A Genome-Wide Association Study of Circulating Galectin-3

Galectin-3 is a lectin involved in fibrosis, inflammation and proliferation. Increased circulating levels of galectin-3 have been associated with various diseases, including cancer, immunological disorders, and cardiovascular disease. To enhance our knowledge on galectin-3 biology we performed the first genome-wide association study (GWAS) using the Illumina HumanCytoSNP-12 array imputed with the HapMap 2 CEU panel on plasma galectin-3 levels in 3,776 subjects and follow-up genotyping in an additional 3,516 subjects. We identified 2 genome wide significant loci associated with plasma galectin-3 levels. One locus harbours the LGALS3 gene (rs2274273; P = 2.35×10⁻¹⁸⁸) and the other locus the ABO gene (rs644234; P = 3.65×10⁻⁴⁷). The variance explained by the LGALS3 locus was 25.6% and by the ABO locus 3.8% and jointly they explained 29.2%. Rs2274273 lies in high linkage disequilibrium with two non-synonymous SNPs (rs4644; r² = 1.0, and rs4652; r² = 0.91) and wet lab follow-up genotyping revealed that both are strongly associated with galectin-3 levels (rs4644; P = 4.97×10⁻⁴⁶⁵ and rs4652 P = 1.50×10⁻⁴²¹) and were also associated with LGALS3 gene-expression. The origins of our associations should be further validated by means of functional experiments.     

Endothelial LGALS9 splice variant expression in endothelial cell biology and angiogenesis

Galectins are carbohydrate binding proteins with versatile functions in tumor progression. Galectin-9, encoded by LGALS9, has been associated with metastasis and immunosuppression. We previously reported on regulation of LGALS9 expression during endothelial cell activation. Here, we show increased galectin-9 protein levels in the endothelium of different tumors, including carcinomas of the lung, liver, breast and kidney. Endothelial cells were found to express five LGALS9 splice variants, two of which have not been reported before. Splicing was found to be confined to exons 5, 6 and 10. Transfection of human microvascular endothelial cells (HMEC) with galectin-9∆5, a specific LGALS9 splice variant, induced a small but significant increase of proliferation, while migration was not affected by any LGALS9 splice variant. Application of recombinant galectin-9∆5 protein dose-dependently reduced proliferation and migration of HMEC as well as human umbilical vein endothelial cells in vitro. Enhanced sprouting and migration of human umbilical vein endothelial cell (HUVEC) towards a galectin-9∆5 gradient were observed. Interestingly, galectin-9∆5 was found to induce a small inhibitory effect on angiogenesis in vivo. Collectively, these data show that endothelial cells regulate the expression and splicing of LGALS9 during angiogenesis. The function of the dominant splice variant, i.e. galectin-9∆5, in endothelial cell biology depends on the concentration and environmental context in which it is presented to the cells.     

Galectin-3 in macrophage-like cells exposed to immunomodulatory drugs

During the last few decades, the effects of immunomodulatory drugs on numerous molecules and biological processes have been widely studied. Nevertheless, the relationship between immunomodulatory drugs and lectin expression/function is still to be elucidated. In this study, we used THP-1-derived macrophages to investigate the effects of non-steroidal anti-inflammatory drugs (aspirin and indomethacin) and glucocorticoids (hydrocortisone and dexamethasone) on galectin-3, a multifunctional beta-galactoside binding lectin, which in general acts as a strong pro-inflammatory signal. The results showed that all immunomodulatory drugs applied in clinically relevant doses affect both the gene (LGALS3) and protein expression level of galectin-3. The provoked changes on protein level are qualitatively and quantitatively different comparing to the effects on galectin-3 mRNA level, and depend on the differentiation state of the cell, drug type and applied concentration as well as on time of the exposure. Our data revealed galectin-3 as a new target molecule of immunomodulatory drugs, thus suggesting an additional pathway of their action on immune response.     

Human galectin-8 isoforms and cancer

Galectins are animal lectins that can specifically bind β-galactosides. Thirteen galectins have already been described. This review focuses on a specific member of this family: galectin-8. This galectin was discovered in prostate cancer cells eight years ago and has been studied extensively in the last few years. The galectin-8 gene (LGALS8) encodes numerous mRNAs by alternate splicing and the presence of three unusual polyadenylation signals. These mRNAs encode six different isoforms of galectin-8: three belong to the tandem-repeat galectin group (with two CRDs linked by a hinge peptide) and three to the prototype group (with one CRD). Various studies showed that galectin-8 is widely expressed in tumor tissues as well as in normal tissues. The level of galectin-8 expression may correlate with the malignancy of human colon cancers and the degree of differentiation of lung squamous cell carcinomas and neuro-endocrine tumors. Recently, the differences in galectin-8 expression levels between normal and tumor tissues have been used as a guide for the selection of strategies for the prevention and treatment of lung squamous cell carcinoma. These experiments are still under investigation, but demonstrate the potential of galectin-8 research to enhance our understanding of, and possibly prevent, the process of neoplastic transformation. Published in 2004.     

Increased LGALS3BP promotes proliferation and migration of oral squamous cell carcinoma via PI3K/AKT pathway

Previous studies showed that lectin galactoside-binding soluble 3 binding protein (LGALS3BP) is an important participant in tumor progression. However, its prognostic value and functional mechanism in oral squamous cell carcinoma (OSCC) are still unclear. In this study, we analyzed LGALS3BP expression in OSCC tissues via Oncomine databases and immunohistochemical staining. LGALS3BP was significantly up-regulated in OSCC tumor tissues. IHC analysis showed that LGALS3BP was predominantly expressed in tumor cells and correlated with poor clinical characteristics. In addition, high LGALS3BP expression predicted poor clinical outcomes and multivariate analysis revealed that LGALS3BP expression was as an independent prognostic factor for OS, DFS and RFS (p < .0001, p = .002, p = .002). Mechanically, LGALS3BP regulated OSCC proliferation and migration via PI3K/AKT pathways, which was abrogated by PI3K inhibitor LY294002 in a dose-dependent manner. Our results suggested that LGALS3BP could be served as a novel independent prognostic factor as well as a potential therapeutic target for OSCC treatment.     

Galectin-3 and metastasis

Galectin-3, a 31 kDa member of the β-galactoside-binding proteins, is an intracellular and extracellular lectin which interacts with intracellular glycoproteins, cell surface molecules and extracellular matrix proteins. Galectin-3 is expressed widely in epithelial and immune cells and its expression is correlated with cancer aggressiveness and metastasis. Galectin-3 is involved in various biological phenomena including cell growth, adhesion, differentiation, angiogenesis and apoptosis. Recent research revealed that galectin-3 is associated with several steps of invasion and metastasis, like angiogenesis, cell-matrix interaction, dissemination through blood flow and extravasation. Recently, we and others have shown that galectin-3 can be a reliable diagnostic marker in certain cancers and one of the target proteins of cancer treatment. In this review, we describe the involvement of galectin-3 in each steps of metastasis and clinical significance of galectin-3. Published in 2004.     

Glucocorticoid‐transactivated TSC22D3 attenuates hypoxia‐ and diabetes‐induced Müller glial galectin‐1 expression via HIF‐1α destabilization

Galectin‐1/LGALS1, a newly recognized angiogenic factor, contributes to the pathogenesis of diabetic retinopathy (DR). Recently, we demonstrated that glucocorticoids suppressed an interleukin‐1β‐driven inflammatory pathway for galectin‐1 expression in vitro and in vivo. Here, we show glucocorticoid‐mediated inhibitory mechanism against hypoxia‐inducible factor (HIF)‐1α‐involved galectin‐1 expression in human Müller glial cells and the retina of diabetic mice. Hypoxia‐induced increases in galectin‐1/LGALS1 expression and promoter activity were attenuated by dexamethasone and triamcinolone acetonide in vitro. Glucocorticoid application to hypoxia‐stimulated cells decreased HIF‐1α protein, but not mRNA, together with its DNA‐binding activity, while transactivating TSC22 domain family member (TSC22D)3 mRNA and protein expression. Co‐immunoprecipitation revealed that glucocorticoid‐transactivated TSC22D3 interacted with HIF‐1α, leading to degradation of hypoxia‐stabilized HIF‐1α via the ubiquitin‐proteasome pathway. Silencing TSC22D3 reversed glucocorticoid‐mediated ubiquitination of HIF‐1α and subsequent down‐regulation of HIF‐1α and galectin‐1/LGALS1 levels. Glucocorticoid treatment to mice significantly alleviated diabetes‐induced retinal HIF‐1α and galectin‐1/Lgals1 levels, while increasing TSC22D3 expression. Fibrovascular tissues from patients with proliferative DR demonstrated co‐localization of galectin‐1 and HIF‐1α in glial cells partially positive for TSC22D3. These results indicate that glucocorticoid‐transactivated TSC22D3 attenuates hypoxia‐ and diabetes‐induced retinal glial galectin‐1/LGALS1 expression via HIF‐1α destabilization, highlighting therapeutic implications for DR in the era of anti‐vascular endothelial growth factor treatment.     

Galectin-3C Inhibits Tumor Growth and Increases the Anticancer Activity of Bortezomib in a Murine Model of Human Multiple Myeloma

Galectin-3 is a human lectin involved in many cellular processes including differentiation, apoptosis, angiogenesis, neoplastic transformation, and metastasis. We evaluated galectin-3C, an N-terminally truncated form of galectin-3 that is thought to act as a dominant negative inhibitor, as a potential treatment for multiple myeloma (MM). Galectin-3 was expressed at varying levels by all 9 human MM cell lines tested. In vitro galectin-3C exhibited modest anti-proliferative effects on MM cells and inhibited chemotaxis and invasion of U266 MM cells induced by stromal cell-derived factor (SDF)-1α. Galectin-3C facilitated the anticancer activity of bortezomib, a proteasome inhibitor approved by the FDA for MM treatment. Galectin-3C and bortezomib also synergistically inhibited MM-induced angiogenesis activity in vitro. Delivery of galectin-3C intravenously via an osmotic pump in a subcutaneous U266 cell NOD/SCID mouse model of MM significantly inhibited tumor growth. The average tumor volume of bortezomib-treated animals was 19.6% and of galectin-3C treated animals was 13.5% of the average volume of the untreated controls at day 35. The maximal effect was obtained with the combination of galectin-3C with bortezomib that afforded a reduction of 94% in the mean tumor volume compared to the untreated controls at day 35. In conclusion, this is the first study to show that inhibition of galectin-3 is efficacious in a murine model of human MM. Our results demonstrated that galectin-3C alone was efficacious in a xenograft mouse model of human MM, and that it enhanced the anti-tumor activity of bortezomib in vitro and in vivo. These data provide the rationale for continued testing of galectin-3C towards initiation of clinical trials for treatment of MM.     

Galectin-3 mRNA level depends on transformation phenotype in ras-transformed NIH 3T3 cells

Summary— The increase in galectin-3 lectin content observed in tumours or in in vitro transformed cells suggests that this lectin is important in the transformation process. In the present study, we investigated the mRNA expression level of the galectin-3, galectin-I and macrophage mannose receptor in normal and ras-transformed NIH 3T3 cells in relation to their transformation state. The galectin3 mRNA content in ras-transformed cells is increased in fully transformed cells, with a maximum in ras-transformed cells that have lost their growth anchorage-dependence. Under the same conditions, the galectin-1 mRNA level which was high in normal cells, increased slightly in transformed cells. The mRNA for the macrophage mannose receptor was not detected in 3T3 cells or in their ras-transformed counterparts.     

Human galectin-8 isoforms and cancer

Galectins are animal lectins that can specifically bind beta-galactosides. Thirteen galectins have already been described. This review focuses on a specific member of this family: galectin-8. This galectin was discovered in prostate cancer cells eight years ago and has been studied extensively in the last few years. The galectin-8 gene ( LGALS8) encodes numerous mRNAs by alternate splicing and the presence of three unusual polyadenylation signals. These mRNAs encode six different isoforms of galectin-8: three belong to the tandem-repeat galectin group (with two CRDs linked by a hinge peptide) and three to the prototype group (with one CRD). Various studies showed that galectin-8 is widely expressed in tumor tissues as well as in normal tissues. The level of galectin-8 expression may correlate with the malignancy of human colon cancers and the degree of differentiation of lung squamous cell carcinomas and neuro-endocrine tumors. Recently, the differences in galectin-8 expression levels between normal and tumor tissues have been used as a guide for the selection of strategies for the prevention and treatment of lung squamous cell carcinoma. These experiments are still under investigation, but demonstrate the potential of galectin-8 research to enhance our understanding of, and possibly prevent, the process of neoplastic transformation.     

On the role of galectin-3 in cancer apoptosis

Galectin-3, a member of the -galactoside-binding gene family, is a multifunctional protein implicated in a variety of biological functions, including tumor cell adhesion, proliferation, differentiation, angiogenesis, cancer progression and metastasis. Recent studies revealed that intracellular galectin-3 exhibits the activity to suppress drug induced apoptosis and anoikis (apoptosis induced by the loss of cell anchorage) that contribute to cell survival. Resistance to apoptosis is essential for cancer cell survival and plays a role in tumor progression. Conversely, it was recently shown that tumor cells secreted galectin-3 induces T-cells apoptosis, thus playing a role in the immune escape mechanism during tumor progression through induction of apoptosis of cancer-infiltrating T-cells. This review summarizes recent evidences on the role of galectin-3 as an anti-apoptotic and/or pro-apoptotic factor in various cell types and discusses the recent understanding of the molecular mechanisms of galectin-3 role in apoptosis. We also suggest potential directions for further analyses of this multifunctional protein.     

Smooth muscle cell differentiation from human bone marrow: Variations in cell type specific markers and Id gene expression in a new model of cell culture

Stromal cells follow a vascular smooth muscle differentiation pathway. However, cell culture models performed from human bone marrow do not allow the obtention of a large proportion of highly differentiated smooth muscle cells (SMC) and their differentiation pathways remain unclear. We have characterized a new model of SMC differentiation from human bone marrow stromal cells by using different factors (bFGF, EGF, insulin and BMP-4). A relative homogeneous population of differentiated SMC was reproducibly obtained in short-term culture with high expression of SMC markers. Id gene expression was investigated and showed that (1) Id2 mRNA expression was upregulated during SMC differentiation without change of Id1 mRNA and (2) Id1 gene expression highly increased concomitantly with a decrease of SMC markers while Id2 mRNA was slightly modulated. Our data suggested that Id genes are potentially implicated in the differentiation pathway of human SMC from bone marrow.     

Involvement of Galectin-3 with Vascular Cell Adhesion Molecule-1 in Growth Regulation of Mouse BALB/3T3 Cells

β-Galactose residues on N-glycans have been implicated to be involved in growth regulation of cells. In the present study we compared the galactosylation of cell surface N-glycans of mouse Balb/3T3 cells between 30 and 100% densities and found the β-1,4-galactosylation of N-glycans increases predominantly in a 100-kDa protein band on lectin blot analysis in combination with digestions by diplococcal β-galactosidase and N-glycanase. When cells at 100% density were treated with jack bean β-galactosidase, the incorporation of 5-bromodeoxyuridine into the cells was stimulated in a dose-dependent manner, suggesting the involvement of the galactose residues in growth regulation of cells. A galactose-binding protein was isolated from the plasma membranes of cells at 100% density by affinity chromatography using an asialo-transferrin-Sepharose column and found to be galectin-3 as revealed by mass spectrometric analysis. The addition of recombinant galectin-3 into cells at 50% density inhibited the incorporation of 5-bromodeoxyuridine in a dose-dependent manner, but the inhibition was prevented with haptenic sugar. An immunocytochemical study showed that galectin-3 is present at the surface of cells at 100% density but not at 30% density where it locates inside the cells. Several glycoproteins bind to a galectin-3-immobilized column, a major of which was identified as vascular cell adhesion molecule (VCAM)-1. Immunocytochemical studies showed that some galectin-3 and VCAM-1 co-localize at the surface of cells at 100% density, indicating that the binding of galectin-3 secreted from cells to VCAM-1 is one of the pathways involved in the growth regulation of Balb/3T3 cells.     

Galectin-3 expression in macrophages is signaled by Ras/MAP kinase pathway and up-regulated by modified lipoproteins

To study the signaling pathway involved in the regulation of galectin-3 expression we used phorbol ester to stimulate macrophage differentiation of THP-1 cells. Treatment with phorbol 12-myristate 13-acetate (PMA) increased significantly the level of expression of galectin-3 in THP-1 cells. PMA-induced galectin-3 overexpression was blocked by: protein kinase C inhibitors staurosporine, calphostin C, and apigenin; tyrosine-specific protein kinase inhibitors genistein and tyrphostin A25; PD 98059, a selective inhibitor of mitogen-activated protein kinase (MAPK) kinase 1 (MEK1 or MKK1); and SB 203580, a specific inhibitor of p38 MAPK. Galectin-3 up-regulation was not affected by exposure to two inhibitors of cAMP-dependent protein kinase (PKA), H-89 and KT5720. Co-transfection of pPG3.5, a plasmid vector containing the rabbit galectin-3 promoter and the constructs pMCL-MKK1 N3 or pRC-RSV-MKK3Glu that constitutively express MKK1 and MKK3, raised the activity of galectin-3 promoter by 185% and 110%, respectively. Co-transfection with a Ha-Ras expression vector stimulated galectin-3 promoter activity approximately 10-fold. Expression of c-Jun or v-Jun raised the level of galectin-3 promoter activity more the three- and fourfold, respectively. Co-transfection of c-Jun and pPG3.5 5'-upstream deletion mutants resulted in a reduction of the galectin-3 promoter activity by 50% to 80%. Transfection of c-Jun, v-Jun or Ha-Ras increased significantly galectin-3 protein in THP-1 cells. These findings indicated that Ras/MEKK1/MKK1-dependent/AP-1 signal transduction pathway plays an important role in the expression of galectin-3 in PMA-stimulated macrophages. We further investigated the effect of modified lipoproteins on galectin-3 expression in macrophages. Murine resident peritoneal macrophages loaded with acetylated low-density lipoprotein (AcLDL) or oxidized LDL (OxLDL) showed increased galectin-3 protein and mRNA. These results showed that treatment of macrophages with PMA or modified lipoproteins results in galectin-3 overexpression. These findings may explain the enhanced expression of galectin-3 in atherosclerotic foam cells and suggest that Ras/MAPK signal transduction pathway is involved in controlling this gene.     

Tim-3 ligand galectin-9 reduces IL-17 level and accelerates Klebsiella pneumoniae infection

T cell immunoglobulin and mucin domain (Tim)-3 is expressed on activated CD4⁺ and CD8⁺ T cells. Identification of galectin-9 as a ligand for Tim-3 has now firmly established the Tim-3/galectin-9 pathway, which results in apoptosis of effector CD4⁺ and CD8⁺ T cells. Moreover, Th17 cells are a recently discovered CD4⁺ effector T cell, which are important in antimicrobial immunity. Whether the Tim-3/galectin-9 pathway affects Th17 immunity has not been elucidated. Here, we demonstrated expression of Tim-3 on Th17 cells by flow cytometry. Th17-skewed cells were sensitive to galectin-9-induced apoptosis. In vitro administration of galectin-9 decreased stimulated Th17 cells and inhibited production of IL-17. Interestingly, Klebsiella pneumoniae (K. pneumoniae) infection led to enhanced IL-17 levels. Recombinant galectin-9 significantly decreased IL-17 in vivo, which resulted in reduced bacterial clearance and high mortality. These observations suggest that the Tim-3/galectin-9 pathway plays an important role in termination of Th17-immune responses, and could be a therapeutic target for inflammatory diseases.