Galectin-3 Up-regulation Research Articles

Methylglyoxal induces endothelial cell apoptosis and coronary microvascular dysfunction through regulating AR-cPLA2 signaling

ObjectiveSince diabetic patients with coronary microvascular dysfunction (CMD) exhibit high cardiac mortality and women have higher prevalence of non-obstructive coronary artery disease than men, we tried to expand the limited understanding about the etiology and the sex difference of diabetic CMD. Approach and resultsAccumulated methylglyoxal (MGO) due to diabetes promotes vascular damage and it was used for mimicking diabetic status. Flow cytometry analysis and isometric tension measurement were performed to evaluate coronary artery endothelial injury. MGO induced apoptosis of coronary endothelial cells, accompanied by downregulation of androgen receptor (AR). Lentivirus-mediated stable expression of AR in coronary endothelial cells increased anti-apoptotic Bcl-2 expression and attenuated MGO-induced cell apoptosis. cPLA2 activation was the downstream of AR downregulation by MGO treatment. Moreover, MGO also activated cPLA2 rapidly to impair endothelium-dependent vasodilation of coronary arteries from mice. Reactive oxygen species (ROS) overproduction was demonstrated to account for MGO-mediated cPLA2 activation and endothelial dysfunction. Importantly, AR blockade increased endothelial ROS production whereas AR activation protected coronary artery endothelial vasodilatory function from the MGO-induced injury. Although galectin-3 upregulation was confirmed by siRNA knockdown in endothelial cells not to participate in MGO-induced endothelial apoptosis, pharmacological inhibitor of galectin-3 further enhanced MGO-triggered ROS generation and coronary artery endothelial impairment. ConclusionsOur data proposed the AR downregulation-ROS overproduction-cPLA2 activation pathway as one of the mechanisms underlying diabetic CMD and postulated a possible reason for the sex difference of CMD-related angina. Meanwhile, MGO-induced galectin-3 activation played a compensatory role against coronary endothelial dysfunction.

Microglia at sites of atrophy restrict the progression of retinal degeneration via galectin-3 and Trem2.

Outer retinal degenerations, including age-related macular degeneration (AMD), are characterized by photoreceptor and retinal pigment epithelium (RPE) atrophy. In these blinding diseases, macrophages accumulate at atrophic sites, but their ontogeny and niche specialization remain poorly understood, especially in humans. We uncovered a unique profile of microglia, marked by galectin-3 upregulation, at atrophic sites in mouse models of retinal degeneration and human AMD. In disease models, conditional deletion of galectin-3 in microglia led to phagocytosis defects and consequent augmented photoreceptor death, RPE damage, and vision loss, indicating protective roles. Mechanistically, Trem2 signaling orchestrated microglial migration to atrophic sites and induced galectin-3 expression. Moreover, pharmacologic Trem2 agonization led to heightened protection but in a galectin-3-dependent manner. In elderly human subjects, we identified this highly conserved microglial population that expressed galectin-3 and Trem2. This population was significantly enriched in the macular RPE-choroid of AMD subjects. Collectively, our findings reveal a neuroprotective population of microglia and a potential therapeutic target for mitigating retinal degeneration.

Galectin-3-centered paracrine network mediates cardiac inflammation and fibrosis upon β-adrenergic insult.

Rapid over-activation of β-adrenergic receptors (β-AR) following acute stress initiates cardiac inflammation and injury by activating interleukin-18 (IL-18), however, the process of inflammation cascades has not been fully illustrated. The present study aimed to determine the mechanisms of cardiac inflammatory amplification following acute sympathetic activation. With bioinformatics analysis, galectin-3 was identified as a potential key downstream effector of β-AR and IL-18 activation. The serum level of galectin-3 was positively correlated with norepinephrine or IL-18 in patients with chest pain. In the heart of mice treated with β-AR agonist isoproterenol (ISO, 5 mg kg-1), galectin-3 expression was upregulated markedly later than IL-18 activation, and Nlrp3-/- and Il18-/- mice did not show ISO-induced galectin-3 upregulation. It was further revealed that cardiomyocyte-derived IL-18 induced galectin-3 expression in macrophages following ISO treatment. Moreover, galectin-3 deficiency suppressed ISO-induced cardiac inflammation and fibrosis without blocking ISO-induced IL-18 increase. Treatment with a galectin-3 inhibitor, but not a β-blocker, one day after ISO treatment effectively attenuated cardiac inflammation and injury. In conclusion, galectin-3 is upregulated to exaggerate cardiac inflammation and injury following acute β-AR activation, a galectin-3 inhibitor effectively blocks cardiac injury one day after β-AR insult.

Modified Citrus Pectin Prevents Blood-Brain Barrier Disruption in Mouse Subarachnoid Hemorrhage by Inhibiting Galectin-3.

Background and Purpose- Plasma levels of galectin-3-a matricellular protein-are increased after aneurysmal subarachnoid hemorrhage (SAH), but the functional significance remains undetermined. This study was conducted to evaluate whether modified citrus pectin (MCP; galectin-3 inhibitor) prevents post-SAH early brain injury, focusing on blood-brain barrier disruption. Methods- C57BL/6 male adult mice (n=251) underwent sham or filament perforation SAH modeling, followed by a random intracerebroventricular injection of vehicle or drug at 30 minutes post-modeling. First, vehicle-treated and 0.8, 4, 16, or 32 µg MCP-treated mice were assessed by neuroscore and brain water content at 24 and 48 hours post-modeling. Second, Evans blue extravasation, Western blotting, coimmunoprecipitation and immunostaining were performed in vehicle-treated or 4 µg MCP-treated mice at 24 hours post-modeling. Third, vehicle or R-galectin-3 (recombinant galectin-3) was administered to SAH mice simultaneously with vehicle or MCP, and neuroscore and Evans blue extravasation were evaluated at 24 hours post-modeling. Fourth, vehicle or R-galectin-3 was administered to MCP-treated SAH mice at 24 hours, and neuroscore and IgG immunostaining were evaluated at 48 hours post-SAH. Results- Among tested dosages, 4 µg MCP showed the best neuroprotective effects as to preventing neurological impairments and brain edema at 24 to 48 hours post-SAH. Four micrograms MCP attenuated post-SAH blood-brain barrier disruption and galectin-3 upregulation in brain capillary endothelial cells, associated with inactivation of ERK (extracellular signal-related kinase) 1/2, STAT (signal transducer and activator of transcription)-3, and MMP (matrix metalloproteinase)-9, and the consequent preservation of a tight junction protein ZO-1 (zonula occludens-1). Coimmunoprecipitation assay demonstrated physical interactions between galectin-3 and TLR (Toll-like receptor) 4. R-galectin-3 blocked the neuroprotective effects of MCP. Conclusions- MCP prevents post-SAH blood-brain barrier disruption possibly by inhibiting galectin-3, of which the mechanisms may include binding to TLR4 and activating ERK1/2, STAT-3, and MMP-9. This study suggests galectin-3 to be a novel therapeutic target against post-SAH early brain injury.

Galectin-3 enhances avian H5N1 influenza A virus-induced pulmonary inflammation by promoting NLRP3 inflammasome activation

Abstract Virus-induced excessive inflammatory response contributes to severe disease and high mortality rates. Galectin-3, a β-galactoside-binding protein widely distributed in immune and epithelial cells, regulates various immune functions and modulates microbial infections. Here we describe galectin-3 upregulation in mouse lung tissue following challenges with the H5N1 influenza virus. We investigated the effects of endogenous galectin-3 on H5N1 infection and found that survival of galectin-3 knockout (Gal-3KO) mice was comparable to Wild-type (WT) mice following infections. Compared to infected WT mice, infected Gal-3KO mice exhibited less inflammation in the lungs and reduced interleukin-1 beta (IL-1β) levels in bronchoalveolar lavage fluid. We also found that bone marrow-derived macrophages (BMMs) from Gal-3KO mice exhibited reduced oligomerization of apoptosis-associated speck-like proteins containing caspase-associated recruitment domains (ASC), and secreted less IL-1β compared to BMMs from WT mice. However, we observed similar levels of the inflammasome component of nucleotide oligomerization domain-like receptor protein 3 (NLRP3) in two genotypes of BMMs. Co-immunoprecipitation data indicate galectin-3 and NLRP3 interaction in BMMs infected with H5N1. We also observed an association between galectin-3 and NLRP3-ASC complex. Our results indicate that galectin-3 promotes host inflammatory responses and regulates IL-1β production by macrophages via interaction with NLRP3. It suggests that endogenous galectin-3 enhances the effects of H5N1 infection.

Correction: Posttranscriptional regulation of Galectin-3 by miR-128 contributes to colorectal cancer progression.

[This corrects the article DOI: 10.18632/oncotarget.14839.].

Galectin-3 Enhances Avian H5N1 Influenza A Virus–Induced Pulmonary Inflammation by Promoting NLRP3 Inflammasome Activation

Highly pathogenic avian influenza A H5N1 virus causes pneumonia and acute respiratory distress syndrome in humans. Virus-induced excessive inflammatory response contributes to severe disease and high mortality rates. Galectin-3, a β-galactoside-binding protein widely distributed in immune and epithelial cells, regulates various immune functions and modulates microbial infections. Here, we describe galectin-3 up-regulation in mouse lung tissue after challenges with the H5N1 influenza virus. We investigated the effects of endogenous galectin-3 on H5N1 infection and found that survival of galectin-3 knockout (Gal-3KO) mice was comparable with wild-type (WT) mice after infections. Compared with infected WT mice, infected Gal-3KO mice exhibited less inflammation in the lungs and reduced IL-1β levels in bronchoalveolar lavage fluid. In addition, the bone marrow-derived macrophages (BMMs) from Gal-3KO mice exhibited reduced oligomerization of apoptosis-associated speck-like proteins containing caspase-associated recruitment domains and secreted less IL-1β compared with BMMs from WT mice. However, similar levels of the inflammasome component of nucleotide oligomerization domain-like receptor protein 3 (NLRP3) were observed in two genotypes of BMMs. Co-immunoprecipitation data indicated galectin-3 and NLRP3 interaction in BMMs infected with H5N1. An association was also observed between galectin-3 and NLRP3/apoptosis-associated speck-like proteins containing caspase-associated recruitment domain complex. Combined, our results suggest that endogenous galectin-3 enhances the effects of H5N1 infection by promoting host inflammatory responses and regulating IL-1β production by macrophages via interaction with NLRP3.

Posttranscriptional regulation of Galectin-3 by miR-128 contributes to colorectal cancer progression.

Here we demonstrated that Galectin-3 protein level was frequently up-regulated in colorectal cancer (CRC) cells and tissues. Galectin-3 up-regulation correlated with CRC progression and predicted a shorter overall survival of CRC patients. Galectin-3 overexpression attenuated the chemo-sensitivity of cancer cells, but enhanced the potential invasiveness. To explore the mechanism for Galectin-3 dysregulation, we found that miR-128 level was frequently down-regulated in CRC and negatively correlated with Galectin-3 level. Using bioinformatics analysis and experimental validation, we showed that miR-128 could directly target Galectin-3 to repress its protein level. MiR-128 decrease associated with CRC progression and predicted a worse overall survival of CRC patients. Ectopic miR-128 expression enhanced the chemo-sensitivity of CRC cells in vitro and in vivo, and inhibited the potential invasiveness. Galectin-3 expression impaired the cancer suppressive effects of miR-128. These data highlighted the role of miR-128/Galectin-3 axis in colorectal cancer.

An increase in galectin-3 causes cellular unresponsiveness to IFN-γ-induced signal transduction and growth inhibition in gastric cancer cells.

Glycogen synthase kinase (GSK)-3β facilitates interferon (IFN)-γ signaling by inhibiting Src homology-2 domain-containing phosphatase (SHP) 2. Mutated phosphoinositide 3-kinase (PI3K) and phosphatase and tensin homolog (PTEN) cause AKT activation and GSK-3β inactivation to induce SHP2-activated cellular unresponsiveness to IFN-γ in human gastric cancer AGS cells. This study investigated the potential role of galectin-3, which acts upstream of AKT/GSK-3β/SHP2, in gastric cancer cells. Increasing or decreasing galectin-3 altered IFN-γ signaling. Following cisplatin-induced galectin-3 upregulation, surviving cells showed cellular unresponsiveness to IFN-γ. Galectin-3 induced IFN-γ resistance independent of its extracellular β-galactoside-binding activity. Galectin-3 expression was not regulated by PI3K activation or by a decrease in PTEN. Increased galectin-3 may cause GSK-3β inactivation and SHP2 activation by promoting PDK1-induced AKT phosphorylation at a threonine residue. Overexpression of AKT, inactive GSK-3βR96A, SHP2, or active SHP2D61A caused cellular unresponsiveness to IFN-γ in IFN-γ-sensitive MKN45 cells. IFN-γ-induced growth inhibition and apoptosis in AGS cells were observed until galectin-3 expression was downregulated. These results demonstrate that an increase in galectin-3 facilitates AKT/GSK-3β/SHP2 signaling, causing cellular unresponsiveness to IFN-γ.

Galectin-3 up-regulation in hypoxic and nutrient deprived microenvironments promotes cell survival.

Galectin-3 (gal-3) is a β-galactoside binding protein related to many tumoral aspects, e.g. angiogenesis, cell growth and motility and resistance to cell death. Evidence has shown its upregulation upon hypoxia, a common feature in solid tumors such as glioblastoma multiformes (GBM). This tumor presents a unique feature described as pseudopalisading cells, which accumulate large amounts of gal-3. Tumor cells far from hypoxic/nutrient deprived areas express little, if any gal-3. Here, we have shown that the hybrid glioma cell line, NG97ht, recapitulates GBM growth forming gal-3 positive pseudopalisades even when cells are grafted subcutaneously in nude mice. In vitro experiments were performed exposing these cells to conditions mimicking tumor areas that display oxygen and nutrient deprivation. Results indicated that gal-3 transcription under hypoxic conditions requires previous protein synthesis and is triggered in a HIF-1α and NF-κB dependent manner. In addition, a significant proportion of cells die only when exposed simultaneously to hypoxia and nutrient deprivation and demonstrate ROS induction. Inhibition of gal-3 expression using siRNA led to protein knockdown followed by a 1.7–2.2 fold increase in cell death. Similar results were also found in a human GBM cell line, T98G. In vivo, U87MG gal-3 knockdown cells inoculated subcutaneously in nude mice demonstrated decreased tumor growth and increased time for tumor engraftment. These results indicate that gal-3 protected cells from cell death under hypoxia and nutrient deprivation in vitro and that gal-3 is a key factor in tumor growth and engraftment in hypoxic and nutrient-deprived microenvironments. Overexpression of gal-3, thus, is part of an adaptive program leading to tumor cell survival under these stressing conditions.

Antiviral activity of aloe-emodin against influenza A virus via galectin-3 up-regulation

Novel influenza A H7N9 virus, which emerged in 2013, and highly pathogenic H5N1 virus, identified since 2003, pose challenges to public health and necessitate quest for new anti-influenza compounds. Anthraquinone derivatives like aloe-emodin, emodin and chrysophanol, reportedly exhibit antiviral activity. This study probes their inhibitory mechanism and effect against influenza A virus. Of three anthraquinone derivatives, aloe-emodin, with a lower cytotoxicity showed concentration-dependently reducing virus-induced cytopathic effect and inhibiting replication of influenza A in MDCK cells. 50% inhibitory concentration value of aloe-emodin on virus yield was less than 0.05μg/ml. Proteomics and Western blot of MDCK cells indicated aloe-emodin up-regulating galectin-3, and thioredoxin as well as down-regulating nucleoside diphosphate kinase A. Western blot and quantitative PCR confirmed aloe-emodin up-regulating galectin-3 expression; recombinant galectin-3 augmented expression of antiviral genes IFN-β, IFN-γ, PKR and 2׳,5׳–OAS in infected cells, agreeing with expression pattern of those treated with aloe-emodin. Galectin-3 also inhibited influenza A virus replication. Proteomic analysis of treated cells indicated galectin-3 up-regulation as one anti-influenza A virus action by aloe-emodin. Since galectin-3 exhibited cytokine-like regulatory actions via JAK/STAT pathways, aloe-emodin also restored NS1-inhibited STAT1-mediated antiviral responses in transfected cells: e.g., STAT1 phosphorylation of interferon (IFN) stimulation response element (ISRE)-driven promoter, RNA-dependent protein kinase (PKR) and 2׳,5׳-oligoadenylate synthetase (2׳,5׳–OAS) expression. Treatment with aloe-emodin could control influenza infection in humans.

Stimulation of transient receptor potential vanilloid 4 channel suppresses abnormal activation of microglia induced by lipopolysaccharide

Microglia are intrinsic immune cells in the brain. In response to neurodegenerative events, excessively activated microglia change their shapes and release various cytokines leading to the pathogenesis of central nervous system (CNS) disease. Because the intracellular mechanisms of this process are still unclear, we have evaluated the functional roles of transient receptor potential vanilloid 4 (TRPV4) channel expressed in the microglia. Robust microglial activation after an injection of lipopolysaccharide (LPS) into the mouse cerebral ventricle was suppressed by concurrent administration of a selective TRPV4 agonist, 4α-phorbol 12,13-didecanoate (4α-PDD). When the mechanism was further investigated using cultured rat microglia intrinsically expressing functional TRPV4, release of tumor necrosis factor-α (TNF-α) and expression of galectin-3 were both increased by LPS. These increases were significantly suppressed by cotreatment with 4α-PDD, and the inhibitory effects of 4α-PDD were abolished by knockdown of TRPV4 or TRPV4 antagonists. The amplitude of voltage-dependent K(+) current, which is augmented during microglial activation, was also suppressed by 4α-PDD treatment. Opening of TRPV4 channels with 4α-PDD induced membrane depolarization mainly by increasing Na(+) influx. In addition, mimicking depolarization with a high-K(+) solution suppressed LPS-induced TNF-α release and galectin-3 upregulation. Both depolarizing treatments with 4α-PDD and high-K(+) solution decreased store-operated Ca(2+) influx caused by thapsigargin. These results suggest that depolarization in response to opening of the TRPV4 channel attenuates the driving force for extracellular Ca(2+) and suppresses microglial activation.

Regulation of adverse remodelling by osteopontin in a genetic heart failure model

Desmin, the muscle-specific intermediate filament protein, is a major target in dilated cardiomyopathy and heart failure in humans and mice. The hallmarks of desmin-deficient (des(-/-)) mice pathology include pronounced myocardial degeneration, extended fibrosis, and osteopontin (OPN) overexpression. We sought to identify the molecular and cellular events regulating adverse cardiac remodelling in des(-/-) mice and their potential link to OPN. In situ hybridization, histology, and immunostaining demonstrated that inflammatory cells and not cardiomyocytes were the source of OPN. RNA profile comparison revealed that activation of inflammatory pathways, sustained by innate immunity mechanisms, predominated among all changes occurring in degenerating des(-/-) myocardium. The expression of the most highly up-regulated genes (OPN: 226×, galectin-3: 26×, osteoactivin/Gpnmb/DC-HIL: 160× and metalloprotease-12: 98×) was associated with heart infiltrating macrophages. To evaluate the role of OPN, we generated des(-/-)OPN(-/-) mice and compared their cardiac function and remodelling indices with those of des(-/-). Osteopontin promoted cardiac dysfunction in this model since des(-/-)OPN(-/-) mice showed 53% improvement of left ventricular function, paralleled to an up to 44% reduction in fibrosis. The diminished fibrotic response in the absence of OPN could be partly mediated by a dramatic reduction in myocardial galectin-3 levels, associated with an impaired galectin-3 secretion by OPN-deficient infiltrating macrophages. Cardiomyocyte death due to desmin deficiency leads to inflammation and subsequent overexpression of a series of remodelling modulators. Among them, OPN seems to be a major regulator of des(-/-) adverse myocardial remodelling and it functions at least by potentiating galectin-3 up-regulation and secretion.

Galectin-3 expression in delayed neuronal death of hippocampal CA1 following transient forebrain ischemia, and its inhibition by hypothermia

The ischemic damage in the hippocampal CA1 sector following transient ischemia, delayed neuronal death, is a typical apoptosis, but the mechanism underlying the delayed neuronal death is still far from fully understood. Galectin-3 is a β-galactosidase-binding lectin which is important in cell proliferation and apoptotic regulation. Galectin-3 is expressed by microglial cells in experimental models of adult stroke. It has been reported that activated microglial cells are widely observed in the brain, including in the hippocampal CA1 region after transient ischemic insult. In the present study, time course expression of galectin-3 following transient forebrain ischemia in gerbils was examined by immunohistochemistry, combined with Iba-1 immunostaining (a specific microglial cell marker), hematoxylin and eosin staining (for morphological observation), and in situ terminal dUTP-biotin nick end labeling of DNA fragments method (for determination of cell death). Following transient ischemia, we observed a transient increase of galectin-3 expression in CA1 region, which was maximal 96h after reperfusion. Galectin-3 expression was predominately localized within CA1 region and observed only in cells which expressed Iba-1. The galectin-3-positive microglial cells emerge after the onset of neuronal cell damage. Expressions of galectin-3 and Iba-1 were strongly reduced by hypothermia during ischemic insult. Prevention of galectin-3 and Iba-1 expression in microglia by hypothermia has led us to propose that hypothermia either inhibits microglial activation or prevents delayed neuronal death itself. Our results indicate that galectin-3 might exert its effect by modulating the neuronal damage in delayed neuronal death.

Galectin-3 Gene Inactivation Reduces Atherosclerotic Lesions and Adventitial Inflammation in ApoE-Deficient Mice

This study has examined the role of galectin-3 (GaL3), a multicompartmented N-acetyllactosamine-binding chimeric lectin, on atherogenesis in the ApoE-deficient mouse model of atherosclerosis. Pathological changes consisting of atheromatous plaques, atherosclerotic microaneurysms extending into periaortic vascular channels, and adventitial and periaortic inflammatory infiltrates were assessed in an equal number (n = 36) of apolipoprotein (Apo)E-deficient mice and ApoE-GaL3 double-knockout mice. These mice were divided into three age groups, 21 to 23 weeks, 25 to 31 weeks, and 36 to 44 weeks of age. Results of this morphological analysis have shown an age-related increase in the incidence of aorta atheromatous plaques and periaortic vascular channels in ApoE-deficient mice. By contrast ApoE/GaL3 double-knockout mice did not show an increase in pathological changes with age. The 36- to 44-week group of ApoE(-/-)/GaL3(-/-) mice had a significantly lower number of atherosclerotic lesions (P < 0.004) and fewer atheromatous plaques (P < 0.008) when compared with ApoE(-/-)/GaL3+/+ mice of the same age. ApoE(-/-)/GaL3(-/-) mice had a lower number of perivascular inflammatory infiltrates and mast cells than those found in ApoE(-/-)/GaL3+/+ mice. The reduced number of perivascular mast cells may have resulted in a low level of interleukin-4 that contributed to the reduction in the morphological parameters of atherogenesis correlated with the lack of GaL3 expression. The effect of GaL3 deficiency on atherogenesis decrease could be related to its function as a multifunctional protein implicated in macrophage chemotaxis, angiogenesis, lipid loading, and inflammation.

Galectin-3 Modulates MUC2 Mucin Expression in Human Colon Cancer Cells at the Level of Transcription via AP-1 Activation

Galectin-3 and MUC2 intestinal mucin each have been correlated with the malignant behavior of colon cancer cells. Galectin-3 modulates expression of MUC2 protein, but the specific regulatory mechanisms are unknown. This study sought to determine how galectin-3 increases MUC2 expression. Galectin-3 levels in human colon cancer cells of high and low metastatic ability were manipulated via expression of galectin-3 complementary DNA in sense or antisense orientation. Galectin-3 and MUC2 protein expression were determined by Western analysis and immunocytochemistry. Transient transfections of promoter reporter constructs were used to monitor MUC2 transcription and AP-1 activity. Electrophoretic mobility shift assays, site-directed mutagenesis, and chromatin immunoprecipitation were used to monitor the participation of AP-1 in MUC2 transcription. Alterations in galectin-3 levels correlated with both MUC2 protein expression and transcription. By using MUC2 promoter constructs of different lengths, galectin-3 responsiveness was found between 1500 and 2186 bp upstream of the translation start site, a region that contains 1 consensus AP-1 binding site. AP-1 activity paralleled MUC2 transcription in the different cell lines. Mutation in the AP-1 site markedly decreased MUC2 promoter activity, and MUC2 transcription was inhibited by cotransfection with a dominant-negative AP-1 vector. Electrophoretic mobility shift assays, co-immunoprecipitation, and chromatin immunoprecipitation analyses suggested an association between galectin-3, c-Jun, and Fra-1 in forming a complex at the AP-1 site on the MUC2 promoter. Galectin-3 up-regulation of MUC2 transcription occurs at the level of transcription through AP-1 activation. This may have important implications for understanding the role of galectin-3 and MUC2 in colon cancer metastasis.

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.