Extracellular Galectin-3 Research Articles

Defining the mechanism of galectin-3–mediated TGF-β1 activation and its role in lung fibrosis

Integrin-mediated activation of the pro-fibrotic mediator transforming growth factor-β1 (TGF-β1), plays a critical role in idiopathic pulmonary fibrosis (IPF) pathogenesis. Galectin-3 is believed to contribute to the pathological wound healing seen in IPF, although its mechanism of action is not precisely defined. We hypothesised that galectin-3 potentiates TGF-β1 activation and/or signaling in the lung to promote fibrogenesis. We show that galectin-3 induces TGF-β1 activation in human lung fibroblasts (HLFs) and specifically that extracellular galectin-3 promotes oleoyl-L-α-lysophosphatidic acid sodium salt (LPA)-induced integrin-mediated TGF-β1 activation. Surface plasmon resonance (SPR) analysis confirmed that galectin-3 binds to αv integrins, αvβ1, αvβ5 and αvβ6 and to the TGFβRII subunit in a glycosylation-dependent manner. This binding is heterogeneous and not a 1:1 binding stoichiometry. Binding interactions were blocked by small molecule inhibitors of galectin-3 which target the carbohydrate recognition domain. Galectin-3 binding to β1 integrin was validated in vitro by co-immunoprecipitation in HLFs. Proximity ligation assays indicated galectin-3 and β1 integrin colocalize closely (≤40 nm) on the cell surface, that colocalization is increased by TGF-β1 treatment and blocked by galectin-3 inhibitors. In the absence of TGF-β1 stimulation, colocalization was detectable only in HLFs from IPF patients suggesting the proteins are inherently more closely associated in the disease state. Galectin-3 inhibitor treatment of precision cut lung slices from IPF patients reduced Col1a1, TIMP1 and HA secretion to a similar degree as TGF-β type I receptor inhibitor. These data suggest galectin-3 promotes TGF-β1 signaling and may induce fibrogenesis by interacting directly with components of the TGF-β1 signaling cascade.

Tumor-Intrinsic Galectin-3 Suppresses Melanoma Metastasis

Melanoma poses a poor prognosis with high mortality rates upon metastasis. Exploring molecular mechanisms governing melanoma progression paves the way for developing novel approaches to control melanoma metastasis and ultimately enhance patient survival rates. Extracellular galectin-3 (Gal-3) has emerged as a pleiotropic promoter of melanoma metastasis, exerting varying activities depending on its interacting partner. However, whether intracellular Gal-3 promotes melanoma aggressive behavior remains unknown. Here, we explored Gal-3 expression in human melanoma tissues as well as murine melanoma models to examine its causal role in metastatic behavior. We found that Gal-3 expression is downregulated in metastatic melanoma tissues compared to its levels in primary melanomas. Enforced silencing of Gal-3 in melanoma cells promoted migration, invasion, colony formation, in vivo xenograft growth and metastasis, and activated canonical oncogenic signaling pathways. Moreover, loss of Gal-3 in melanoma cells resulted in upregulated expression of the pro-metastatic transcription factor, nuclear factor of activated T cells (NFAT1), and its downstream metastasis-associated proteins, matrix metalloproteinase-3 (MMP-3) and interleukin-8 (IL-8). Overall, our findings implicate melanoma intracellular Gal-3 as a major determinant of its metastatic behavior and reveal a negative regulatory role for Gal-3 on the expression of NFAT1 in melanoma cells.

Galectin-1 and Galectin-3 in B-Cell Precursor Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemias arising from the malignant transformation of B-cell precursors (BCP-ALLs) are protected against chemotherapy by both intrinsic factors as well as by interactions with bone marrow stromal cells. Galectin-1 and Galectin-3 are lectins with overlapping specificity for binding polyLacNAc glycans. Both are expressed by bone marrow stromal cells and by hematopoietic cells but show different patterns of expression, with Galectin-3 dynamically regulated by extrinsic factors such as chemotherapy. In a comparison of Galectin-1 x Galectin-3 double null mutant to wild-type murine BCP-ALL cells, we found reduced migration, inhibition of proliferation, and increased sensitivity to drug treatment in the double knockout cells. Plant-derived carbohydrates GM-CT-01 and GR-MD-02 were used to inhibit extracellular Galectin-1/-3 binding to BCP-ALL cells in co-culture with stromal cells. Treatment with these compounds attenuated migration of the BCP-ALL cells to stromal cells and sensitized human BCP-ALL cells to vincristine and the targeted tyrosine kinase inhibitor nilotinib. Because N-glycan sialylation catalyzed by the enzyme ST6Gal1 can regulate Galectin cell-surface binding, we also compared the ability of BCP-ALL wild-type and ST6Gal1 knockdown cells to resist vincristine treatment when they were co-cultured with Galectin-1 or Galectin-3 knockout stromal cells. Consistent with previous results, stromal Galectin-3 was important for maintaining BCP-ALL fitness during chemotherapy exposure. In contrast, stromal Galectin-1 did not significantly contribute to drug resistance, and there was no clear effect of ST6Gal1-catalysed N-glycan sialylation. Taken together, our results indicate a complicated joint contribution of Galectin-1 and Galectin-3 to BCP-ALL survival, with different roles for endogenous and stromal produced Galectins. These data indicate it will be important to efficiently block both extracellular and intracellular Galectin-1 and Galectin-3 with the goal of reducing BCP-ALL persistence in the protective bone marrow niche during chemotherapy.

Abstract 997: γ-Tocotrienol inhibition of metastatic phenotypic behavior is associated with a decrease in galectin-3 expression and distribution in the highly malignant mouse +SA & TS/A mammary tumor cells

Abstract γ-Tocotrienol is a rare natural isoform of vitamin E that displays potent apoptotic and anti-metastatic activity against breast cancer. Galectin-3 is a protein that plays an important role in metastasis by binding to glycoconjugates on receptors and extracellular matrix proteins. Studies were conducted to investigate the effects of γ-tocotrienol on galectin-3 expression and morphology in highly malignant +SA, TS/A MDA-MB-231 tumor cells. Computer-aided molecular modeling studies compared anticancer agents, salirasib and γ-tocotrienol, with the active site of galectin-3 and β-lactose (a known ligand). Results showed that γ-tocotrienol displays similar galectin-3 amino acid interactions as β-lactose and salirasib. Protein docking show that salirasib (active binding -2.8) and γ- tocotrienol (active binding -2.87) had similar binding scores and binding moieties. Immunocytochemical fluorescent staining show that γ-tocotrienol treatment disrupted galectin-3 expression and distribution, and a corresponding reduction in +SA cellular migration similar to that induced by β-lactose. Western blot analysis shows that γ-tocotrienol treatment induces a downregulation of galectin-3 expression. Immunocytochemistry studies shows that galectin-3 is highly expressed in +SA and TS/A cells, and γ-tocotrienol greatly reduced galectin-3 expression and distribution Furthermore, galectin-3 was found to form extracellular structures on the outer membrane of +SA, TS/A, and MDA-MB-231 cells, and γ-tocotrienol greatly attenuated galectin-3 and fibronectin distribution. γ-Tocotrienol treatment decreased f-actin stress fibers distribution in all cell lines. Furthermore, treatment with 1.5 μM histamine significantly stimulated +SA mammary tumor cell migration, whereas combination treatment with 5 μM γ-tocotrienol completely blocked histamine-induced +SA cell migration. In TS/A cells, 6-15 μM tocotrienol induced a dose-responsive increase in doxorubicin staining inside the nucleus. In MDA-MB-231 cells, syncytial cells were associated with galectin-3 expression and this was suppressed by 5-15 μM γ-tocotrienol. In summary, these results demonstrate that γ-tocotrienol treatment inhibits extracellular galectin-3 expression and disrupts galectin-3 carbohydrate binding and activation of +SA, TS/A and MDA-MB-231 breast cancer cells. These findings also suggest that γ-tocotrienol may provide significant benefits in the prevention and/or treatment of metastatic breast cancer. This study was supported in part by funding from the Louisiana Cancer Foundation. Citation Format: Paul W. Sylvester, Jessie J. Grazier. γ-Tocotrienol inhibition of metastatic phenotypic behavior is associated with a decrease in galectin-3 expression and distribution in the highly malignant mouse +SA & TS/A mammary tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 997.

Galectin-3 Promotes Müller Glia Clearance Phagocytosis via MERTK and Reduces Harmful Müller Glia Activation in Inherited and Induced Retinal Degeneration.

Clearance phagocytosis is a documented function of Müller glia in the retina. However, the molecular mechanisms of Müller glia phagocytosis remain largely undefined. Here, we show that extracellular galectin-3 and protein S promote clearance phagocytosis by immortalized human MIO-M1 Müller cells in an additive, saturable manner. Galectin-3 promotes phagocytosis by primary Müller glia from wild-type (WT) mice but not from mice that lack the engulfment receptor MERTK and therefore develop postnatal photoreceptor degeneration. Probing a possible functional link between Müller galectin-3 and MERTK, we discovered that mertk−/− Müller glia in situ show excess galectin-3 at postnatal day 20 (P20), an age prior to detectable photoreceptor degeneration. Moreover, double knockout (DKO) mice lacking both galectin-3 and MERTK show increased activation of Müller cells (but not of microglia) at P20 and more pronounced photoreceptor loss at P35 compared to mice lacking MERTK alone. Exploring the well-established sodium iodate injury model, we also found more severe activation specifically of Müller glia, and worse retinal damage in mice lacking galectin-3 compared to WT mice. Indeed, galectin-3 deficiency significantly increased sensitivity to injury, yielding Müller activation and retinal damage at a sodium iodate concentration that had no effect on the WT retina. Altogether, our results from both inherited and acutely induced models of retinal degeneration agree that eliminating galectin-3 exacerbates Müller cell activation and retinal degeneration. These data identify an important protective role for the MERTK ligand galectin-3 in the retina in restraining Müller glia activation.

O-GlcNAcylation and Regulation of Galectin-3 in Extraembryonic Endoderm Differentiation.

The regulation of proteins through the addition and removal of O-linked β-N-acetylglucosamine (O-GlcNAc) plays a role in many signaling events, specifically in stem cell pluripotency and the regulation of differentiation. However, these post-translational modifications have not been explored in extraembryonic endoderm (XEN) differentiation. Of the plethora of proteins regulated through O-GlcNAc, we explored galectin-3 as a candidate protein known to have various intracellular and extracellular functions. Based on other studies, we predicted a reduction in global O-GlcNAcylation levels and a distinct galectin expression profile in XEN cells relative to embryonic stem (ES) cells. By conducting dot blot analysis, XEN cells had decreased levels of global O-GlcNAc than ES cells, which reflected a disbalance in the expression of genes encoding O-GlcNAc cycle enzymes. Immunoassays (Western blot and ELISA) revealed that although XEN cells (low O-GlcNAc) had lower concentrations of both intracellular and extracellular galectin-3 than ES cells (high O-GlcNAc), the relative secretion of galectin-3 was significantly increased by XEN cells. Inducing ES cells toward XEN in the presence of an O-GlcNAcase inhibitor was not sufficient to inhibit XEN differentiation. However, global O-GlcNAcylation was found to decrease in differentiated cells and the extracellular localization of galectin-3 accompanies these changes. Inhibiting global O-GlcNAcylation status does not, however, impact pluripotency and the ability of ES cells to differentiate to the XEN lineage.

Abstract 2889: γ-Tocotrienol inhibition of metastatic phenotypic behavior is associated with a decrease in galectin-3 expression and distribution in the highly malignant mouse +SA & TS/A mammary tumor cells

Abstract γ-Tocotrienol is a rare natural isoform of vitamin E that displays potent apoptotic and anti-metastatic activity against breast cancer. Galectin-3 is a protein that plays an important role in metastasis by binding to glycoconjugates on receptors and extracellular matrix proteins. Studies were conducted to investigate the effects of γ-tocotrienol on galectin-3 expression and activity in highly malignant mouse +SA mammary tumor cells. Computer-aided molecular modeling studies were carried out within the active site of galectin-3 based on the crystal structure (5EXO) obtained from the protein data bank with a known ligand, and anticancer agents (salirasib, γ-tocotrienol, and β-lactose) galectin-3 docking scores and amino acid interactions were determine. Treatment effects on galectin-3 expression and distribution was determined by Western blot analysis and immunocytochemical fluorescent staining, respectively. Treatment effects on +SA cellular migration and invasion was also determined. Results show that salirasib and β-lactose (known inhibitors of galectin-3) that share some pharmacophoric properties with γ- tocotrienol, with protein docking studies showing that salirasib (active binding -2.8) and γ- tocotrienol (active binding -2.87) had a similar binding score and binding moieties. Western blot analysis shows that γ-tocotrienol treatment induces a downregulation of galectin-3 expression, whereas immunocytochemistry studies shows that galectin-3 is highly expressed in +SA and TS/A cells, and treatment with 5 μM & 6 μM γ-tocotrienol greatly reduced galectin-3 expression in these cells, respectively. Furthermore, galectin-3 was found to form globular structures on the outer membrane of +SA and TS/A cells. In treated and control +SA cells, fibronectin and galectin-3 were present on the outside of cells, and γ-tocotrienol greatly attenuated galectin-3 and fibronectin distribution. In TS/A cells, galectin-3 distribution changed in control and treatment groups. Furthermore, treatment with 1.5 μM histamine significantly stimulated +SA mammary tumor cell migration, whereas combined treatment with 5 μM γ-tocotrienol completely blocked histamine-induced +SA cell migration. In summary, these results demonstrate for the first time that γ-tocotrienol treatment inhibits extracellular galectin-3 expression and disrupts galectin-3 carbohydrate binding and activation of +SA and TS/A mammary tumor cells. These findings also suggest that γ-tocotrienol may provide significant benefits in the prevention and/or treatment of metastatic breast cancer. Citation Format: Jessie J. Grazier, Paul W. Sylvester. γ-Tocotrienol inhibition of metastatic phenotypic behavior is associated with a decrease in galectin-3 expression and distribution in the highly malignant mouse +SA & TS/A mammary tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2889.

The therapeutic potential of galectin-3 inhibition in fibrotic disease.

Galectin-3 is a beta-galactoside-binding mammalian lectin and part of the 15 member galectin family that are evolutionarily highly conserved. It is the only chimeric protein with a C-terminal carbohydrate recognition domain (CRD) linked to a proline, glycine, and tyrosine rich additional N-terminal domain. Galectin-3 binds several cell surface glycoproteins via its CRD domain as well as undergoing oligomerization, via binding at the N-terminal or the CRD, resulting in the formation of a galectin-3 lattice on the cell surface. The galectin-3 lattice has been regarded as being a crucial mechanism whereby extracellular galectin-3 modulates cellular signalling by prolonging retention time or retarding lateral movement of cell surface receptors in the plasma membrane. As such galectin-3 can regulate various cellular functions such as diffusion, compartmentalization and endocytosis of plasma membrane glycoproteins and glycolipids and the functionality of membrane receptors. In multiple models of organ fibrosis, it has been demonstrated that galectin-3 is potently pro-fibrotic and modulates the activity of fibroblasts and macrophages in chronically inflamed organs. Increased galectin-3 expression also activates myofibroblasts resulting in scar formation and may therefore impact common fibrotic pathways leading to fibrosis in multiple organs. Over the last decade there has been a marked increase in the scientific literature investigating galectin-3 in a range of fibrotic diseases as well as the clinical development of new galectin-3 inhibitors. In this review we will examine the role of galectin-3 in fibrosis, the therapeutic strategies for inhibiting galectin-3 in fibrotic disease and the clinical landscape to date.

Abstract 5015: γ-Tocotrienol inhibition of metastatic phenotypic behavior is associated with a decrease in galectin-3 expression and distribution in the highly malignant mouse +SA mammary tumor cells

Abstract γ-Tocotrienol is a rare natural isoform of vitamin E that displays potent apoptotic and anti-metastatic activity against breast cancer. Galectin-3 is a protein that plays an important role in metastasis by binding to glycoconjugates on receptors and extracellular matrix proteins. Studies were conducted to investigate the effects of γ-tocotrienol on galectin-3 expression and activity in highly malignant mouse +SA mammary tumor cells. Computer-aided molecular modeling studies were carried out within the active site of galectin-3 based on the crystal structure (5EXO) obtained from the protein data bank with a known ligand, and anticancer agents (salirasib, γ-tocotrienol, and β-lactose) galectin-3 docking scores and amino acid interactions were determine. Treatment effects on galectin-3 expression and distribution was determined by Western blot analysis and immunocytochemical fluorescent staining, respectively. Treatment effects on +SA cellular migration and invasion was also determined. Results show that salirasib and β-lactose (known inhibitors of galectin-3) that share some pharmacophoric properties with γ- tocotrienol, with protein docking studies showing that salirasib (active binding -2.8) and γ- tocotrienol (active binding -2.87) had a similar binding score and binding moieties. Western blot analysis shows that γ-tocotrienol treatment induces a downregulation of galectin-3 expression, whereas immunocytochemistry studies shows that galectin-3 is highly expressed in +SA cells, and treatment with 5 μM γ-tocotrienol greatly reduced galectin-3 expression in these cells. Furthermore, galectin-3 was found to form globular structures on the outer membrane of +SA cells in the vehicle-treated control group, and +SA group, and a high level of fibronectin on the outside of +SA cells, and γ-tocotrienol greatly attenuated galectin-3 expression and distribution in +SA cells. Furthermore, treatment with 1.5 μM histamine significantly stimulated +SA mammary tumor cell migration, whereas combined treatment with 5 μM γ-tocotrienol completely blocked histamine-induced +SA cell migration. In summary, these results demonstrate for the first time that γ-tocotrienol treatment inhibits extracellular galectin-3 expression and disrupts galectin-3 carbohydrate binding, activation and migration of +SA mammary tumor cells. These findings also suggest that γ-tocotrienol may provide significant benefits in the prevention and/or treatment of metastatic breast cancer. This study was supported in part by funding from the Louisiana Cancer Foundation. Citation Format: Paul William Sylvester, Jessie Grazier, Mohammed R. Anwar. γ-Tocotrienol inhibition of metastatic phenotypic behavior is associated with a decrease in galectin-3 expression and distribution in the highly malignant mouse +SA mammary tumor cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5015.

Galectin-3 enhances neutrophil motility and extravasation into the airways during Aspergillus fumigatusinfection.

Aspergillus fumigatus is an opportunistic mold that infects patients who are immunocompromised or have chronic lung disease, causing significant morbidity and mortality in these populations. While the factors governing the host response to A. fumigatus remain poorly defined, neutrophil recruitment to the site of infection is critical to clear the fungus. Galectin-3 is a mammalian β-galactose-binding lectin with both antimicrobial and immunomodulatory activities, however the role of galectin-3 in the defense against molds has not been studied. Here we show that galectin-3 expression is markedly up-regulated in mice and humans with pulmonary aspergillosis. Galectin-3 deficient mice displayed increased fungal burden and higher mortality during pulmonary infection. In contrast to previous reports with pathogenic yeast, galectin-3 exhibited no antifungal activity against A. fumigatus in vitro. Galectin-3 deficient mice exhibited fewer neutrophils in their airways during infection, despite normal numbers of total lung neutrophils. Intravital imaging studies confirmed that galectin-3 was required for normal neutrophil migration to the airspaces during fungal infection. Adoptive transfer experiments demonstrated that stromal rather than neutrophil-intrinsic galectin-3 was necessary for normal neutrophil entry into the airspaces. Live cell imaging studies revealed that extracellular galectin-3 directly increases neutrophil motility. Taken together, these data demonstrate that extracellular galectin-3 facilitates recruitment of neutrophils to the site of A. fumigatus infection, and reveals a novel role for galectin-3 in host defense against fungal infections.

The Examination of Viral Characteristics of HIV-1 CRF07_BC and Its Potential Interaction with Extracellular Galectin-3.

HIV-1 CRF07_BC is a B’ and C subtype recombinant emerging virus and many of its viral characteristics remain unclear. Galectin-3 (Gal3) is a β-galactose binding lectin that has been reported as a pattern recognition receptor (PRR) and is known to mediate adhesion between cells and microbes. This study aims to examine the viral characteristics of HIV-1 CRF07_BC virus and the role of extracellular galectin-3 in HIV-1 CRF07_BC infection. A total of 28 HIV-1+ injecting drug users (IDUs) were recruited and 24 (85.7%) were identified as HIV-1 CRF07_BC. Results indicate that significant higher serum galectin-3 was measured in CRF07_BC infected patients and CRF07_BC infection triggered significant galectin-3 expression (p < 0.01). Viral characteristics demonstrate that CRF07_BC virions display a higher level of envelope gp120 spikes. The virus infectivity assay demonstrated that co-treatment with galectin-3 significantly promoted CRF07_BC attachment and internalization (p < 0.01). A co-immunoprecipitation assay showed that pulldown galectin-3 co-precipitated both CD4 and gp120 proteins. Results from an enzyme-linked immunosorbent assay (ELISA) indicate that the galectin-3 promoting effect occurs through enhancement of the interaction between gp120 and CD4. This study suggests that CRF07_BC was predominant in HIV-1+ IDUs and CRF07_BC utilized extracellular galectin-3 to enhance its infectivity via stabilization of the gp120-CD4 interaction.

Non-classical role of Galectin-3 in cancer progression: translocation to nucleus by carbohydrate-recognition independent manner

Galectin-3 is a carbohydrate-binding protein and regulates diverse functions, including cell proliferation and differentiation, mRNA splicing, apoptosis induction, immune surveillance and inflammation, cell adhesion, angiogenesis, and cancer-cell metastasis. Galectin-3 is also recommended as a diagnostic or prognostic biomarker of various diseases, including heart disease, kidney disease, and cancer. Galectin-3 exists as a cytosol, is secreted in extracellular spaces on cells, and is also detected in nuclei. It has been found that galectin-3 has different functions in cellular localization: (i) Extracellular galectin-3 mediates cell attachment and detachment. (ii) cytosolic galectin-3 regulates cell survival by blocking the intrinsic apoptotic pathway, and (iii) nuclear galectin-3 supports the ability of the transcriptional factor for target gene expression. In this review, we focused on the role of galectin-3 on translocation from cytosol to nucleus, because it happens in a way independent of carbohydrate recognition and accelerates cancer progression. We also suggested here that intracellular galecin-3 could be a potent therapeutic target in cancer therapy.

Elevated plasma galectin-3 levels and their correlation with disease activity in adult-onset Still's disease.

With galectin-3 playing an important role in inflammatory responses, elevated galectin-3 levels have been shown in patients with autoimmune diseases. However, there are limited data regarding galectin-3 expression in patients with autoinflammatory diseases such as adult-onset Still's disease (AOSD). This study aimed to investigate the extracellular galectin-3 expression and examine its association with activity parameters and disease outcome in AOSD patients. Plasma levels of galectin-3 and inflammasome downstream cytokines including interleukin (IL)-1β and IL-18 were determined by ELISA in 42 active AOSD patients and 20 healthy controls (HC). The protein levels of galectin-3 and cytokines were determined using immunoblotting. Plasma levels of galectin-3 and inflammasome downstream cytokines including IL-1β and IL-18 were significantly higher in AOSD patients (median 5.02ng/ml, interquartile range [IQR] 3.12-7.88ng/ml; 3.42pg/ml, IQR 1.48-6.70pg/ml; and 5758pg/ml, IQR 859-11,895pg/ml, respectively) compared with HC (1.86ng/ml, IQR 1.09-2.89ng/ml; 0.99pg/ml, IQR 0.62-1.35pg/ml; and 129pg/ml, IQR 71-155pg/ml, respectively, all p < 0.001). Plasma galectin-3 levels were positively correlated with clinical activity scores, inflammatory parameters values, and the levels of IL-1β and IL-18 in AOSD patients. AOSD patients with systemic pattern had significantly higher galectin-3 levels (median 6.08ng/ml, IQR 4.01-9.54ng/ml) compared with those with chronic articular pattern (3.56ng/ml, IQR 3.04-4.98ng/ml, p < 0.05). After 6-month therapy, galectin-3 levels significantly declined, paralleling the decreases in clinical activity scores and plasma levels of IL-1β and IL-18. Elevated galectin-3 levels and their positive correlation with disease activity scores, inflammatory parameter, and inflammasome downstream cytokines suggest the involvement of galectin-3 in AOSD pathogenesis.Key Points• We revealed for the first time the association of plasma galectin-3 levels with AOSD activity parameters.• We explored the link between galectin-3 levels and NLRP3-inflammasome downstream cytokines in AOSD disease.

Blocking extracellular Galectin-3 in patients with osteoarthritis

ObjectiveThis pilot clinical trial examined the efficacy of blocking extracellular Galectin-3 (Gal-3) with modified citrus pectin (MCP), in patients suffering from knee osteoarthritis (OA). Methods50 patients were randomized in a 1:1 ratio to receive MCP or placebo at a dose of 4 g (5 capsules) twice daily for 12 weeks. Serum Gal-3 levels and OA severity were evaluated at baseline and 12 weeks. Gal-3 levels were detected by sandwich ELISA and OA severity was determined using WOMAC-knee, SF-36, and RAPID3 surveys during these visits. MCP tolerability was assessed by a basic metabolic panel during a week 6 follow up visit. ResultsPatients enrolled in both the MCP treatment and placebo groups shared similar baseline characteristics in OA severity, serum Gal-3 levels, and pain management. Improvement across all surveys was noted independent of supplement or placebo treatment. No significant change in Gal-3 levels were observed in either cohort over the 12-week study. ConclusionTreatment of knee OA with a 12-week course of MCP did not significantly improve disease burden compared to placebo.

Calreticulin and Galectin-3 Opsonise Bacteria for Phagocytosis by Microglia.

Opsonins are soluble, extracellular proteins, released by activated immune cells, and when bound to a target cell, can induce phagocytes to phagocytose the target cell. There are three known classes of opsonin: antibodies, complement factors and secreted pattern recognition receptors, but these have limited access to the brain. We identify here two novel opsonins of bacteria, calreticulin, and galectin-3 (both lectins that can bind lipopolysaccharide), which were released by microglia (brain-resident macrophages) when activated by bacterial lipopolysaccharide. Calreticulin and galectin-3 both bound to Escherichia coli, and when bound increased phagocytosis of these bacteria by microglia. Furthermore, lipopolysaccharide-induced microglial phagocytosis of E. coli bacteria was partially inhibited by: sugars, an anti-calreticulin antibody, a blocker of the calreticulin phagocytic receptor LRP1, a blocker of the galectin-3 phagocytic receptor MerTK, or simply removing factors released from the microglia, indicating this phagocytosis is dependent on extracellular calreticulin and galectin-3. Thus, calreticulin and galectin-3 are opsonins, released by activated microglia to promote clearance of bacteria. This innate immune response of microglia may help clear bacterial infections of the brain.

Extracellular and intracellular small-molecule galectin-3 inhibitors

Galectin-3 is a carbohydrate binding protein which has important roles in cancer and immunity. Potent galectin-3 inhibitors have been synthesized, for experimental purposes and potential clinical use. As galectin-3 is implicated in both intra- and extracellular activities, permeability of galectin-3 inhibitors is an important parameter determining biological effects. We compared the cellular uptake of galectin-3 inhibitors and their potency in the intracellular or extracellular space. The inhibitors differed in their polar surface area (PSA), but had similar affinities for galectin-3. Using a well-established permeability assay, we confirmed that the uptake was significantly higher for the inhibitor with the lowest PSA, as expected. To analyze intracellular activity of the inhibitors, we developed a novel assay based on galectin-3 accumulation around damaged intracellular vesicles. The results show striking differences between the inhibitors intracellular potency, correlating with their PSAs. To test extracellular activity of the inhibitors, we analyzed their potency to block binding of galectin-3 to cell surfaces. All inhibitors were equally able to block galectin-3 binding to cells and this was proportional to their affinity for galectin-3. These inhibitors may serve as useful tools in exploring biological roles of galectin-3 and may further our understanding of intracellular versus extracellular roles of galectin-3.

Galectin-3 Activation and Inhibition in Heart Failure and Cardiovascular Disease: An Update.

Galectin-3 is a versatile protein orchestrating several physiological and pathophysiological processes in the human body. In the last decade, considerable interest in galectin-3 has emerged because of its potential role as a biotarget. Galectin-3 is differentially expressed depending on the tissue type, however its expression can be induced under conditions of tissue injury or stress. Galectin-3 overexpression and secretion is associated with several diseases and is extensively studied in the context of fibrosis, heart failure, atherosclerosis and diabetes mellitus. Monomeric (extracellular) galectin-3 usually undergoes further “activation” which significantly broadens the spectrum of biological activity mainly by modifying its carbohydrate-binding properties. Self-interactions of this protein appear to play a crucial role in regulating the extracellular activities of this protein, however there is limited and controversial data on the mechanisms involved. We therefore summarize (recent) literature in this area and describe galectin-3 from a binding perspective providing novel insights into mechanisms by which galectin-3 is known to be “activated” and how such activation may be regulated in pathophysiological scenarios.

Galectin-3 captures interferon-gamma in the tumor matrix reducing chemokine gradient production and T-cell tumor infiltration

The presence of T cells in tumors predicts overall survival for cancer patients. However, why most tumors are poorly infiltrated by T cells is barely understood. T-cell recruitment towards the tumor requires a chemokine gradient of the critical IFNγ-induced chemokines CXCL9/10/11. Here, we describe how tumors can abolish IFNγ-induced chemokines, thereby reducing T-cell attraction. This mechanism requires extracellular galectin-3, a lectin secreted by tumors. Galectins bind the glycans of glycoproteins and form lattices by oligomerization. We demonstrate that galectin-3 binds the glycans of the extracellular matrix and those decorating IFNγ. In mice bearing human tumors, galectin-3 reduces IFNγ diffusion through the tumor matrix. Galectin antagonists increase intratumoral IFNγ diffusion, CXCL9 gradient and tumor recruitment of adoptively transferred human CD8+ T cells specific for a tumor antigen. Transfer of T cells reduces tumor growth only if galectin antagonists are injected. Considering that most human cytokines are glycosylated, galectin secretion could be a general strategy for tumor immune evasion.

Galectin-3 as a novel regulator of osteoblast-osteoclast interaction and bone homeostasis

Bone tissue undergoes permanent and lifelong remodeling with a concerted action of bone-building osteoblasts and bone-resorbing osteoclasts. A precise cooperation between those two cell types is critical in the complex process of bone renewal. Galectin-3 is a member of the β-galactoside-binding lectin family playing multiple roles in cell growth, differentiation and aggregation. As it has been described to be expressed in bone, galectin-3 might influence bone homeostasis by regulating the function and/or interplay of osteoblasts and osteoclasts. Here, we investigated the role of galectin-3 in osteoclastogenesis and osteoblast-osteoclast interactions. Bone histomorphometric analysis and μCT measurements revealed a decreased trabecular bone volume and an increased osteoclast number in 12weeks old male galectin-3 knockout mice compared to wildtype littermates. Galectin-3 deficient bone marrow cells displayed a higher osteoclastogenic capacity in ex vivo differentiation assays, associated with elevated TRAF6 mRNA levels, suggesting an intrinsic inhibition of osteoclastogenesis by galectin-3 interfering with RANKL-mediated signaling. Furthermore, the addition of extracellular galectin-3 to murine or human osteoclastogenesis assays inhibited osteoclast formation and osteoclast numbers were higher in co-culture assays with galectin-3 deficient osteoblasts. In conclusion, our data suggest the secretion of galectin-3 as a novel mechanism for osteoblasts to control osteoclastogenesis and to maintain trabecular bone homeostasis independently of the RANKL/OPG-axis.

Extracellular galectin-3 counteracts adhesion and exhibits chemoattraction in Helicobacter pylori-infected gastric cancer cells.

Galectin-3 (Gal-3) is a β-galactoside lectin that is upregulated and rapidly secreted by gastric epithelial cells in response to Helicobacter pylori infection. An earlier study reported the involvement of H. pylori cytotoxin-associated gene A (cagA) in the expression of intracellular Gal-3. However, the role of extracellular Gal-3 and its functional significance in H. pylori-infected cells remains uncharacterized. Data presented here demonstrate secretion of Gal-3 is an initial host response event in gastric epithelial cells during H. pylori infection and is independent of CagA. Previously, Gal-3 was shown to bind to H. pylori LPS. The present study elaborates the significance of this binding, as extracellular recombinant Gal-3 (rGal-3) was shown to inhibit the adhesion of H. pylori to the gastric epithelial cells. Interestingly, a decrease in H. pylori adhesion to host cells also resulted in a decrease in apoptosis. Furthermore, the study also demonstrated a chemoattractant role of extracellular rGal-3 in the recruitment of THP-1 monocytes. This study outlines the previously unidentified roles of extracellular Gal-3 where it acts as a negative regulator of H. pylori adhesion and apoptosis in gastric epithelial cells, and as a chemoattractant to THP-1 monocytes. Our findings could contribute to the better understanding of how Gal-3 acts as a modulator under H. pylori-induced pathological conditions.