Identification and characterization of ligands for L-selectin in the kidney. II. Expression of chondroitin sulfate and heparan sulfate proteoglycans reactive with L-selectin

Ligands for the leukocyte adhesion molecule L-selectin are expressed not only in lymph node high endothelial venules (HEV) but also in the renal distal tubuli. Here we report that L-selectin-reactive molecules in the kidney are chondroitin sulfate and heparan sulfate proteoglycans of 500-1000 kDa, unlike those in HEV bearing sialyl Lewis X-like carbohydrates. Binding of L-selectin to these molecules was mediated by the lectin domain of L-selectin and required divalent cations. Binding was inhibited by chondroitinase and/or heparitinase but not sialidase. Thus, L-selectin can recognize chondroitin sulfate and heparan sulfate glycosaminoglycans structurally distinct from sialyl Lewis X-like carbohydrates.     

Selectin blocking activity of a fucosylated chondroitin sulfate glycosaminoglycan from sea cucumber. Effect on tumor metastasis and neutrophil recruitment

Heparin is an excellent inhibitor of P- and L-selectin binding to the carbohydrate determinant, sialyl Lewis(x). As a consequence of its anti-selectin activity, heparin attenuates metastasis and inflammation. Here we show that fucosylated chondroitin sulfate (FucCS), a polysaccharide isolated from sea cucumber composed of a chondroitin sulfate backbone substituted at the 3-position of the beta-D-glucuronic acid residues with 2,4-disulfated alpha-L-fucopyranosyl branches, is a potent inhibitor of P- and L-selectin binding to immobilized sialyl Lewis(x) and LS180 carcinoma cell attachment to immobilized P- and L-selectins. Inhibition occurs in a concentration-dependent manner. Furthermore, FucCS was 4-8-fold more potent than heparin in the inhibition of the P- and L-selectin-sialyl Lewis(x) interactions. No inhibition of E-selectin was observed. FucCS also inhibited lung colonization by adenocarcinoma MC-38 cells in an experimental metastasis model in mice, as well as neutrophil recruitment in two models of inflammation (thioglycollate-induced peritonitis and lipopolysaccharide-induced lung inflammation). Inhibition occurred at a dose that produces no significant change in plasma activated partial thromboplastin time. Removal of the sulfated fucose branches on the FucCS abolished the inhibitory effect in vitro and in vivo. Overall, the results suggest that invertebrate FucCS may be a potential alternative to heparin for blocking metastasis and inflammatory reactions without the undesirable side effects of anticoagulant heparin.     

Gangliosides expressed on breast cancer cells are E-selectin ligands

Cancer cell adhesion to vascular endothelium is a critical process in hematogenous metastasis. We hypothesized that breast cancer cells express ligands that bind under blood flow conditions to E-selectin expressed by endothelial cells. At a hemodynamic wall shear rate, BT-20 and MDA-MB-468 breast cancer cells adhered to cytokine-activated human umbilical cord vein endothelial cells (HUVECs) but not to anti-E-selectin monoclonal antibody treated HUVECs, demonstrating that adhesion was specifically mediated by E-selectin. Characterization of glycans expressed on breast cancer cells by a panel of antibodies revealed that BT-20 cells expressed sialyl Lewis X (sLe^x) and sialyl Lewis A (sLe^a) but MDA-MB-468 cells did not, suggesting that the former possess classical glycans involved in E-selectin mediated adhesion while the latter have novel binding epitopes. Protease treatment of the breast cancer cells failed to significantly alter the carbohydrate expression profiles, binding to soluble E-selectin–Ig chimera, or the ability of the cells to tether and roll on E-selectin expressed by HUVECs, indicating that glycosphingolipids are functional E-selectin ligands on these cells. Furthermore, extracted breast cancer cell gangliosides supported binding of E-selectin–Ig chimera and adhesion of E-selectin transfected cells under physiological flow conditions. In summary, our results demonstrate that breast cancer cells express sialylated glycosphingolipids (gangliosides) as E-selectin ligands that may be targeted for prevention of metastasis.     

Sialyl Lewis(a): a tumor-associated carbohydrate antigen involved in adhesion and metastatic potential of cancer cells

Neoplastic transformation is often associated with characteristic changes in the expression of the sialyl Lewis(a) and sialyl Lewis(x) antigens, representing typical tumor-associated carbohydrate antigens. High amounts of sialyl Lewis(a) are present in human adenocarcinomas of the colon, pancreas and stomach. A growing amount of data suggests that this carbohydrate structure is the ligand for E-selectin. Sialylated Lewis structures present on the surface of tumor cells are carried by the carbohydrate chains of glycoproteins and glycolipids. There are several lines of evidence showing that sialyl Lewis(a) is responsible for the adhesion of human cancer cells to endothelium. E-selectin present on endothelial cells mediates these interactions. Selectins and their carbohydrate ligands can thus play an important role in the selective homing of tumor cells during metastasis. However, the presence of sialyl Lewis(a) antigen on the surface of tumor cells and their adhesion to E-selectin-expressing cells in in vitro adhesion assay by itself can not be directly related to metastatic properties of all cancer cells.     

Sialyl Lewis(x)/E-selectin-mediated rolling in a cell-free system.

Selections mediate transient adhesion of neutrophils to stimulated endothelial cells at sites of inflammation by binding counter-receptors that present carbohydrates such as sialyl Lewis(x). We have developed a cell-free adhesion assay using sialyl Lewis(x)-coated microspheres and E-selection-IgG chimera-coated substrates to investigate the premise that rolling primarily results from functional properties of selection-carbohydrate bonds, whereas cellular morphology and signaling act as secondary effects. Sialyl Lewis(x)-coated microspheres attach to and roll over E-selectin-IgG chimera-coated substrates between the physiological wall shear stresses of 0.7 and 2 dynes/cm2. Rolling velocities vary with time and depend on E-selectin-IgG chimera site density and wall shear stress. Our results show that sialyl Lewis(x) is a minimal functional recognition element required for rolling on E-selectin under flow.     

Regulation of sialyl Lewis antigen expression in colon cancer cells by sialidase NEU4

Sialyl Lewis antigens, sialyl Lewis a and sialyl Lewis x, are utilized as tumor markers, and their increase in cancer is associated with tumor progression by enhancement of cancer cell adhesion to endothelial E-selectin. However, regulation mechanisms are not fully understood. We previously demonstrated that NEU4 is the only sialidase efficiently acting on mucins and it is down-regulated in colon cancer. To elucidate the significance of NEU4 down-regulation, we investigated sialyl Lewis antigens as endogenous substrates for the sialidase. NEU4 was found to hydrolyze the antigens in vitro and decrease cell surface levels much more effectively than other sialidases. Western blot, thin layer chromatography, and metabolic inhibition studies of desialylation products revealed NEU4 to preferentially catalyze sialyl Lewis antigens expressed on O-glycans. Cell adhesion to and motility and growth on E-selectin were significantly reduced by NEU4. E-selectin stimulation of colon cancer cells enhanced cell motility through activation of the p38/Hsp27/actin reorganization pathway, whereas NEU4 attenuated the signaling. On immunocytochemical analysis, some NEU4 molecules were localized at cell surfaces. Under hypoxia conditions whereby the antigens were increased concomitantly with several sialyl- and fucosyltransferases, NEU4 expression was markedly decreased. These results suggest that NEU4 plays an important role in control of sialyl Lewis antigen expression and its impairment in colon cancer.     

Effect of sialyl Lewis X-glycoliposomes on the inhibition of E-selectin-mediated tumour cell adhesion in vitro

The aim of this study was to evaluate the potential of different types of sialyl Lewis X-conjugated liposomes as competitive inhibitors for tumour cell adhesion to endothelial E-selectin. Sterically stabilised liposomes with the sLeX ligand at the terminal end of the polyethyleneglycol (PEG) chain, as well as vesicles that had the ligand embedded within the PEG-layer, were compared to ligand-bearing liposomes without sterical stabilisation. First, 14 different tumour cell lines were characterised for their expression of sialyl Lewis X and/or A. Tumour cell adhesion was characterised in three static assays in vitro using: (i) immobilised E-selectin, (ii) CHO cells, transfected to express E-selectin and (iii) human umbilical vein endothelial cells (HUVEC). Sterically stabilised liposomes with the ligand at the terminal end of the polyethylene chain were the most effective inhibitors in all three assays and inhibited the adhesion of HT29 colon- and Lewis lung (LL) carcinoma cells by about 60-80%. The binding was not affected by a PEG-coating of the liposomes. Sterical stabilisation, on the other hand, completely prevented macrophage uptake (J774 cell line) independently of the presence of the ligand, while plain liposomes were taken up in an amount of 5.4 nmol liposomal lipids/10(6) macrophages.     

Role of calcium in E-selectin induced phenotype of T84 colon carcinoma cells

The adhesion of cancer cells to the endothelium during the metastatic process involves the interaction of specific cell-cell adhesion receptors on the cell surface. E-selectin on endothelial cells and sialyl Lewis X carbohydrate component on tumor cells are mainly implicated in the adhesion of colon carcinoma cells to the endothelium of target organ. In this paper we show that binding of E-selectin to T84 colon tumor cells causes approximately a twofold increase in intracellular calcium concentration. In particular, using two inhibitors of receptor operated calcium channels, CAI and SK&F 96365, we present evidences that the augmentation in cytoplasmic calcium originates from ionic influx from extracellular sources. Furthermore, we demonstrated that modulation of [Ca2+]i by engagement of E-selectin receptor starts signal transduction pathways that affect cell spreading, tyrosine phosphorylation signaling, and cancer cell motility.     

E-selectin modulates the malignant properties of T84 colon carcinoma cells

The extravasation of metastatic cells is regulated by molecular events involving the initial adhesion of tumor cells to the endothelium and subsequently the migration of cells in the host connective tissue. E-selectin on endothelial cells and sialyl Lewis X carbohydrate component on tumor cells are mainly involved in the adhesion of colon carcinoma cells to the endothelium of target organ. Interaction of T84 colon cancer cells to purified E-selectin in vitro caused an increase in the tyrosine phosphorylation of a number of proteins as well as the modulation of cellular properties correlated to the metastatic phenotype. Specifically, E-selectin-stimulated actin reorganization, increased collagenase secretion, and induced cell migration. Treatment of T84 cells with herbimycin A inhibited cell adhesion as well as selectin-induced increase of cell migration, and cytoskeleton assembly. Our data demonstrate that binding of cancer cells to E-selectin starts signal transduction pathways which may affect the tumor metastatic abilities.     

Carbohydrate-mediated cell adhesion involved in hematogenous metastasis of cancer

The carbohydrate determinants, sialyl Lewis A and sialyl Lewis X, which are frequently expressed on human cancer cells, serve as ligands for a cell adhesion molecule of the selectin family, E-selectin, which is expressed on vascular endothelial cells. These carbohydrate determinants are involved in the adhesion of cancer cells to vascular endothelium and thus contribute to hematogenous metastasis of cancer. The initial adhesion mediated by these molecules triggers activation of integrin molecules through the action of several cytokines and leads to the extravasation of cancer cells. Cancer cells also produce humoral factors that facilitate E-selectin expression on endothelial cells. The degree of expression of the carbohydrate ligands at the surface of cancer cells is well correlated with the frequency of hematogenous metastasis and prognostic outcome of patients with cancers. The alteration of glycosyltransferase activities that leads to the enhanced expression of these carbohydrate ligands on cancer cell surface are currently being investigated. This revised version was published online in November 2006 with corrections to the Cover Date.     

Incomplete synthesis of the Sda/Cad blood group carbohydrate in gastrointestinal cancer

Cancer-associated changes in cell surface carbohydrates, including incomplete synthesis of normal carbohydrate epitopes, strongly affect malignant and metastatic potential. Here, we report that compensating for the cancer-associated loss of a single glycosyltransferase, beta1,4N-acetylgalactosaminyltransferase T2, dramatically decreased cell surface expression of both E-selectin ligands (sialyl Lewis(x) and sialyl Lewis(a)). This modification was associated with elevated expression of the Sd(a) carbohydrate determinant, which is expressed in normal gastrointestinal mucosa and is strikingly downregulated in cancer tissues. Loss of E-selectin ligands resulted in decreased adhesion of cancer cells to activated human endothelial cells in vitro and eventually suppressed metastatic potential in vivo.     

The three members of the selectin receptor family recognize a common carbohydrate epitope, the sialyl Lewis(x) oligosaccharide

The selectins (lectin-EGF-complement binding-cell adhesion molecules [LEC-CAMs]) are a family of mammalian receptors implicated in the initial interactions between leukocytes and vascular endothelia, leading to lymphocyte homing, platelet binding, and neutrophil extravasation. The three known selectins, L-selectin (leukocyte adhesion molecule-1 [LECAM-1]), E-selectin (endothelial-leukocyte adhesion molecule-1 [ELAM-1]), and P-selectin (GMP-140) share structural features that include a calcium-dependent lectin domain. The sialyl Lewis(x) carbohydrate epitope has been reported as a ligand for both E- and P-selectins. Although L-selectin has been demonstrated to bind to carbohydrates, structural features of potential mammalian carbohydrate ligand(s) have not been well defined. Using an ELISA developed with a sialyl Lewis(x)-containing glycolipid and an E-selectin-IgG chimera, we have demonstrated the direct binding of the L-selectin-IgG chimera to sialyl Lewis(x). This recognition was calcium dependent, and could be blocked by Mel-14 antibody but not by other antibodies. Recognition was confirmed by the ability of cells expressing the native L-selectin to adhere to immobilized sialyl Lewis(x). These data suggest that the sialyl Lewis(x) oligosaccharide may form the basis of a recognition domain common to all three selectins.     

Spectrum of sialylated and nonsialylated fuco-oligosaccharides bound by the endothelial-leukocyte adhesion molecule E-selectin. Dependence of the carbohydrate binding activity on E-selectin density.

Carbohydrate recognition by the human endothelial-leukocyte adhesion molecule, E-selectin, has been investigated by binding studies using 3H-labeled Chinese hamster ovary cells expressing different levels of the transfected full-length adhesion molecule and a series of structurally defined oligosaccharides linked to the lipid phosphatidylethanolamine dipalmitoate (neoglycolipids) and synthetic glycolipids chromatographed on silica gel plates or immobilized on plastic wells. Evidence is presented for density-dependent binding of the membrane-associated E-selectin not only to 3'-sialyl-lacto-N-fucopentaose II (3'-S-LNFP-II) and 3'-sialyl-lacto-N-fucopentaose III (3'-S-LNFP-III) which express the sialyl Le(a) and sialyl Le(x) antigens, respectively, but also to the nonsialylated analogue LNFP-II; there is a threshold density of E-selectin required for binding to these sialylated sequences, and binding to the nonsialylated analogue is a property only of cells with the highest density of E-selectin expression. The presence of fucose linked to subterminal rather than to an internal N-acetylglucosamine is shown to be a requirement for E-selectin binding, and although the presence of sialic acid 3-linked to the terminal galactose of the LNFP-II or LNFP-III sequences substantially enhances E-selectin binding, the presence of 6-linked sialic acid abolishes binding. E-selectin binding is unaffected in the presence of the blood group H fucose (alpha 1-2 linked to galactose to form the Le(b) antigen). However, the binding is abolished when in addition alpha 1-3-linked N-acetylgalactosamine to the galactose (blood group A antigen) is present. These results indicate that some E-selectin-mediated adhesive events may be influenced by blood group status.     

Binding of L-selectin to its vascular and extravascular ligands is differentially regulated by pH

Ligands for L-selectin, a leukocyte adhesion molecule, are expressed in high endothelial venules (HEVs) in lymph nodes and extravascular tissues, such as renal tubules. Here, we report that the binding of L-selectin to its vascular and extravascular ligands is differentially regulated by pH. The optimal L-selectin-dependent binding of leukocytes to HEVs was observed at pH 7.4, a physiological pH in the blood. In contrast, the optimal binding of leukocytes to the renal tubules was observed at pH 5.6. Consistently, optimal binding of soluble recombinant L-selectin to a major vascular ligand, 6-sulfo sialyl Lewis X, was observed at pH 7.4. Binding to extravascular ligands, such as chondroitin sulfate (CS) B, CS E and heparan sulfate, occurred at pH 5.6. Under physiological shear stress ranging from 1 to 2 dynes/cm², maximal leukocyte rolling on vascular ligands was observed at pH 6.8 to 7.4, and no rolling was detected at pH conditions below 5.6. These findings suggest that the pH environment is one important factor that determines leukocyte trafficking under physiological and pathological conditions.     

P- and E-selectins recognize sialyl 6-sulfo Lewis X, the recently identified L-selectin ligand

Recently we identified sialyl 6-sulfo Le(x) as a major L-selectin ligand on high endothelial venules of human peripheral lymph nodes. In this study we investigated the ligand activity of sialyl 6-sulfo Le(x) to E- and P-selectins and compared it with the binding activity of conventional sialyl Le(x), by using cultured human lymphoid cells expressing both carbohydrate determinants. The results of the recombinant selectin binding studies and the nonstatic monolayer cell adhesion assays indicated that both sialyl 6-sulfo Le(x) and conventional sialyl Le(x) served as ligand for E- and P-selectins, while L-selectin was quite specific to sialyl 6-sulfo Le(x). Anti-PSGL-1 antibodies as well as O-sialoglycoprotein endopeptidase treatment almost completely abrogated the binding of P-selectin but barely affected the binding of E-selectin, indicating that these carbohydrate determinants carried by O-glycans of PSGL-1 selectively serves as a ligand for P-selectin, while the ligand for E-selectin is not restricted to PSGL-1 nor to O-sialoglycoprotein endopeptidase-sensitive glycans. The binding of L-selectin was markedly reduced by O-sialoglycoprotein endopeptidase treatment but only minimally affected by anti-PSGL-1 antibodies, indicating that O-glycans carrying sialyl 6-sulfo Le(x) were the major L-selectin ligands, while PSGL-1 was only a minor core protein for L-selectin in these cells. These results indicated that each member of the selectin family has a distinct ligand binding specificity.     

Significant decrease in alpha1,3-linked fucose in association with increase in 6-sulfated N-acetylglucosamine in peripheral lymph node addressin of FucT-VII-deficient mice exhibiting diminished lymphocyte homing

Lymphocyte homing is mediated by binding of L-selectin on lymphocytes with L-selectin ligands present on high-endothelial venules (HEV) of peripheral and mesenteric lymph nodes. L-selectin ligands are specific O-linked carbohydrates, 6-sulfo sialyl Lewis X, composed of sialylated, fucosylated, and sulfated glycans. Abrogation of fucosyltransferase-VII (FucT-VII) results in almost complete loss of lymphocyte homing, but structural analysis of carbohydrates has not been carried out on FucT-VII null mice. To determine whether functional losses seen in FucT-VII null mice are caused by structural changes in carbohydrates, we elucidated the carbohydrate structure of GlyCAM-1, a major L-selectin counter-receptor. Our results show that most alpha1,3-fucosylated structures in 6-sulfo sialyl Lewis X are absent and 6-sulfo N-acetyllactosamine is increased in the mutant mice. Surprisingly, the amount of 6'-sulfated galactose (Gal) that bound to Sumbucus nigra agglutinin column was also increased. We found that structures of those oligosaccharides containing 6'-sulfated Gal are almost identical to those synthesized by keratan sulfate sulfotransferase (KSST). We then showed that overexpression of KSST suppresses the expression of sialyl Lewis X on Chinese hamster ovary (CHO) cells engineered to express sialyl Lewis X. Moreover, KSST expression in those cells suppressed lymphocyte rolling compared with mock-transfected CHO cells expressing 6-sulfo sialyl Lewis X. 6'-Sulfo sialyl Lewis X can neither be found in GlyCAM-1 from CHO cells expressing both KSST and FucT-VII nor be found in GlyCAM-1 from HEV of mice. These results combined together suggest that KSST competes with FucT-VII for the same acceptor substrate and downregulates the synthesis of L-selectin ligand by inhibiting alpha1,3-fucosylation.     

Formation of dermatan sulfate by cultured human skin fibroblasts. Effects of sulfate concentration on proportions of dermatan/chondroitin

[3H,35S]Dermatan/chondroitin sulfate glycosaminoglycans produced during culture of fibroblasts in medium containing varying concentrations of sulfate were tested for their susceptibility to chondroitin ABC lyase and chondroitin AC lyase. Chondroitin ABC lyase completely degraded [3H]hexosamine-labeled and [35S] sulfate-labeled dermatan/chondroitin sulfate to disaccharides. Chondroitin AC lyase treatment of the labeled glycosaminoglycans produced different results. With this enzyme, dermatan/chondroitin sulfate formed at high concentrations of sulfate yielded small glycosaminoglycans and larger oligosaccharides but almost no disaccharide. This indicated that the dermatan/chondroitin sulfate co-polymer contained mostly iduronic acid with only an occasional glucuronic acid. As the medium sulfate concentration was progressively lowered, there was a concomitant increase in the susceptibility to degradation by chondroitin AC lyase. Thus, the labeled glycosaminoglycans formed at the lowest concentration of sulfate yielded small oligosaccharides including substantial amounts of disaccharide. The smaller chondroitin AC lyase-resistant [3H,35S]dermatan/chondroitin sulfate oligosaccharides were analyzed by gel filtration. Results indicated that, in general, the iduronic acid-containing disaccharide residues present in the undersulfated [3H,35S]glycosaminoglycan were sulfated, whereas the glucuronic acid-containing disaccharide residues were non-sulfated. This work confirms earlier reports that there is a relationship between epimerization and sulfation. Moreover, it demonstrates that medium sulfate concentration is critical in determining the proportions of dermatan to chondroitin (iduronic/glucuronic acid) produced by cultured cells.     

Dual roles of sialyl Lewis X oligosaccharides in tumor metastasis and rejection by natural killer cells

Aberrant expression of cell surface carbohydrates such as sialyl Lewis X is associated with tumor formation and metastasis. In order to determine the roles of sialyl Lewis X in tumor metastasis, mouse melanoma B16-F1 cells were stably transfected with alpha1, 3-fucosyltransferase III to express sialyl Lewis X structures. The transfected B16-F1 cells, B16-FTIII, were separated by cell sorting into three different groups based on the expression levels of sialyl Lewis X. When these transfected cells were injected into tail veins of C57BL/6 mice, B16-FTIII.M cells expressing moderate amounts of sialyl Lewis X in poly-N-acetyllactosamines produced large numbers of lung tumor nodules. Surprisingly, B16-FTIII.H cells expressing the highest amount of sialyl Lewis X in shorter N-glycans died in lung blood vessels, producing as few lung nodules as B16-FTIII.N cells which lack sialyl Lewis X. In contrast, B16-FIII.H cells formed more tumors in beige mice and NK cell-depleted C57BL/6 mice than did B16-FTIII.M cells. B16-FTIII.H cells bound to E-selectin better than did B16-FTIII.M cells, but both cells grew at the same rate. These results indicate that excessive expression of sialyl Lewis X in tumor cells leads to rejection by NK cells rather than tumor formation facilitated by attachment to endothelial cells.     

Indoxyl sulfate induces leukocyte-endothelial interactions through up-regulation of E-selectin

Despite a positive correlation between chronic kidney disease and atherosclerosis, the causative role of uremic toxins in leukocyte-endothelial interactions has not been reported. We thus examined the effects of indoxyl sulfate, a uremic toxin, on leukocyte adhesion to activated endothelial cells and the underlying mechanisms. Pretreatment of human umbilical vein endothelial cells (HUVEC) with indoxyl sulfate significantly enhanced the adhesion of human monocytic cells (THP-1 cell line) to TNF-α-activated HUVEC under physiological flow conditions. Treatment with indoxyl sulfate enhanced the expression level of E-selectin, but not that of ICAM-1 or VCAM-1, in HUVEC. Indoxyl sulfate treatment enhanced the activation of JNK, p38 MAPK, and NF-κB in TNF-α-activated HUVEC. Inhibitors of JNK and NF-κB attenuated indoxyl sulfate-induced E-selectin expression in HUVEC and subsequent THP-1 adhesion. Furthermore, treatment with the NAD(P)H oxidase inhibitor apocynin and the glutathione donor N-acetylcysteine inhibited indoxyl sulfate-induced enhancement of THP-1 adhesion to HUVEC. Next, we examined the in vivo effect of indoxyl sulfate in nephrectomized chronic kidney disease model mice. Indoxyl sulfate-induced leukocyte adhesion to the femoral artery was significantly reduced by anti-E-selectin antibody treatment. These findings suggest that indoxyl sulfate enhances leukocyte-endothelial interactions through up-regulation of E-selectin, presumably via the JNK- and NF-κB-dependent pathway.