Ichiro Kuwabara

Human Galectin-3 Is a Novel Chemoattractant for Monocytes and Macrophages

Galectin-3 is a β-galactoside-binding protein implicated in diverse biological processes. We found that galectin-3 induced human monocyte migration in vitro in a dose-dependent manner, and it was chemotactic at high concentrations (1.0 μM) but chemokinetic at low concentrations (10–100 nM). Galectin-3-induced monocyte migration was inhibited by its specific mAb and was blocked by lactose and a C-terminal domain fragment of the protein, indicating that both the N-terminal and C-terminal domains of galectin-3 are involved in this activity. Pertussis toxin (PTX) almost completely blocked monocyte migration induced by high concentrations of galectin-3. Galectin-3 caused a Ca2+ influx in monocytes at high, but not low, concentrations, and both lactose and PTX inhibited this response. There was no cross-desensitization between galectin-3 and any of the monocyte-reactive chemokines examined, including monocyte chemotactic protein-1, macrophage inflammatory protein-1α, and stromal cell-derived factor-1α. Cultured human macrophages and alveolar macrophages also migrated toward galectin-3, but not monocyte chemotactic protein-1. Finally, galectin-3 was found to cause monocyte accumulation in vivo in mouse air pouches. These results indicate that galectin-3 is a novel chemoattractant for monocytes and macrophages and suggest that the effect is mediated at least in part through a PTX-sensitive (G protein-coupled) pathway.

Critical role of galectin-3 in phagocytosis by macrophages

Galectin-3 is a member of a large family of animal lectins. This protein is expressed abundantly by macrophages, but its function in this cell type is not well understood. We have studied the effect of galectin-3 gene targeting on phagocytosis, a major function of macrophages. Compared with wild-type macrophages, galectin-3-deficient (gal3-/-) cells exhibited reduced phagocytosis of IgG-opsonized erythrocytes and apoptotic thymocytes in vitro. In addition, gal3-/- mice showed attenuated phagocytic clearance of apoptotic thymocytes by peritoneal macrophages in vivo. These mice also exhibited reduced IgG-mediated phagocytosis of erythrocytes by Kupffer cells in a murine model of autoimmune hemolytic anemia. Additional experiments indicate that extracellular galectin-3 does not contribute appreciably to the phagocytosis-promoting function of this protein. Confocal microscopic analysis of macrophages containing phagocytosed erythrocytes revealed localization of galectin-3 in phagocytic cups and phagosomes. Furthermore, gal3-/- macrophages exhibited a lower degree of actin rearrangement upon Fcgamma receptor crosslinkage. These results indicate that galectin-3 contributes to macrophage phagocytosis through an intracellular mechanism. Thus, galectin-3 may play an important role in both innate and adaptive immunity by contributing to phagocytic clearance of microorganisms and apoptotic cells.

Wedgelike glycodendrimers as inhibitors of binding of mammalian galectins to glycoproteins, lactose maxiclusters, and cell surface glycoconjugates.

Galectins are mammalian carbohydrate‐binding proteins that are involved in cell–cell and cell–matrix adhesion, cell migration, and growth regulation with relevance to inflammation and tumor spread. These important functions account for the interest to design suitable low molecular weight inhibitors that match the distinct modes of presentation of the carbohydrate recognition domains of the different galectin subfamilies. Using 3,5‐di‐(2‐aminoethoxy)benzoic acid as the branching unit, wedgelike glycodendrimers with two, four, and eight lactose moieties (G1–G3) were synthesized. They were tested in solid‐phase competition assays with lactose maxiclusters and various N‐glycan branching profiles (miniclusters) as the matrix and also in cell assays. Prototype galectins‐1 and ‐7, chimera‐type galectin‐3, a plant (AB)2 toxin, and a lactose‐binding immunoglobulin G fraction from human serum were the carbohydrate‐binding targets. Potent inhibition and remarkable cluster effects were seen for the homodimeric galectin‐1, especially in combination with biantennary N‐glycans as the matrix. Remarkably, for the tetravalent G2 glycodendrimer, the inhibitory potency of each lactose unit reached a maximum value of 1667 relative to free lactose. In haemagglutination experiments as a model for cell adhesion, galectin‐3 was markedly sensitive to increased sugar valency and a relative potency per lactose of 150 was reached. The spatial orientation of the carbohydrate recognition domains of the endogenous lectins and the branching pattern of the carbohydrates of the glycoprotein matrices used are both important factors in the design and synthesis of glycodendrimers with galectin‐selective properties.

Galectins-3 and -7, but not Galectin-1, Play a Role in Re-epithelialization of Wounds

Disorders of wound healing characterized by impaired or delayed re-epithelialization are a serious medical problem. These conditions affect many tissues, are painful, and are difficult to treat. In this study, using cornea as a model, we demonstrate for the first time the importance of carbohydrate-binding proteins galectins-3 and -7 in re-epithelialization of wounds. In two different models of corneal wound healing, re-epithelialization of wounds was significantly slower in galectin-3-deficient (gal3−/−) mice compared with wild-type (gal3+/+) mice. In contrast, there was no difference in corneal epithelial wound closure rates between galectin-1-deficient and wild-type mice. Quantitation of the bromodeoxyuridine-labeled cells in gal3+/+ and gal3−/− corneas revealed that corneal epithelial cell proliferation rate is not perturbed in gal3−/− corneas. Exogenous galectin-3 accelerated re-epithelialization of wounds in gal3+/+ mice but, surprisingly, not in the gal3−/− mice. Gene expression analysis using cDNA microarrays revealed that healing corneas of gal3−/− mice contain markedly reduced levels of galectin-7 compared with those of gal3+/+ mice. More importantly, unlike galectin-3, galectin-7 accelerated re-epithelialization of wounds in both gal3−/− and gal3+/+ mice. In corresponding experiments, recombinant galectin-1 did not stimulate the corneal epithelial wound closure rate. The extent of acceleration of re-epithelialization of wounds with both galectin-3 and galectin-7 was greater than that observed in most of the published studies using growth factors. These findings have broad implications for developing novel therapeutic strategies for treating nonhealing wounds.

Homodimeric galectin-7 (p53-induced gene 1) is a negative growth regulator for human neuroblastoma cells

The extracellular functions of galectin-7 (p53-induced gene 1) are largely unknown. On the surface of neuroblastoma cells (SK-N-MC), the increased GM1 density, a result of upregulated ganglioside sialidase activity, is a key factor for the switch from proliferation to differentiation. We show by solid-phase and cell assays that the sugar chain of this ganglioside is a ligand for galectin-7. In serum-supplemented proliferation assays, galectin-7 reduced neuroblastoma cell growth without the appearance of features characteristic for classical apoptosis. The presence of galectin-3 blocked this effect, which mechanistically resembles that of galectin-1. By virtue of carbohydrate binding, galectin-7 thus exerts neuroblastoma growth control similar to galectin-1 despite their structural differences. In addition to p53-linked proapoptotic activity intracellularly, galectin-7, acting as a lectin on the cell surface, appears to be capable of reducing cancer cell proliferation in susceptible systems.

An Anti-Apoptotic Role for Galectin-3 in Diffuse Large B-Cell Lymphomas

Increased resistance to apoptosis promotes lymphomagenesis with aberrant expression of cell survival proteins such as BCL-2 and c-MYC occurring in distinct lymphoma subtypes. Galectin-3 is an anti-apoptotic protein that protects T cells, macrophages, and breast carcinoma cells from death triggered by a variety of agents. We have found high levels of galectin-3 protein expression in a subset of B-cell neoplasms including diffuse large B-cell lymphoma (DLBCL), primary effusion lymphoma (PEL), and multiple myeloma (MM), in both cell lines and patient samples. However, we failed to detect galectin-3 in Burkitt lymphoma (BL), follicular lymphoma (FL), marginal zone lymphoma (MZL), MALT lymphoma or B-small lymphocytic lymphoma (B-SLL) cell lines or patient samples. To determine whether galectin-3 expression protects B cells from apoptosis, galectin-3-negative BL cells were transfected with a galectin-3 expressing plasmid, which resulted in markedly increased resistance to anti-Fas-induced cell death. In contrast, galectin-3-positive PEL cells transfected with an amino-terminal truncated galectin-3 vector showed increased sensitivity to anti-Fas induced apoptosis. During normal B-cell development, galectin-3 expression was lowest in germinal center and plasma B cells, from which DLBCL, PEL, and MM derive, and highest in long-lived naïve and memory B cells. This pattern of expression suggests that aberrantly increased galectin-3 levels in specific B-cell populations may yield a protective advantage during transformation and/or progression of certain B-cell neoplasms.

Glycoprotein 90K/MAC‐2BP interacts with galectin‐1 and mediates galectin‐1–induced cell aggregation

The glycoprotein 90K was originally described as a tumor‐secreted antigen and subsequently found to have immunostimulatory activity as well as other possible functions. This protein interacts with an endogenous lectin, galectin‐3, and may play a role in tumor metastasis through this interaction. Because 90K is heavily glycosylated, it may also interact with other members of the galectin family, which would contribute to the multifunctionality of 90K. To test this possibility, we studied the recognition of 90K by galectin‐1, which, like galectin‐3, has been associated with neoplastic transformation. In a solid‐phase binding assay, human recombinant galectin‐1 bound immobilized human recombinant 90K in a fashion that was inhibitable by lactose. Galectins 1 and 3 appeared to bind to separate sites on 90K because they did not affect the binding of each other. The dissociation constant of galectin‐1 to 90K was on the order of 10−7 M. Galectin‐1 also induced aggregation of a human melanoma cell line, A375, in a carbohydrate‐dependent manner, and this appeared to be mediated, at least in part, by 90K expressed on A375 cells, since it was inhibitable by a specific anti‐90K monoclonal antibody. We conclude that 90K interacts with both galectin‐1 and galectin‐3 and both interactions contribute to the formation of multicell aggregates. Because both of these galectins as well as 90K are often over‐expressed in neoplasm, these interactions may occur in the setting of various carcinomas and contribute to their progression and metastasis.

Hepatocyte Growth Factor Suppresses Profibrogenic Signal Transduction via Nuclear Export of Smad3 With Galectin-7

BACKGROUND & AIMS Hepatocyte growth factor (HGF) and transforming growth factor-beta (TGF-beta) regulate diversified cellular functions and often act antagonistically against each other. For example, TGF-beta is the most potent factor accelerating liver fibrosis, whereas HGF treatment prevents its progression. Here, we propose a novel molecular mechanism by which HGF counter represses TGF-beta-stimulated profibrogenic signal transduction. METHODS Effects of HGF on TGF-beta-responsive gene transcription of type I collagen, the major matrix component of fibrotic liver, were examined by using cultured hepatic stellate cells (HSC) and transgenic mice harboring alpha2(I) collagen gene (COL1A2) promoter. Expression and subcellular localization of Smad3 were determined by Western blot analyses and immunofluorescence staining, respectively. A mass spectrometric analysis was employed to identify immunoprecipitated proteins with antiphospho-Smad2/3 antibodies. RESULTS Over expression of HGF inhibited COL1A2 transcription in cultured HSC and suppressed activation of COL1A2 promoter in liver tissue induced by carbon tetrachloride administration. A mass spectrometric analysis identified galectin-7 as one of the immunoprecipitated proteins with antiphospho-Smad2/3 antibodies following HGF treatment. HGF accelerated nuclear export of Smad3 by enhancing its interaction with galectin-7. Transfection of cells with galectin-7 small interfering RNA inhibited nuclear export of Smad3 and abolished suppressive effect of HGF on expression of TGF-beta-responsive genes such as COL1A2 and plasminogen activator inhibitor-1. On the other hand, over expression of galectin-7 suppressed TGF-beta-stimulated expression of those target genes. CONCLUSIONS These results reveal a novel function of intracellular galectin-7 as a transcriptional regulator via its interaction with Smad3 and provide a molecular basis for the antifibrotic effect of HGF.

Suppression of Tumor Growth by Galectin-7 Gene Transfer

Galectin-7 is a β-galactoside-binding animal lectin specifically expressed in stratified epithelia. Its expression is inducible by p53 and is down-regulated in squamous cell carcinomas. Other investigators previously showed that galectin-7 is a proapoptotic protein, and we showed that ectopic expression of galectin-7 in HeLa cells renders the cells more sensitive to a variety of apoptotic stimuli. In the present study, we showed that ectopic expression of galectin-7 in the human colon carcinoma cell line DLD-1 also made the cells more sensitive to apoptosis under various conditions. We also found that galectin-7-transfected DLD-1 (DLD-1-Gal7) cells grew significantly more slowly than control transfectants (DLD-1-V) under normal culture conditions in the absence of apoptosis. Moreover, a significantly lower number of colonies were formed from DLD-1-Gal7 cells than from DLD-1-V cells under anchorage-independent cell growth conditions. Most importantly, tumor formation from DLD-1-Gal7 cells was dramatically reduced compared with DLD-1-V cells when these cells were inoculated s.c. into severe combined immunodeficient mice. DLD-1-Gal7 tumors showed a significantly lower proliferation rate than DLD-1-V tumors as determined by in vivo 5-bromo-2′-deoxyuridine incorporation. DLD-1-Gal7 tumors also contained a lower density of blood vessels than DLD-1-V tumors, suggesting that ectopic expression of galectin-7 suppresses angiogenesis. This may partially account for the greater suppressive effect of galectin-7 on tumor growth in vivo than in vitro. Our results show that galectin-7 has a suppressive effect on tumor growth, suggesting that galectin-7 gene transfer or other means of specifically inducing galectin-7 expression may be a new approach for management of cancers.

Changes in Galectin-7 and Cytokeratin-19 Expression during the Progression of Malignancy in Thyroid Tumors: Diagnostic and Biological Implications

Galectin-7 is associated with p53-dependent onset of apoptosis and proliferation control/differentiation in keratinocyte development. It is also up-regulated in chemically induced rat mammary carcinogenesis. Because the levels of expression of galectin-7 have never been investigated in thyroid tumors (in contrast to those of galectin-1 and -3 associated with malignancy), we initiated analysis of the expression of galectin-7 in benign and malignant thyroid lesions together with that of cytokeratin-19 (CK19), a marker already demonstrated to be useful in diagnosing this kind of lesion. The immunohistochemical expression levels were quantitatively determined by means of computer-assisted microscopy on a series of 84 thyroid lesions including 10 multinodular goiters, 32 adenomas, and 42 carcinomas. Our data clearly indicate a marked down-regulation of galectin-7 expression in a large proportion of adenomas (including the normomacrofollicular, microfollicular, and trabecular variants) if compared with carcinomas. In accordance with results of previous studies, a marked up-regulation of CK19 expression was observed in the thyroid carcinomas, and this contrasted in particular with the low CK19 expression observed in the microfollicular adenomas. Of importance for diagnostic implications, the combination of these two markers enabled our series of microfollicular adenomas (characterized by low galectin-7 and CK19 expression) to be efficiently distinguished from the encapsulated follicular variant of papillary thyroid carcinomas (high galectin-7 and CK19 expression).