Effect Of Gal-1 Research Articles

Galectin-1 promotes human neutrophil migration

An important step of innate immune response is the recruitment of polymorphonuclear leukocytes (PMN) to injured tissues through chemotactic molecules. Galectins, a family of endogenous lectins, participate in numerous functions such as lymphoid cell migration, homing, cell-cell and cell-matrix interactions. Particularly, galectin-3 (Gal-3) and -9 have been implicated in the modulation of acute and chronic inflammation by inducing the directional migration of monocytes/macrophages and eosinophils, whereas Gal-1 is considered to function as an anti-inflammatory molecule, capable of inhibiting the influx of PMN to the site of injury. In this study, we assessed the effect of Gal-1 on neutrophil recruitment, in the absence of additional inflammatory insults. Contrasting with its capacity to inhibit cell trafficking and modulate the release of mediators described in models of acute inflammation and autoimmunity, we evidenced that Gal-1 has the capacity to induce neutrophil migration both in vitro and in vivo. This effect is not mediated through a G-protein-coupled receptor but potentially through the sialoglycoprotein CD43, via carbohydrate binding and through the p38 mitogen-activated protein kinase pathway. These results suggest a novel biological function for CD43 on neutrophils and highlight that depending on the environment, Gal-1 can act either as chemoattractant or, as a molecule that negatively regulates migration under acute inflammatory conditions, underscoring the potential of Gal-1 as a target for innovative drug development.

Differential expression of galectin‐1 and its interactions with cells and laminins in the intervertebral disc

Galectin-1 (Gal-1), an endogenous β-galactoside-binding protein, binds to laminins, which are highly expressed in the nucleus pulposus (NP) of the intervertebral disc (IVD). The objective of this study is to evaluate the expression of Gal-1 protein in IVD tissues during aging and the effect of Gal-1 on IVD cell adhesion to laminins. Tissues from rat, porcine, and human (scoliosis or disc degeneration) IVDs were used to evaluate Gal-1 expression via immunostaining, RT-PCR, and Western blot analysis. Attachment of isolated IVD cells (porcine and human) on select laminin isoforms (LM-111 and LM-511) was compared with/without pre-incubation with exogenous Gal-1. A biotinylated Gal-1(B-Gal-1) was used to evaluate for binding to IVD cells and to select for IVD cells by magnetic activated cell sorting (MACS). NP cells expressed high levels of Gal-1 protein as compared to anulus fibrosus (AF) cells in immature tissues, while exogenous Gal-1 increased both NP and AF cell attachment to laminins and exhibited a similar binding to both cell types in vitro. With aging, Gal-1 levels in NP tissue appeared to decrease. In addition, incubation with B-Gal-1 was able to promote the retention of more than 50% of IVD cells via MACS. Our results provide new findings for the presence and functional role of Gal-1 within IVDs. Similar staining patterns for Gal-1 and LM-511 in IVD tissue suggest that Gal-1 may serve as an adhesion molecule to interact with both cells and laminins. This MACS protocol may be useful for selecting pure IVD cells from mixed cells of pathological tissue.

Abstract 2229: Galectin-1 reduces the efficacy of PI3K pathway inhibitors in glioblastoma cells

Abstract Galectin-1 (Gal-1), is a small ∼14kD carbohydrate binding protein that is known to be significantly upregulated in gliomas, and has also been closely associated with patient survival. PI3K/AKT signaling is important in cancer cells because upregulation of this pathway is associated with increased cell survival and growth. Specifically in glioblastomas there is a signaling alteration in the RTK/PI3K/RAS/PTEN axis in nearly 90% of patients leading to therapeutic resistance and poor prognosis. While Gal-1 has a defined role in activating MAPK signaling, the effect of Gal-1 on PI3K signaling is unclear. The purpose of these experiments was to study the role that Gal-1 has in cellular survival signaling, primarily the PI3K pathway. Previous experiments from our laboratory have demonstrated a significant role for Gal-1 in PI3K signaling and AKT activation. An in vitro PI3K reaction showed that Gal-1 can increase PI3K enzyme activity, even in the presence of a PI3K inhibitor. Gal-1 was also shown to specifically interact with the PI3K enzyme in an in vitro Co-IP assay. Based on these results, we investigated the role of Gal-1 in protecting cells from PI3K pathway inhibition. LN229, U87, and LN18 glioma cell lines were used for these experiments. Cells were treated with multiple different inhibitors for specific proteins in the PI3K signaling pathway (EGFR - Gefitinib, AKT - MK-2206, PI3K - LY294002 and PI-103, mTor - AZD 8055 and Everolimus). Initially, control cells were compared with cells of different Gal-1 status for PI3K pathway activation by western blot. We looked at multiple levels of activation including pAKT, p-mTOR, and p-p70S6K. Following this analysis, proliferation was examined by MTS assay and clonogenic survival was also tested. Our results indicate that in the absence of Gal-1, all of the different inhibitors listed above showed increased signaling inhibition in each of the cell lines tested. In the control cells, higher concentrations of these inhibitors were required to cause similar reduction in pathway activation markers such as pAKT. Based on these results we examined proliferation rates and clonogenic survival for several of the tested inhibitors. In these experiments, we observed that Gal-1 increases baseline survival signaling in cells and leads to an increased resistance to many of the tested inhibitors. In conclusion Gal-1 has demonstrated an ability to protect cancer cells from the effects of multiple PI3K pathway inhibitors (i.e. PI3K, EGFR, and mTOR). Likely, this effect is due to the increased baseline survival signaling seen in Gal-1 expressing cells. While the mechanism for Gal-1's affect on inhibitor activity is still under investigation, this research highlights that Gal-1 status might serve as a predictive biomarker for the effectiveness of many different PI3K pathway inhibitors that are currently showing limited efficacy in clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2229. doi:1538-7445.AM2012-2229

Abstract 1293: Galectin-3 facilitates the stability of p21 protein through carbohydrate recognition domain in prostate cancer cells

Abstract Galectin-3 (Gal-3), a β-galactoside-binding protein, has multiple functions such as regulating cell growth, apoptosis, angiogenesis, tumor metastasis and so on. p21cip/waf-1, a cyclin-dependent kinase inhibitor, also regulates apoptosis and cell growth as well. Gal-3 and p21 have some similar functions, and both of them play important roles in tumor progression. Here, we try to understand how Gal-3 regulates p21 protein in prostate cancer cells. Our study showed that the changes of expressions of Gal-3 and p21 in prostate cancer cells are parallel. Functions of Gal-3 in regulating apoptosis and cell growth are partially mediated by p21. Gal-3 protein helps to prevent the degradation of p21 protein and delay the turnover of p21 in prostate cancer cells. The protective effect of Gal-3 on the stability of p21 protein is attenuated by Gal-3 inhibitor lactose and modified citrus pectin, which suggests that carbohydrate recognition domain of Gal-3 is responsible for its effect on the stability of p21. Our study is helpful to further understand the molecular mechanisms of Gal-3 functions and demonstrate the crosslink between Gal-3 and p21 pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1293. doi:1538-7445.AM2012-1293

Abstract 3874: Galectin-3 Enhances Post Ischemic Angiogenesis Via Modulation Of Integrin And Akt Pathways.

Introduction: As restoration of blood flow is critical for improved functional recovery following ischemic stroke, enhancing cerebral angiogenesis might be immensely helpful. Galectin-3 (gal-3) is a β-galactoside-binding lectin that plays key roles in inflammation, survival, and angiogenesis. Methods: We used transient middle cerebral artery occlusion (MCAO), oxygen-glucose deprivation (OGD) model, western blot analysis, RT-PCR, Immunofluorescence staining. Results: We currently report that intra-cerebroventricular infusion of recombinant gal-3 in rats subjected to transient MCAO increases the post-ischemic functional recovery. We further show that gal-3 mRNA and protein expression is up-regulated in vitro when activated microglial BV2 cells were subjected to either oxygen-glucose deprivation (OGD) or LPS (lipo-polysaccharide) stimulation. Immunofluorescence staining showed increased cytoplasmic gal-3 that is known to have pro-survival function. This increase in gal-3 is associated with increased number of pro-angiogenic structures in a 3D human vein umbilical cord endothelial (HUVEC) and microglia co-culture model. The pro-angiogenic effect of gal-3 was suppressed by treatment with gal-3 siRNA indicating the specific role of gal-3 in promoting angiogenesis. Exogenous gal-3 treatment further augmented the angiogenic potential of BV2 microglia cells. In addition, gal-3 increased survival of microglial BV2 cells, HUVEC, and neuro-progenitor cells. Gal-3 levels were also directly correlated with increased levels of Integrin-linked kinase 1(ILK1), pro-survival factor AKT and their down stream effector pro-angiogenic factors bFGF and MMP2. Conclusion: Taken together, our studies emphasize the importance of gal-3 in enhancing angiogenesis that is critical for neuron survival and neurogenesis in the post-stroke brain.

Galectin-1 and Galectin-8 Have Redundant Roles in Promoting Plasma Cell Formation

Galectin (Gal) family members are a type of soluble lectin, and they play important roles in immunomodulation. Their redundant roles have been proposed. We previously found that Gal-1 promotes the formation of Ab-secreting plasma cells, but B cells from Gal-1-deficient and control animals produce comparable amounts of Abs. In the current study, we used synthetic sulfomodified N-acetyllactosamine (LacNAc) analogs and short hairpin RNAs for Gal-8 to demonstrate a redundancy in the effects of Gal-1 and Gal-8 on plasma cell formation. Gal-1 and Gal-8 were both expressed during plasma cell differentiation, and both Gals promoted the formation of plasma cells. Gal-1 and Gal-8 bound better to mature B cells than to plasma cells, and the expression of glycosyltransferase enzymes changed during differentiation, with a decrease in mannosyl (α-1,6-)-glycoprotein β-1,6-N-acetyl-glucosaminyltransferase and N-acetylglucosaminyltransferase-1 mRNAs in plasma cells. Synthetic sulfomodified Galβ1-3GlcNAc disaccharides (type 1 LacNAcs) selectively prevented Gal-8 binding, leading to a blockade of Ab production in Gal-1-deficient B cells. Furthermore, synthetic type 1 LacNAcs that were able to block the binding of both Gals greatly reduced the effect of exogenously added recombinant Gal-1 and Gal-8 on promoting Ab production. These results reveal a novel role for Gal-8 in collaboration with Gal-1 in plasma cell formation, and suggest the possibility of using distinct LacNAc ligands to modulate the function of Gals.

Is galectin-1 a trigger for trophoblast cell fusion?: the MAP-kinase pathway and syncytium formation in trophoblast tumour cells BeWo

Galectin-1 (gal-1), a member of the mammalian β-galactoside-binding proteins, exerts biological effects by recognition of glycan ligands, including those involved in cell adhesion and growth regulation. In a previous study, we demonstrated that gal-1 induces cell differentiation processes on the membrane of choriocarcinoma cells BeWo, including the receptor tyrosine kinases, REarranged during transfection, janus kinase 2 and vascular endothelial growth factor receptor 3. Within this study, we examined which mitogen-activated protein kinases (MAPK) and serine/threonine kinases were phoshorylated by gal-1. Out of a number of 21 different MAPKs and other serine/threonine kinases, the stimulation of BeWo cells with gal-1 showed a significant alteration of signal intensity in extracellular-regulated kinases 1/2 (ERK1/2), Akt-3, Akt-pan and glycogen synthase kinase-α/β (GSK-3α/β). We demonstrated that gal-1 significantly inhibited ERK1/2, Akt-3/pan and GSK-3α/β phosphorylation in BeWo cells and in addition induced Elk1 transcription factor activation. In contrast to gal-1 effects, MAPK inhibitor U0126 reduced syncytium formation of BeWo cells. The results of our data showed that phosphorylation of MAP kinases are involved in gal-1-induced signal transduction processes in BeWo cells. Additional results obtained with MAPK inhibitor U0126 close the gap between syncytium formation induced by gal-1 and MAPK activation in trophoblast cells. Furthermore, we demonstrated that gal-1 induces the activation of Elk1, a transcription factor that is activated by MAPK pathways.

Novel roles of galectin-1 in hepatocellular carcinoma cell adhesion, polarization, and in vivo tumor growth

Galectin-1 (Gal-1), a widely expressed β-galactoside-binding protein, exerts pleiotropic biological functions. Gal-1 is up-regulated in hepatocarcinoma cells, although its role in liver pathophysiology remains uncertain. We investigated the effects of Gal-1 on HepG2 hepatocellular carcinoma (HCC) cell adhesion and polarization. Soluble and immobilized recombinant Gal-1 (rGal-1) promoted HepG2 cell adhesion to uncoated plates and also increased adhesion to laminin. Antibody-mediated blockade experiments revealed the involvement of different integrins as critical mediators of these biological effects. In addition, exposure to rGal-1 markedly accelerated the development of apical bile canaliculi as shown by TRITC-phalloidin labeling and immunostaining for multidrug resistance associated-protein 2 (MRP2). Notably, rGal-1 did not interfere with multidrug resistance protein 1/P-glycoprotein or MRP2 apical localization, neither with transfer nor secretion of 5-chloromethylfluorescein diacetate through MRP2. Stimulation of cell adhesion and polarization by rGal-1 was abrogated in the presence of thiodigalactoside, a galectin-specific sugar, suggesting the involvement of protein-carbohydrate interactions in these effects. Additionally, Gal-1 effects were abrogated in the presence of wortmmanin, PD98059 or H89, suggesting involvement of phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinase and cyclic adenosine monophosphate-dependent protein kinase signaling pathways in these functions. Finally, expression levels of this endogenous lectin correlated with HCC cell adhesion and polarization and up-regulation of Gal-1-favored growth of hepatocarcinoma in vivo. Our results provide the first evidence of a role of Gal-1 in modulating HCC cell adhesion, polarization, and in vivo tumor growth, with critical implications in liver pathophysiology.

Targeting Galectin-1 in Carcinoma-Associated Fibroblasts Inhibits Oral Squamous Cell Carcinoma Metastasis by Downregulating MCP-1/CCL2 Expression

Carcinoma-associated fibroblasts (CAFs) in tumor stroma play an important role in tumor progression and have been associated with a poor prognosis in oral squamous cell carcinoma (OSCC). However, how CAFs influence OSCC malignancy and whether normalizing CAFs inhibits cancer progression remain unclear. The relationship between the expression of Galectin-1 (Gal-1) and alpha-smooth muscle actin (α-SMA, a CAF marker) in OSCC patient samples and primary cultured CAFs was examined by quantitative real-time PCR, Western blotting, and immunofluorescence. To examine the effect of Gal-1 on CAF activation and CAF-mediated tumor invasion and migration in vitro, Gal-1 expression was knocked down by small hairpin RNA. Finally, cancer cells and CAFs were coimplanted into SCID mice to evaluate the effect of Gal-1 on CAF-modulated tumor progression in vivo. Gal-1 expression is positively associated with α-SMA in the stroma of OSCC specimens. Gal-1 knockdown decreases activated CAF characteristics, resulting in a decrease in α-SMA expression and extracellular matrix protein production. Notably, blocking Gal-1 expression significantly inhibits CAF-conditioned medium-induced tumor cell migration and invasion, possibly by reducing the production of monocyte chemotactic protein-1 (MCP-1/CCL2). MCP-1 induces the migration of OSCC cells by binding to the receptor CCR2; adding an MCP-1 antibody to CAF-conditioned medium that inhibits the interaction between MCP-1 and CCR2 abolishes migration. Finally, we found that Gal-1 knockdown in CAFs significantly reduces CAF-augmented tumor growth and metastasis in vivo. Our findings demonstrate that Gal-1 regulates CAF activation and indicate that targeting Gal-1 in CAFs inhibits OSCC metastasis by modulating MCP-1 expression.

Synergistic effects of galectin-1 and reactive astrocytes on functional recovery after contusive spinal cord injury

Galectin-1 (Gal-1), a carbohydrate-binding protein, is differentially expressed by various normal and pathological tissues and appears to be functionally polyvalent. Recent evidence indicates that Gal-1 is involved in the proliferation of adult neural progenitor cells in neurogenic regions during adulthood. However, localization and functional roles of Gal-1 in the adult spinal cord have not been clarified. Here, we investigated the spatio-temporal profile of endogenous Gal-1 expression by in situ hybridization before and after experimental adult spinal cord injury and examined the correlation of Gal-1 with the fate of dividing cells in vivo, using double-labeling methods. Gal-1 mRNA was detectable at a relatively low level in uninjured spinal cord, but was markedly increased in the gray matter and/or white matter and in the ependyma rostral and caudal to the lesion site after injury. Co-localization results revealed that Gal-1 was expressed predominantly by GFAP-positive reactive astrocytes. In addition, intrathecal infusion of recombinant Gal-1 enhanced cell division and reactive astrocytosis in the adult spinal cord. To explore further whether Gal-1 and reactive astrocytes provide a synergistic effect on neurological recovery following SCI, we investigated the differences in behavioral analysis between wild-type (WT) and reactive astrocyte-deficient transgenic mice after injury and found neuroprotective effects of Gal-1 appeared to be specifically mediated through reactive astrocytes. These results indicate that Gal-1 exhibits great potential as a novel neuroprotective agent for the treatment of SCI.

Stimulation of Syncytium Formation in vitro in Human Trophoblast Cells by Galectin-1

Background Galectin-1 (gal-1), a member of the mammalian β-galactoside-binding proteins, exerts biological effects by recognition of glycan ligands, including those involved in cell adhesion and growth regulation. In trophoblast cells, gal-1 binds to cell surface glycoproteins (e.g., Mucin1). It has been demonstrated that gal-1 recognizes appropriate glycotopes on the syncytiotrophoblast and extravillous trophoblast from second trimester human placenta and choriocarcinoma cells BeWo, which reveal two coexisting phenotypes, the cytotrophoblast-like and the syncytiotrophoblast-like phenotype. So the aim of this study was to investigate the effect of gal-1 on syncytium formation in BeWo and human villous trophoblasts (HVT) cells. Materials and methods The effect of gal-1 on syncytium formation was investigated with immunocytochemical and double immunofluorescence stainings, cell-labelling and Real-time RT-PCR. BeWo choriocarcinoma and HVT cells were incubated in vitro for 24 and 48 h in the absence (controls) and presence of gal-1 and forskolin and stained with antibodies against Ki67, β-catenin, E-cadherin and syncytin. BeWo and HVT cells were incubated with 60 μg/ml gal-1 for 48 h (BeWo) or 96 h (HVT) and cell fusion was detected by fluorescent cell-labelling solution. Finally, BeWo cells were incubated for 1 h or 48 h in the absence and presence of 60 μg/ml gal-1 and Real-time RT-PCR was performed. Results We showed with immunocytochemical staining a downregulation of β-catenin expression in the 24 h BeWo cell culture and with double immunofluorescence staining an inhibition of the β-catenin and E-cadherin expression in the 48 h BeWo cell culture stimulated with gal-1 or forskolin. The inhibition of E-cadherin was demonstrated on mRNA level in the 1 h BeWo cell culture too. Increased cell fusion was also showed with DiO and DiI fluorescent cell-labelling solution in the 48 h BeWo cell culture. In addition, we demonstrated the downregulation of Ki67 protein expression in the 24 h BeWo cell culture and on mRNA level in the 1 h BeWo cell culture. We also showed the upregulation of syncytin protein and mRNA expression after incubation of the 48 h BeWo cell culture with gal-1 or forskolin. Similar results were obtained with HVT cells: the amount of cell fusion was significantly increased in the gal-1 treated 48 h HVT cell culture in vitro compared to untreated cells as demonstrated with β-catenin and E-cadherin double immunofluorescence staining. This increase was also shown by fluorescent cell-labelling with DiO and DiI in the 96 h HVT cell culture compared to untreated cells. Conclusion Our data suggest that gal-1 stimulates the syncytium formation in choriocarcinoma cells BeWo and HVT cells in vitro and inhibits the expression of β-catenin, E-cadherin and in addition Ki67 in BeWo cells. Therefore gal-1 may be a major trigger for the process of trophoblast cell fusion.

Activation of RAW264.7 macrophages by oxidized galectin-1

Galectin-1, a member of the β-galactoside-binding lectin family, exists in both reduced and oxidized states. Oxidized galectin-1 (Gal-1/Ox), which lacks lectin activity, has been shown to promote axonal regeneration after injury by activating macrophages, which causes the release of factors that enhance Schwann cell migration and neurite outgrowth. However, the mechanism of macrophage activation by Gal-1/Ox remains unknown. In this study, we examined the effects of Gal-1/Ox on RAW264.7 macrophages and RT4-D6P2T Schwann cells. Gal-1/Ox stimulated migration of RT4-D6P2T Schwann cells directly and by activating RAW264.7 macrophages to release factors that promoted cell migration. Gal-1/Ox inhibited nitric oxide (NO) production induced by interferon-γ by suppressing expression of inducible NO synthase in RAW264.7 macrophages and not by arginase activation and cell death. Furthermore, Gal-1/Ox-activated extracellular signal-regulated protein kinase 1/2 (ERK1/2) in RAW264.7 macrophages, although the mitogen-activated protein kinase (MEK)/ERK1/2 pathway was not involved in release of factors that promoted Schwann cell migration. On the other hand, Gal-1/Ox-induced RT4-D6P2T Schwann cell migration appeared to be mediated by the MEK/ERK1/2 pathway. These results suggest that Gal-1/Ox inhibits inflammatory responses in macrophages and promotes Schwann cell migration directly and by macrophage activation.

Inhibiton of RET and JAK2 Signals and Upregulation of VEGFR3 Phosphorylation in Vitro by Galectin-1 in Trophoblast Tumor Cells BeWo

Background Galectin-1 (gal-1), a member of the mammalian β-galactoside-binding proteins, binds to cell surface glycoproteins (Mucin-1) on trophoblast cells. Although it has been demonstrated that gal-1 induces cell differentiation processes in these cells, no information on its signal transduction processes is available so far. As tyrosine phosphorylation is a major mechanism that controls multiple biological processes including cell differentiation, survival and proliferation, the aim of this study was to examine which human receptor tyrosine kinases (RTKs) were phosphorylated in trophoblast cells by gal-1. Materials and Methods BeWo choriocarcinoma cells were incubated for 24 h in the absence (controls) and presence of 60 μg/ml galectin-1. With the RayBio ® Human RTK Phosphorylation Antibody Array 1, the relative levels of phosphorylation of different human RTKs could be detected simultaneously. The signal intensities were compared and quantified with the Quantity One Version 4.5.2 program. Gal-1-treated and non-treated cells were incubated with antibodies against REarranged during Transfection (RET) and phosphorylated RET Y905. Staining reaction was performed with the avidin-biotinylated peroxidase complex (ABC) reagent. Results We demonstrated that gal-1 inhibited RET and Janus Kinase 2 (JAK2) signals and upregulated Vascular endothelial growth factor receptor 3 (VEGFR3) signal in BeWo cells. We also showed the downregulation of phosphorylation on RET phosphotyrosine residue 905 in BeWo cells with phosphorylation specific antibodies and immunocytochemistry. Conclusion Out of a number of 71 different RTKs, the stimulation of BeWo cells with gal-1 showed a significant alteration of signal intensity in only 3 RTKs: JAK2, RET and VEGFR3. Our data suggest that phosphorylation of these RTKs could be involved in cell differentiation processes that could be responsible for the already known effect of gal-1 on BeWo cells, the inhibition of proliferation.

Increased expression of galectin-1 is associated with human oral squamous cell carcinoma development

Galectin-1 (Gal-1) is a newly found immunoregulatory carbohydrate-binding protein in cancer biology. The purpose of this study was to evaluate the effects of Gal-1 on oral squamous cell carcinoma (OSCC) development. Immunohistochemistry, Western blotting and RT-PCR were carried out in 62 primary OSCC, 38 oral leukoplakia (OPL) tissues to detect the Gal-1 expression in both protein and mRNA levels. Ten normal oral mucosa (NOM) tissues were used as control. Gal-1 protein was significantly overexpressed in OSCC cancer cells and OPL prickle cells compared to NOM (P<0.05). In accordance with Gal-1 protein, Gal-1 mRNA was also up-regulated in OSCC tissues and OPL tissues. Furthermore, both the Gal-1 protein and mRNA in OSCC tissues were higher than in OPL tissues (P<0.05). Our data supports the important roles of Gal-1 in OSCC development and suggests that Gal-1 upregulation in the OSCC and OPL tissues might be a predictor of early oral carcinogenesis.

Suppression of Autoimmune Diabetes by Soluble Galectin-1

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that targets the beta-cells of the pancreas. We investigated the ability of soluble galectin-1 (gal-1), an endogenous lectin that promotes T cell apoptosis, to down-regulate the T cell response that destroys the pancreatic beta-cells. We demonstrated that in nonobese diabetic (NOD) mice, gal-1 therapy reduces significantly the amount of Th1 cells, augments the number of T cells secreting IL-4 or IL-10 specific for islet cell Ag, and causes peripheral deletion of beta-cell-reactive T cells. Administration of gal-1 prevented the onset of hyperglycemia in NOD mice at early and subclinical stages of T1D. Preventive gal-1 therapy shifted the composition of the insulitis into an infiltrate that did not invade the islets and that contained a significantly reduced number of Th1 cells and a higher percentage of CD4(+) T cells with content of IL-4, IL-5, or IL-10. The beneficial effects of gal-1 correlated with the ability of the lectin to trigger apoptosis of the T cell subsets that cause beta-cell damage while sparing naive T cells, Th2 lymphocytes, and regulatory T cells in NOD mice. Importantly, gal-1 reversed beta-cell autoimmunity and hyperglycemia in NOD mice with ongoing T1D. Because gal-1 therapy did not cause major side effects or beta-cell toxicity in NOD mice, the use of gal-1 to control beta-cell autoimmunity represents a novel alternative for treatment of subclinical or ongoing T1D.

Sialylation of β1 Integrins Blocks Cell Adhesion to Galectin-3 and Protects Cells against Galectin-3-induced Apoptosis

Sialylation of β1 Integrins Blocks Cell Adhesion to Galectin-3 and Protects Cells against Galectin-3-induced Apoptosis

Sialylation of 1 Integrins Blocks Cell Adhesion to Galectin-3 and Protects Cells against Galectin-3-induced Apoptosis

In previous studies, we determined that beta1 integrins from human colon tumors have elevated levels of alpha2-6 sialylation, a modification added by beta-galactosamide alpha-2,6-sialyltranferase I (ST6Gal-I). Intriguingly, the beta1 integrin is thought to be a ligand for galectin-3 (gal-3), a tumor-associated lectin. The effects of gal-3 are complex; intracellular forms typically protect cells against apoptosis through carbohydrate-independent mechanisms, whereas secreted forms bind to cell surface oligosaccharides and induce apoptosis. In the current study, we tested whether alpha2-6 sialylation of the beta1 integrin modulates binding to extracellular gal-3. Herein we report that SW48 colonocytes lacking alpha2-6 sialylation exhibit beta1 integrin-dependent binding to gal-3-coated tissue culture plates; however, binding is attenuated upon forced expression of ST6Gal-I. Removal of alpha2-6 sialic acids from ST6Gal-I expressors by neuraminidase treatment restores gal-3 binding. Additionally, using a blot overlay approach, we determined that gal-3 binds directly and preferentially to unsialylated, as compared with alpha2-6-sialylated, beta1 integrins. To understand the physiologic consequences of gal-3 binding, cells were treated with gal-3 and monitored for apoptosis. Galectin-3 was found to induce apoptosis in parental SW48 colonocytes (unsialylated), whereas ST6Gal-I expressors were protected. Importantly, gal-3-induced apoptosis was inhibited by function blocking antibodies against the beta1 subunit, suggesting that beta1 integrins are critical transducers of gal-3-mediated effects on cell survival. Collectively, our results suggest that the coordinate up-regulation of gal-3 and ST6Gal-I, a feature that is characteristic of colon carcinoma, may confer tumor cells with a selective advantage by providing a mechanism for blockade of the pro-apoptotic effects of secreted gal-3.

Novel insights into the inhibitory effects of Galectin-1 on neutrophil recruitment under flow

Galectin-1 (Gal-1) is a beta-galactoside-binding protein endowed with anti-inflammatory properties. The purpose of this study was to investigate the effects of endogenous and exogenous Gal-1 on neutrophil recruitment onto TNF-treated endothelium. The effect of human recombinant (hr)Gal-1 on markers of neutrophil activation (CD11b expression, P-selectin glycoprotein ligand 1, and L-selectin shedding) was also assessed. Gal-1 inhibited the platelet-activating factor-induced increase in CD11b expression in a concentration-dependent manner, as assessed by flow cytometry. To determine the effects of Gal-1 on neutrophil recruitment, an in vitro flow chamber was used: Preincubation of neutrophils with hrGal-1 significantly decreased the extent of capture, rolling, and adhesion on activated endothelial monolayers. This inhibition was shared with the endogenous protein, as knockdown of endothelial Gal-1 using small interfering RNA resulted in a significant increase in the number of cells captured and rolling. To verify the effects of Gal-1 in an in vivo system, intravital microscopy of Gal-1 null mice and their wild-type counterparts was performed. Leukocyte adhesion and emigration were increased significantly in the cremasteric circulation of Gal-1 null mice inflamed with IL-1beta. These findings indicate that Gal-1 functions to limit neutrophil recruitment onto a TNF-treated endothelium, a property that may underline its inhibitory effects in acute inflammation.

Extracellular localization of galectin-3 has a deleterious role in joint tissues

In this study we examine the extracellular role of galectin-3 (gal-3) in joint tissues. Following intra-articular injection of gal-3 or vehicle in knee joints of mice, histological evaluation of articular cartilage and subchondral bone was performed. Further studies were then performed using human osteoarthritic (OA) chondrocytes and subchondral bone osteoblasts, in which the effect of gal-3 (0 to 10 μg/ml) was analyzed. Osteoblasts were incubated in the presence of vitamin D3 (50 nM), which is an inducer of osteocalcin, encoded by an osteoblast terminal differentiation gene. Genes of interest mainly expressed in either chondrocytes or osteoblasts were analyzed with real-time RT-PCR and enzyme immunoassays. Signalling pathways regulating osteocalcin were analyzed in the presence of gal-3. Intra-articular injection of gal-3 induced knee swelling and lesions in both cartilage and subchondral bone. On human OA chondrocytes, gal-3 at 1 μg/ml stimulated ADAMTS-5 expression in chondrocytes and, at higher concentrations (5 and 10 μg/ml), matrix metalloproteinase-3 expression. Experiments performed with osteoblasts showed a weak but bipolar effect on alkaline phosphatase expression: stimulation at 1 μg/ml or inhibition at 10 μg/ml. In the absence of vitamin D3, type I collagen alpha 1 chain expression was inhibited by 10 μg/ml of gal-3. The vitamin D3induced osteocalcin was strongly inhibited in a dose-dependent manner in the presence of gal-3, at both the mRNA and protein levels. This inhibition was mainly mediated by phosphatidylinositol-3-kinase. These findings indicate that high levels of extracellular gal-3, which could be encountered locally during the inflammatory process, have deleterious effects in both cartilage and subchondral bone tissues.

Mice deficient in galectin-1 exhibit attenuated physiological responses to chronic hypoxia-induced pulmonary hypertension

Pulmonary hypertension (PH) is characterized by sustained vasoconstriction, with subsequent extracellular matrix (ECM) production and smooth muscle cell (SMC) proliferation. Changes in the ECM can modulate vasoreactivity and SMC contraction. Galectin-1 (Gal-1) is a hypoxia-inducible beta-galactoside-binding lectin produced by vascular, interstitial, epithelial, and immune cells. Gal-1 regulates SMC differentiation, proliferation, and apoptosis via interactions with the ECM, as well as immune system function, and, therefore, likely plays a role in the pathogenesis of PH. We investigated the effects of Gal-1 during hypoxic PH by quantifying 1) Gal-1 expression in response to hypoxia in vitro and in vivo and 2) the effect of Gal-1 gene deletion on the magnitude of the PH response to chronic hypoxia in vivo. By constructing and screening a subtractive library, we found that acute hypoxia increases expression of Gal-1 mRNA in isolated pulmonary mesenchymal cells. In wild-type (WT) mice, Gal-1 immunoreactivity increased after 6 wk of hypoxia. Increased expression of Gal-1 protein was confirmed by quantitative Western analysis. Gal-1 knockout (Gal-1(-/-)) mice showed a decreased PH response, as measured by right ventricular pressure and the ratio of right ventricular to left ventricular + septum wet weight compared with their WT counterparts. However, the number and degree of muscularized vessels increased similarly in WT and Gal-1(-/-) mice. In response to chronic hypoxia, the decrease in factor 8-positive microvessel density was similar in both groups. Vasoreactivity of WT and Gal-1(-/-) mice was tested in vivo and with use of isolated perfused lungs exposed to acute hypoxia. Acute hypoxia caused a significant increase in RV pressure in wild-type and Gal-1(-/-) mice; however, the response of the Gal-1(-/-) mice was greater. These results suggest that Gal-1 influences the contractile response to hypoxia and subsequent remodeling during hypoxia-induced PH, which influences disease progression.