Galectin-1 Knockdown Research Articles

Human multipotent mesenchymal stromal cells use galectin-1 to inhibit immune effector cells

AbstractHuman multipotent mesenchymal stromal cells (MSCs) suppress proliferation and alloreactivity of T cells. Several signaling molecules and enzymes contribute to this effect. We focused on carbohydrate-protein interactions and investigated whether lectins are involved in immune modulation by MSC. Gene expression profiling of MSCs revealed that one of the most important lectins in this setting, galectin-1, was highly expressed. Galectin-1 protein was detected intracellularly and on the cell surface of MSCs. In addition, galectin-1 was released into the cell culture supernatant by MSCs. To analyze the functional role of galectin-1, a stable knockdown of galectin-1 in MSCs with use of a retroviral transfection system was established. Galectin-1 knockdown in MSCs resulted in a significant loss of their immunomodulatory properties, compared with MSCs infected with nontargeting control sequences. The galectin-1 knockdown partially restored the proliferation of CD4+ and CD8+ T cells. By contrast, the effect of MSCs on nonalloreactive natural killer (NK) cells was unaffected by down-regulation of galectin-1 expression. Furthermore, MSC-derived galectin-1 significantly modulated the release of cytokines involved in graft-versus-host disease (GVHD) and autoimmunity (eg, tumor necrosis factor-α [TNFα], IFNγ, interleukin-2 [IL-2], and IL-10. These results identify galectin-1 as the first lectin mediating the immunomodulatory effect of MSCs on allogeneic T cells.

Hypoxia inducible factor-1 mediates expression of galectin-1: the potential role in migration/invasion of colorectal cancer cells

The expression of galectin-1, one of the most important lectins participating in the malignant tumor development, has been shown to be regulated by hypoxia, but its exact mechanism remains elusive. Here, we find that ectopically expressed hypoxia-inducible factor (HIF) 1alpha protein, an oxygen-sensitive subunit of HIF-1 that is a master factor for cellular response to hypoxia, significantly increases galectin-1 expression in both messenger RNA and protein levels in all four colorectal cancer (CRC) cell lines tested. However, hypoxia-induced galectin-1 expression cannot be seen in sentrin/SUMO-specific protease 1 homozygous-null mouse embryonic fibroblasts that fail to accumulate HIF-1alpha protein. Furthermore, silence of HIF-1alpha or HIF-1beta expression by specific short hairpin RNAs (shRNAs) antagonizes hypoxia-induced galectin-1 expression. All these results propose that galectin-1 is a direct target of transcriptional factor HIF-1. Applying luciferase reporter assay and chromatin immunoprecipitation, we identify that two hypoxia-responsive elements located at -441 to -423 bp upstream to transcriptional start site of galectin-1 gene are essential for HIF-1-mediated galectin-1 expression. Finally, the knockdown of galectin-1 by its specific shRNA can significantly reduce hypoxia-induced invasion and migration of CRC cell line, and the ectopic expression of galectin-1 can remarkably restore invasion and migration abilities of HIF-1alpha-knocked SW620 cells, proposing that galectin-1 mediates the HIF-1-induced migration and invasion of CRC cells during hypoxia. Taken together, our results shed new light for understanding mechanism for hypoxia/HIF-1-mediated migration/invasion of CRC cells.

Intra-articular lentivirus-mediated delivery of galectin-3 shRNA and galectin-1 gene ameliorates collagen-induced arthritis

Different members of the galectin family may have inhibitory or stimulatory roles in controlling immune responses and regulating inflammatory reactions in autoimmune diseases such as rheumatoid arthritis (RA). A hypothetical model of a cross talk between galectin-1 and galectin-3 has been established in the circumstance of rheumatoid joints. As galectin-3 is a positive regulator and galectin-1 is a negative regulator of inflammation and autoimmune responses, in this study we evaluated the effects of local knockdown of galectin-3 or overexpression of galectin-1 on ameliorating collagen-induced arthritis (CIA) in rats. Lentiviral vectors encoding galectin-3 small hairpin RNA (shRNA) and galectin-1, as well as two control vectors expressing luciferase shRNA and green fluorescent protein, were individually injected intra-articularly into the ankle joints of rats with CIA, and their treatment responses were monitored by measuring the clinical, radiological and histological changes. Our results show that both knockdown of galectin-3 and overexpression of galectin-1 induced higher percentages of antigen-induced T-cell death in the lymph node cells from arthritic rats. Furthermore, these treatments significantly reduced articular index scores, radiographic scores and histological scores, accompanied with decreased T-cell infiltrates and reduced microvessel density in the ankle joints. Our findings implicate galectin-3 and galectin-1 as potential therapeutic targets for the treatment of RA.

Abstract 301: Galectin-1 modulates PI3 kinase activity

Abstract The purpose of these experiments was to study the role that Galectin-1 (GAL1) has in Phosphoinositide 3-kinase (PI3K) signaling. GAL1 protein levels have been identified to be significantly upregulated in gliomas. GAL1 levels have also been closely associated with patient survival. The PI3K enzyme converts phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol (3,4,5)-trisphosphate (PIP3). PIP3 serves as a membrane anchor leading to the activation of AKT. PI3K/AKT signaling is important in cancer cell biology because induction of this pathway is associated with increased cell survival, cell cycle progression, and growth. A PIP3 Mass ELISA was performed on cell lysates isolated from wild type (WT) and GAL1 knockdown cells with and without Inslulin-like growth factor 1 (IGF1) stimulation. Briefly, cells were stimulated with 5mM IGF1 followed by cell lysis. Lysates were compared by ELISA for PIP3 levels. Following this an in vitro PI3K assay was done. This assay is designed to measure the activity of a purified PI3K enzyme by detecting PIP2 to PIP3 conversion. Enzyme was incubated with Gal1 alone, LY294002 PI3K inhibitor, or with both Gal1 and the inhibitor. Controls included enzyme only, enzyme with inhibitor, no enzyme, and no substrate. In order to investigate whether a physical interaction was occurring between Gal1 and PI3K, Co-IP experiments were performed. These experiments involved isolation of cell lysates from WT LN229 cells and GAL1 knockdown LN229 cells. Following pulldown, Co-IP's were run on an SDS-PAGE gel, transferred to blots, and probed for GAL1 and p85 α or β. Previous experiments demonstrated a possible role for GAL1 in PI3K signaling and AKT activation. In the PIP3 mass ELISA experiment, WT cells demonstrated elevated cellular PIP3 levels in response to IGF1 stimulation, as expected. GAL1 knockdown cells did not show any increase in PIP3 levels in response to stimulation. We next examined if GAL1 was having a direct effect on PI3K activity. In vitro PI3K reactions showed a specific upregulation of PI3K activity in response to addition of GAL1. In response to 1000-fold changes in GAL1 concentration there was an approximate 2-fold increase in PI3K (p85α/p110α) activity. GAL1 also demonstrated an ability to overcome LY294002 inhibition of the enzyme. Enzyme incubated with both inhibitor and 5uM GAL1 showed 4 times the conversion as compared to enzyme with inhibitor alone. Interaction studies indicate that there is not a physical interaction between GAL1 and either p85 α or β. Future studies characterizing the role of GAL1 in PI3K signaling include repeating the PI3K assay with other PI3K class I isoforms to examine differences. Fluorescence Resonance Energy Transfer (FRET) experiments will also be performed to further test for a physical interaction between PI3K and GAL1. In conclusion GAL1 has demonstrated an ability to effect PI3K activity, and thereby PI3K signaling. However, a physical interaction between GAL1 and PI3K was not observed. 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 301.

Evidence for the Involvement of Galectin-3 in Mesenchymal Stem Cell Suppression of Allogeneic T-Cell Proliferation

Human bone marrow-derived mesenchymal stem cells (MSC) are multipotent non-hematopoietic progenitors that have regulatory activity on immune cells. NOD- and Toll-like receptors (NLR, TLR) have several roles in immunity, including those relevant to pathogen recognition and shaping the course of immune responses by controlling gene expression. We have shown that these innate immune receptors are expressed by hematopoietic CD34+ progenitors and MSC. To uncover genes critical in MSC function, first we have used microarray to screen for potential transcripts whose levels are altered in response to NOD-1 and TLR-2 activation, and second we validated some candidate genes using real-time RT-PCR, Western blots and cellular assays. Amongst the altered genes, galectin-3 was upregulated at both mRNA and protein levels in response to TLR-2 activation. Interestingly, MSC secreted galectin-3, a protein known to modulate T-cell proliferation, gene expression, cell adhesion and migration. Knockdown of galectin-3 in MSC using small interfering RNA (siRNA) reduced the immunosuppressive effect of MSC on mixed lymphocyte cultures when compared to cells treated with an irrelevant siRNA (P < 0.05). Collectively, the data emphasize a new role of galectin-3 in the immunomodulatory function of MSC and indicate that NOD signalling pathway is also functional in these cells.

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

beta-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 beta-1,4-galactosylation of N-glycans increases predominantly in a 100-kDa protein band on lectin blot analysis in combination with digestions by diplococcal beta-galactosidase and N-glycanase. When cells at 100% density were treated with jack bean beta-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.

The Potential Role of Galectin-1 in Hypoxia Induced AML Cell Differentiation through C/EBPαa Transcription Regulation.

Abstract 3120Poster Board III-57Recently we reported that cobalt chloride-simulated hypoxia and mild hypoxia could induce the differentiation of human acute myeloid leukemic (AML) cells probably through hypoxia inducible factor-1 alpha (HIF-1αa) independent of its transcription activity. In this study, we used differential gel electrophoresis to compare the HIF-1αa regulated proteins between conditional induction and absence of HIF-1αa protein in U937T cells. Among all the up-regulated proteins indentified, the mRNA and protein level of galectin-1 was most remarkably changed. Knockdown of galectin-1 partially blocked the differentiation induced by HIF-1αa overexpression in U937T cells as assessed by morphological criteria and differentiation associated-antigens. Notably, reduced expression of C/EBPαa, a critical transcription factor for granulocyte differentiation, could inhibit the up-regulation of galectin-1 induced by HIF-1αa, and inducible expression of C/EBPαa in U937T cells also increased galectin-1 expression. Furthermore, by using the luciferase assay and chromatin immunoprecipitation we localized the binding site of C/EBPαa in the promoter of galectin-1. Finally the role of galectin-1 during chloride-simulated hypoxia and mild hypoxia was addressed by decreasing the expression of galectin-1 with small interfering RNA. Taken together, these results conclude that galectin-1 is required for hypoxia triggered C/EBPαa dependent AML cell differentiation. DisclosuresNo relevant conflicts of interest to declare.

Galectin-3 regulates myofibroblast activation and hepatic fibrosis

Central to fibrogenesis and the scarring of organs is the activation of fibroblasts into matrix-secreting myofibroblasts. We demonstrate that Galectin-3 expression is up-regulated in established human fibrotic liver disease and is temporally and spatially related to the induction and resolution of experimental hepatic fibrosis. Disruption of the Galectin-3 gene blocks myofibroblast activation and procollagen (I) expression in vitro and in vivo, markedly attenuating liver fibrosis. Addition of exogenous recombinant Galectin-3 in vitro reversed this abnormality. The reduction in hepatic fibrosis observed in the Galectin-3(-/-) mouse occurred despite equivalent liver injury and inflammation, and similar tissue expression of TGF-beta. TGF-beta failed to transactivate Galectin-3(-/-) hepatic stellate cells, in contrast with WT hepatic stellate cells; however, TGF-beta-stimulated Smad-2 and -3 activation was equivalent. These data suggest that Galectin-3 is required for TGF-beta mediated myofibroblast activation and matrix production. Finally, in vivo siRNA knockdown of Galectin-3 inhibited myofibroblast activation after hepatic injury and may therefore provide an alternative therapeutic approach to the prevention and treatment of liver fibrosis.

Potential involvement of galectin-3 and SNAP23 in Aeromonas hydrophila cytotoxic enterotoxin-induced host cell apoptosis

We investigated the potential of the cytotoxic enterotoxin (Act) of Aeromonas hydrophila to bind to 1869 human and 4319 yeast proteins, using protein microarray technology. Act was capable of binding nine different human proteins, including the SNARE complex scaffolding protein synaptosomal-associated protein 23 (SNAP23), galectin-3, and guanylate kinase 1 (GUK-1). Act was also able to bind to four of the yeast proteins examined, which included the vesicle tethering protein Vsp52. We verified interaction of Act with murine and human SNAP23, galectin-3, and GUK-1 by sandwich Western blot analysis. In order to determine the physiological relevance of Act binding to these three proteins, we performed small interfering RNA (siRNA) gene knockdown experiments in RAW 264.7 cells, a murine macrophage cell line in which Act-induced signaling and cell death is well characterized. Based on real-time reverse transcriptase-polymerase chain reaction, siRNA transfection of RAW 264.7 cells with specific oligonucleotides reduced the expression of genes encoding SNAP23, galectin-3, and GUK-1 by 62, 63, and 99%, respectively. Knockdown of galectin-3 and SNAP23, but not GUK-1, significantly reduced Act-induced apoptosis of host cells, as determined by TUNEL (TdT-mediated dUTP nick end labeling) assay, lactate dehydrogenase release, Giemsa staining, and reduction in activation of caspase 3, compared to toxin-treated macrophages that were transfected with a random sequence control siRNA. We also performed these assays using a human intestinal epithelial cell line (HT-29) and observed a similar trend of galectin-3 and SNAP23 association with Act-induced apoptosis. This is the first report of putative protein binding partners for this toxin and potential mediators/regulators of Act-induced apoptosis.