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.     

Galectin-3 modulates phagocytosis-induced stellate cell activation and liver fibrosis in vivo

Hepatic stellate cells (HSC), the key fibrogenic cells of the liver, transdifferentiate into myofibroblasts upon phagocytosis of apoptotic hepatocytes. Galectin-3, a β-galactoside-binding lectin, is a regulator of the phagocytic process. In this study, our aim was to study the mechanism by which extracellular galectin-3 modulates HSC phagocytosis and activation. The role of galectin-3 in engulfment was evaluated by phagocytosis and integrin binding assays in primary HSC. Galectin-3 expression was studied by real-time PCR and enzyme-linked immunosorbent assay, and in vivo studies were done in wild-type and galectin-3(-/-) mice. We found that HSC from galectin-3(-/-) mice displayed decreased phagocytic activity, expression of transforming growth factor-β1, and procollagen α1(I). Recombinant galectin-3 reversed this defect, suggesting that extracellular galectin-3 is required for HSC activation. Galectin-3 facilitated the α(v)β(3) heterodimer-dependent binding, indicating that galectin-3 modulates HSC phagocytosis via cross-linking this integrin and enhancing the tethering of apoptotic cells. Blocking integrin α(v)β(3) resulted in decreased phagocytosis. Galectin-3 expression and release were induced in active HSC engulfing apoptotic cells, and this was mediated by the nuclear factor-κB signaling. The upregulation of galectin-3 in active HSC was further confirmed in vivo in bile duct-ligated (BDL) rats. Galectin-3(-/-) mice displayed significantly decreased fibrosis, with reduced expression of α-smooth muscle actin and procollagen α1(I) following BDL. In summary, extracellular galectin-3 plays a key role in liver fibrosis by mediating HSC phagocytosis, activation, and subsequent autocrine and paracrine signaling by a feedforward mechanism.     

Expression of Galectin-7 Is Induced in Breast Cancer Cells by Mutant p53

Galectin-7 was initially described as a marker of epithelial differentiation expressed in the stratified epithelium of various tissues. Like other members of the galectin family, its expression level is often significantly altered in cancer cells. In breast cancer, its expression is significantly augmented in aggressive molecular subtypes, most notably in estrogen receptor-negative tumors and in cell lines with a basal-like phenotype. Studies using experimental mouse models have further shown high expression of galectin-7 was sufficient to increase the metastatic behavior of poorly metastatic breast cancer cells, rendering them more resistant to apoptosis. This expression pattern in breast cancer cells is unexpected because galectin-7 was originally identified as a p53-induced gene. To address this paradox, we have examined the molecular mechanisms regulating galectin-7 in breast cancer cells. Our results showed that transfection of breast cancer cells with expression vectors encoding mutant p53 was sufficient to induce galectin-7 at both mRNA and protein levels. Doxorubicin treatment of breast cancer cells harboring a mutant p53 also induced galectin-7. This induction was specific since knockdown of endogenous mutant p53 inhibited doxorubicin-induced galectin-7 expression. The p53-induced galectin-7 expression in breast cancer cells correlated with increased NF-κB activity and was inhibited by NF-κB inhibitors, indicating that the ability of mutant p53 to induce galectin-7 was dependent on NF-κB activity. The implication of NF-κB was further supported by data showing that NF-κB bound to the endogenous galectin-7 promoter and that TNFα-induced galectin-7 expression was abolished by NF-κB inhibitors. Taken together, our data provide an explanation to the observed high galectin-7 expression levels in cancer cells and suggest that galectin-7 could be part of a common pathway used by mutant p53 to promote cancer progression.     

TGFβ regulates Galectin-3 expression through canonical Smad3 signaling pathway in nucleus pulposus cells: implications in intervertebral disc degeneration

Galectin-3 is highly expressed in notochordal nucleus pulposus (NP) and thought to play important physiological roles; however, regulation of its expression remains largely unexplored. The aim of the study was to investigate if TGFβ regulates Galectin-3 expression in NP cells. TGFβ treatment resulted in decreased Galectin-3 expression. Bioinformatic analysis using JASPAR and MatInspector databases cross-referenced with published ChIP-Seq data showed nine locations of highly probable Smad3 binding in the LGALS3 proximal promoter. In NP cells, TGFβ treatment resulted in decreased activity of reporters harboring several 5′ deletions of the proximal Galectin-3 promoter. While transfection of NP cells with constitutively active (CA)-ALK5 resulted in decreased promoter activity, DN-ALK5 blocked the suppressive effect of TGFβ on the promoter. The suppressive effect of Smad3 on the Galectin-3 promoter was confirmed using gain- and loss-of-function studies. Transfection with DN-Smad3 or Smad7 blocked TGFβ mediated suppression of promoter activity. We also measured Galectin-3 promoter activity in Smad3 null and wild type cells. Noteworthy, promoter activity was suppressed by TGFβ only in wild type cells. Likewise, stable silencing of Smad3 in NP cells using sh-Smad3 significantly blocked TGFβ-dependent decrease in Galectin-3 expression. Treatment of human NP cells isolated from tissues with different grades of degeneration showed that Galectin-3 expression was responsive to TGF-β-mediated suppression. Importantly, Galectin-3 synergized effects of TNF-α on inflammatory gene expression by NP cells. Together these studies suggest that TGFβ, through Smad3 controls Galectin-3 expression in NP cells and may have implications in the intervertebral disc degeneration.     

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.     

Developmental aspects of galectin-3 expression in the lens

In order to investigate the temporal and spatial expression pattern of the lectin galectin-3 during lens development we performed immunohistochemical studies using monoclonal and polyclonal antibodies against galectin-3 on paraffin sections of human, mouse and rat eyes. Galectin-3 has been shown to be involved in various biological functions related to cell adhesion, proliferation, apoptosis and differentiation in other tissues. In the human lens, galectin-3 shows a selective expression pattern during lens development. It is present in all cells of the early lens vesicle and at later stages it is strongly expressed during the elongation phase in differentiating primary lens fibres. From about 7 weeks onwards the anterior lens epithelium fails to express galectin-3. Adult lenses, however, exhibit immunoreactivity in the anterior epithelial cells and in the early differentiating secondary fibres of the lens' outer cortex prior to the onset of degradation of the nuclei. In contrast to the observed expression pattern in prenatal human lenses, mouse and rat lenses exhibited immunoreactivity for galectin-3 during postnatal and adult stages only. At these stages, the expression pattern closely resembles that seen in the corresponding human lenses. The spatiotemporal pattern of galectin-3 distribution during lens development favours a role of this lectin in adhesion processes and in the regulation of programmed organelle elimination during lens cell differentiation.     

Galectin-3 and metastasis

Galectin-3, a 31 kDa member of the beta-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.     

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 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.     

Multifaceted role of galectin-3 on human glioblastoma cell motility

Astrocytic tumors' aggressiveness results from an imbalance between cell proliferation and cell death favoring growth, but also from the propensity of tumor cells to detach from the primary tumor site, migrate, and invade the surrounding parenchyma. Astrocytic tumor progression is known to be associated with an increased expression of galectin-3. We investigated in cell culture how galectin-3 expression affects astrocytoma cell motility. Galectin-3 deficient cells were obtained by stable transfection of the U373 glioblastoma cell line with a specific expression antisense plasmid. Cultured galectin-3 deficient glioblastoma cells showed increased motility potential on laminin and modifications in the cytoskeleton reorganization. In addition, c-DNA microarrays and quantitative immunofluorescence analysis showed that galectin-3 deficient U373 cells have an increased expression of integrins-alpha6 and -beta1, proteins known to be implicated in the regulation of cell adhesion.     

Transfer to in vitro conditions influences expression and intracellular distribution of galectin-3 in murine peritoneal macrophages

Galectin-3 is a beta-galactoside-binding lectin that has been implicated in numerous physiological processes, including mRNA splicing, cell differentiation, tumor metastasis and the stress response. We have studied effects of transfer of resident murine peritoneal macrophages to in vitro conditions on galectin-3 in different cell compartments. Galectin-3 was purified by immunoprecipitation with rat monoclonal antibody M3/38, and analyzed by immunoblotting using the same antibody. Transfer to in vitro conditions nearly doubled the total amount of galectin-3 in cells, and caused significant alterations in its intracellular distribution, indicating that galectin-3 is involved in the adaptation of peritoneal macrophages to in vitro conditions.     

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。