The involvement of galectin-1 in skeletal muscle determination, differentiation and regeneration

The dogma that a cell is rigidly committed to one tissue type has been heavily challenged over the past few years with numerous reports of transdifferentiation of cells between different lineages. Cells capable of entering lineages other than that of their tissue of origin have been identified in several diverse tissues. Recently we have focussed on a non-committed myogenic cell within the dermis that is capable, under certain conditions, of expressing muscle specific markers and even fusing to the terminally differentiated stage of muscle cell development. We have identified galectin-1 as being a potent factor implicated in this process. In this review we discuss our findings and consider the involvement of galectin-1 in muscle determination, differentiation and regeneration. Published in 2004.     

Activation of the neutrophil nicotinamide adenine dinucleotide phosphate oxidase by galectin-1

Galectins are a group of lactose-binding proteins widely distributed in nature. Twelve mammalian galectins have so far been identified, but their functions are to a large extent unknown. In this work we study galectin-1 in its interaction with human neutrophils, with regard to both cell surface binding and activation of the superoxide-producing NADPH-oxidase. We show that galectin-1 is able to activate the neutrophil NADPH-oxidase, provided that the cells have been primed by extravasation from the blood into the tissue, an activation pattern that is similar to that of galectin-3. Using in vitro priming protocols, the galectin-1 responsiveness was found to correlate to granule mobilization and galectin-1 binding to the cells, suggesting the presence of granule-localized receptors that are up-regulated to the cell surface upon priming. By galectin-1 overlay of fractionated neutrophils we identified potential galectin-1 receptor candidates localized in the membranes of the secretory vesicle and gelatinase granules. The binding of galectin-1 and galectin-3 to neutrophil proteins was compared, as were the dose dependencies for activation by the two lectins. The results suggest that, although similarities are found between the two galectins, they appear to activate the NADPH-oxidase using different receptors. In conclusion, galectin-1 appears to have proinflammatory functions, mediated through activation of the neutrophil respiratory burst.     

Galectin-3 Stimulates Cell Proliferation

Galectin-3, a β-galactoside-binding protein, has been shown to be involved in multiple biological processes through interaction with its complementary glycoconjugates. Here we provide the first evidence of galectin-3 as a mitogen. Incubation of quiescent cultures of normal human lung fibroblast IMR-90 cells with recombinant galectin-3 (rgalectin-3) stimulated DNA synthesis as well as cell proliferation in a dose-dependent manner. This mitogenic activity was dependent on the lectin property of galectin-3, as it could be significantly inhibited by lactose, a disaccharide competitive for carbohydrate-binding by galectin-3. Chemical cross-linking and affinity-purification experiments identified binding of rgalectin-3 to cell surface glycoproteins, which were not recognized by antibodies directed against lysosome-associated membrane proteins (LAMPs), putative cellular ligands for galectin-3. Moreover, pulse–chase analysis revealed no secretion of galectin-3 by IMR-90 cells. These results indicate that galectin-3 is a mitogen capable of stimulating fibroblast cell proliferation in a paracrine fashion through interaction with cell surface glycoconjugates different from LAMPs and suggest a possible involvement of galectin-3 in tissue remodeling.     

Selective striatal connections of midbrain dopaminergic nuclei in the chick (Gallus domesticus)

Histochemical monitoring of developmental processes is presently centered on protein-protein interactions. However, oligosaccharides have the potential to store and transmit biological information. Carbohydrate chains of cellular glycoconjugates present determinants for binding of endogenous lectins. This interaction can be relevant for developmental processes. In fact, beta-galactosides and their derivatives serve as ligands for members of the lectin family of galectins. Since it is unclear to what extent functions of different galectins differ or overlap, hereby introducing redundancy into this system, monitoring of galectin presence during tissue maturation should include more than one type of galectin (galectin fingerprinting). Here, we focus on the two most frequently described ones, namely the homodimeric prototype galectin-1 and the chimera-type galectin-3, the latter one so far not characterized from bovine tissue. In the first step, we have detected its presence biochemically in addition to the abundant galectin-1 in bovine respiratory and digestive tracts during development. Evidently, diversification of the primitive foregut will not lead to an alteration of this property. Immunohistochemistry revealed clear differences in the galectins' localization profiles. Galectin-1 expression is strong in mesenchymal cells, especially smooth muscle cells, while epithelial lining harbors galectin-3. A gradual increase in staining intensity with development is especially observed in the case of galectin-3. Notably, this change is accompanied by a shift from primarily nuclear localization to the cytoplasm, an alteration not seen for galectin-1. However, nuclear presence of galectin-1 is encountered. Thus, the delineation of differences in expression of galectin-1 and -3 with respect to cell types and in the developmental course of subcellular localization argues in favor of mediation of nonoverlapping functions by these two homologous, endogenous lectins.     

Galectin-1 expression in innervated and denervated skeletal muscle

Galectin-1 is a soluble carbohydrate-binding protein with a particularly high expression in skeletal muscle. Galectin-1 has been implicated in skeletal muscle development and in adult muscle regeneration, but also in the degeneration of neuronal processes and/or in peripheral nerve regeneration. Exogenously supplied oxidized galectin-1, which lacks carbohydrate-binding properties, has been shown to promote neurite outgrowth after sciatic nerve sectioning. In this study, we compared the expression of galectin-1 mRNA and immunoreactivity in innervated and denervated mouse and rat hind-limb and hemidiaphragm muscles. The results show that galectin-1 mRNA expression and immunoreactivity are up-regulated following denervation. The galectin-1 mRNA is expressed in the extrasynaptic and perisynaptic regions of the muscle, and its immunoreactivity can be detected in both regions by Western blot analysis. The results are compatible with a role for galectin-1 in facilitating reinnervation of denervated skeletal muscle.     

The Human Endometrium Expresses the Glycoprotein Mucin-1 and Shows Positive Correlation for Thomsen-Friedenreich Epitope Expression and Galectin-1 Binding

Mucin 1 (MUC1) is a glycoprotein in human endometrium and is abundant at the luminal epithelial surface in the receptive phase. It has a highly glycosylated ecto-domain that contains keratan sulfate chains, that disappears at the time of implantation. In addition, the glycoforms on MUC1 differ in fertile and infertile women. Therefore the aims of this study were investigations on glycosylation of MUC1 with the Thomsen-Friedenreich (TF) epitope on normal human endometrium throughout the menstrual cycle and binding of galectin-1 on the TF epitope in the endometrium and the expression of galectin-1 on the human oocyte. Human endometrial tissue was obtained from 54 premenopausal patients and was immunohistochemically analyzed with monoclonal antibodies against MUC1, TF epitope, galectin-1, and biotinylated galectin-1. In addition, human oocytes were analyzed for TF, galectin-1 expression, and galectin-1 binding. We identified a significant upregulation of MUC1 and TF epitope and, in addition, galectin-1 binding in glandular epithelium and epithelial apical surface tissue from proliferative to secretory phase. With double staining experiments, we identified a coexpression of TF and MUC1 in the early secretory phase and galectin-1 binding to TF during the same period of time. In addition we identified TF epitope and galectin-1 expression plus binding on the human oocyte and irregularly fertilized oocytes. Upregulation of TF epitope on the glandular epithelium and epithelial apical surface tissue in the secretory phase and binding of galectin-1 at the same time show the possibility of galectin-1–mediated trophectoderm binding to the endometrium within the window of implantation. (J Histochem Cytochem 57:871–881, 2009)     

Muscle-derived stem cells isolated as non-adherent population give rise to cardiac, skeletal muscle and neural lineages

Stem cells with the ability to differentiate in specialized cell types can be extracted from a wide array of adult tissues including skeletal muscle. Here we have analyzed a population of cells isolated from skeletal muscle on the basis of their poor adherence on uncoated or collagen-coated dishes that show multi-lineage differentiation in vitro. When analysed under proliferative conditions, these cells express stem cell surface markers Sca-1 (65%) and Bcrp-1 (80%) but also MyoD (15%), Neuronal beta III-tubulin (25%), GFAP (30%) or Nkx2.5 (1%). Although capable of growing as non-attached spheres for months, when given an appropriate matrix, these cells adhere giving rise to skeletal muscle, neuronal and cardiac muscle cell lineages. A similar cell population could not be isolated from either bone marrow or cardiac tissue suggesting their specificity to skeletal muscle. When injected into damaged muscle, these non-adherent muscle-derived cells are retrieved expressing Pax7, in a sublaminar position characterizing satellite cells and participate in forming new myofibers. These data show that a non-adherent stem cell population can be specifically isolated and expanded from skeletal muscle and upon attachment to a matrix spontaneously differentiate into muscle, cardiac and neuronal lineages in vitro. Although competing with resident satellite cells, these cells are shown to significantly contribute to repair of injured muscle in vivo supporting that a similar muscle-derived non-adherent cell population from human muscle may be useful in treatment of neuromuscular disorders.     

Expression of galectin-1 and galectin-3 in human fetal thyroid gland.

High levels of expression of galectin-1 and galectin-3, the beta-galactoside-binding proteins, have been recently described in malignant thyroid tumors but not in adenomas nor in normal thyroid tissue. However, there are no data about the expression of these galectins during fetal thyroid development. In this study we analyzed immunohistochemically the presence of galectin-1 and galectin-3 in human fetal thyroid glands (16-37 weeks of gestation). Weak to moderate cytoplasmic staining for galectin-1 was observed in follicular cells of all fetal thyroids. Galectin-3 could not be detected in thyroid follicular cells of any fetal thyroid investigated. Both galectins were detected in stromal tissue, but staining for galectin-1 was more intense. The absence of galectin-3 in thyroid cells during fetal development suggests that galectin-3 is expressed de novo during malignant transformation of thyroid epithelium, and that galectin-1 could be considered an oncofetal antigen. The results obtained indicated potential roles for galectin-1 and galectin-3 during the investigated period of human fetal thyroid gland development. Both galectins might participate in developmental processes regarding stromal fetal thyroid tissue organization, whereas galectin-1 might have a function in thyroid epithelium maturation.     

Sialylation regulates galectin-3/ligand interplay during mammary tumour progression--a case of targeted uncloaking

Galectin-3 is involved both in facilitating detachment of cells from primary tumour sites and favouring cancer cell adhesion and survival to anoikis in the blood stream. The mechanisms behind these apparently contradictory roles of the lectin have not yet been resolved. In order to investigate possible interplays between galectin-3 and its ligands underlying their role in the metastatic process, we examined mucin-1 (MUC1) and epidermal growth factor receptor (EGFR), well-known galectin-3 ligands, as well as galectin-3-binding site expression in a series of spontaneous canine malignant mammary tumours (CMMT) and a metastatic CMMT cell line. Despite the fact that CMMT cells expressed MUC1 and EGFR homogeneously over their plasma membrane, intravascular tumour cells, positive for galectin-3, expressed MUC1 and EGFR in a more focal membrane localization. Moreover, MUC1 overexpression in primary CMMT was present in parallel with down-regulation of galectin-3. Furthermore, in the CMT-U27 cell line, galectin-3 knock-down led to increased MUC1 expression, while MUC1 knock-down led to down-regulation of the lectin. Finally, removal of sialic acid from both CMMT and CMT-U27 xenograft samples exposed galectin-3-ligands throughout the tumour tissue, whereas these ligands were only present in galectin-3-positive invading cells in untreated samples. Interestingly indeed, we show that in vessel-invading cells, there is interaction between galectin-3 and the T antigen in vivo. We therefore hypothesized that loss of galectin-3 and sialylation-related masking of its ligands, in conjunction with their overexpression in specific tumour cell subpopulations, are crucial in regulating adhesive/de-adhesive events in the progression and invasive capacity of metastatic cells.     

Combined systemic and local delivery of stem cell inducing/recruiting factors for in situ tissue regeneration

Whereas the conventional tissue engineering strategy involves the use of scaffolds combined with appropriate cell types to restore normal functions, the concept of in situ tissue regeneration uses host responses to a target-specific scaffold to mobilize host cells to a site of injury without the need for cell seeding. For this purpose, local delivery of bioactive molecules from scaffolds has been generally used. However, this approach has limited stem cell recruitment into the implants. Thus, we developed a combination of systemic delivery of substance P (SP) and local release of stromal-derived factor-1α (SDF-1α) from an implant. In this study, we examined whether this combined system would significantly enhance recruitment of host stem cells into the implants. Flow cytometry and immunohistochemistry for CD29/CD45, CD146/α-smooth muscle actin, and c-kit demonstrated that this system significantly increased the number of stem cell-like cells within the implants when compared with other systems. In vitro culture of the cells that had infiltrated into the scaffolds from the combined system confirmed that host stem cells were recruited into these implants and indicated that they were capable of differentiation into multiple lineages. These results indicate that this combined system may lead to more efficient tissue regeneration.     

Lack of galectin-3 disturbs mesenteric lymph node homeostasis and B cell niches in the course of Schistosoma mansoni infection

Galectin-3 is a β-galactoside-binding protein that has been shown to regulate pathophysiological processes, including cellular activation, differentiation and apoptosis. Recently, we showed that galectin-3 acts as a potent inhibitor of B cell differentiation into plasma cells. Here, we have investigated whether galectin-3 interferes with the lymphoid organization of B cell compartments in mesenteric lymph nodes (MLNs) during chronic schistosomiasis, using WT and galectin-3(-/-) mice. Schistosoma mansoni synthesizes GalNAcβ1-4(Fucα1-3)GlcNAc(Lac-DiNAc) structures (N-acetylgalactosamine β1-4 N-acetylglucosamine), which are known to interact with galectin-3 and elicit an intense humoral response. Antigens derived from the eggs and adult worms are continuously drained to MLNs and induce a polyclonal B cell activation. In the present work, we observed that chronically-infected galectin-3(-/-) mice exhibited a significant reduced amount of macrophages and B lymphocytes followed by drastic histological changes in B lymphocyte and plasma cell niches in the MLNs. The lack of galectin-3 favored an increase in the lymphoid follicle number, but made follicular cells more susceptible to apoptotic stimuli. There were an excessive quantity of apoptotic bodies, higher number of annexin V(+)/PI(-) cells, and reduced clearance of follicular apoptotic cells in the course of schistosomiasis. Here, we observed that galectin-3 was expressed in non-lymphoid follicular cells and its absence was associated with severe damage to tissue architecture. Thus, we convey new information on the role of galectin-3 in regulation of histological events associated with B lymphocyte and plasma cell niches, apoptosis, phagocytosis and cell cycle properties in the MLNs of mice challenged with S.mansoni.     

Association of galectin-1 and galectin-3 with Gemin4 in complexes containing the SMN protein

In previous studies we showed that galectin-1 and galectin-3 are factors required for the splicing of pre-mRNA, as assayed in a cell-free system. Using a yeast two-hybrid screen with galectin-1 as bait, Gemin4 was identified as a putative interacting protein. Gemin4 is one component of a macromolecular complex containing approximately 15 polypeptides, including SMN (survival of motor neuron) protein. Rabbit anti-galectin-1 co-immunoprecipitated from HeLa cell nuclear extracts, along with galectin-1, polypeptides identified to be in this complex: SMN, Gemin2 and the Sm polypeptides of snRNPs. Direct interaction between Gemin4 and galectin-1 was demonstrated in glutathione S-transferase (GST) pull-down assays. We also found that galectin-3 interacted with Gemin4 and that it constituted one component of the complex co-immunoprecipitated with galectin-1. Indeed, fragments of either Gemin4 or galectin-3 exhibited a dominant negative effect when added to a cell-free splicing assay. For example, a dose-dependent inhibition of splicing was observed in the presence of exogenously added N-terminal domain of galectin-3 polypeptide. In contrast, parallel addition of either the intact galectin-3 polypeptide or the C-terminal domain failed to yield the same effect. Using native gel electrophoresis to detect complexes formed by the splicing extract, we found that with addition of the N-terminal domain the predominant portion of the radiolabeled pre-mRNA was arrested at a position corresponding to the H-complex. Inasmuch as SMN-containing complexes have been implicated in the delivery of snRNPs to the H-complex, these results provide strong evidence that galectin-1 and galectin-3, by interacting with Gemin4, play a role in spliceosome assembly in vivo.     

Analogs of tetrahydroisoquinoline natural products that inhibit cell migration and target galectin-3 outside of its carbohydrate-binding site

Cell migration is central to a number of normal and disease processes. Small organic molecules that inhibit cell migration have potential as both research probes and therapeutic agents. We have identified two tetrahydroisoquinoline natural product analogs with antimigratory activities on Madin-Darby canine kidney epithelial cells: a semisynthetic derivative of quinocarmycin (also known as quinocarcin), DX-52-1, and a more complex synthetic molecule, HUK-921, related to the naphthyridinomycin family. It has been assumed that the cellular effects of reactive tetrahydroisoquinolines result from the alkylation of DNA. We have reported previously that the primary target of DX-52-1 relevant to cell migration appears to be the membrane-cytoskeleton linker protein radixin. Here we extend the analysis of the protein targets of DX-52-1, reporting that the multifunctional carbohydrate-binding protein galectin-3 is a secondary target of DX-52-1 that may also be relevant to the antimigratory effects of both DX-52-1 and HUK-921. All known inhibitors of galectin-3 target its beta-galactoside-binding site in the carbohydrate recognition domain. However, we found that DX-52-1 and HUK-921 bind galectin-3 outside of its beta-galactoside-binding site. Intriguingly HUK-921, although a less potent inhibitor of cell migration than DX-52-1, had far greater selectivity for galectin-3 over radixin, exhibiting little binding to radixin, both in vitro and in cells. Overexpression of galectin-3 in cells led to a dramatic increase in cell adhesion on different extracellular matrix substrata as well as changes in cell-cell adhesion and cell motility. Galectin-3-overexpressing cells had greatly reduced sensitivity to DX-52-1 and HUK-921, and these compounds caused a change in localization of the overexpressed galectin-3 and reversion of the cells to a more normal morphology. The converse manipulation, RNA interference-based silencing of galectin-3 expression, resulted in reduced cell-matrix adhesion and cell migration. In aggregate, the data suggest that DX-52-1 and HUK-921 inhibit a carbohydrate binding-independent function of galectin-3 that is involved in cell migration.     

Cell surface counter receptors are essential components of the unconventional export machinery of galectin-1

Galectin-1 is a component of the extracellular matrix as well as a ligand of cell surface counter receptors such as beta-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 beta-galactoside binding, does not get exported from CHO cells. We conclude that the beta-galactoside binding site represents the primary targeting motif of galectins defining a galectin export machinery that makes use of beta-galactoside-containing surface molecules as export receptors for intracellular galectin-1.     

CD7 delivers a pro-apoptotic signal during galectin-1-induced T cell death

Galectin-1, an endogenous lectin expressed in lymphoid organs and immune-privileged sites, induces death of human and murine thymocytes and T cells. Galectin-1 binds to several glycoproteins on the T cell surface, including CD7. However, the T cell surface glycoprotein receptors responsible for delivering the galectin-1 death signal have not been identified. We show that CD7 is required for galectin-1-mediated death. This demonstrates a novel function for CD7 as a death trigger and identifies galectin-1/CD7 as a new biologic death signaling pair.     

Stem cells for spine surgery

In the past few years, stem cells have become the focus of research by regenerative medicine professionals and tissue engineers. Embryonic stem cells, although capable of differentiating into cell lineages of all three germ layers, are limited in their utilization due to ethical issues. In contrast, the autologous harvest and subsequent transplantation of adult stem cells from bone marrow, adipose tissue or blood have been experimentally utilized in the treatment of a wide variety of diseases ranging from myocardial infarction to Alzheimer’s disease. The physiologic consequences of stem cell transplantation and its impact on functional recovery have been studied in countless animal models and select clinical trials. Unfortunately, the bench to bedside translation of this research has been slow. Nonetheless, stem cell therapy has received the attention of spinal surgeons due to its potential benefits in the treatment of neural damage, muscle trauma, disk degeneration and its potential contribution to bone fusion.     

Induction of cell adhesion by galectin-8 and its target molecules in Jurkat T-cells

We previously showed that tandem-repeat type galectin-8, which has two covalently linked carbohydrate recognition domains (CRDs), induces neutrophil-adhesion through binding to integrin alphaM. Here, we analysed the function of galectin-8 in Jurkat T-cells. Galectin-8, as well as tandem-repeat galectin-9, and several multivalent plant lectins, induced Jurkat T-cell adhesion to a culture plate, whereas single-CRD galectins-1 and -3 did not. Galectin-8 also induced the adhesion of peripheral blood leucocytes to human umbilical vein endothelial cells. These results suggest that the di- or multi-valent structure of galectin-8 is essential for the induction of cell adhesion and that this ability exhibits broad specificity for leucocytes. The galectin-8-induced cell adhesion was accompanied by stress fibre formation, which suggests that intracellular signalling is required. We have identified integrin alpha4 as one of the candidate target molecules associated with the induction of cell adhesion. Indeed, inhibition of the function of integrin alpha4 by treating cells with a blocking-antibody reduced the sensitivity to galectin-8. Also, the phosphorylation of Pyk and ERK1/2, indicators of integrin-mediated signalling, was up-regulated on treatment with galectin-8. Thus, a primary target of galectin-8 must be the sugar chains on members of the integrin family, which are abundantly expressed on the surface of leucocytic cells.     

Characterization of the interaction between galectin-1 and lymphocyte glycoproteins CD45 and Thy-1

Galectin-1 is a sugar binding protein specific for beta-galactosides and not requiring metal ions for binding activity. It exists as a soluble protein which forms a noncovalent homodimer and is expressed with a broad tissue distribution. Recently, galectin-1 has been shown to play a possible role in the immune system mediating apoptosis of activated T cells with indirect evidence suggesting that galectin-1 interacts with the heavily glycosylated, transmembrane, protein phosphotyrosine phosphatase CD45. The interaction of galectin-1 with purified lymphocyte cell surface proteins was studied using surface plasmon resonance in a BIAcoretrade mark. Galectin-1 was shown to bind CD45 and Thy-1 in a carbohydrate-dependent manner. Several galectin-1 molecules could bind each CD45 molecule. The dissociation constant of dimeric galectin-1 binding to CD45 was measured at approximately 5 microM, indicating the concentration at which cross-linking of cell surface glycoproteins by galectin-1 would occur. A possible role for galectin-1 in the organization of cell surface glycoproteins is discussed.