we demonstrated that alpha-toxin interacts with the hosts' ECM protein's receptor, beta1-integrin, which indicates that beta1-integrin may be a potential receptor of alpha-toxin on epithelial cells. Taken together, our results indicate that exported alpha-toxin inhibits the adhesion and internalization of S. aureus by interfering with integrin-mediated pathogen-host cell interactions.

Bacterial Adhesion↗
PubMed2001

Laminin-1 and alpha6beta1 integrin regulate acinar morphogenesis of normal and malignant human prostate epithelial cells.

BACKGROUND: Cell-matrix interactions via integrin receptors are critical for acinar morphogenesis. The non-tumorigenic, human prostate epithelial cell line RWPE-1 was used in a three-dimensional (3D) cell culture model to identify the matrix protein and its integrin receptor required for acinar morphogenesis. METHODS: 3D cultures, immunostaining, confocal microscopy, and Western blot analysis were used to examine acinar formation on matrix proteins and to determine integrin receptor expression. RESULTS: RWPE-1 cells differentiate into acini of polarized cells with a distinct lumen in 3D Matrigel culture. In contrast, the malignant WPE1-NB26 prostate epithelial cells form solid cell masses. In 3D gels of laminin-1, type IV collagen, or fibronectin, RWPE-1 cells form acini only in laminin-1. Anti-laminin-1 antibody reduces acinar formation in a dose-dependent manner. Polarized RWPE-1 cells showed basal expression of alpha6 and beta1 integrin subunits. Blocking antibodies to alpha6 or beta1 reduced acinar formation to 9 and 6 percent of control, respectively. The beta1 integrin colocalized with focal adhesion kinase (FAK). Inhibition of extracellular signal-regulated kinase kinase activity significantly reduced acinar formation to 38 percent of control, suggesting that beta1 integrin-mediated signal transduction may be regulated through a FAK pathway. CONCLUSIONS: While basal expression of alpha6beta1 integrin in RWPE-1 cells correlates with their ability to polarize and form acini, a decrease or loss of alpha6, and diffused beta1 expression in WPE1-NB26 cells correlates with loss of acinar-forming ability. Results show that laminin-1 and a functional alpha6beta1 integrin receptor are required for acinar morphogenesis. This novel 3D cell culture model is useful for elucidating regulation of acinar morphogenesis and its loss during prostate carcinogenesis.

Antibodies↗