Syndecans promote integrin-mediated adhesion of mesenchymal cells in two distinct pathways

Syndecans are transmembrane proteoglycans that support integrin-mediated adhesion. Well documented is the contribution of syndecan-4 that interacts through its heparan sulphate chains to promote focal adhesion formation in response to fibronectin domains. This process has requirements for integrin and signaling through the cytoplasmic domain of syndecan-4. Here an alternate pathway mediated by the extracellular domains of syndecans-2 and -4 is characterized that is independent of both heparan sulphate and syndecan signaling. This pathway is restricted to mesenchymal cells and was not seen in any epithelial cell line tested, apart from vascular endothelia. The syndecan ectodomains coated as substrates promoted integrin-dependent attachment, spreading and focal adhesion formation. Syndecan-4 null cells were competent, as were fibroblasts compromised in heparan sulphate synthesis that were unable to form focal adhesions in response to fibronectin. Consistent with actin cytoskeleton organization, the process required Rho-GTP and Rho kinase. While syndecan-2 and -4 ectodomains could both promote integrin-mediated adhesion, their pathways were distinct, as shown by competition assays. Evidence for an indirect interaction of beta1 integrin with both syndecan ectodomains was obtained, all of which suggests a distinct mechanism of integrin-mediated adhesion.     

Bacillus anthracis internalization by human fibroblasts and epithelial cells

The current model for Bacillus anthracis dissemination in vivo focuses on macrophages as carriers. However, recent evidence suggested that other host cells may also play a role in the process. Here, we tested the possibility of B. anthracis being internalized by a human fibroblast cell line, HT1080 and an epithelial cell line, Caco-2. A combination of gentamicin protection assays, scanning and transmission electron microscopy (EM) and fluorescence microscopy was used. The results demonstrated for the first time that both spores and vegetative cells of B. anthracis Sterne strain 7702 were able to adhere to and be internalized by cultured HT1080 and Caco-2 cells. Spore adherence to and internalization by HT1080 cells were not affected by a germination inhibitor. This suggested that certain features on dormant spores were sufficient for these processes. Vegetative cell adherence to and internalization by both cell lines were growth phase-dependent. EM images suggested that vegetative cells may have the ability to escape phagocytic vacuoles. Finally, we showed that internalization of both spores and vegetative cells required active functions of the host cell cytoskeleton. These results raised the possibility that B. anthracis may disseminate in vivo by directly infecting non-phagocytic cells.     

Rho GTPase activity modulates Pseudomonas aeruginosa internalization by epithelial cells

The Gram-negative pathogen Pseudomonas aeruginosa invades epithelial cells in vivo and in vitro . We have examined the pathway(s) by which epithelial cells internalize P. aeruginosa strain PA103 using Madin-Darby canine kidney (MDCK) cells. We have recently demonstrated that P. aeruginosa internalization occurs by an actin-dependent Toxin B-inhibited pathway which becomes downregulated as epithelial cells become polarized, suggesting that one or more of the Rho family GTPases is involved in bacterial internalization. Here, we demonstrate that activation of the Rho family GTPases by cytotoxic necrotizing factor 1 (CNF-1) stimulates P. aeruginosa internalization. Examination of the roles of the individual Rho family GTPases in internalization shows that expression of a constitutively active allele of RhoA (RhoAV14), but not of constitutively active Rac1 (Rac1V12) or Cdc42 (Cdc42V12), is sufficient to increase uptake of PA103 pscJ . This relative increase persists when bacterial infection is established at the basolateral surface of polarized cells, suggesting that the effect of RhoAV14 is not simply due to its known ability to disrupt tight junction integrity in polarized cells. RhoAV14-mediated stimulation of bacterial uptake is actin dependent as it is abrogated by exposure to latrunculin A. We also find that endogenous Rho GTP levels in epithelial cells are increased by infection with an internalized strain of P. aeruginosa; conversely, a poorly internalized isogenic strain expressing the bacterial anti-internalization protein ExoT causes decreased Rho GTP levels. Experimental inhibition of Rho, either by expressing dominant negative RhoAN19 or by inhibiting native Rho using a membrane permeable fusion construct of a Rho-specific inhibitor, C3 ADP-ribosyltransferase, does not inhibit PA103 pscJ internalization in MDCK or HeLa cells. Models consistent with these data are presented.     

Loss of proliferation and antigen presentation activity following internalization of polydispersed carbon nanotubes by primary lung epithelial cells

Interactions between poly-dispersed acid functionalized single walled carbon nanotubes (AF-SWCNTs) and primary lung epithelial (PLE) cells were studied. Peritoneal macrophages (PMs, known phagocytic cells) were used as positive controls in this study. Recovery of live cells from cultures of PLE cells and PMs was significantly reduced in the presence of AF-SWCNTs, in a time and dose dependent manner. Both PLE cells as well as PMs could take up fluorescence tagged AF-SWCNTs in a time dependent manner and this uptake was significantly blocked by cytochalasin D, an agent that blocks the activity of acto-myosin fibers and therefore the phagocytic activity of cells. Confocal microscopic studies confirmed that AF-SWCNTs were internalized by both PLE cells and PMs. Intra-trachially instilled AF-SWCNTs could also be taken up by lung epithelial cells as well as alveolar macrophages. Freshly isolated PLE cells had significant cell division activity and cell cycling studies indicated that treatment with AF-SWCNTs resulted in a marked reduction in S-phase of the cell cycle. In a previously standardized system to study BCG antigen presentation by PLE cells and PMs to sensitized T helper cells, AF-SWCNTs could significantly lower the antigen presentation ability of both cell types. These results show that mouse primary lung epithelial cells can efficiently internalize AF-SWCNTs and the uptake of nanotubes interfered with biological functions of PLE cells including their ability to present BCG antigens to sensitized T helper cells.     

Cellular internalization of lactoferrin in intestinal epithelial cells

We studied the cellular internalization of lactoferrin (Lf) in an intestinal epithelial cell line, Caco-2, to investigate the mechanism of biological actions of ingested Lf. RT-PCR and Western blotting analyses revealed that differentiated Caco-2 cells express LfR mRNA and its protein with a 34 kD molecular weight under reducing conditions. Biotin-labeled Lf showed specific binding to the cellular membrane of differentiated Caco-2 cells with a dissociation constant (Kd) of 0.16 microM. The cellular internalization of Lf was studied in differentiated Caco-2 cells grown as monolayers on Transwell inserts, and compared to that of human transferrin (Tf). After labeling with fluorescent dye, either Lf or Tf was added to Caco-2 cells from the apical side or the basolateral one. Laser scanning confocal microscopy showed that labeled Lf was internalized only from the apical side and localized to the nuclei. On the other hand, labeled Tf was internalized from the basolateral side, not from the apical side, and localized in the cytoplasm. The internalization of labeled Lf was inhibited by excess of unlabeled Lf, but not of Tf. The internalization of labeled Lf, but not of labeled Tf, was also suppressed by heparin. This indicates that a heparin-binding site in the N-terminal region of Lf could be important for the internalization of Lf. These findings suggest that ingested Lf might be internalized by the intestinal epithelium in a manner different from Tf and might function in the nucleus.     

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.     

Pseudomonas aeruginosa internalization by corneal epithelial cells involves MEK and ERK signal transduction proteins

Invasion of epithelial cells represents a potential pathogenic mechanism for Pseudomonas aeruginosa. We explored the role of mitogen-activated protein kinase kinases (MEK 1/2) and the extracellular signal-regulated kinases (ERK 1/2) in P. aeruginosa invasion. Treatment of corneal epithelial cells with MEK inhibitors, PD98059 (20 microM) or UO126 (100 microM), reduced P. aeruginosa invasion by approximately 60% without affecting bacterial association with the cells (P=0.0001). UO124, a negative control for UO126, had no effect on bacterial internalization. Infection of cells with an internalization-defective flhA mutant of P. aeruginosa was associated with less ERK 1/2 tyrosine phosphorylation than infection with wild-type invasive P. aeruginosa. An ERK-2 inhibitor, 5-iodotubercidin (20 microM), reduced P. aeruginosa invasion by approximately 40% (P=0.035). Together, these data suggest that P. aeruginosa internalization by epithelial cells involves a pathway(s) that includes MEK and ERK signaling proteins.     

Binding and internalization of VIP in rat intestinal epithelial cells.

We have prepared villous cells from the jejunum of the rat small intestine and studied the effects of divalent cations and bacitracin on the binding and internalization of VIP. Villous epithelial cells (4 x 10(6) cells/ml) were suspended in a Hepes-NaCl buffer with 1.0% BSA, (pH 7.4) and the cells were incubated for varying periods of time with 125I-VIP at 24 degrees C. Specific binding of radiolabeled VIP was maximal within 10 min (10%) and slowly declined to 9.0 percent after 30 min. In the presence of 1.0 mg/ml bacitracin, however, maximal specific binding of VIP was only 2.7 percent (P less than or equal to 0.001). The addition of CA2+ or Mg2+ to the buffer significantly decreased binding of VIP in a concentration dependent manner. At 8.0, 4.0, 2.0 and 1.0 mM Ca2+, binding of 125I-VIP decreased by 70, 60, 40 and 25 percent, whereas in the presence of the same concentrations of Mg2+ binding was decreased to 50, 38, 25 and 10 percent (P less than or equal to 0.01). To determine if epithelial cells internalize VIP, we bound 125I-VIP to villous cells and then differentiated surface-bound and internalized radioactivity by treating with trypsin (150 micrograms/ml). Surface bound radioligand was the same at both 24 and 4 degrees C (5.3%), while internalized 125I-VIP was 4.0% at 24 degrees C compared to only 1.0% at 4 degrees C (P less than or equal to 0.001). At 24 and 4 degrees C, both Ca2+ (4.0 mM) and Mg2+ (8.0 mM) decreased surface bound radioligand by 60 percent (P less than or equal to 0.01) and lowered internalized radioactivity. These data demonstrate that (1) bacitracin decreases the binding of VIP to small intestinal epithelial cells, (2) both Ca2+ and Mg2+ affect the binding of VIP to its surface receptor and (3) VIP is internalized into epithelial cells.     

Trefoil peptides promote restitution of wounded corneal epithelial cells

The ocular surface shares many characteristics with mucosal surfaces. In both, healing is regulated by peptide growth factors, cytokines, and extracellular matrix proteins. However, these factors are not sufficient to ensure most rapid healing. Trefoil peptides are abundantly expressed epithelial cell products which exert protective effects and are key regulators of gastrointestinal epithelial restitution, the critical early phase of cell migration after mucosal injury. To assess the role of trefoil peptides in corneal epithelial wound healing, the effects of intestinal trefoil factor (ITF/TFF3) and spasmolytic polypeptide (SP/TFF2) on migration and proliferation of corneal epithelial cells were analyzed. Both ITF and SP enhanced restitution of primary rabbit corneal epithelial cells in vitro. While the restitution-enhancing effects of TGF-alpha and TGF-beta were both inhibited by neutralizing anti-TGF-beta-antibodies, trefoil peptide stimulation of restitution was not. Neither trefoil peptide significantly affected proliferation of primary corneal epithelial cells. ITF but not SP or pS2 mRNA was present in rabbit corneal and conjunctival tissues. In summary, the data indicate an unanticipated role of trefoil peptides in healing of ocular surface and demand rating their functional actions beyond the gastrointestinal tract.     

Induction of p53 by urokinase in lung epithelial cells

Urokinase plasminogen activator (uPA) is a serine protease that catalyzes the conversion of plasminogen to plasmin. The plasminogen/plasmin system includes the uPA, its receptor, and its inhibitor (plasminogen activator inhibitor-1). Interactions between these molecules regulate cellular proteolysis as well as adhesion, cellular proliferation, and migration, processes germane to the pathogenesis of lung injury and neoplasia. In previous studies, we found that uPA regulates cell surface fibrinolysis by regulating its own expression as well as that of the uPA receptor and plasminogen activator inhibitor-1. In this study, we found that uPA alters expression of the tumor suppressor protein p53 in Beas2B airway epithelial cells in both a time- and concentration-dependent manner. These effects do not require uPA catalytic activity because the amino-terminal fragment of uPA lacking catalytic activity was as potent as two chain active uPA. Single chain uPA also enhanced p53 expression to the same extent as intact two chain active uPA and the amino-terminal fragment. Pretreatment of cells with anti-beta1 integrin antibody blocked uPA-induced p53 expression. uPA-induced p53 expression occurs without increased p53 mRNA expression. However, uPA induced oncoprotein MDM2 in a concentration-dependent manner. uPA-induced p53 expression does not require activation of tyrosine kinases. Inactivation of protein-tyrosine phosphatase SHP-2 inhibits both basal and uPA-induced p53 expression. Plasmin did not alter uPA-mediated p53 expression. The induction of p53 expression by exposure of lung epithelial cells to uPA is a newly recognized pathway by which urokinase may influence the proliferation of lung epithelial cells. This pathway could regulate pathophysiologic alterations of p53 expression in the setting of lung inflammation or neoplasia.     

Expression of airway secretory epithelial functions by lung carcinoma cells

We examined 12 non-small cell lung carcinoma cell lines for expression of airway goblet, serous, and mucous cell characteristics. The cells expressed some ultrastructural traits of secretory epithelial cells but none contained secretory granules typical of the airway secretory cells. Using immunocytochemistry and cell-specific monoclonal antibodies, we identified heterogeneous expression of goblet, mucous, and serous cell markers among the cell lines. After metabolic radiolabeling, cells incorporated isotope into high molecular weight material. Incubation of pulse-radiolabeled cells with a number of known mucus secretogogues revealed that 5 of the 12 cell lines released radiolabeled material in response to the agonists. However, in each cell line only one of the receptor-activated pathways tested was intact. Although we did not identify a single cell line expressing a phenotype similar to normal airway secretory cells, particular functions retained by some of these cell lines may make them useful for specific studies of mucus production or secretion.     

Mechanotransduction of stretch-induced prostanoid release by fetal lung epithelial cells

Mechanical ventilation is the primary supportive treatment for infants and adults suffering from severe respiratory failure. Adverse mechanical ventilation (overdistension of the lung) triggers a proinflammatory response. Along with cytokines, inflammatory mediators such as bioactive lipids are involved in the regulation of the inflammatory response. The arachidonic acid pathway is a key source of bioactive lipid mediators, including prostanoids. Although ventilation has been shown to influence the production of prostanoids in the lung, the mechanotransduction pathways are unknown. Herein, we established that cyclic stretch of fetal lung epithelial cells, but not fibroblasts, can evoke an extremely sensitive, rapid alteration in eicosanoid metabolism through a cyclooxygenase (COX)-2 dependent mechanism. Cyclic stretch significantly increased PGI(2), PGF(2alpha), PGD(2), PGE(2), and thromboxane B(2) levels in the media of epithelial cells, but did not alter leukotriene B(4) or 12-hydroxyeicosatetraenoic acid levels. Inhibition of COX-2, but not COX-1, attenuated the cyclic stretch-induced PG increase in the media, suggesting that cyclic stretch primarily affected PG synthesis. Substrate (free arachidonic acid) availability for PG generation was increased because of a cyclic stretch-induced activation of cytosolic phospholipase A(2) (cPLA(2)) via an influx of extracellular calcium and phosphorylation by mitogen-activated protein kinase, p44/42MAPK. The data are compatible with cPLA(2) and COX-2 being intimately involved in regulating the injury response to adverse mechanical ventilation.     

Regulation of the mouse mammary tumor virus receptor by phosphorylation and internalization in mammary epithelial cells

The mouse mammary tumor virus enters mammary epithelial cells via a plasma membrane protein that binds to a viral envelope glycoprotein, gp52. In intact cells, this gp52 receptor can be phosphorylated by activators of protein kinase A and protein kinase C (PKC), but this modification does not occur in response to epidermal growth factor, whose receptor is a tyrosine kinase, or to gp52. Phosphorylation of the gp52 receptor rapidly leads to internalization and gradual loss of binding activity. Both the phosphorylation and the intrnalization induced by PKC are abolished by prior downregulation of this kinase. Although the physiological function of the gp52 receptor is unknown, its binding to gp52 can stimulate several biological activities, including amino acid accumulation. Receptor processingimpairs this gp52-induced amino acid uptake, as well as viral infection, by depleting the binding protein at the cell surface. In contrast, PKC augments insulin-induced amino acid transport, and PKC downregulation abolishes the action of insulin, suggesting that insulin and gp52 utlize partially separate pathways leading to amino acid transport. These data further suggest that PKC may be involved in this insulin-stimulated activity. © 1994 Wiley-Liss, Inc.     

CpG oligodeoxynucleotides promote phospholipase D dependent phagolysosome maturation and intracellular mycobacterial killing in M. tuberculosis infected type II alveolar epithelial cells

CpG oligodeoxynucleotides have been previously shown to enhance antimycobacterial response in human monocytes/macrophages. The present study reports evidences showing the capability of CpG oligodeoxynucleotides to induce (i) host phospholipase D (PLD) activation, (ii) PLD dependent reactive oxygen intermediate production, (iii) PLD dependent phagolysosome maturation and (iv) PLD dependent intracellular mycobacterial killing in type II alveolar epithelial cells. These are the first evidences showing that alveolar epithelial cells may represent efficient effecter cells during primary innate antimycobacterial immune response.     

Detyrosinated microtubule protrusions in suspended mammary epithelial cells promote reattachment

Breast tumor cells enter the bloodstream long before the development of clinically evident metastasis. However, the early presence of such bloodborne cells predicts poor patient outcome. Nearly 90% of human breast tumors arise as carcinomas from mammary epithelial cells, so it is important to study how these cells respond to the detached conditions that they would experience in the bloodstream. We report here that mammary epithelial cell lines produce long and dynamic protrusions of the plasma membrane when detached. Although human and mouse mammary epithelial cell lines die by apoptosis within 16 h of detachment, this protrusive response persists for days in cells overexpressing either Bcl-2 or Bcl-xL. Unlike actin-dependent invadopodia and podosomes, these protrusions are actually enhanced by actin depolymerization with Cytochalasin-D or Latrunculin-A. Immunofluorescence and Western blotting demonstrate that the protrusions are enriched in detyrosinated Glu-tubulin, a post-translationally modified form of alpha-tubulin that is found in stabilized microtubules. Video microscopy indicates that these protrusions promote cell-cell attachment, and inhibiting microtubule-based protrusions correlates with reduced extracellular matrix attachment. Since bloodborne metastasis depends on both cell-cell and cell-matrix attachment, microtubule-based protrusions in detached mammary epithelial cells provide a novel mechanism that could influence the metastatic spread of breast tumors.     

Microrheology of human lung epithelial cells measured by atomic force microscopy

Lung epithelial cells are subjected to large cyclic forces from breathing. However, their response to dynamic stresses is poorly defined. We measured the complex shear modulus (G(*)(omega)) of human alveolar (A549) and bronchial (BEAS-2B) epithelial cells over three frequency decades (0.1-100 Hz) and at different loading forces (0.1-0.9 nN) with atomic force microscopy. G(*)(omega) was computed by correcting force-indentation oscillatory data for the tip-cell contact geometry and for the hydrodynamic viscous drag. Both cell types displayed similar viscoelastic properties. The storage modulus G'(omega) increased with frequency following a power law with exponent approximately 0.2. The loss modulus G"(omega) was approximately 2/3 lower and increased similarly to G'(omega) up to approximately 10 Hz, but exhibited a steeper rise at higher frequencies. The cells showed a weak force dependence of G'(omega) and G"(omega). G(*)(omega) conformed to the power-law model with a structural damping coefficient of approximately 0.3, indicating a coupling of elastic and dissipative processes within the cell. Power-law behavior implies a continuum distribution of stress relaxation time constants. This complex dynamics is consistent with the rheology of soft glassy materials close to a glass transition, thereby suggesting that structural disorder and metastability may be fundamental features of cell architecture.     

Induction of COX-2 by acrolein in rat lung epithelial cells

Acrolein is a highly reactive alpha, beta-unsaturated aldehyde, and a product of lipid peroxidation reactions. Acrolein is also an environmental pollutant and a key component of cigarette smoke, and has been implicated in multiple respiratory diseases. Lung tissue is a primary target for acrolein toxicity in smokers and may lead to chronic lung inflammation and lung cancer. Chronic inflammation, associated with expression of cyclooxygenase-2 (COX-2) and prostaglandins, are predisposing factors for malignancy. In this study, we investigated the induction of COX-2 by acrolein in rat lung epithelial cells and its related signaling cascade. Induction of COX-2 by acrolein was significant at 6 h post-treatment and was dependent upon NFκB activation. The activation of NFκB by acrolein was induced as a result of degradation of IκBα over the time of treatment. In addition, the upstream signaling cascade involved Raf-1/ERK activation by acrolein in the COX-2 induction and was inhibited by GW5074 (a Ras/Raf-1/ERK inhibitor), thereby providing evidence for the role of this cascade in this process. The results of these studies offer an explanation for the mechanism of COX-2 induction by acrolein in rat lung epithelial cells.