Vascular adhesion protein-1 mediates adhesion and transmigration of lymphocytes on human hepatic endothelial cells

Vascular adhesion protein-1 (VAP-1) is an amine oxidase and adhesion receptor that is expressed by endothelium in the human liver. The hepatic sinusoids are perfused by blood at low flow rates, and sinusoidal endothelium lacks selectin expression and has low levels of CD31, suggesting that VAP-1 may play a specific role in lymphocyte recruitment to the liver. In support of this we now report the constitutive expression of VAP-1 on human hepatic sinusoidal endothelial cells (HSEC) in vitro and demonstrate that VAP-1 supports adhesion and transmigration of lymphocytes across these cells under physiological shear stress. These are the first studies to report the function of VAP-1 on primary human endothelial cells. Under static conditions lymphocyte adhesion to unstimulated HSEC was dependent on VAP-1 and ICAM-2, whereas adhesion to TNF-alpha-stimulated HSEC was dependent on ICAM-1, VCAM-1, and VAP-1. Under conditions of flow, blocking VAP-1 reduced lymphocyte adhesion to TNF-alpha-treated HSEC by 50% and significantly reduced the proportion of adherent lymphocytes that transmigrated across cytokine or LPS-activated endothelium. In addition, inhibition of the amine oxidase activity of VAP-1 reduced both adhesion and transmigration of lymphocytes to a level similar to that seen with VAP-1 Ab. Thus, VAP-1 can support transendothelial migration as well as adhesion, and both functions are dependent on its enzymatic activity. In the absence of selectins and CD31, VAP-1 may play a specific role in lymphocyte recruitment via hepatic sinusoidal endothelium. Moreover, since VAP-1 is induced on nonhepatic endothelium in response to inflammation, its ability to support lymphocyte transendothelial migration may be an important systemic function of VAP-1.     

Ninjurin1 is expressed in myeloid cells and mediates endothelium adhesion in the brains of EAE rats

Ninjurin1 (nerve injury-induced protein, Ninj1) is an adhesion molecule that is essential for cell-to-cell interactions. However, little is known about the function of Ninj1 in the central nervous system (CNS). To address its role in the CNS, we analyzed the expression pattern of Ninj1 in normal rats and in an experimental autoimmune encephalomyelitis (EAE) model. Ninj1 was expressed in three major compartments of brains, meninges, the choroid plexus, and parenchymal perivascular spaces. In the EAE brains, Ninj1 was strongly expressed in myeloid cells (macrophages/monocytes and neutrophils) and partially expressed in endothelial cells (ECs). Furthermore, Ninj1 enhanced adhesion between BV2 cells (murine monocyte lineage microglia) and HBMECs (human brain microvascular endothelial cells). Collectively, our findings suggest that Ninj1 may mediate the entry of myeloid cells into the CNS in normal and EAE brains, and it is a potential therapeutic target for regulating myeloid cell trafficking across the blood-brain barrier (BBB) in CNS immune processes.     

P-selectin mediates metastatic progression through binding to sulfatides on tumor cells

Hematogenous carcinoma metastasis is associated with tumor cell emboli formation, which is now known to be facilitated by selectins. P-selectin-mediated interactions of platelets with cancer cells are based mostly on mucin- and glycosaminoglycan-type selectin ligands. We previously showed that mouse colon carcinoma cells (MC-38) carry P-selectin ligands of nonmucin origin, which were not identified. Here we show that P-selectin ligands recognized on MC-38 cells are sulfated glycolipids, thereby facilitating experimental metastasis in a syngeneic mouse model. Metabolic inhibition of sulfation by incubation of cells with sodium chlorate almost completely abrogated P-selectin binding. Metabolic labeling of MC-38 cells with (35)S sulfate revealed only a single band as detected by high-performance thin layer chromatography analysis of a total lipid extract. Matrix-assisted laser desorption/ionization tandem time-of-flight/time-of-flight analysis (MALDI-TOF-TOF) analysis of the purified sulfate-containing lipid fraction identified the selectin ligand to be a sulfated galactosylceramide SM4 (HSO(3)-3Galbeta-1Cer). Modulation of glycolipid biosynthesis in MC-38 cells altered P-selectin binding, thereby confirming sulfoglycolipids to be major P-selectin ligands. In addition, P-selectin was also found to recognize lactosylceramide sulfate SM3 (HSO(3)-3Galbeta-4Glcbeta-1Cer) and gangliotriaosylceramide sulfate SM2 [GalNAcbeta-4(HSO(3)-3)Galbeta-4Glcbeta-1Cer] in human hepatoma cells. Finally, the enzymatic removal of sulfation from the cell surface of MC-38 cells resulted in decreased P-selectin binding and led to attenuation of metastasis. Thus, SM4 sulfatide serves as a native ligand for P-selectin contributing to cell-cell interactions and to facilitation of metastasis.     

The homophilic binding of junctional adhesion molecule-C mediates tumor cell-endothelial cell interactions

The junctional adhesion molecule C (JAM-C) was recently shown to undergo a heterophilic interaction with the leukocyte beta2 integrin Mac-1, thereby mediating interactions between vascular cells in inflammatory cell recruitment. Here, the homophilic interaction of JAM-C is presented and functionally characterized to mediate tumor cell-endothelial cell interactions. Recombinant soluble JAM-C in fluid phase bound to immobilized JAM-C as assessed in a purified system; moreover, JAM-C-transfected Chinese hamster ovary (CHO) cells adhered to immobilized JAM-C. The homophilic interaction of JAM-C was mediated by the isolated amino-terminal Ig domain (D1), but not the carboxyl-terminal Ig domain (D2), of the molecule. Dimerization of JAM-A is dependent on the sequence RVE in the amino-terminal Ig domain. This motif is conserved in JAM-C (Arg64-Ile65-Glu66), and a single amino acid mutation in this motif (E66R) abolished the homophilic interaction of JAM-C. The lung carcinoma cell line NCI-H522 was found to express JAM-C. NCI-H522 cells adhered to immobilized JAM-C, as well as to JAM-C-transfected CHO cells, but not to mock-transfected CHO cells or to CHO cells transfected with the JAM-C mutant (E66R). Adhesion of NCI-H522 cells to JAM-C protein or JAM-C-transfected CHO cells was abolished in the presence of soluble JAM-C or the isolated D1. Furthermore, the adhesion of NCI-H522 cells to endothelial cells was significantly blocked by soluble JAM-C or the isolated D1. Thus, JAM-C undergoes a homophilic interaction via the Arg64-Ile65-Glu66 motif on the membrane-distal Ig domain of the molecule. The homophilic interaction of JAM-C can mediate tumor cell-endothelial cell interactions and may thereby be involved in the process of tumor cell metastasis.     

Inhibition of basal and tumor necrosis factor-enhanced binding of murine tumor cells to murine endothelium by transforming growth factor-beta 1.

The adherence of cells to microvascular endothelium is important in a number of processes, including inflammatory responses and metastasis. It has been demonstrated that in human models, cytokines such as TNF, IL-1, IFN-gamma increase the adhesiveness of endothelium for cells of the immune and inflammatory system by stimulating the expression of cell adhesion molecules on endothelial cell surfaces. We and others have shown similar cytokine-induced endothelial adhesiveness for tumor cells in murine and human models. In contrast to the effect of those modulators, transforming growth factor-beta (TGF-beta) has been shown to inhibit the binding of human neutrophils and T lymphocytes to human endothelium, although the mechanism of TGF-beta action remains unknown. Little is known about the effect of TGF-beta on tumor cell-endothelial interaction. In the present study, we demonstrate that TGF-beta inhibits basal and TNF-enhanced binding of murine P815 mastocytoma cells to murine microvascular endothelium (MME). The alterations in MME mediated by TGF-beta, also lead to the inhibition of adherence of murine splenocytes, thymocytes, and human lymphoblastoid cells but do not inhibit adherence of murine B16 melanoma cells. The effect of TGF-beta is transient and inhibition of the endothelial adhesive phenotype is strongest 12 to 24 h after addition of the factor to MME. The TGF-beta-mediated inhibition of P815 basal binding to endothelium is dependent on protein synthesis because cycloheximide reverses the TGF-beta effect. TGF-beta does not appear to activate classical signal transduction pathways. Inhibitors of G proteins do not abolish TGF-beta action, protein kinase C and protein kinase A activators elicit an effect opposite to that of the factor, TGF-beta does not increase intracellular cAMP levels, and finally calcium-mobilizing agents do not mimic, but rather inhibit the effect of TGF-beta. However, TGF-beta-mediated inhibition of both basal binding and TNF-enhanced P815 binding to MME is completely abolished in the presence of the protein phosphatase inhibitor okadaic acid which suggests that TGF-beta may elicit its effect by stimulating protein phosphatase activity.     

Wnt-1 modulates cell-cell adhesion in mammalian cells by stabilizing beta-catenin binding to the cell adhesion protein cadherin

Wnt-1 homologs have been identified in invertebrates and vertebrates and play important roles in cellular differentiation and organization. In Drosophila, the products of the segment polarity genes wingless (the Wnt-1 homolog) and armadillo participate in a signal transduction pathway important for cellular boundary formation in embryonic development, but functional interactions between the proteins are unknown. We have examined Wnt-1 function in mammalian cells in which armadillo (beta-catenin and plakoglobin) is known to bind to and regulate cadherin cell adhesion proteins. We show that Wnt-1 expression results in the accumulation of beta-catenin and plakoglobin. In addition, binding of beta-catenin to the cell adhesion protein, cadherin, is stabilized, resulting in a concomitant increase in the strength of calcium-dependent cell-cell adhesion. Thus, a consequence of the functional interaction between Wnt-1 and armadillo family members is the strengthening of cell-cell adhesion, which may lead to the specification of cellular boundaries.     

MUC1 mediates Pneumocystis murina binding to airway epithelial cells

Previous studies have shown that Pneumocystis binds to pneumocytes, but the proteins responsible for binding have not been well defined. Mucins are the major glycoproteins present in mucus, which serves as the first line of defence during airway infection. MUC1 is the best characterised membrane‐tethered mucin and is expressed on the surface of most airway epithelial cells. Although by electron microscopy Pneumocystis primarily binds to type I pneumocytes, it can also bind to type II pneumocytes. We hypothesized that Pneumocystis organisms can bind to MUC1 expressed by type II pneumocytes. Overexpression of MUC1 in human embryonic kidney HEK293 cells increased Pneumocystis binding, while knockdown of MUC1 expression by siRNA in A549 cells, a human adenocarcinoma‐derived alveolar type II epithelial cell line, decreased Pneumocystis binding. Immunofluorescence labelling indicated that MUC1 and Pneumocystis were co‐localised in infected mouse lung tissue. Incubation of A549 cells with Pneumocystis led to phosphorylation of ERK1/2 that increased with knockdown of MUC1 expression by siRNA. Pneumocystis caused increased IL‐6 and IL‐8 secretion by A549 cells, and knockdown of MUC1 further increased their secretion in A549 cells. Taken together, these results suggest that binding of Pneumocystis to MUC1 expressed by airway epithelial cells may facilitate establishment of productive infection.     

Lipoxygenase metabolites of arachidonic and linoleic acids modulate the adhesion of tumor cells to endothelium via regulation of protein kinase C.

12(S)-hydroxyeicosatetraenoic acid (12[S]-HETE) and 13(S)-hydroxyoctadecadienoic acid (13[S]-HODE), lipoxygenase metabolites of arachidonic acid and linoleic acid, respectively, previously have been suggested to regulate tumor cell adhesion to endothelium during metastasis. Adhesion of rat Walker carcinosarcoma (W256) cells to a rat endothelial cell monolayer was enhanced after treatment with 12(S)-HETE and this 12(S)-HETE enhanced adhesion was blocked by 13(S)-HODE. Protein kinase inhibitors, staurosporine, calphostin C, and 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine, inhibited the 12(S)-HETE enhanced W256 cell adhesion. Depleting W256 cells of protein kinase C (PKC) with phorbol 12-myristate-13-acetate abolished their ability to respond to 12(S)-HETE. Treatment of W256 cells with 12(S)-HETE induced a 100% increase in membrane-associated PKC activity whereas 13(S)-HODE inhibited the effect of 12(S)-HETE on PKC translocation. High-performance liquid chromatographic analysis revealed that in W256 cells 12-HETE and 13-HODE were two of the major lipoxygenase metabilites of arachidonic acid and linoleic acid, respectively. Therefore, these two metabolites may provide an alternative signaling pathway for the regulation of PKC. Further, these findings suggest that the regulation of tumor cell adhesion to endothelium by 12(S)-HETE and 13(S)-HODE may be a PKC-dependent process.     

MAC-1 mediates adherence of human monocytes to endothelium via a protein kinase C dependent mechanism

Leukocyte adherence is mediated by a superfamily of glycoproteins denoted LFA-1 (the lymphocyte function-associated antigen-1), Mac-1 (macrophage antigen-1) and p150,95. The relative importance of these in mediating human monocyte adherence to endothelium, and the biochemical mechanisms which modulate these events, are not understood. In this report, the role of protein kinase C (pkC) in regulating human monocyte adherence to endothelial cells has been investigated. Addition of phorbol 12,13-dibutyrate (PDBu), which specifically stimulates pkC, caused a dose-dependent increase in their adherence to monolayers of bovine aortic endothelial cells. 4 alpha-phorbol didecanoate (4 alpha-PDD), a structural analogue of PDBu which does not stimulate pkC, failed to increase monocyte adhesion. PDBu also produced a dose-dependent increase in the expression of both Mac-1 and p150,95. The pkC-stimulated adherence of monocytes to endothelium was inhibited by the presence of a monoclonal antibody to Mac-1, while monoclonal antibodies to p150,95 and LFA-1 did not influence adherence. It is concluded that monocyte adherence to endothelial cells is regulated through a pkC-dependent mechanism; moreover, this process is mediated primarily via the Mac-1 adhesion glycoprotein.     

Chloroplastic protein NRIP1 mediates innate immune receptor recognition of a viral effector

Plant innate immunity relies on the recognition of pathogen effector molecules by nucleotide-binding-leucine-rich repeat (NB-LRR) immune receptor families. Previously we have shown the N immune receptor, a member of TIR-NB-LRR family, indirectly recognizes the 50 kDa helicase (p50) domain of Tobacco mosaic virus (TMV) through its TIR domain. We have identified an N receptor-interacting protein, NRIP1, that directly interacts with both N's TIR domain and p50. NRIP1 is a functional rhodanese sulfurtransferase and is required for N to provide complete resistance to TMV. Interestingly, NRIP1 that normally localizes to the chloroplasts is recruited to the cytoplasm and nucleus by the p50 effector. As a consequence, NRIP1 interacts with N only in the presence of the p50 effector. Our findings show that a chloroplastic protein is intimately involved in pathogen recognition. We propose that N's activation requires a prerecognition complex containing the p50 effector and NRIP1.     

Vascular adhesion protein 1 (VAP-1) functions as a molecular brake during granulocyte rolling and mediates recruitment in vivo.

Granulocyte extravasation from the blood into tissues is a prerequisite for a proper inflammatory response. It is regulated by a multistep adhesion cascade consisting of successive contacts between leukocyte surface receptors and their endothelial ligands on vessels. Vascular adhesion protein 1 (VAP-1) is an endothelial surface glycoprotein with two functions. It is an enzyme (monoamine oxidase) and an adhesion molecule for lymphocytes. Its function in binding of granulocytes or in leukocyte trafficking into sites of inflammation in vivo has remained unknown. Here we show that treatment of rabbits with anti-VAP-1 monoclonal antibodies abrogates approximately 70% of granulocyte extravasation into a site of an experimental inflammation. Using intravital microscopy, VAP-1 blockade is shown to increase the velocity of the rolling granulocytes and the frequency of their jerky skippings during the rolling. In addition, the number of firmly bound leukocytes decreased by 44% when VAP-1 was rendered nonfunctional. Our results suggest that VAP-1 functions as a molecular brake early in the adhesion cascade and consequently decreases the firm adherence; it may also directly influence the transmigration step. These data elucidate a new interplayer in the granulocyte extravasation process and provide a novel physiological function for a member of the monoamine oxidase family.     

Helicobacter pylori-downregulated tumor necrosis factor receptor-associated protein 1 mediates apoptosis of human gastric epithelial cells

Heat shock proteins (HSPs) are crucial proteins in maintaining the homeostasis of human gastric epithelial cells. Tumor necrosis factor receptor-associated protein 1 (TRAP1), a member of the HSP90 family, has been shown to be involved in various crucial physiological processes, particularly against apoptosis. However, the regulation and function of TRAP1 in Helicobacter pylori infection is still unknown. Here, we found that TRAP1 expression was downregulated on human gastric epithelial cells during H. pylori infection by real-time polymerase chain reaction (PCR) and western blot analysis. Through virulence factors mutant H. pylori strains infection and inhibitors screening, we found that H. pylori vacuolating cytotoxin A ( vacA), but not cytotoxin-associated gene A ( cagA) protein, induced human gastric epithelial cells to downregulate TRAP1 via P38MAPK pathway by real-time PCR and western blot analysis. Furthermore, downregulation of TRAP1 with lentivirus carrying TRAP1 short hairpin RNA constructs impairs mitochondrial function, and increases apoptosis of gastric epithelial cells. The results indicate that H. pylori vacA downregulated TRAP1 is involved in the regulation of gastric epithelial cell apoptosis.     

A single binding site mediates resistance- and disease-associated activities of the effector protein NIP1 from the barley pathogen Rhynchosporium secalis

The effector protein NIP1 from the barley (Hordeum vulgare) pathogen Rhynchosporium secalis specifically induces the synthesis of defense-related proteins in cultivars of barley expressing the complementary resistance gene, Rrs1. In addition, it stimulates the activity of the barley plasma membrane H(+)-ATPase in a genotype-unspecific manner and it induces necrotic lesions in leaf tissues of barley and other cereal plant species. NIP1 variants type I and II, which display quantitative differences in their activities as elicitor and H(+)-ATPase stimulator, and the inactive mutant variants type III* and type IV*, were produced in Escherichia coli. Binding studies using (125)I-NIP1 type I revealed a single class of binding sites with identical binding characteristics in microsomes from near-isogenic resistant (Rrs1) and susceptible (rrs1) barley. Binding was specific, reversible, and saturable, and saturation ligand-binding experiments yielded a K(d) of 5.6 nm. A binding site was also found in rye (Secale cereale) and the nonhost species wheat (Triticum aestivum), oat (Avena sativa), and maize (Zea mays), but not in Arabidopsis (Arabidopsis thaliana). For NIP1 types I and II, equilibrium competition-binding experiments revealed a correlation between the difference in their affinities to the binding site and the differences in their elicitor activity and H(+)-ATPase stimulation, indicating a single target molecule to mediate both activities. In contrast, the inactive proteins type III* and type IV* are both characterized by high affinities similar to type I, suggesting that binding of NIP1 to this target is not sufficient for its activities.     

Fatty acid modulation of tumor cell adhesion to microvessel endothelium and experimental metastasis

Tumor cell interaction with the endothelium of the vessel wall is a rate limiting step in metastasis. The fatty acid modulation of this interaction was investigated in low (LM) and high (HM) metastatic B16 amelanotic melanoma (B16a) cells. 12(S)-HETE increased the adhesion of LM cells to endothelium derived from pulmonary microvessels. All other monohydroxy and dihydroxy fatty acids were ineffective. LTB₄ induced a modest stimulation but LTC₄, LTD₄, LTE₄ as well as LXA₄ and LXB₄ were ineffective. The 12(S)-HETE enhanced adhesion of B16a cells was inhibited by pretreatment with 13(S)-HODE but not by 13(R)-, 9(S)-HODE or 13-OXO-ODE. 13(S)-HODE decreased adhesion of HM B16a cells to endothelium. 12(S)-HETE enhanced surface expression of integrin αIIbβ₃ and monoclonal antibodies against this integrin but not against α₅β₁, blocked enhanced but not basal adhesion to endothelium. Intravenous injection of 12(S)-HETE treated LM cells resulted in increased lung colonization (experimental metastasis). This effect was specific for 12(S)-HETE and was inhibited by 13(S)-HODE but not by other HODE's. 12(S)-HETE also enhanced lung colonization by HM cells and 13(S)-HODE decreased lung colonization by HM cells. Our results suggest a highly specific bidirectional modulation of metastatic phenotype and lung colonization by 12(S)-HETE and 13(S)-HODE.     

Haptoglobin-related protein mediates trypanosome lytic factor binding to trypanosomes

Trypanosome lytic factor (TLF-1) is an unusual high density lipoprotein (HDL) found in human serum that is toxic to Trypanosoma brucei brucei and may be critical in preventing human infections by this parasite. TLF-1 is composed of four major apolipoproteins: apolipoprotein AI, apolipoprotein AII, paraoxonase, and the primate-specific haptoglobin-related protein (Hpr). Hpr is greater than 90% homologous to haptoglobin (Hp), an abundant acute phase serum protein. Killing of trypanosomes by TLF-1 requires cell surface binding, endocytosis, and subsequent lysosomal targeting. Low temperature binding studies reveal two receptors for TLF-1: one that is high affinity/low capacity (K(d) approximately 12 nm, 350 receptors per cell) and another that binds with low affinity/high capacity (K(d) approximately 1 microm, 60,000 receptors per cell). The low affinity binding is competed by nonlytic human HDL and is likely to be apolipoprotein AI-mediated. Purified human Hpr and human Hp bind to trypanosomes, are internalized, and are targeted to the lysosome. Furthermore, Hpr shows competition for TLF-1 binding, and a monoclonal antibody against Hpr prevents both TLF-1 uptake and trypanosome killing. Based on these results, we propose that Hpr mediates the high affinity binding of TLF-1 to T. b. brucei through a haptoglobin-like receptor.     

Bartonella effector protein C mediates actin stress fiber formation via recruitment of GEF-H1 to the plasma membrane

Bartonellae are Gram-negative facultative-intracellular pathogens that use a type-IV-secretion system (T4SS) to translocate a cocktail of Bartonella effector proteins (Beps) into host cells to modulate diverse cellular functions. BepC was initially reported to act in concert with BepF in triggering major actin cytoskeletal rearrangements that result in the internalization of a large bacterial aggregate by the so-called ‘invasome’. Later, infection studies with bepC deletion mutants and ectopic expression of BepC have implicated this effector in triggering an actin-dependent cell contractility phenotype characterized by fragmentation of migrating cells due to deficient rear detachment at the trailing edge, and BepE was shown to counterbalance this remarkable phenotype. However, the molecular mechanism of how BepC triggers cytoskeletal changes and the host factors involved remained elusive. Using infection assays, we show here that T4SS-mediated transfer of BepC is sufficient to trigger stress fiber formation in non-migrating epithelial cells and additionally cell fragmentation in migrating endothelial cells. Interactomic analysis revealed binding of BepC to a complex of the Rho guanine nucleotide exchange factor GEF-H1 and the serine/threonine-protein kinase MRCKα. Knock-out cell lines revealed that only GEF-H1 is required for mediating BepC-triggered stress fiber formation and inhibitor studies implicated activation of the RhoA/ROCK pathway downstream of GEF-H1. Ectopic co-expression of tagged versions of GEF-H1 and BepC truncations revealed that the C-terminal ‘Bep intracellular delivery’ (BID) domain facilitated anchorage of BepC to the plasma membrane, whereas the N-terminal ‘filamentation induced by cAMP’ (FIC) domain facilitated binding of GEF-H1. While FIC domains typically mediate post-translational modifications, most prominently AMPylation, a mutant with quadruple amino acid exchanges in the putative active site indicated that the BepC FIC domain acts in a non-catalytic manner to activate GEF-H1. Our data support a model in which BepC activates the RhoA/ROCK pathway by re-localization of GEF-H1 from microtubules to the plasma membrane.     

Prostate tumor CXC-chemokine profile correlates with cell adhesion to endothelium and extracellular matrix

Though chemokines of the CXC family are thought to play key roles in neoplastic transformation and tumor invasion, information about CXC chemokines in prostate cancer is sparse. To evaluate the involvement of CXC chemokines in prostate cancer, we analyzed the CXC coding mRNA of both chemokine ligands (CXCL) and chemokine receptors (CXCR), using the prostate carcinoma cell lines PC-3, DU-145 and LNCaP. CXCR proteins were further evaluated by Western blot, CXCR surface expression by flow cytometry and confocal microscopy. The expression pattern was correlated to adherence of the tumor cells to an endothelial cell monolayer or to extracellular matrix components. Based on growth and adhesion capacity, PC-3 and DU-145 were identified to be highly aggressive tumor cells (PC-3>DU-145), whereas LNCaP belonged to the low aggressive phenotype. CXCL1, CXCL3, CXCL5 and CXCL6 mRNA, chemokines with pro-angiogenic activity, were strongly expressed in DU-145 and PC-3, but not in LNCaP. CXCR3 and CXCR4 surface level differed in the following order: LNCaP>DU-145>PC-3. The differentiation factor, fatty acid valproic acid, induced intracellular CXCR accumulation. Therefore, prostate tumor malignancy might be accompanied by enhanced synthesis of angiogenesis stimulating CXC chemokines. Further, shifting CXCR3 and CXCR4 from the cell surface to the cytoplasm might activate pro-tumoral signalling events and indicate progression from a low to a highly aggressive phenotype.     

Galectin-9 suppresses tumor metastasis by blocking adhesion to endothelium and extracellular matrices

We previously described an inverse correlation between galectin-9 (Gal-9) expression and metastasis in patients with malignant melanoma and breast cancer. This study verified the ability of Gal-9 to inhibit lung metastasis in experimental mouse models using highly metastatic B16F10 melanoma and Colon26 colon cancer cells. B16F10 cells transfected with a secreted form of Gal-9 lost their metastatic potential. Intravenous Gal-9 administration reduced the number of metastases of both B16F10 and Colon26 cells in the lung, indicating that secreted Gal-9 suppresses metastasis. Analysis of adhesive molecule expression revealed that B16F10 cells highly express CD44, integrin alpha1, alpha 4, alpha V, and beta1, and that Colon26 cells express CD44, integrin alpha2, alpha 5, alpha V, and beta1, suggesting that Gal-9 may inhibit the adhesion of tumor cells to vascular endothelium and the extracellular matrix (ECM) by binding to such adhesive molecules. Indeed, Gal-9 suppressed the binding of hyaluronic acid to CD44 on both B16F10 and Colon26 cells, and also suppressed the binding of vascular cell adhesion molecule-1 to very late antigen-4 on B16F10 cells. Furthermore, Gal-9 inhibited the binding of tumor cells to ECM components, resulting in the suppression of tumor cell migration. The present results suggest that Gal-9 suppresses both attachment and invasion of tumor cells by inhibiting the binding of adhesive molecules on tumor cells to ligands on vascular endothelium and ECM.     

A mucus adhesion promoting protein, MapA, mediates the adhesion of Lactobacillus reuteri to Caco-2 human intestinal epithelial cells

Lactobacillus reuteri is one of the dominant lactobacilli found in the gastrointestinal tract of various animals. A surface protein of L. reuteri 104R, mucus adhesion promoting protein (MapA), is considered to be an adhesion factor of this strain. We investigated the relation between MapA and adhesion of L. reuteri to human intestinal (Caco-2) cells. Quantitative analysis of the adhesion of L. reuteri strains to Caco-2 cells showed that various L. reuteri strains bind not only to mucus but also to intestinal epithelial cells. In addition, purified MapA bound to Caco-2 cells, and this binding inhibited the adhesion of L. reuteri in a concentration-dependent manner. Based on these observations, the adhesion of L. reuteri appears due to the binding of MapA to receptor-like molecules on Caco-2 cells. Further, far-western analysis indicated the existence of multiple receptor-like molecules in Caco-2 cells.