Galectin-4 is a carbohydrate-binding protein belonging to the galectin family. Here we provide novel evidence that galectin-4 is selectively expressed and secreted by intestinal epithelial cells and binds potently to activated peripheral and mucosal lamina propria T-cells at the CD3 epitope. The carbohydrate-dependent binding of galectin-4 at the CD3 epitope is fully functional and inhibited T cell activation, cycling and expansion. Galectin-4 induced apoptosis of activated peripheral and mucosal lamina propria T cells via calpain-, but not caspase-dependent, pathways. Providing further evidence for its important role in regulating T cell function, galectin-4 blockade by antisense oligonucleotides reduced TNF-alpha inhibitor induced T cell death. Furthermore, in T cells, galectin-4 reduced pro-inflammatory cytokine secretion including IL-17. In a model of experimental colitis, galectin-4 ameliorated mucosal inflammation, induced apoptosis of mucosal T-cells and decreased the secretion of pro-inflammatory cytokines. Our results show that galectin-4 plays a unique role in the intestine and assign a novel role of this protein in controlling intestinal inflammation by a selective induction of T cell apoptosis and cell cycle restriction. Conclusively, after defining its biological role, we propose Galectin-4 is a novel anti-inflammatory agent that could be therapeutically effective in diseases with a disturbed T cell expansion and apoptosis such as inflammatory bowel disease.
Galectin-3 is a human lectin involved in many cellular processes including differentiation, apoptosis, angiogenesis, neoplastic transformation, and metastasis. We evaluated galectin-3C, an N-terminally truncated form of galectin-3 that is thought to act as a dominant negative inhibitor, as a potential treatment for multiple myeloma (MM). Galectin-3 was expressed at varying levels by all 9 human MM cell lines tested. In vitro galectin-3C exhibited modest anti-proliferative effects on MM cells and inhibited chemotaxis and invasion of U266 MM cells induced by stromal cell-derived factor (SDF)-1α. Galectin-3C facilitated the anticancer activity of bortezomib, a proteasome inhibitor approved by the FDA for MM treatment. Galectin-3C and bortezomib also synergistically inhibited MM-induced angiogenesis activity in vitro. Delivery of galectin-3C intravenously via an osmotic pump in a subcutaneous U266 cell NOD/SCID mouse model of MM significantly inhibited tumor growth. The average tumor volume of bortezomib-treated animals was 19.6% and of galectin-3C treated animals was 13.5% of the average volume of the untreated controls at day 35. The maximal effect was obtained with the combination of galectin-3C with bortezomib that afforded a reduction of 94% in the mean tumor volume compared to the untreated controls at day 35. In conclusion, this is the first study to show that inhibition of galectin-3 is efficacious in a murine model of human MM. Our results demonstrated that galectin-3C alone was efficacious in a xenograft mouse model of human MM, and that it enhanced the anti-tumor activity of bortezomib in vitro and in vivo. These data provide the rationale for continued testing of galectin-3C towards initiation of clinical trials for treatment of MM.
Galectins are a family of β-galactoside-binding lectins that exert diverse extracellular and intracellular effects. Galectin-7 and galectin-1 show opposing effects on proliferation and survival in different cell types. Galectin-7 is a p53-induced gene and an enhancer of apoptosis, whereas galectin-1 induces tumorigenicity and resistance to apoptosis in several types of cancers. We show here that in cells derived from neurofibromin-deficient (Nf1−/−) malignant peripheral nerve sheath tumors (MPNSTs), Ras inhibition by S-trans,trans-farnesylthiosalicylic-acid (FTS; Salirasib) shifts the pattern of galectin expression. Whereas FTS decreased levels of both active Ras and galectin-1 expression, it dramatically increased both the mRNA and protein expression levels of galectin-7. Galectin-7 accumulation was mediated through JNK inhibition presumably resulting from the observed induction of p53, and was negatively regulated by the AP-1 inhibitor JDP2. Expression of galectin-7 by itself decreased Ras activation in ST88-14 cells and rendered them sensitive to apoptosis. This observed shift in galectin expression pattern together with the accompanying shift from cell proliferation to apoptosis represents a novel pattern of Ras inhibition by FTS. This seems likely to be an important phenomenon in view of the fact that both enhanced cell proliferation and defects of apoptosis constitute major hallmarks of human cancers and play a central role in the resistance of MPNSTs to anti-cancer treatments. These findings suggest that FTS, alone or in combination with chemotherapy agents, may be worth developing as a possible treatment for MPNSTs.
apoptosis; c-jun; farnesylthiosalicylic acid; FTS; galectin-1; galectin -7; JDP2; NF1; p53; Ras; Salirasib
Galectins regulate cellular functions by binding to glycan ligands on cell surface glycoprotein receptors. Prototype galectins, such as galectin-1, are one carbohydrate recognition domain (CRD) monomers that noncovalently dimerize, whereas tandem-repeat galectins, such as galectin-9, have two non-identical CRDs connected by a linker domain. Dimerization of prototype galectins, or both CRDs in tandem-repeat galectins, is typically required for the crosslinking of glycoprotein receptors and subsequent cellular signaling. Several studies have found that tandem-repeat galectins are more potent than prototype galectins in triggering many cell responses, including cell death. These differences could be due to CRD specificity, the presence or absence of a linker domain between CRDs, or both. To interrogate the basis for the increased potency of tandem-repeat galectins compared with prototype galectins in triggering cell death, we created three tandem-repeat galectin constructs with different linker regions joining identical galectin-1 CRDs, so that any differences we observed would be due to the contribution of the linker region rather than due to CRD specificity. We found that random-coil or rigid α-helical linkers that permit separation of the two galectin-1 CRDs facilitated the formation of higher-order galectin multimers and that these galectins were more potent in binding to glycan ligands and cell surface glycoprotein receptors, as well as triggering T cell death, compared with native galectin-1 or a construct with a short rigid linker. Thus, the increased potency of tandem-repeat galectins compared with prototype galectins is likely due to the ability of the linker domain to permit intermolecular CRD interactions, resulting in the formation of higher-order multimers with increased valency, rather than differences in CRD specificity.
apoptosis; galectin; glycan microarray; lattice; tandem repeat
The epidermal growth factor receptor (EGFR)-mediated signaling pathways are important in a variety of cellular processes, including cell migration and wound re-epithelialization. Intracellular trafficking of EGFR is critical for maintaining EGFR surface expression. Galectin-3, a member of an animal lectin family, has been implicated in a number of physiological and pathological processes. Through studies of galectin-3-deficient mice and cells isolated from these mice, we demonstrated that absence of galectin-3 impairs keratinocyte migration and skin wound re-epithelialization. We have linked this pro-migratory function to a crucial role of cytosolic galectin-3 in controlling intracellular trafficking and cell surface expression of EGFR after EGF stimulation. Without galectin-3, the surface levels of EGFR are dramatically reduced and the receptor accumulates diffusely in the cytoplasm. This is associated with reduced rates of both endocytosis and recycling of the receptor. We have provided evidence that this novel function of galectin-3 may be mediated through interaction with its binding partner Alix, which is a protein component of the endosomal sorting complex required for transport (ESCRT) machinery. Our results suggest that galectin-3 is potentially a critical regulator of a number of important cellular responses through its intracellular control of trafficking of cell surface receptors.
Galectin-1 is a component of the extracellular matrix as well as a ligand of cell surface counter receptors such as β-galactoside–containing glycolipids, however, the molecular mechanism of galectin-1 secretion has remained elusive. Based on a nonbiased screen for galectin-1 export mutants we have identified 26 single amino acid changes that cause a defect of both export and binding to counter receptors. When wild-type galectin-1 was analyzed in CHO clone 13 cells, a mutant cell line incapable of expressing functional galectin-1 counter receptors, secretion was blocked. Intriguingly, we also find that a distant relative of galectin-1, the fungal lectin CGL-2, is a substrate for nonclassical export from Chinese hamster ovary (CHO) cells. Alike mammalian galectin-1, a CGL-2 mutant defective in β-galactoside binding, does not get exported from CHO cells. We conclude that the β-galactoside binding site represents the primary targeting motif of galectins defining a galectin export machinery that makes use of β-galactoside–containing surface molecules as export receptors for intracellular galectin-1.
Galectin-12, a member of the galectin family of animal lectins, is preferentially expressed in adipocytes. We recently reported that this galectin is localized on lipid droplets, specialized organelles for fat storage. Galectin-12 regulates lipid degradation (lipolysis) by modulating lipolytic protein kinase A (PKA) signaling. Mice deficient in galectin-12 exhibit enhanced adipocyte lipolysis, increased mitochondria respiration, reduced adiposity and ameliorated insulin resistance associated with weight gain. The results suggest that galectin-12 may be a useful target for treatment of obesity-related metabolic conditions, such as insulin resistance, metabolic syndrome, and type 2 diabetes. Most previously described galectins largely reside in the cytosol, although they can also be induced to become associated with membrane-containing structures. Along with an in-depth characterization of galectin-12, this mini-review comments on this first report of a galectin normally localized specifically in an organelle that performs an important intracellular function. Further studies will help shed light on how this protein regulates cellular homeostasis, especially energy homeostasis, and provide additional insight into the intracellular functions of galectins.
adipocyte; adipose tissue; galectin; galectin-12; insulin sensitivity; lipid metabolism; lipolysis
In the following experiments, we sought to understand the triggering mechanism which propels galectin-3 to be secreted into the extracellular compartment from its intracellular stores in breast carcinoma cells. We also wanted to analyze in greater details, the role of galectin-3 in cellular adhesion and spreading. To do this, we made use of two pairs of breast carcinoma cell lines where one of the pair has high expression of galectin-3 and the other low expression of the lectin. We determined that galectin-3 secreted into the conditioned medium of sub confluent and spread cells in culture was quite low, almost negligible. However, once the cells were detached and rounded up, a mechano-sensing mechanism triggered the rapid secretion of galectin-3 into the conditioned medium. The secretion was constitutive as long as the cells remained detached. Galectin-3 was shown to be actively taken up from the conditioned medium by spreading cells. The cells which express and secrete high levels of galectin-3 adhered and spread much faster on plastic than those with reduced expression. The uptake of galectin-3 according to our data was important in cell spreading because if this process was compromised significantly, cells failed to spread. The data suggested that galectin-3 uptake modulates the adhesion plaques in that cells which express high levels of galectin-3 have thin-dot like plaques that may be suited for rapid adhesion and spreading while cells in which galectin-3 expression is reduced or knocked-down, have thick and elongated plaques which may be suited for a firmer adhesion to the substratum. Recombinant galectin-3 added exogenously reduced the thickness of the adhesion plaques of tumor cells with reduced galectin-3 expression. Taken together, the present data suggest that galectin-3 once externalized, is a powerful modulator of cellular adhesion and spreading in breast carcinoma cells.
Galectin-3; secretion; uptake; extra cellular; adhesion plaques; mechano- transduction; integrin; breast; carcinoma; growth factor
Galectins, a family of β-galactoside binding lectins, have recently emerged as novel regulators of tissue homeostasis. Galectin-7 is predominantly expressed in stratified epithelia, especially in epidermis. We report here the generation of galectin-7–deficient mice that are viable and do not display phenotypical abnormalities in skin structure or expression of epidermal markers. However, these mice show unique defects in the maintenance of epidermal homeostasis in response to environmental challenges. First, after UVB irradiation in vivo, the apoptotic response is prematurely triggered and lasts longer in the mutant epidermis. This result contrasts with the proapoptotic role that had been proposed for galectin-7. Second, wound-healing experiments in vivo revealed that galectin-7–deficient mice displayed a reduced reepithelialization potential compared with wild-type littermates. This effect could be attributed to a defect in cell migration. Because galectin-7 is located in the podosomes of keratinocytes migrating out of skin explants in culture, we propose that this glycan-binding protein may directly influence cell/extracellular matrix interactions. Finally, we also detected an unexpected intense hyperproliferative reaction consecutive to both types of stress in galectin-7–deficient mice. Together, these studies provide the first genetic evidence showing that galectin-7 can modulate keratinocyte apoptosis, proliferation, and migration during skin repair.
Galectin-3 is highly expressed in epithelial cells including keratinocytes and is involved in the pathogenesis of inflammatory skin diseases by affecting the functions of immune cells. For example, galectin-3 can contribute to atopic dermatitis (AD) by promoting polarization toward a Th2 immune response by regulating dendritic cell (DC) and T cell functions. In addition, galectin-3 may be involved in the development of contact hypersensitivity by regulating the migratory capacity of antigen presenting cells. Galectin-3 may act as a regulator of epithelial tumor progression and development through various signaling pathways, such as inhibiting keratinocyte apoptosis through regulation of the activation status of extracellular signal-regulated kinase (ERK) and activated protein kinase B (AKT). Galectin-3 is detected at different stages of melanoma development. In contrast, a marked decrease in the expression of galectin-3 is observed in non-melanoma skin cancers, such as squamous cell carcinoma (SCC) and basal cell carcinoma (BCC). Galectin-3 may play an important role in tumor cell growth, apoptosis, cell motility, invasion, and metastasis. Galectin-3 may be a novel therapeutic target for a variety of skin diseases.
Podoplanin is a small, mucin-like membrane glycoprotein highly expressed by lymphatic but not by blood vascular endothelial cells. Although it was shown to be indispensable for the correct formation and function of the lymphatic vasculature, its precise molecular function has remained unknown. In the present study, we identified the mammalian lectin galectin-8 as a novel, glycosylation-dependent interaction partner of podoplanin. Galectin-8 is a tandem-repeat type galectin, which interacts with cell surface glycoproteins, including certain integrins, as well as with extracellular matrix molecules such as fibronectin. Here we show that, similar to podoplanin, galectin-8 is more highly expressed by lymphatic than by blood vascular endothelial cells, and that it promotes lymphatic endothelial cell adhesion as well as haptotactic migration when immobilized onto a surface, while inhibiting the formation of tube-like structures by lymphatic endothelial cells in a collagen matrix when incorporated into the matrix. Importantly, functions of blood vascular endothelial cells, which lack podoplanin expression, are not affected by galectin-8. These data suggest a role for galectin-8 and podoplanin in supporting the connection of the lymphatic endothelium to the surrounding extracellular matrix, most likely in cooperation with other glycoproteins on the surface of lymphatic endothelial cells.
podoplanin; galectin-8; lymphatic endothelial cell; lymphangiogenesis
Galectin-1 has been implicated in regulating T cell survival, function, and Th1/Th2 balance in several mouse models, though the molecular and cellular basis of its immuno-modulatory activity has not been completely elucidated. Therefore, we examined galectin-1 expression and activity within differentiated murine Th1 and Th2 subsets. While recombinant galectin-1 specifically bound to both T cell subsets, Th1 and Th2 T cells expressed distinct combinations of galectin-1 reactive epitopes and were differentially responsive to galectin-1 exposure. Indeed, Th1 cells were more susceptible to galectin-1 induced death than Th2 cells. Th2 protection from apoptosis was correlated with expression of anti-apoptotic galectin-3. Further, galectin-1 promoted TCR-induced type 2 cytokine production by Th2 cells. Differentiated Th2 cells constitutively expressed high levels of galectin-1 and can be induced to produce even higher levels of galectin-1 with restimulation, whereas comparable levels of galectin-1 in Th1 cells were only observed after re-stimulation. Co-culturing experiments using galectin-1−/− and galectin-1+/+ Th1 and Th2 T cells demonstrated that Th2-derived galectin-1 induced Th1 apoptosis, whereas Th1-derived galectin-1 promoted Th2 cytokine production. These studies identify galectin-1 as a cross-regulatory cytokine that selectively antagonizes Th1 survival, while promoting TCR-induced Th2 cytokine production.
Apoptosis; CD4 T cells; cell differentiation; cytokines; galectin-1
The galectins comprise a family of 14 members of β-galactoside-binding proteins, characterized by their affinity for β-galactosides and by a conserved sequence in the carbohydrate recognition domain that bind to the carbohydrate portion of cell surface glycoproteins or glycolipids. Galectin-3, a 31 kDa gene product, is a multifunctional oncogenic protein which regulates cell growth, cell adhesion, cell proliferation, angiogenesis, and apoptosis. Recent studies have revealed that galectin-3 demonstrates anti-apoptotic effects which contribute to cell survival in several types of cancer cells. Intracellular galectin-3 in particular, which contains the NWGR anti-death motif of the Bcl-2 family, inhibits cell apoptosis induced by chemotherapeutic agent such as cisplatin and etoposide in some types of cancer cells. We have also reported that nuclear export of phosphorylated galectin-3 regulates its anti-apoptotic activity in response to chemotherapeutic drugs. Here, we will describe the role of galectin-3 as an anti-apoptotic factor in response to chemotherapeutic drugs and will discuss recent data on its molecular mechanism that contribute to drug resistance. We suggest that targeting galectin-3 could improve the efficacy of anticancer drug chemotherapy in several types of cancer.
Galectin-3; Apoptosis; Cancer chemotherapy; Drug resistance
Galectin-1 is a lectin recognized by galactoside-containing glycoproteins, and is involved in cancer progression and metastasis. The role of galectin-1 in radiosensitivity has not previously been investigated. Therefore, this study tests whether galectin-1 is involved in the radiosensitivity mediated by the H-Ras signaling pathway using cervical carcinoma cell lines. A knockdown of galectin-1 expression in HeLa cells decreased clonogenic survival following irradiation. The clonogenic survival increased in both HeLa and C33A cells with galectin-1 overexpression. The overexpression or knockdown of galectin-1 did not alter radiosensitivity, whereas H-Ras was silenced in both cell lines. Whereas K-Ras was knocked down, galectin-1 restored the radiosensitivity in HeLa cells and C33A cells. The knockdown of galectin-1 increased the high-dose radiation-induced cell death of HeLa cells transfected by constitutively active H-Ras. The knockdown of galectin-1 inhibited the radiation-induced phosphorylation of Raf-1 and ERK in HeLa cells. Overexpression of galectin-1 enhanced the phosphorylation of Raf-1 and ERK in C33A cells following irradiation. Galectin-1 decreased the DNA damage detected using comet assay and γ-H2AX in both cells following irradiation. These findings suggest that galectin-1 mediates radioresistance through the H-Ras-dependent pathway involved in DNA damage repair.
galectin-1; cervical cancer; radiosensitivity; radioresistance; H-Ras
Expression of galectin-3 is associated with sarcoma progression, invasion and metastasis. Here we determined the role of extracellular galectin-3 on migration of sarcoma cells on laminin-111. Cell lines from methylcholanthrene-induced sarcomas from both wild type and galectin-3−/− mice were established. Despite the presence of similar levels of laminin-binding integrins on the cell surface, galectin-3−/− sarcoma cells were more adherent and less migratory than galectin-3+/+ sarcoma cells on laminin-111. When galectin-3 was transiently expressed in galectin-3−/− sarcoma cells, it inhibited cell adhesion and stimulated the migratory response to laminin in a carbohydrate-dependent manner. Extracellular galectin-3 led to the recruitment of SHP-2 phosphatase to focal adhesion plaques, followed by a decrease in the amount of phosphorylated FAK and phospho-paxillin in the lamellipodia of migrating cells. The promigratory activity of extracellular galectin-3 was inhibitable by wortmannin, implicating the activation of a PI-3 kinase dependent pathway in the galectin-3 triggered disruption of adhesion plaques, leading to sarcoma cell migration on laminin-111.
Disassembly and phagocytic removal of dying cells is critical to maintain immune homeostasis. The factors regulating fragmentation and uptake of dying lymphocytes are not well understood. Degradation of fodrin, a cytoskeletal linker molecule that attaches CD45 to the actin cytoskeleton, has been described in apoptotic cells, although no specific initiator of fodrin degradation has been identified. CD45 is a glycoprotein receptor for galectin-1, an endogenous lectin that can trigger lymphocyte apoptosis. CD45 is not required for membrane changes or DNA degradation during galectin-1 death. However, here we show that fodrin degradation occurs during galectin-1 T cell death, and CD45 is essential for fodrin degradation to occur. In the absence of CD45 and fodrin degradation, cell death is not accompanied by membrane blebbing, indicating that fodrin degradation occurs via a distinct pathway compared to the pathway that initiates apoptotic membrane changes and DNA degradation. Moreover, there is slower phagocytic uptake of cells in which fodrin degradation is blocked relative to cells in which CD45-mediated fodrin degradation occurs. These studies identify a novel role for CD45 in regulating cellular disassembly and promoting phagocytic clearance during galectin-1 induced T cell death.
Galectin-3 is expressed and secreted by immune cells and has been implicated in multiple aspects of the inflammatory response. It is a glycan binding protein which can exert its functions within cells or exogenously by binding cell surface ligands, acting as a molecular bridge or activating signaling pathways. In addition, this lectin has been shown to bind to microorganisms. In this study we investigated the interaction between galectin-3 and Neisseria meningitidis, an important extracellular human pathogen, which is a leading cause of septicaemia and meningitis. Immunohistochemical analysis indicated that galectin-3 is expressed during meningococcal disease and co-localises with bacterial colonies in infected tissues from patients. We show that galectin-3 binds to N. meningitidis and we demonstrate that this interaction requires full length, intact lipopolysaccharide molecules. We found that neither exogenous nor endogenous galectin-3 contributes to phagocytosis of N. meningitidis; instead exogenous galectin-3 increases adhesion to monocytes and macrophages but not epithelial cells. Finally we used galectin-3 deficient (Gal-3−/−) mice to evaluate the contribution of galectin-3 to meningococcal bacteraemia. We found that Gal3−/−mice manifested significantly lower levels of bacteriaemia compared with wild-type mice after challenge with live bacteria, indicating that galectin-3 confers an advantage to N.meningitidis during systemic infection.
Galectin-9, a beta-galactoside binding lectin, has recently been isolated from murine embryonic kidney. In this study, its biological functions and expression in embryonic, newborn, and adult mice tissues were investigated. By Northern blot analyses, it was found widely distributed and its expression was developmentally regulated. In situ hybridization studies revealed an accentuated expression of galectin-9 in liver and thymus of embryonic mice. In postnatal mice, antigalectin-9 immunoreactivity was observed in various tissues, including thymic epithelial cells. The high expression of galectin-9 in the thymus led us to investigate its role in the clonal deletion of thymocytes. Fusion proteins were generated, which retained lactose-binding activity like the endogenous galectin-9. Galectin-9, at 2.5 microM concentration, induced apoptosis in approximately 30% of the thymocytes, as assessed by terminal deoxytransferase-mediated dUTP nick end labeling method. The apoptotic effect was dose dependent and lactose inhibitable. At higher concentrations, it induced homotypic aggregation of the thymocytes. Electron microscopy revealed approximately 60% of the thymocytes undergoing apoptosis in the presence of galectin-9. By immunofluorescence microscopy, some of the thymocytes undergoing apoptosis had plasmalemmal bound galectin-9. Galectin-9 failed to induce apoptosis in hepatocytes. Taken together, these findings indicate that galectin-9, a developmentally regulated lectin, plays a role in thymocyte-epithelial interactions relevant to the biology of the thymus.
Mitochondria play a pivotal role in apoptosis in multicellular organisms by releasing apoptogenic factors such as cytochrome c that activate the caspases effector pathway, and apoptosis-inducing factor (AIF) that is involved in a caspase-independent cell death pathway. Here we report that cell death in the single-celled organism Dictyostelium discoideum involves early disruption of mitochondrial transmembrane potential (ΔΨm) that precedes the induction of several apoptosis-like features, including exposure of the phosphatidyl residues at the external surface of the plasma membrane, an intense vacuolization, a fragmentation of DNA into large fragments, an autophagy, and the release of apoptotic corpses that are engulfed by neighboring cells. We have cloned a Dictyostelium homolog of mammalian AIF that is localized into mitochondria and is translocated from the mitochondria to the cytoplasm and the nucleus after the onset of cell death. Cytoplasmic extracts from dying Dictyostelium cells trigger the breakdown of isolated mammalian and Dictyostelium nuclei in a cell-free system, and this process is inhibited by a polyclonal antibody specific for Dictyostelium discoideum apoptosis-inducing factor (DdAIF), suggesting that DdAIF is involved in DNA degradation during Dictyostelium cell death. Our findings indicate that the cell death pathway in Dictyostelium involves mitochondria and an AIF homolog, suggesting the evolutionary conservation of at least part of the cell death pathway in unicellular and multicellular organisms.
β-Galactoside-binding lectin 9 (galectin-9) is a tandem repeat-type member of the galectin family. It was initially characterized as an eosinophil chemoattractant and an inducer of apoptosis in thymocytes. Subsequently, galectin-9 was identified as a ligand for transmembrane immunoglobulin mucin domain 3 (Tim-3), a type I glycoprotein induced on T cells during chronic inflammation. Work in autoimmune diseases and chronic viral infections have led to the current hypothesis that the function of Tim-3 is to limit immune responses. However, it is still not known to what degree these effects are due to the galectin-9/Tim-3 interaction. In this study, we show that galectin-9 is not limited to the role of a pro-apoptotic agent, but that it can also induce the production of pro-inflammatory cytokines from T helper cells. This effect is dose-dependent and does not require Tim-3. These findings suggest that the effects of galectin-9 on T cells are more complex than previously thought and are mediated by additional receptors apart from Tim-3.
apoptosis; cytokines; galectin; mucin; T helper cells
Rationale: Idiopathic pulmonary fibrosis (IPF) is a chronic dysregulated response to alveolar epithelial injury with differentiation of epithelial cells and fibroblasts into matrix-secreting myofibroblasts resulting in lung scaring. The prognosis is poor and there are no effective therapies or reliable biomarkers. Galectin-3 is a β-galactoside binding lectin that is highly expressed in fibrotic tissue of diverse etiologies.
Objectives: To examine the role of galectin-3 in pulmonary fibrosis.
Methods: We used genetic deletion and pharmacologic inhibition in well-characterized murine models of lung fibrosis. Further mechanistic studies were performed in vitro and on samples from patients with IPF.
Measurements and Main Results: Transforming growth factor (TGF)-β and bleomycin-induced lung fibrosis was dramatically reduced in mice deficient in galectin-3, manifest by reduced TGF-β1–induced EMT and myofibroblast activation and collagen production. Galectin-3 reduced phosphorylation and nuclear translocation of β-catenin but had no effect on Smad2/3 phosphorylation. A novel inhibitor of galectin-3, TD139, blocked TGF-β–induced β-catenin activation in vitro and in vivo and attenuated the late-stage progression of lung fibrosis after bleomycin. There was increased expression of galectin-3 in the bronchoalveolar lavage fluid and serum from patients with stable IPF compared with nonspecific interstitial pneumonitis and controls, which rose sharply during an acute exacerbation suggesting that galectin-3 may be a marker of active fibrosis in IPF and that strategies that block galectin-3 may be effective in treating acute fibrotic exacerbations of IPF.
Conclusions: This study identifies galectin-3 as an important regulator of lung fibrosis and provides a proof of principle for galectin-3 inhibition as a potential novel therapeutic strategy for IPF.
fibrosis; epithelial cells; fibroblasts
In the adult mammalian brain, neural stem cells (NSCs) proliferate in the dentate gyrus (DG) of the hippocampus and generate new neurons throughout life. A multimodal protein, Galectin-1, is expressed in neural progenitor cells (NPCs) and implicated in the proliferation of the NPCs in the DG. However, little is known about its detailed expression profile in the NPCs and functions in adult neurogenesis in the DG.
Our immunohistochemical and morphological analysis showed that Galectin-1 was expressed in the type 1 and 2a cells, which are putative NSCs, in the subgranular zone (SGZ) of the adult mouse DG. To study Galectin-1's function in adult hippocampal neurogenesis, we made galectin-1 knock-out mice on the C57BL6 background and characterized the effects on neurogenesis. In the SGZ of the galectin-1 knock-out mice, increased numbers of type 1 cells, DCX-positive immature progenitors, and NeuN-positive newborn neurons were observed. Using triple-labeling immunohistochemistry and morphological analyses, we found that the proliferation of the type-1 cells was increased in the SGZ of the galectin-1 knock-out mice, and we propose that this proliferation is the mechanism for the net increase in the adult neurogenesis in these knock-out mice DG.
Galectin-1 is expressed in the neural stem cells and down-regulates neurogenesis in the adult hippocampus.
High expression of galectin 3 at sites of joint destruction in rheumatoid arthritis (RA) suggests that galectin 3 plays a role in RA pathogenesis. Previous studies have demonstrated the effects of galectins on immune cells, such as lymphocytes and macrophages. This study was undertaken to investigate the hypothesis that galectin 3 induces proinflammatory effects in RA by modulating the pattern of cytokine and chemokine production in synovial fibroblasts.
Matched samples of RA synovial and skin fibroblasts were pretreated with galectin 3 or tumor necrosis factor α (TNFα), and the levels of a panel of cytokines, chemokines, and matrix metalloproteinases (MMPs) were determined using enzyme-linked immunosorbent assays and multiplex assays. Specific inhibitors were used to dissect signaling pathways, which were confirmed by Western blotting and NF-κB activation assay.
Galectin 3 induced secretion of interleukin-6 (IL-6), granulocyte–macrophage colony-stimulating factor, CXCL8, and MMP-3 in both synovial and skin fibroblasts. By contrast, galectin 3–induced secretion of TNFα, CCL2, CCL3, and CCL5 was significantly greater in synovial fibroblasts than in skin fibroblasts. TNFα blockade ruled out autocrine TNFα-stimulated induction of chemokines. The MAPKs p38, JNK, and ERK were necessary for IL-6 production, but phosphatidylinositol 3-kinase (PI 3-kinase) was required for selective CCL5 induction. NF-κB activation was required for production of both IL-6 and CCL5.
Our findings indicate that galectin 3 promotes proinflammatory cytokine secretion by tissue fibroblasts. However, galectin 3 induces the production of mononuclear cell–recruiting chemokines uniquely from synovial fibroblasts, but not matched skin fibroblasts, via a PI 3-kinase signaling pathway. These data provide further evidence of the role of synovial fibroblasts in regulating the pattern and persistence of the inflammatory infiltrate in RA and suggest a new and important functional consequence of the observed high expression of galectin 3 in the rheumatoid synovium.
Many apoptotic signaling pathways are directed to mitochondria, where they initiate the release of apoptogenic proteins and open the proposed mitochondrial permeability transition (PT) pore that ultimately results in the activation of the caspase proteases responsible for cell disassembly. BNIP3 (formerly NIP3) is a member of the Bcl-2 family that is expressed in mitochondria and induces apoptosis without a functional BH3 domain. We report that endogenous BNIP3 is loosely associated with mitochondrial membrane in normal tissue but fully integrates into the mitochondrial outer membrane with the N terminus in the cytoplasm and the C terminus in the membrane during induction of cell death. Surprisingly, BNIP3-mediated cell death is independent of Apaf-1, caspase activation, cytochrome c release, and nuclear translocation of apoptosis-inducing factor. However, cells transfected with BNIP3 exhibit early plasma membrane permeability, mitochondrial damage, extensive cytoplasmic vacuolation, and mitochondrial autophagy, yielding a morphotype that is typical of necrosis. These changes were accompanied by rapid and profound mitochondrial dysfunction characterized by opening of the mitochondrial PT pore, proton electrochemical gradient (Δψm) suppression, and increased reactive oxygen species production. The PT pore inhibitors cyclosporin A and bongkrekic acid blocked mitochondrial dysregulation and cell death. We propose that BNIP3 is a gene that mediates a necrosis-like cell death through PT pore opening and mitochondrial dysfunction.
Detergent-insoluble complexes prepared from pig small intestine are highly enriched in several transmembrane brush border enzymes including aminopeptidase N and sucrase-isomaltase, indicating that they reside in a glycolipid-rich environment in vivo. In the present work galectin-4, an animal lectin lacking a N-terminal signal peptide for membrane translocation, was discovered in these complexes as well, and in gradient centrifugation brush border enzymes and galectin-4 formed distinct soluble high molecular weight clusters. Immunoperoxidase cytochemistry and immunogold electron microscopy showed that galectin-4 is indeed an intestinal brush border protein; we also localized galectin-4 throughout the cell, mainly associated with membraneous structures, including small vesicles, and to the rootlets of microvillar actin filaments. This was confirmed by subcellular fractionation, showing about half the amount of galectin-4 to be in the microvillar fraction, the rest being associated with insoluble intracellular structures. A direct association between the lectin and aminopeptidase N was evidenced by a colocalization along microvilli in double immunogold labeling and by the ability of an antibody to galectin-4 to coimmunoprecipitate aminopeptidase N and sucrase-isomaltase. Furthermore, galectin-4 was released from microvillar, right-side-out vesicles as well as from mucosal explants by a brief wash with 100 mM lactose, confirming its extracellular localization. Galectin-4 is therefore secreted by a nonclassical pathway, and the brush border enzymes represent a novel class of natural ligands for a member of the galectin family. Newly synthesized galectin-4 is rapidly “trapped” by association with intracellular structures prior to its apical secretion, but once externalized, association with brush border enzymes prevents it from being released from the enterocyte into the intestinal lumen.