Galectin-3 expression in response to LPS, immunomodulatory drugs and exogenously added galectin-3 in monocyte-like THP-1 cells

Galectin-3, a structurally unique beta-galactoside-binding lectin, through the specific protein?Cprotein and protein?Ccarbohydrate interactions participates in numerous biological processes, such as cell proliferation and apoptosis, adhesion and activation. Its expression and secretion by until now an unknown mechanism are modulated by diverse molecules and are dependent on different physiological and pathophysiological conditions. By autocrine and paracrine actions, galectin-3 modulates many immune reactions and affects various immune cells, particularly those of monocyte?Cmacrophage lineage. This is why galectin-3 has recently become an attractive therapeutic target. However, molecular mechanisms of its actions as well as regulatory mechanism of its expression and activation are still largely unknown. In this study, we show that lipopolysaccharide (LPS) provokes upregulation of galectin-3 expression on both gene and protein level in monocyte-like THP-1 cells, which can be inhibited by dexamethasone, but not with non-steroidal anti-inflammatory drugs aspirin and indomethacin. Resting and LPS-challenged monocyte-like THP-1 cells do not have detectable amount of surface-bound galectin-3, but are able to bind exogenously added galectin-3 with the same capacity. Although galectin-3 is generally considered to be a pro-inflammatory molecule, here we show that the exogenously added galectin-3 does not affect interleukin (IL)-1??, IL-6, IL-8, IL-10, IL-12p70 and TNF-?? production in resting and LPS-activated monocyte-like THP-1 cells nor influences its own gene expression level in those cells.     

半乳糖苷凝集素-3与肥胖及2型糖尿病

肥胖及2型糖尿病是代谢紊乱相关的慢性低度系统炎症状态。半乳糖苷凝集素-3（galectin-3）是一种β-半乳糖苷结合蛋白,在炎症、信号转导、细胞增殖与分化等过程中发挥重要作用。新近的研究表明,半乳糖苷凝集素-3在患肥胖和2型糖尿病的人及鼠类体内高表达,对鼠类体脂的沉积、脂肪细胞分化、血糖浓度、胰岛素敏感性、葡萄糖耐受性和系统炎症等具有重要影响。本文综述了半乳糖苷凝集素-3的结构、分布及其对肥胖和2型糖尿病的调控作用与分子机制,以期为研发针对半乳糖苷凝集素-3靶点的新药提供重要思路和参考。     

Cell-type specific regulation of galectin-3 expression by glucocorticoids in lung Clara cells and macrophages

Bronchiolar Clara cells are integral components of lung homeostasis, predominantly distributed in distal airways. In addition to the 16 kDa Clara cell protein, a major secretory product with anti-inflammatory effects, rat Clara cells express the glycan-binding protein galectin-3 and secrete it into the airways. Given the essential role of galectin-3 in the control of inflammation and the well-established function of glucocorticoids (GCs) in lung physiology, here we investigated whether galectin-3 is a target of the regulatory effects of GCs. Adult male rats were subjected to bilateral adrenalectomy and the lungs were processed for light and transmission electron microscopy, immunoelectron microscopy and Western blot analysis. Profound changes in bronchiolar Clara cells and macrophage morphology could be observed by electron microscopy after adrenalectomy. While specific galectin-3 staining was detected in the nucleus and cytoplasm of Clara cells and macrophages from control animals, cytoplasmic galectin-3 expression was dramatically reduced after adrenalectomy in both cell types. This effect was cell-specific as it did not affect expression of this lectin in ciliated cells. After dexamethasone treatment, galectin-3 expression increased significantly in the nucleus and cytoplasm of macrophages and Clara cells. Western blot analysis showed a clear decrease in galectin-3 expression in ADX animals, which was recovered after a 7-day treatment with dexamethasone. In peritoneal macrophages, galectin-3 expression was also dependent on the effects of GCs both in vivo and in vitro. Our results identify a cell type-specific control of galectin-3 synthesis by GCs in lung bronchiolar Clara cells and interstitial macrophages, which may provide an alternative mechanism by which GCs contribute to modulate the inflammatory response.     

Cyclin-dependent kinase inhibitors and JNK act as molecular switches, regulating the choice between growth arrest and apoptosis induced by galectin-8

Galectin-8, a mammalian beta-galactoside binding lectin, functions as an extracellular matrix protein that forms high affinity interactions with integrins. Here we demonstrated that soluble galectin-8 inhibits cell cycle progression and induces growth arrest. These effects cannot be attributed to interference with cell adhesion but can be attributed to a 4-5-fold increase in the cellular content of the cyclin-dependent kinase inhibitor p21, which was already evident following a 4-h incubation of H1299 cells with galectin-8. The increase in p21 levels was preceded by a 3-5-fold increase in JNK and protein kinase B (PKB) activities. Accordingly, SP600125, the inhibitor of JNK, and wortmannin, the inhibitor of phosphatidylinositol 3-kinase, which is the upstream activator of PKB, inhibited the increase in the cellular content of p21. Furthermore, overexpression of a dominant inhibitory form of SEK1, the upstream kinase regulator of JNK, inhibited both JNK activation and p21 accumulation. When p21 expression was inhibited by cycloheximide, galectin-8 directed the cells toward apoptosis, which involves induction of poly(ADP-ribose) polymerase cleavage. Indeed, galectin-8-induced apoptosis was 2-fold higher in HTC (p21-null) cells when compared with parental HTC cells. Because overexpression of galectin-8 attenuates the rate of DNA synthesis, stable colonies that overexpress and secrete galectin-8 can be generated only in cells overexpressing a growth factor receptor, such as the insulin receptor. These results implicate galectin-8 as a modulator of cellular growth through up-regulation of p21. This process involves activation of JNK, which enhances the synthesis of p21, combined with the activation of PKB, which inhibits p21 degradation. These effects of the lectin depended upon protein-sugar interactions and were induced when galectin-8 was present as a soluble ligand or when it was overexpressed in cells.     

Galectin-3 Silencing Inhibits Epirubicin-Induced ATP Binding Cassette Transporters and Activates the Mitochondrial Apoptosis Pathway via β-Catenin/GSK-3β Modulation in Colorectal Carcinoma

Multidrug resistance (MDR), an unfavorable factor compromising the treatment efficacy of anticancer drugs, involves the upregulation of ATP binding cassette (ABC) transporters and induction of galectin-3 signaling. Galectin-3 plays an anti-apoptotic role in many cancer cells and regulates various pathways to activate MDR. Thus, the inhibition of galectin-3 has the potential to enhance the efficacy of the anticancer drug epirubicin. In this study, we examined the effects and mechanisms of silencing galectin-3 via RNA interference (RNAi) on the β-catenin/GSK-3β pathway in human colon adenocarcinoma Caco-2 cells. Galectin-3 knockdown increased the intracellular accumulation of epirubicin in Caco-2 cells; suppressed the mRNA expression of galectin-3, β-catenin, cyclin D1, c-myc, P-glycoprotein (P-gp), MDR-associated protein (MRP) 1, and MRP2; and downregulated the protein expression of P-gp, cyclin D1, galectin-3, β-catenin, c-Myc, and Bcl-2. Moreover, galectin-3 RNAi treatment significantly increased the mRNA level of GSK-3β, Bax, caspase-3, and caspase-9; remarkably increased the Bax-to-Bcl-2 ratio; and upregulated the GSK-3β and Bax protein expressions. Apoptosis was induced by galectin-3 RNAi and/or epirubicin as demonstrated by chromatin condensation, a higher sub-G1 phase proportion, and increased caspase-3 and caspase-9 activity, indicating an intrinsic/mitochondrial apoptosis pathway. Epirubicin-mediated resistance was effectively inhibited via galectin-3 RNAi treatment. However, these phenomena could be rescued after galectin-3 overexpression. We show for the first time that the silencing of galectin-3 sensitizes MDR cells to epirubicin by inhibiting ABC transporters and activating the mitochondrial pathway of apoptosis through modulation of the β-catenin/GSK-3β pathway in human colon cancer cells.     

Human galectin-2: expression profiling by RT-PCR/immunohistochemistry and its introduction as a histochemical tool for ligand localization

Sugar-encoded information of glyco-conjugates is translated into cellular responses by endogenous lectins. Galectins stand out against other lectin families due to their wide range of functions including cell adhesion, tissue invasion or growth regulation exerted at extracellular, membrane, cytoplasmic and nuclear sites. This remarkable versatility warrants close scrutiny of their emerging network, in this study with focus on homodimeric human galectin-2. We first detected presence of specific mRNA in various tissue types by processing post mortem and surgical specimens by RT-PCR protocols. Overlap of gene expression was noted with proto-type galectins-1 and -7 and also family members from the other two subgroups. To monitor expression on the level of protein a polyclonal anti-galectin-2 antibody was raised. Immunopositivity was semi-quantitatively assessed in sections of 209 human samples establishing an array both of normal tissues and samples with inflammation or benign/malignant growth. In general, positivity was predominantly epithelial without restriction of staining to certain tissue types, as fittingly indicated by our RT-PCR analysis. Staining was not limited to the cytoplasm but also included nuclear sites. To examine the suitability of the labeled lectin as a histochemical probe we biotinylated galectin-2 under activity-preserving conditions and introduced it to tissue profiling. Specific cytoplasmic staining proved the validity of the concept. Our results encourage systematic histopathologic studies by immuno- and lectin histochemistry, especially by adding galectin-2 as study object to galectin fingerprinting which has already yielded prognostic information on galectins-1, -3, -4 and -8 and hereby contributed to define functional overlap/divergence in this lectin family.     

Galectin-7 (PIG1) exhibits pro-apoptotic function through JNK activation and mitochondrial cytochrome c release.

Galectin-7 is normally expressed in all types of stratified epithelia, but is significantly down-regulated in squamous cell carcinomas. This protein was recently found to be highly inducible by p53 in a colon carcinoma cell line, DLD-1, and designated as PIG1 (for p53-induced gene 1). We studied transfectants of HeLa and DLD-1 cells ectopically expressing this protein and found that they were more susceptible to apoptosis than control transfectants. This was observed in apoptosis induced by mechanistically distinct stimuli, suggesting that galectin-7 acts on a common point in the apoptosis signaling pathways. Further analyses of actinomycin D-induced apoptosis demonstrated that galectin-7 expression causes enhanced caspase-3 activity and poly(ADP-ribose) polymerase cleavage, and the potentiation of apoptosis by galectin-7 was completely abrogated by a caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone. In addition, galectin-7 transfectants displayed accelerated mitochondrial cytochrome c release and up-regulated JNK activity upon apoptosis induction. Several lines of evidence indicate that the effect on apoptosis is not due to the lectin functioning extracellularly through interactions with cell surface glycoconjugates. In fact, this lectin is found to localize in nuclei and cytoplasm of the transfectants and the transformed keratinocyte line HaCaT. Therefore, galectin-7 is a pro-apoptotic protein that functions intracellularly upstream of JNK activation and cytochrome c release. DNA microarray analysis revealed genes that are differentially expressed between galectin-7 and control transfectants. Some of them are potentially contributory to this lectin's proapoptotic function and these include redox-related genes monoamine oxidase B, ryanodine receptor 2, and glutathione S-transferase Mu 3.     

Sialylation regulates galectin-3/ligand interplay during mammary tumour progression--a case of targeted uncloaking

Galectin-3 is involved both in facilitating detachment of cells from primary tumour sites and favouring cancer cell adhesion and survival to anoikis in the blood stream. The mechanisms behind these apparently contradictory roles of the lectin have not yet been resolved. In order to investigate possible interplays between galectin-3 and its ligands underlying their role in the metastatic process, we examined mucin-1 (MUC1) and epidermal growth factor receptor (EGFR), well-known galectin-3 ligands, as well as galectin-3-binding site expression in a series of spontaneous canine malignant mammary tumours (CMMT) and a metastatic CMMT cell line. Despite the fact that CMMT cells expressed MUC1 and EGFR homogeneously over their plasma membrane, intravascular tumour cells, positive for galectin-3, expressed MUC1 and EGFR in a more focal membrane localization. Moreover, MUC1 overexpression in primary CMMT was present in parallel with down-regulation of galectin-3. Furthermore, in the CMT-U27 cell line, galectin-3 knock-down led to increased MUC1 expression, while MUC1 knock-down led to down-regulation of the lectin. Finally, removal of sialic acid from both CMMT and CMT-U27 xenograft samples exposed galectin-3-ligands throughout the tumour tissue, whereas these ligands were only present in galectin-3-positive invading cells in untreated samples. Interestingly indeed, we show that in vessel-invading cells, there is interaction between galectin-3 and the T antigen in vivo. We therefore hypothesized that loss of galectin-3 and sialylation-related masking of its ligands, in conjunction with their overexpression in specific tumour cell subpopulations, are crucial in regulating adhesive/de-adhesive events in the progression and invasive capacity of metastatic 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.     

Purification of galectin-3 from ovine placenta: developmentally regulated expression and immunological relevance

Galectins, beta-galactoside-binding lectins, are extensively distributed in the animal kingdom and share some basic molecular properties. Galectin-3, a member of this family, is generally associated with differentiation, morphogenesis, and metastasis. In this study, galectin-3 was isolated from ovine placental cotyledons round the middle of the gestation period by lactose extraction followed by affinity chromatography on lactosyl-agarose, and separated from galectin-1 by size exclusion chromatography on a Superose 12 column. Under native conditions this lectin behaved as a monomer with an apparent molecular weight of approximately 29,000 and an isoelectric point of 9.0. The partial amino acid sequence of the peptides obtained by tryptic digestion of this protein followed by HPLC separation showed striking homology with other members of the galectin-3 subfamily. Furthermore, ovine placental galectin-3 exhibited specific mitogenic activity toward rat spleen mononuclear cells. Besides, this protein strongly reacted with a rabbit antiserum raised against a chicken galectin. Results obtained by Western blot analysis showed that its expression was greatly decreased in term placenta with respect to the middle of the gestation period, suggesting a regulated expression throughout development.      

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.     

Expression and clinical significance of galectin-3 in osteosarcoma

Galectin-3 is a multifunctional β-galactoside-binding protein which has been shown to play a role in carcinogenesis. However, the involvement of galectin-3 in osteosarcoma remains unclear. In this study, we aimed to examine the serum level of galectin-3 in osteosarcoma patients and healthy controls, and the protein expression of galectin-3 in osteosarcoma tissues and their adjacent non-malignant tissues. We further aimed to investigate the clinical significance of galectin-3 serum and protein expression levels. Galectin-3 serum level was evaluated using ELISA in 132 osteosarcoma patients and 184 healthy controls, while the protein expression of galectin-3 was determined using immunohistochemistry in the malignant and the surrounding non-malignant tissues of the same 132 osteosarcoma patients. Our results showed that the mean galectin-3 serum level was significantly higher in patients than in controls (2.35 ± 0.91 ng/ml vs. 0.86 ± 0.20 ng/ml) (p < 0.0001). Among patients, a higher galectin-3 serum level was significantly associated with the Enneking stage of cancer (p < 0.0001). In addition, we found a significant overrepresentation of high galectin-3 expression in osteosarcoma tissues than in non-malignant tissues (p < 0.0001). Galectin-3 expression in osteosarcoma tissues was also found to be correlated with the Enneking stage of cancer (p < 0.0001) and the occurrence of metastasis (p < 0.0001). In conclusion, galectin-3 could serve as a useful prognostic marker in osteosarcoma.     

Synthesis of an endogeneous lectin, galectin-1, by human endothelial cells is up-regulated by endothelial cell activation

The pattern of expression of an endogenous lectin, galectin-1, was examined in human lymphoid tissue. Galectin-1 was detected in the endothelial cells lining specialized vessels, termed high endothelial venules, in activated lymphoid tissue, but not in a resting lymph node. Cultured endothelial cells (human aortic and umbilical vein endothelial cells (HAECs and HUVECs)) expressed galectin-1. Activation of the cultured endothelial cells increased the level of galectin-1 expression, as determined by ELISA, Northern blot analysis and high throughput cDNA sequencing. These results suggest that galectin-1 expressed by endothelial cells may bind to and affect the trafficking of cells emigrating from blood into tissues.