E-selectin Ligand Research Articles

E-selectin ligand–1 regulates growth plate homeostasis in mice by inhibiting the intracellular processing and secretion of mature TGF-β

The majority of human skeletal dysplasias are caused by dysregulation of growth plate homeostasis. As TGF-beta signaling is a critical determinant of growth plate homeostasis, skeletal dysplasias are often associated with dysregulation of this pathway. The context-dependent action of TFG-beta signaling is tightly controlled by numerous mechanisms at the extracellular level and downstream of ligand-receptor interactions. However, TGF-beta is synthesized as an inactive precursor that is cleaved to become mature in the Golgi apparatus, and the regulation of this posttranslational intracellular processing and trafficking is much less defined. Here, we report that a cysteine-rich protein, E-selectin ligand-1 (ESL-1), acts as a negative regulator of TGF-beta production by binding TGF-beta precursors in the Golgi apparatus in a cell-autonomous fashion, inhibiting their maturation. Furthermore, ESL-1 inhibited the processing of proTGF-beta by a furin-like protease, leading to reduced secretion of mature TGF-beta by primary mouse chondrocytes and HEK293 cells. In vivo loss of Esl1 in mice led to increased TGF-beta/SMAD signaling in the growth plate that was associated with reduced chondrocyte proliferation and delayed terminal differentiation. Gain-of-function and rescue studies of the Xenopus ESL-1 ortholog in the context of early embryogenesis showed that this regulation of TGF-beta/Nodal signaling was evolutionarily conserved. This study identifies what we believe to be a novel intracellular mechanism for regulating TGF-beta during skeletal development and homeostasis.

Core 1-derived O-glycans are essential E-selectin ligands on neutrophils

Neutrophils roll on E-selectin in inflamed venules through interactions with cell-surface glycoconjugates. The identification of physiologic E-selectin ligands on neutrophils has been elusive. Current evidence suggests that P-selectin glycoprotein ligand-1 (PSGL-1), E-selectin ligand-1 (ESL-1), and CD44 encompass all glycoprotein ligands for E-selectin; that ESL-1 and CD44 use N-glycans to bind to E-selectin; and that neutrophils lacking core 2 O-glycans have partially defective interactions with E-selectin. These data imply that N-glycans on ESL-1 and CD44 and O-glycans on PSGL-1 constitute all E-selectin ligands, with neither glycan subset having a dominant role. The enzyme T-synthase transfers Gal to GalNAcalpha1-Ser/Thr to form the core 1 structure Galbeta1-3GalNAcalpha1-Ser/Thr, a precursor for core 2 and extended core 1 O-glycans that might serve as selectin ligands. Here, using mice lacking T-synthase in endothelial and hematopoietic cells, we found that E-selectin bound to CD44 and ESL-1 in lysates of T-synthase-deficient neutrophils. However, the cells exhibited markedly impaired rolling on E-selectin in vitro and in vivo, failed to activate beta2 integrins while rolling, and did not emigrate into inflamed tissues. These defects were more severe than those of neutrophils lacking PSGL-1, CD44, and the mucin CD43. Our results demonstrate that core 1-derived O-glycans are essential E-selectin ligands; that some of these O-glycans are on protein(s) other than PSGL-1, CD44, and CD43; and that PSGL-1, CD44, and ESL-1 do not constitute all glycoprotein ligands for E-selectin.

Abstract 5184: Breast cancer cells express glycoprotein and glycolipid ligands for E-selectin

Abstract E-selectin, an adhesion molecule expressed by endothelial cells, has been implicated in various cancer metastatic processes. The hypothesis of the present study is that endothelial E-selectin is involved in the adhesion of breast cancer cells under physiological flow conditions. The characterization of E-selectin ligands expressed by BT-20 and MDA-MB-468 human breast cancer cells was performed. E-selectin ligands were broadly classified into glycoprotein and glycolipid ligands. Purified fractions of glycolipids containing sialic acid residues, known as gangliosides, were found to be E-selectin ligands at wall shear stress of 0.80 dynes/cm2. Immunostaining of breast cancer cells for various proteins known for E-selectin activity on non breast cancer cells revealed that P-selectin glycoprotein ligand-1 (PSGL-1), CD-43, podocalyxin like protein (PCLP), carcinoembryonic antigen (CEA) were absent, but CD44 was expressed by both breast cancer cell lines. Dynamic antigen capture assay at wall shear stress of 0.80 dynes/cm2 indicated that CD44 expressed by breast cancer cells is an E-selectin ligand; however, E-selectin ligand activity of CD44 was weak compared to LS-174T colon carcinoma cells. Efforts were then made to characterize novel glycoprotein ligands using immunoprecipitation and western blotting techniques. BT-20 cells showed presence of protein E-selectin ligands. The characterization of glycans decorating protein E-selectin ligands was performed by immunoprecipitation and western blot staining with various carbohydrate antibodies. In summary breast cancer cells express CD44 and novel glycoproteins as well as glycolipids as E-selectin ligands. Further characterization and identification of novel E-selectin ligands can lead to new diagnostic and prognostic approaches. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5184.

Abstract 5174: Characterization of glycolipid ligands for E-selectin

Abstract The prevention, treatment, and curing of cancer are three of the most researched and highly anticipated areas of modern medicine. One of the greatest barriers to making advances within these fields is the lack of understanding of the molecular mechanisms responsible for cancer metastasis. Head and neck cancer (HNC) is the fifth most diagnosed cancer in the United States, according to the American Cancer Society, and claims the lives of many of its victims because it is often not diagnosed until metastasis has already occurred. After the onset of metastasis, the five-year survival rate drops to below thirty-five percent. Therefore, the study of how metastasis occurs is essential to finding a treatment for HNC. It is believed that metastasis occurs when cancer cells break free from the primary tumor and travel through the blood stream, attaching themselves onto endothelium of blood vessel walls and in turn, forming secondary tumors in new organs. The cell adhesion mediated by glycolipids on cancer cells to E-selectin on vascular endothlelium may be a significant contributor to cancer metastasis. E-selectin plays a significant role in the first two steps of the adhesion cascade, tethering and rolling, which initiate the recruitment process. In our preliminary studies, it has been shown that HNC cells express glycolipids that can bind to E-selectin. Flow cytometry shows that HNC cells express sialofucosylated molecules that could act as E-selectin ligands, and flow chamber adhesion assays confirm that HNC cells bind to cytokine-stimulated endothelial cells in a wholly E-selectin-dependent manner. Enzyme treatment further reveals that sialofucosylated glycolipids are major adhesive ligand(s) for E-selectin. Through the use of specialized flow chambers mimicking physiological conditions, optical tweezers, gas chromatography-mass spectrometry, the mediators of these adhesive interactions can be studied in depth, leading to the discovery of characteristics of cancer glycolipids that promote adhesion. The purpose of this investigation is to gain further insight as to which specific glycolipids located on HNCcells are potent ligands for E-selectin binding. With increased understanding of selectin-ligand adhesion interactions, the identity and properties of molecules responsible for metastasis can be used to determine possible therapeutic methods. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5174.

CD8 T Cells Activated in Distinct Lymphoid Organs Differentially Express Adhesion Proteins and Coexpress Multiple Chemokine Receptors

Previous work from this laboratory showed that generation of memory CD8 T cells by different immunization routes correlates with control of tumors growing in distinct sites. We hypothesized that effector CD8 T cell expression of adhesion proteins and chemokine receptors would be influenced by activation in different secondary lymphoid organs. In this report, CD8 T cells were activated by immunization with bone marrow-derived dendritic cells via i.p., i.v., or s.c. routes. Three distinct populations of activated CD8 T cells arise in mesenteric, axillary/brachial, and mediastinal lymph nodes and spleen based on differential expression of alpha4beta7 integrin, E-selectin ligand, and alpha4beta1 integrin, respectively. In contrast, three subsets of CD8 T cells defined by differential expression of P-selectin ligand and chemokine receptors were induced irrespective of activation site. The majority of activated CD8 T cells expressed CXCR3, with one subset additionally expressing P-selectin ligand, and another subset additionally expressing CCR3, CCR4, CCR5, CCR6, and CCR9. In the mesenteric lymph node, a fourth subset expressed CCR9 and CXCR3 in the absence of CCR5. Similar homing receptor profiles were induced in the same sites after localized vaccinia immunization. Homing receptor expression on CD8 T cells activated in vitro was distinct, revealing influences of both dendritic cells and the lymphoid microenvironment. Collectively, these results identify previously undescribed populations of activated CD8 T cells based on adhesion protein expression and coexpression of chemokine receptors that arise after activation in distinct secondary lymphoid organs.

Abstract 5178: Homing of circulating prostate cancer cells to bone is regulated by alpha 1,3 fucosyltransferase 7

Abstract Circulating cancer cells are known to bind vascular surfaces and then to extravasate into target tissues. How such binding occurs is an essential, yet ill-defined step in metastasis. We and others have proposed that cancer cells exploit identical adhesive determinants as hematopoietic cells in order to bind microvascular endothelial cells. In particular, sialyl Lewis X (sLe(X)) on prostate cancer (PCa) cells is thought to promote metastasis by mediating PCa cell binding to microvascular endothelial (E)-selectin. Nonetheless, regulation of sLe(X) and related E-selectin ligand expression in PCa cells is a poorly understood factor in PCa metastasis. Here, we describe an important glycobiological mechanism regulating E-selectin-mediated adhesion and metastatic potential of PCa cells to the bone marrow. We demonstrate that alpha1, 3 fucosyltransferases (FT) 3, 6, and 7 are markedly elevated in bone- and liver-metastatic PCa tissue and dictate synthesis of sLe(X) and E-selectin ligands on metastatic PCa cells. Upregulated FT3, FT6, or FT7 expression induced robust PCa PC-3 cell adhesion to bone marrow endothelium and to inflamed postcapillary venules in an E-selectin-dependent manner. Membrane proteins, CD44, carcinoembryonic antigen (CEA), podocalyxin-like protein (PCLP), and melanoma cell adhesion molecule (MCAM) were major scaffolds presenting E-selectin-binding determinants on FT-upregulated PC-3 cells. Strikingly, elevated FT7 expression promoted PC-3 cell trafficking to and retention in bone marrow through an E-selectin dependent event. These results highlight alpha1, 3 FTs as potent enhancers of PCa cell metastatic efficiency to the bone marrow via an E-selectin-dependent trafficking mechanism. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5178.

Inflammatory Biomarkers in 70 Depressed Inpatients With and Without the Metabolic Syndrome

Chronic subclinical inflammation may be associated with the metabolic syndrome as well as with depression. We examined the impact of the metabolic syndrome on concentrations of inflammatory biomarkers in major depression. Data for 70 inpatients with major depressive disorder (diagnosed according to ICD-10 and DSM-IV), and with or without the metabolic syndrome, were assessed 4 to 5 weeks after admission to the clinic of the Department of Psychiatry, Charité-University Medicine, Berlin, between 2005 and 2007. The metabolic syndrome was defined according to the criteria of the International Diabetes Federation (2005). Immunologic biomarkers assessed included adiponectin, resistin, serum amyloid A (SAA), C-reactive protein (CRP), fibrinogen, interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), soluble E-selectin, and CD40 ligand (CD40L). Severity of depression was measured with the 17-item Hamilton Depression Rating Scale. After regressional correction for confounding variables and covariates, a 2-factorial analysis of variance (metabolic syndrome x time) revealed that the metabolic syndrome's presence affected adiponectin (F(43,1) = 5.56; P < .05) and IL-6 levels (F(25,1) = 6.80; P < .05) significantly. There was also a trend for effects on fibrinogen levels (F(47,1) = 3.66; P = .06). This is the first study to evaluate the putative additive effect of the metabolic syndrome on a panel of 9 inflammatory biomarkers in depression. Our findings support an additive effect on some (adiponectin, IL-6, and trendwise for fibrinogen) markers. Patients with the metabolic syndrome and major depression are at higher risk for more frequent and more severe cardiovascular side effects than their counterparts without the metabolic syndrome.

Platelets Control Leukocyte Recruitment in a Murine Model of Cutaneous Arthus Reaction

Platelets have been shown to be important in inflammation, but their role in the cutaneous Arthus reaction remains unclear. To assess the role of platelets in this pathogenetic process, the cutaneous Arthus reaction was examined in wild-type mice and mice lacking E-selectin, P-selectin, or P-selectin glycoprotein ligand-1 (PSGL-1) with or without platelet depletion by busulfan, a bone marrow precursor cell-specific toxin. Edema and hemorrhage induced by immune complex challenge significantly decreased in busulfan-treated wild-type mice compared with untreated mice. Busulfan treatment did not affect edema and hemorrhage in P-selectin- or PSGL-1-deficient mice, suggesting that the effect by busulfan is dependent on P-selectin and PSGL-1 expression. The inhibited edema and hemorrhage paralleled reduced infiltration of neutrophils and mast cells and reduced levels of circulating platelets. Increased cutaneous production of interleukin-6, tumor necrosis factor-alpha, and platelet-derived chemokines during Arthus reaction was inhibited in busulfan-treated wild-type mice relative to untreated mice, which paralleled the reduction in cutaneous inflammation. Flow cytometric analysis showed that immune complex challenge generated blood platelet-leukocyte aggregates that decreased by busulfan treatment. In thrombocytopenic mice, the cutaneous inflammation after immune complex challenge was restored by platelet infusion. These results suggest that platelets induce leukocyte recruitment into skin by forming platelet-leukocyte aggregates and secreting chemokines at inflamed sites, mainly through the interaction of P-selectin on platelets with PSGL-1 on leukocytes.

NF κB Activation in Embryonic Endothelial Progenitor Cells Enhances Neovascularization via PSGL-1 Mediated Recruitment: Novel Role for LL37

Embryonal endothelial progenitor cells (eEPCs) are capable of inducing therapeutic angiogenesis in a chronic hind limb model. However, the proportion of eEPCs recruited to the ischemic tissue appears to be a limiting step for the induction of cell-based therapeutic neovascularization. In the present study, we primed eEPCs with the human cathelicidin LL37 (hCAP-18) ex vivo to selectively enhance the eEPC-dependent gain of perfusion in vivo and elucidated the mechanism of action of LL37 on eEPCs. Seven days after femoral artery excision, 5 x 10(6) eEPCs (wt, wild type; p65t, transiently p65 transient; p65s, stable p65-transfected; LL37-eEPCs, LL37 peptide preincubated) were retroinfused into the anterior tibial vein. Recruitment of diI-labeled eEPCs in the ischemic gastrocnemic muscle was investigated 2 days later, whereas collateral growth and perfusion score (obtained by fluorescent microspheres) were assessed at day 7 and day 35 and are given as percentage of day 7 level. Capillary/muscle fiber ratio in the ischemic lower limb was obtained at day 35. Embryonic EPC recruitment in vitro and in vivo was found elevated after LL37 and p65t pretreatment, but not in p65s-eEPCs displaying increased IkappaBalpha or after LL37 in IkappaB-DN overexpressing eEPCs. Using LL37- and p65t-eEPCs, collateral growth (181 +/- 10% and 165 +/- 8%, respectively) surpassed that of wt-eEPCs (135 +/- 7%), increasing perfusion ratio (208 +/- 20% and 210 +/- 17% vs. 142 +/- 12% in wt-eEPCs, respectively), whereas p65s-eEPCs exerted no additive effect (collateral growth 130 +/- 8%; perfusion ratio 155 +/- 15%). Moreover, p65t-eEPC-induced neovascularization was abrogated by blocking antibodies against E-selectin and P-selectin glycoprotein ligand-1 (PSGL-1). We conclude that NF kappaB activation by LL37 or transient p65-transfection increases functionally relevant eEPC recruitment to ischemic muscle tissue via induction of PSGL-1 and E-selectin.

Abstract C19: C2-O-sLeX glycans on human carcinoma cells are high-affinity selectin ligands that mediate tumor adhesion to vascular endothelium and invasion

Abstract The family of selectin adhesion molecules, CD62-E and P-selectin expressed on vascular endothelium, and L-selectin expressed on leukocytes, bind sialyl-Lewis X (sLeX)-related glycans and are critical for leukocyte trafficking to lymphoid tissues and sites of inflammation. The sLeX-modified core 2 O-glycans (C2-O-sLeX) on leukocytes promote significantly higher affinity selectin binding than sLeX. Our objectives were to determine whether C2-O-sLeX glycans conferred carcinoma cells with high binding activity to the selectins and participated in metastasis. We investigated C2-O-sLeX expression in human colorectal adenocarcinomas and metastatic liver tumors by immunohistochemistry and RT-PCR. C2-O-sLeX was abundantly expressed in adenocarcinomas and mRNA expression of C2GnT1, the enzyme responsible for C2-O-sLeX synthesis, was greater than 15-fold upregulated. C2-O-sLeX was also present in metastatic liver tumors but not normal colorectal or liver tissues. We evaluated human lung (H1299), pancreatic (AsPC-1), colon (LS174T), and hepatic (HepG2) carcinoma cell lines for their expression of C2-O-sLeX or sLeX glycans and ability to bind to selectins. We also examined mRNA expression of genes coding for the enzymes alpha-2,3 fucosyltransferases (FucT-III) and core 2 beta-1,6 N-acetylglucosaminyltransferase (C2GnT1). FucT-III is responsible for sLeX synthesis and the formation of the core 2 branched O-glycan structures is dependent on C2GnT1. The activity of both enzymes is necessary for C2-O-sLeX synthesis. All four carcinoma cell lines expressed similar levels of C2GnT1 mRNA but FucT-III mRNA levels were lower in H1299 and AsPC-1 cells by RT-PCR. LS174T and HepG2 cell lines were 70 to 90% positive for cell surface expression of C2-O-sLeX and between 70 to 97% positive for sLeX by flow cytometry. C2-O-sLeX and sLeX were also detected in these cells by Western blotting. H1299 and AsPC-1 cells did not express these glycans. H1299 and AsPC-1 cells stably transfected with FucT-III were 43% and 7% positive for C2-O-sLeX respectively. Transfected (H1299/FucT-III and AsPC-1/FucTIII) and LS174T and HepG2 cell lines bound avidly to E-selection but not P- and L-selectin. We used shRNA vectors to silence gene expression of C2GnT1 in H1299/FucT-III and LS174T cells. We observed a 40–90% decrease in C2-O-sLeX but not sLeX expression and a 30–50% decrease in binding to E-selectin on cells containing C2GnT1 shRNA compared to unmanipulated cells. Migration of H1299/FucT-III cells across a reconstituted basement membrane was significantly increased compared to untransfected cells using a matrigel invasion assay. In contrast, the migration of H1299 cells containing C2GnT1 shRNA was reduced. Similarly, LS174T cells transfected with C2GnT1 shRNA exhibited significantly reduced migration and proliferation. Our findings demonstrate that C2-O-sLeX glycans are tumor-associated antigens expressed on carcinoma cells. Importantly, C2-O-sLeX glycans are high affinity ligands for E-selectin and play a role in the adhesion of carcinoma cells to endothelium and tumor invasiveness. These findings are relevant to the development of novel biomarkers and allow further insight into the role of sLeX-containing glycans in carcinoma metastasis. C2-O-sLeX may prove useful to predict patient prognosis and survival. This work was supported in part by funds from the National Institutes of Health/National Cancer Institute grant K08 CA111829-04. Citation Information: Cancer Res 2009;69(23 Suppl):C19.

Alpha 1,3 fucosyltransferases are master regulators of prostate cancer cell trafficking

How cancer cells bind to vascular surfaces and extravasate into target organs is an underappreciated, yet essential step in metastasis. We postulate that the metastatic process involves discrete adhesive interactions between circulating cancer cells and microvascular endothelial cells. Sialyl Lewis X (sLe(X)) on prostate cancer (PCa) cells is thought to promote metastasis by mediating PCa cell binding to microvascular endothelial (E)-selectin. Yet, regulation of sLe(X) and related E-selectin ligand expression in PCa cells is a poorly understood factor in PCa metastasis. Here, we describe a glycobiological mechanism regulating E-selectin-mediated adhesion and metastatic potential of PCa cells. We demonstrate that alpha1,3 fucosyltransferases (FT) 3, 6, and 7 are markedly elevated in bone- and liver-metastatic PCa and dictate synthesis of sLe(X) and E-selectin ligands on metastatic PCa cells. Upregulated FT3, FT6, or FT7 expression induced robust PCa PC-3 cell adhesion to bone marrow (BM) endothelium and to inflamed postcapillary venules in an E-selectin-dependent manner. Membrane proteins, CD44, carcinoembryonic antigen (CEA), podocalyxin-like protein (PCLP), and melanoma cell adhesion molecule (MCAM) were major scaffolds presenting E-selectin-binding determinants on FT-upregulated PC-3 cells. Furthermore, elevated FT7 expression promoted PC-3 cell trafficking to and retention in BM through an E-selectin dependent event. These results indicate that alpha1,3 FTs could enhance metastatic efficiency of PCa by triggering an E-selectin-dependent trafficking mechanism.

Abstract 5270: Vascular Function and Vascular Inflammation in Polycystic Kidney Disease

Background: Polycystins, which have been localized to vascular smooth muscle cells, may lead to vascular dysfunction and contribute to cardiovascular disease in autosomal dominant polycystic kidney disease (ADPKD). We examined vascular function and vascular inflammation in ADPKD. Methods: This is an ancillary study to the Halt Progression in PKD Study, which is examining the effect of renin-angiotensin-aldosterone blockade on progression of ADPKD. We used peripheral arterial tonometry to assess augmentation index (AIx), a measure of arterial stiffness and wave reflection, in 52 ADPKD patients with hypertension and glomerular filtration rate (GFR) 25– 60 ml/min/1.73 m 2 , 50 ADPKD patients with hypertension and GFR &gt;60 ml/min/1.73 m 2 , and 51 healthy controls. We measured serum levels of soluble intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (VCAM-1), p-selectin, e-selectin, soluble Fas, and Fas ligand (FasL) as markers of vascular inflammation. Results: AIx, sICAM, p-selectin, and Fas were higher, and FasL was lower in subjects with ADPKD (p&lt;0.05, Table ). Adjusting for age, race, height, heart rate, and mean arterial pressure had no effect on group differences in AIx (healthy control = 8%, ADPKD GFR &gt;60 = 21%, ADPKD GFR 25– 60 = 20%). Group means were significantly different even after adjustment for vascular inflammatory markers (healthy control = 3%, ADPKD GFR &gt;60 = 24%, ADPKD GFR 25– 60 = 24%). There were no differences in AIx, sICAM, p-selectin, e-selectin, or FasL between the two ADPKD groups (Table 1 , p&gt;0.05). Conclusion: Vascular dysfunction and vascular inflammation are evident even in early stages of ADPKD with preserved GFR. Further research is needed to examine the relative contributions of genetic aberrations, hypertension, and reduced kidney function to the pathophysiology of vascular dysfunction in ADPKD. Table. Subject information.

Multivalent Display of Quinic Acid Based Ligands for Targeting E-Selectin Expressing Cells

The site-specific expression of molecular markers on endothelial cells of blood vessels during inflammatory response and angiogenesis provides an opportunity to target drugs and imaging molecules to the vascular endothelium of diseased tissues. This paper describes an innovative strategy for selective delivery of polymer conjugates to E- and P-selectin expressing cells using a series of quinic acid (Qa) based non-carbohydrate analogues of the natural ligand sialyl Lewis(x) (sLe(x)) as targeting moieties. We demonstrate that such analogues antagonize the adhesion of sLe(x) expressing HL-60 cells to both E- and P-selectin. Significantly, the apparent avidity of polymer conjugates carrying multiple Qa copies has increased by 3 orders of magnitude relative to their monomeric forms. Furthermore, we found that the major mechanism of copolymer entry and delivery into E-selectin expressing cells is endocytosis. These selectin-targetable copolymers provide the foundation to support controlled delivery of anticancer drugs and imaging agents to tumor vasculature for therapeutic and diagnostic applications.

Circulating Galectin-3 Promotes Metastasis by Modifying MUC1 Localization on Cancer Cell Surface

Adhesion of circulating tumor cells to the blood vessel endothelium is a critical step in cancer metastasis. We show in this study that galectin-3, the concentration of which is greatly increased in the circulation of cancer patients, increases cancer cell adhesion to macrovascular and microvascular endothelial cells under static and flow conditions, increases transendothelial invasion, and decreases the latency of experimental metastasis in athymic mice. These effects of galectin-3 are shown to be a consequence of its interaction with cancer-associated MUC1, which breaks the "protective shield" of the cell-surface MUC1 by causing MUC1 polarization, leading to exposure of smaller cell-surface adhesion molecules/ligands including CD44 and ligand(s) for E-selectin. Thus, the interaction in the bloodstream of cancer patients between circulating galectin-3 and cancer cells expressing MUC1 bearing the galectin-3 ligand TF (Galbeta1,3GalNAc-) promotes metastasis. This provides insight into the molecular regulation of metastasis and has important implications for the development of novel therapeutic strategies for prevention of metastasis.

P-Selectin Glycoprotein Ligand-1 Contributes to Wound Healing Predominantly as a P-Selectin Ligand and Partly as an E-Selectin Ligand

Cell adhesion molecules are critical to wound healing through leukocyte recruitment. Although P-selectin glycoprotein ligand-1 (PSGL-1) regulates leukocyte rolling by binding P-selectin, but also binding E- and L-selectins with lower affinity, little is known about a role of PSGL-1 in wound healing. To clarify a role of PSGL-1 and its interaction with E- and P-selectins in wound healing, we investigated cutaneous wound healing in PSGL-1-deficient (PSGL-1(-/-)) mice in comparison with E-selectin(-/-), P-selectin(-/-), and P-selectin(-/-) mice treated with an anti-E-selectin antibody. PSGL-1 deficiency inhibited early wound healing, which was accompanied by decreased inflammatory cell infiltration and growth factor expression. By contrast, E-selectin deficiency did not affect wound healing. In general, the inhibitory effect of PSGL-1 deficiency on wound healing was similar to that of P-selectin deficiency either alone or with E-selectin blockade. However, early granulation tissue formation, late angiogenesis, and early infiltration of neutrophils and macrophages in PSGL-1(-/-) mice were inhibited beyond the inhibition in P-selectin(-/-) mice, but to a similar level of inhibition in P-selectin(-/-) mice with E-selectin blockade. These results suggest that PSGL-1 contributes to wound healing predominantly as a P-selectin ligand and partly as an E-selectin ligand by mediating infiltration of inflammatory cells.

In Vivo Detection of Inflammation Using Pegylated Iron Oxide Particles Targeted at E-Selectin

Ultrasmall particles of iron oxide (USPIO) possess superparamagnetic properties and are used as negative contrast agent in magnetic resonance imaging (MRI) because of their strong T(2) and T(2)* effects. Besides this method, electron paramagnetic resonance (EPR) offers the unique capability to quantify these particles. The objective of this study was to evaluate a molecular marker for non invasive diagnosis and monitoring of inflammation. During inflammation cell adhesion molecules such as E-selectin are expressed on the endothelial cell surface. An E-selectin ligand was coupled to pegylated USPIO particles. Inflammation was induced by intramuscular injection of Freund's Complete Adjuvant in male NMRI mice. After intravenous injection of grafted or ungrafted USPIO particles, iron concentration in inflamed muscles was quantified ex vivo by X-band EPR. Particle accumulation was also assessed in vivo by L-Band EPR, as well as by T(2)-weighted MRI. We determined the mean iron oxide concentration in inflamed muscles after injection of grafted or ungrafted UPSIO particles, which was 0.8% and 0.4% of the initially injected dose, respectively. By L-band EPR, we observed that the concentration of the grafted USPIO particles in inflamed muscles was twice higher than for the ungrafted particles. Using MRI experiments, a higher signal loss was clearly observed in the inflamed muscle when grafted particles were injected in comparison with the ungrafted particles. Even taking into account a non specific accumulation of iron oxides, the targeting of USPIO particles with E-selectin ligands significantly improved the sensitivity of detection of inflamed tissues.

Role of selectins in the pathogenesis of multiple myeloma

11103 Background: Multiple myeloma (MM) is characterized by the disseminated involvement of the bone-marrow (BM), and its progression involves a continuous circulation of the MM cells (MMCs) in the peripheral blood and homing back to the BM. Selectins are adhesion molecules involved in the primary interaction of lymphocytes with the endothelial cells (ECs) of blood vessels. In this study we studied the role selectins in the pathogenesis of MM. Methods: We have characterized the expression of E, L and P-selectins and their ligands on MM cell lines, patient sample and plasma cells from normal subjects (NPCs). We have tested the effect of blockade of each of the selectins and selectin-ligands on the interaction of MMCs with ECs. Moreover, we tested the effect of a pan selectin inhibitor on MMCs adhesion to ECs, and trans-well (through filter) and trans-endothelial SDF1-induced migration in vitro, and characterized its effect on cytoskeletal signaling induced by the interaction of MMCs and ECs. Moreover, we have tested the effect of the inhibitor on homing of MMCs to the BM in mice using in vivo flow cytometry to detect the number of circulating cells, and in vivo confocal microscopy to directly visualize the homing. Results: All MM cell lines and patient samples had low expression of all selectins and high expression of L and P, but not E, selectin ligands. While NPCs showed low expression of all selectins and ligands. Blockade of L and P-selectin ligands reduced the interaction of MMCs with ECs in vitro, while blockade of E-selectin ligand or any of selectins did not show any effect. The pan-selectin inhibitor reduced the interaction of MMCs with ECs in vitro, did not alter their SDF1-induced migration through filter, but reduced significantly the migration through ECs. The inhibitor inhibited the activation of FAK and ERK induced by interaction of MMCs and ECs. Moreover, the selectin inhibitor extending the circulation time of MM cells in mice, and reduced the homing of MMCs. Conclusions: We found that L and P selectin ligands are highly expressed in MMCs compared to NPCs, and that those play a major role in homing of MMCs to the BM. Moreover, the pan-selectin inhibitor prevented the homing of MMCs to the BM. This provides a basis for testing the effect of the inhibitor on MM tumor progression and initiation. No significant financial relationships to disclose.

The E-Selectin Ligand Basigin/CD147 Is Responsible for Neutrophil Recruitment in Renal Ischemia/Reperfusion

E-selectin and its ligands are essential for extravasation of leukocytes in inflammation. Here, we report that basigin (Bsg)/CD147 is a ligand for E-selectin that promotes renal inflammation in ischemia/reperfusion. Compared with wild-type mice, Bsg-deficient (Bsg(-/-)) mice demonstrated striking suppression of neutrophil infiltration in the kidney after renal ischemia/reperfusion. Although E-selectin expression increased similarly between the two genotypes, Bsg(-/-) mice exhibited less renal damage, suggesting that Bsg on neutrophils contribute to renal injury in this model. Neutrophils expressed Bsg with N-linked polylactosamine chains and Bsg(-)(/)(-) neutrophils showed reduced binding to E-selectin. Bsg isolated from HL-60 cells bound to E-selectin, and tunicamycin treatment to abolish N-linked glycans from Bsg abrogated this binding. Furthermore, Bsg(-)(/)(-) neutrophils exhibited reduced E-selectin-dependent adherence to human umbilical vein endothelial cells in vitro. Injection of labeled neutrophils into mice showed that Bsg(-)(/)(-) neutrophils were less readily recruited to the kidney after renal ischemia/reperfusion than Bsg(+/+) neutrophils, regardless of the recipient's genotype. Taken together, these results indicate that Bsg is a physiologic ligand for E-selectin that plays a critical role in the renal damage induced by ischemia/reperfusion.

Serum 3′-sulfo-Le a indication of gastric cancer metastasis

Background 3′-Sulfo-Le a is known to be the potent ligand of E-selectin which is important in cell adhesion and migration. Yet the significance of serum 3′-sulfo-Le a has not been explored and reported. Methods Serum 3′-sulfo-Le a was analyzed by enzyme-linked immunosorbent assay. SPSS software was used for statistics analysis. Cell adhesion to HUVEC and sL-selectin, and cell migration were performed in gastric cancer cells SCG7901 with 3′-sulfo-Le a silence by Gal3ST-2 RNAi. Results Through analysis, the mean levels of serum 3′-sulfo-Le a antigen were found significantly higher in 108 patients with gastric cancer than that in 74 healthy volunteers. Depth of tumor invasion, lymph node metastasis, and differentiation were noted to be significantly correlated with the expression of this antigen in gastric carcinoma. After treatment with 5-FU (5-fluorouracil) and ATRP (N-all-trans-retinoyl- l-proline), the expression of 3′-sulfo-Le a antigen was markedly down regulated in SCG7901 gastric cancer cells. After transfection of Gal3ST-2 RNAi, the expression of 3′-sulfo-Le a was silenced and the cell adhesion to HUVEC or sL-selectin, and cell migration were suppressed. Conclusion Serum 3′-sulfo-Le a antigen can provide important information in patients with primary gastric cancer, which might be useful as a predictive marker especially for the detection of tumor metastasis.

Maintenance of Peripheral Tolerance through Controlled Tissue Homing of Antigen-Specific T Cells in K14-mOVA Mice

Immunological tolerance is crucial to avoid autoimmune and inflammatory diseases; however, the mechanisms involved are incompletely understood. To study peripheral tolerance to skin-associated Ags, we generated new transgenic mice expressing a membrane-bound form of OVA in skin under the human keratin 14 (K14) promoter (K14-mOVA mice). In contrast to other transgenic mice expressing similar self-Ags in skin, adoptive transfer of Ag-specific T cells does not induce inflammatory skin disease in our K14-mOVA mice. OVA-specific T cells transferred into K14-mOVA mice are activated in lymphoid tissues, undergo clonal expansion, and eventually acquire effector function. Importantly, these Ag-specific T cells selectively up-regulate expression of E-selectin ligand in cutaneous lymph nodes but not in mesenteric lymph nodes and spleen, demonstrating that expression of endogenous self-Ags in skin dictates imprinting of skin tissue homing in vivo. However, an additional inflammatory signal, here induced by tape stripping, is required in K14-mOVA mice to induce T cell migration to skin and development of inflammatory skin disease. Depletion of regulatory CD4(+)CD25(+) T cells did not provoke homing of transferred T cells to skin under steady-state conditions, indicating that these cells are not the key regulators for inhibiting T cell homing in K14-mOVA mice. Both skin-derived and lymph node-resident CD8alpha(+) dendritic cells are responsible for Ag presentation in vivo and induce tolerance to skin Ags, as we show by selective depletion of langerin(+) and CD11c(+) dendritic cells. Taken together, controlled skin homing of T cells is critical for the maintenance of peripheral immune tolerance to epidermal self-Ags.