Galectin-7 reprograms skin carcinogenesis by fostering innate immune evasive programs.

Non-melanoma skin cancer (NMSC) has risen dramatically as a result of chronic exposure to sunlight ultraviolet (UV) radiation, climatic changes and clinical conditions associated with immunosuppression. In spite of considerable progress, our understanding of the mechanisms that control NMSC development and their associated molecular and immunological landscapes is still limited. Here we demonstrated a critical role for galectin-7 (Gal-7), a ß-galactoside-binding protein preferentially expressed in skin tissue, during NMSC development. Transgenic mice (Tg46) overexpressing Gal-7 in keratinocytes showed higher number of papillomas compared to WT mice or mice lacking Gal-7 (Lgals7-/-) when subjected to a skin carcinogenesis protocol, in which tumor initiator 7,12-dimethylbenz[a]anthracene (DMBA) and tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were sequentially administered. RNAseq analysis of Tg46 tumor lesions revealed a unique profile compatible with cells of the myelomonocytic lineage infiltrating these tumors, an effect that was substantiated by a higher number of CD11b+Gr1+ cells in tumor-draining lymph nodes. Heightened c-Met activation and Cxcl-1 expression in Tg46 lesions suggested a contribution of this pathway to the recruitment of these cells. Remarkably, Gal-7 bound to the surface of CD11b+Ly6ChiLy6Glo monocytic myeloid cells and enhanced their immunosuppressive activity, as evidenced by increased IL-10 and TGF-ß1 secretion, and higher T-cell inhibitory activity. In vivo, carcinogen-treated Lgals7-/- animals adoptively transferred with Gal-7-conditioned monocytic myeloid cells developed higher number of papillomas, whereas depletion of these cells in Tg46-treated mice led to reduction in the number of tumors. Finally, human NMSC biopsies showed increased LGALS7 mRNA and Gal-7 protein expression and displayed transcriptional profiles associated with myeloid programs, accompanied by elevated CXCL1 expression and c-Met activation. Thus, Gal-7 emerges as a critical mediator of skin carcinogenesis and a potential therapeutic target in human NMSC.

Untangling Galectin-Mediated Circuits that Control Hypoxia-Driven Angiogenesis.

Development of an aberrant vascular network is a hallmark of the multistep pathological process of tumor growth and metastasis. In response to hypoxia, several pro-angiogenic factors are synthesized to support vascularization programs required for cancer progression. Emerging data indicate the involvement of glycans and glycan-binding proteins as critical regulators of vascular circuits in health and disease. Galectins may be regulated by hypoxic conditions and control angiogenesis in different physiopathological settings. These ß-galactoside-binding proteins may promote sprouting angiogenesis by interacting with different glycosylated receptors and triggering distinct signaling pathways. Understanding the role of galectins in tumor neovascularization will contribute to the design of novel anti-angiogenic therapies aimed at complementing current anti-cancer modalities and overcoming resistance to these treatments. Here we describe selected strategies and methods used to study the role of hypoxia-regulated galectins in the regulation of blood vessel formation.

Circulating Galectin-1 and Galectin-3 in Sera From Patients With Systemic Sclerosis: Associations With Clinical Features and Treatment.

Systemic Sclerosis (SSc) is a rheumatic disease characterized by fibrosis, microvascular damage and immune dysregulation. Two major subsets, limited cutaneous systemic sclerosis (lcSSc) and diffuse cutaneous systemic sclerosis (dcSSc) can be defined, according to the extent of skin involvement. Increasing evidence indicates a role for galectins in immune and vascular programs, extracellular matrix remodeling and fibrosis, suggesting their possible involvement in SSc. Here, we determined serum levels of galectin (Gal)-1 and Gal-3 in 83 SSc patients (dcSSc n = 17; lcSSc n = 64; ssSSc n = 2), and evaluated their association with clinical manifestations of the disease. Patients with dcSSc showed lower Gal-3 levels, compared to lcSSc (p = 0.003), whereas no considerable difference in Gal-1 levels was detected between groups. Remarkably, higher concentrations of Gal-1 were associated with the presence of telangiectasias (p = 0.015), and higher concentrations Gal-3 were associated with telangiectasias (p = 0.021), diarrhea (p = 0.039) and constipation (p = 0.038). Moreover, lower Gal-3 levels were associated with the presence of tendinous retractions (p = 0.005). Patients receiving calcium blockers (p = 0.048), methotrexate (p = 0.046) or any immunosuppressive treatment (p = 0.044) presented lower concentrations of Gal-3 compared to those not receiving such treatments. The presence of telangiectasia and the type of SSc maintained their statistical association with Gal-3 (ß 0.25; p = 0.022 and ß 0.26; p = 0.017, respectively) in multiple linear regression models. In conclusion, serum levels of Gal-3 are associated with clinical manifestations of SSc. Among them, the presence of telangiectasias could be explained by the central role of this lectin in the vascularization programs.

Galectin-1 impacts on glucose homeostasis by modulating pancreatic insulin release.

Type-2 diabetes mellitus (T2DM) is an expanding global health problem, involving defective insulin secretion by pancreatic ß-cells and peripheral insulin resistance, leading to impaired glucose regulation. Galectin-1-an endogenous lectin with affinity for N-acetyllactosamine (LacNAc)-containing glycans-has emerged as a regulator of inflammatory and metabolic disorders. However, the role of galectin-1 in glucose homeostasis and pancreatic ß-cell function, independently of hypercaloric diets, has not been explored. Here, we identified a phenotype compatible with T2DM, involving alterations in glucose metabolism and pancreatic insulin release, in female but not male mice lacking galectin-1 (Lgals1-/-). Compared with age-matched controls, Lgals1-/- female mice exhibited higher body weight and increased food intake ad libitum as well as after fasting and acute re-feeding. Although fasted serum insulin levels and insulin sensitivity were similar in both genotypes, Lgals1-/- female mice presented altered glucose tolerance and higher basal glucose levels depending on the fasting period. Insulin response to glucose overload was impaired, while pancreatic insulin content was enhanced in the absence of galectin-1. Accordingly, recombinant galectin-1 enhanced glucose-stimulated insulin release in vitro. Our study identifies a role for galectin-1 in regulating glucose metabolism through modulation of pancreatic insulin secretion, highlighting novel opportunities to control T2DM.

An adipose tissue galectin controls endothelial cell function via preferential recognition of 3-fucosylated glycans.

Upon overnutrition, adipocytes activate a homeostatic program to adjust anabolic pressure. An inflammatory response enables adipose tissue (AT) expansion with concomitant enlargement of its capillary network, and reduces energy storage by increasing insulin resistance. Galectin-12 (Gal-12), an endogenous lectin preferentially expressed in AT, plays a key role in adipocyte differentiation, lipolysis, and glucose homeostasis. Here, we reveal biochemical and biophysical determinants of Gal-12 structure, including its preferential recognition of 3-fucosylated structures, a unique feature among members of the galectin family. Furthermore, we identify a previously unanticipated role for this lectin in the regulation of angiogenesis within AT. Gal-12 showed preferential localization within the inner side of lipid droplets, and its expression was upregulated under hypoxic conditions. Through glycosylation-dependent binding to endothelial cells, Gal-12 promoted in vitro angiogenesis. Moreover, analysis of in vivo AT vasculature showed reduced vascular networks in Gal-12-deficient (Lgals12-/-) compared to wild-type mice, supporting a role for this lectin in AT angiogenesis. In conclusion, this study unveils biochemical, topological, and functional features of a hypoxia-regulated galectin in AT, which modulates endothelial cell function through recognition of 3-fucosylated glycans. Thus, glycosylation-dependent programs may control AT homeostasis by modulating endothelial cell biology with critical implications in metabolic disorders and inflammation.

Galectins in Intestinal Inflammation: Galectin-1 Expression Delineates Response to Treatment in Celiac Disease Patients.

Galectins, a family of animal lectins characterized by their affinity for N-acetyllactosamine-enriched glycoconjugates, modulate several immune cell processes shaping the course of innate and adaptive immune responses. Through interaction with a wide range of glycosylated receptors bearing complex branched N-glycans and core 2-O-glycans, these endogenous lectins trigger distinct signaling programs thereby controling immune cell activation, differentiation, recruitment and survival. Given the unique features of mucosal inflammation and the differential expression of galectins throughout the gastrointestinal tract, we discuss here key findings on the role of galectins in intestinal inflammation, particularly Crohn's disease, ulcerative colitis, and celiac disease (CeD) patients, as well as in murine models resembling these inflammatory conditions. In addition, we present new data highlighting the regulated expression of galectin-1 (Gal-1), a proto-type member of the galectin family, during intestinal inflammation in untreated and treated CeD patients. Our results unveil a substantial upregulation of Gal-1 accompanying the anti-inflammatory and tolerogenic response associated with gluten-free diet in CeD patients, suggesting a major role of this lectin in favoring resolution of inflammation and restoration of mucosal homeostasis. Thus, a coordinated network of galectins and their glycosylated ligands, exerting either anti-inflammatory or proinflammatory responses, may influence the interplay between intestinal epithelial cells and the highly specialized gut immune system in physiologic and pathologic settings.

Galectin-1: A Jack-of-All-Trades in the Resolution of Acute and Chronic Inflammation.

Regulatory signals provide negative input to immunological networks promoting resolution of acute and chronic inflammation. Galectin-1 (Gal-1), a member of a family of evolutionarily conserved glycan-binding proteins, displays broad anti-inflammatory and proresolving activities by targeting multiple immune cell types. Within the innate immune compartment, Gal-1 acts as a resolution-associated molecular pattern by counteracting the synthesis of proinflammatory cytokines, inhibiting neutrophil trafficking, targeting eosinophil migration and survival, and suppressing mast cell degranulation. Likewise, this lectin controls T cell and B cell compartments by modulating receptor clustering and signaling, thus serving as a negative-regulatory checkpoint that reprograms cellular activation, differentiation, and survival. In this review, we discuss the central role of Gal-1 in regulatory programs operating during acute inflammation, autoimmune diseases, allergic inflammation, pregnancy, cancer, and infection. Therapeutic strategies aimed at targeting Gal-1-glycan interactions will contribute to overcome cancer immunosuppression and reinforce antimicrobial immunity, whereas stimulation of Gal-1-driven immunoregulatory circuits will help to mitigate exuberant inflammation.

Expression of fragile X mental retardation protein and Fmr1 mRNA during folliculogenesis in the rat.

Fragile X mental retardation protein (FMRP) belongs to a small family of RNA-binding proteins. Its absence or inactivity is responsible for fragile X syndrome, the most common cause of inherited mental retardation. Despite its ubiquitous expression, FMRP function and expression remain almost understudied in non-neuronal tissues, though previous studies on germline development during oogenesis may suggest a special function of this protein also in ovarian tissue. In addition, the well-documented association of FMR1 premutation state with fragile X-related premature ovarian insufficiency adds interest to the role of FMRP in ovarian physiology. The aim of the present work was to investigate the expression of Fmr1 mRNA and its protein, FMRP, at different stages of rat follicular development. By immunohistochemical studies we demonstrated FMRP expression in granulosa, theca and germ cells in all stages of follicular development. In addition, changes in Fmr1 expression, both at the protein and mRNA levels, were observed. FMRP levels increased upon follicular development while preantral and early antral follicles presented similar levels of Fmr1 transcripts with decreased expression in preovulatory follicles. These observations suggest that Fmr1 expression in the ovary is regulated at different and perhaps independent levels. In addition, our results show expression of at least four different isoforms of FMRP during all stages of follicular growth with expression patterns that differ from those observed in brain and testis. Our study shows a regulated expression of Fmr1, both at mRNA and protein levels, during rat follicular development.

Targeting galectin-1 overcomes breast cancer-associated immunosuppression and prevents metastatic disease.

Galectin-1 (Gal1), an evolutionarily conserved glycan-binding protein, contributes to the creation of an immunosuppressed microenvironment at sites of tumor growth. In spite of considerable progress in elucidating its role in tumor-immune escape, the mechanisms underlying the inhibitory functions of Gal1 remain obscure. Here, we investigated the contribution of tumor Gal1 to tumor growth, metastasis, and immunosuppression in breast cancer. We found that the frequency of Gal1(+) cells in human breast cancer biopsies correlated positively with tumor grade, while specimens from patients with benign hyperplasia showed negative or limited Gal1 staining. To examine the pathophysiologic relevance of Gal1 in breast cancer, we used the metastatic mouse mammary tumor 4T1, which expresses and secretes substantial amounts of Gal1. Silencing Gal1 expression in this model induced a marked reduction in both tumor growth and the number of lung metastases. This effect was abrogated when mice were inoculated with wild-type 4T1 tumor cells in their contralateral flank, suggesting involvement of a systemic modulation of the immune response. Gal1 attenuation in 4T1 cells also reduced the frequency of CD4(+)CD25(+) Foxp3(+) regulatory T (T(reg)) cells within the tumor, draining lymph nodes, spleen, and lung metastases. Further, it abrogated the immunosuppressive function of T(reg) cells and selectively lowered the expression of the T-cell regulatory molecule LAT (linker for activation of T cells) on these cells, disarming their suppressive activity. Taken together, our results offer a preclinical proof of concept that therapeutic targeting of Gal1 can overcome breast cancer-associated immunosuppression and can prevent metastatic disease.

When galectins recognize glycans: from biochemistry to physiology and back again.

In the past decade, increasing efforts have been devoted to the study of galectins, a family of evolutionarily conserved glycan-binding proteins with multifunctional properties. Galectins function, either intracellularly or extracellularly, as key biological mediators capable of monitoring changes occurring on the cell surface during fundamental biological processes such as cellular communication, inflammation, development, and differentiation. Their highly conserved structures, exquisite carbohydrate specificity, and ability to modulate a broad spectrum of biological processes have captivated a wide range of scientists from a wide spectrum of disciplines, including biochemistry, biophysics, cell biology, and physiology. However, in spite of enormous efforts to dissect the functions and properties of these glycan-binding proteins, limited information about how structural and biochemical aspects of these proteins can influence biological functions is available. In this review, we aim to integrate structural, biochemical, and functional aspects of this bewildering and ancient family of glycan-binding proteins and discuss their implications in physiologic and pathologic settings.

Cell-type specific regulation of galectin-3 expression by glucocorticoids in lung Clara cells and macrophages.

Bronchiolar Clara cells are integral components of lung homeostasis, predominantly distributed in distal airways. In addition to the 16 kDa Clara cell protein, a major secretory product with anti-inflammatory effects, rat Clara cells express the glycan-binding protein galectin-3 and secrete it into the airways. Given the essential role of galectin-3 in the control of inflammation and the well-established function of glucocorticoids (GCs) in lung physiology, here we investigated whether galectin-3 is a target of the regulatory effects of GCs. Adult male rats were subjected to bilateral adrenalectomy and the lungs were processed for light and transmission electron microscopy, immunoelectron microscopy and Western blot analysis. Profound changes in bronchiolar Clara cells and macrophage morphology could be observed by electron microscopy after adrenalectomy. While specific galectin-3 staining was detected in the nucleus and cytoplasm of Clara cells and macrophages from control animals, cytoplasmic galectin-3 expression was dramatically reduced after adrenalectomy in both cell types. This effect was cell-specific as it did not affect expression of this lectin in ciliated cells. After dexamethasone treatment, galectin-3 expression increased significantly in the nucleus and cytoplasm of macrophages and Clara cells. Western blot analysis showed a clear decrease in galectin-3 expression in ADX animals, which was recovered after a 7-day treatment with dexamethasone. In peritoneal macrophages, galectin-3 expression was also dependent on the effects of GCs both in vivo and in vitro. Our results identify a cell type-specific control of galectin-3 synthesis by GCs in lung bronchiolar Clara cells and interstitial macrophages, which may provide an alternative mechanism by which GCs contribute to modulate the inflammatory response.

Regulated expression of galectin-3, a multifunctional glycan-binding protein, in haematopoietic and non-haematopoietic tissues.

Galectin-3 belongs to a family of highly conserved animal lectins characterized by their ability to recognize multiple N-acetyllactosamine sequences, which can be displayed on both N- and O-glycans on cell surface glycoconjugates. Although first identified in macrophages, galectin-3 (also called "Mac-2, εBP, CBP35 or L-29") has been found to be widely distributed in several tissues and developmental stages where, depending on its extracellular or intracellular localization, it can display a broad diversity of biological functions including immunomodulation, host-pathogen interactions, embryogenesis, angiogenesis, cell migration, wound healing and apoptosis. In spite of the existence of several reviews describing the multifunctional properties of galectin-3, an integrated view of the regulated expression of this glycan-binding protein in different normal tissues is lacking. Here we attempt to summarize and integrate available information on galectin-3 distribution in normal haematopoietic and non-haematopoietic tissues, mainly in adulthood, with only a brief reference to its expression during embryonic stages. In addition, given the multiplicity of biological roles attributed to this protein, a brief description of galectin-3 functions is also included. Understanding how galectin-3 is regulated in normal tissues will contribute to a rational design of approaches aimed at modulating galectin-3 expression and subcellular localization for experimental and therapeutic purposes.

Alpha-enolase: a novel autoantigen in patients with premature ovarian failure.

OBJECTIVE: Although controversial, the presence of circulating antiovarian antibodies (AOA) may be considered a marker of autoimmune premature ovarian failure (POF). The purpose of the present work was to evaluate the presence of AOA in POF patients, and to identify a possible autoantigen in order to develop a reliable diagnostic tool that might help to determine the real prevalence of autoimmune POF. DESIGN: Non-randomised study. Blood sampling for determination of circulating AOA. PATIENTS: One hundred and ten patients with POF and 60 normally menstruating women with no record of autoimmune diseases (controls). MEASUREMENTS: Presence of circulating AOA was assessed by Western-blot, using cytosolic fraction from human ovarian homogenate as antigen. RESULTS: Twenty-one of 110 women with POF presented circulating antibodies directed toward an antigen of approximately 50 kD. Sixty control subjects proved negative. After purification and analysis by mass spectrometry, the antigen was identified as alpha-enolase. CONCLUSION: Determination of the presence of circulating antialpha-enolase antibodies might be instrumental in identifying those patients who may present a putative defect in immunoregulation and therefore a possible autoimmune aetiolgy for POF.

Controversial role of inhibin alpha-subunit gene in the aetiology of premature ovarian failure.

BACKGROUND: Premature ovarian failure (POF) is characterized by hypergonadotropic amenorrhoea before the age of 40. Inhibin alpha-subunit (INHalpha) gene is proposed as a candidate gene due to its role in negative feedback control of FSH. METHODS: Polymorphism -16C>T of INHalpha gene was studied in 61 POF patients and 82 controls above 40 years old (C > 40). Substitution 769G>A was studied in 59 POF patients, 76 C > 40 and 73 controls below 40 years old (C < 40). RESULTS: No significant difference in risk of POF development for -16T allele was found when comparing idiopathic POF (I-POF) with C > 40 (Odds ratio = 1.46; 95% confidence interval = 0.63-3.19). Implication of -16C>T polymorphism in serum inhibin levels was analysed in 46 controls, and no significant differences (P > 0.05) were found between CC and CT + TT genotype groups when comparing either mid-follicular phase Pro-alphaC and inhibin B values or mid-luteal phase Pro-alphaC and inhibin A values. Heterozygosity for substitution 769G>A was found in 1 of 59 POF woman, 2 of 76 C > 40 and 6 of 73 C < 40. Presence of this substitution in a relevant number of control subjects is herein described for the first time. CONCLUSION: Our results indicate that -16C>T and 769G>A variants in INHalpha gene may not be associated to POF disease.

Screening of FSH receptor gene in Argentine women with premature ovarian failure (POF).

Diverse mutations in FSH-receptor (FSHR) gene have been described as possible cause of premature ovarian failure (POF). To investigate the presence of mutations and/or polymorphisms in FSHR gene, DNA from 20 POF, 5 of which were diagnosed as resistant ovary syndrome (ROS), and from 44 controls was isolated from peripheral lymphocytes. The complete coding sequence was analysed by PCR followed by SSCP, direct sequencing or restriction enzyme analysis. No mutations in FSHR gene were identified in the patients studied. The two already described polymorphisms in exon 10, A919G and A2039G, cosegregated in all the homozygous individuals, indicating that FSHR presents two isoforms: Ala307-Ser680 and Thr307-Asn680. OR results suggest that the 919G-2039G allelic variant or the homozygous genotype is not associated to disease risk. In addition, a heterozygous substitution T1022C (Val341Ala) was found in two control subjects. We suggest that mutations in FSHR gene are rare in women with POF in Argentine. Presence of a particular FSHR isoform does not appear to be associated with this disease.

Circulating immunoglobulins that inhibit the binding of follicle-stimulating hormone to its receptor: a putative diagnostic role in resistant ovary syndrome?

OBJECTIVE: To evaluate the presence of circulating immunoglobulins that inhibit FSH binding to its receptor (Ig-FSHR) in patients with premature ovarian failure (POF). DESIGN: Non-randomized study. Blood sampling for determination of circulating immunoglobulins. patients Two hundred and forty-seven patients with POF and 60 normally menstruating women (controls). measurements Circulating immunoglobulins that inhibit FSH binding to its receptor were assessed by FSH-binding inhibition assay. RESULTS: Twenty-three out of 247 women with POF presented circulating immunoglobulins that inhibit FSH binding to its receptor. These patients had been previously diagnosed as ROS. Sixty control subjects proved negative. CONCLUSION: Determination of the presence of circulating immunoglobulins that inhibit FSH binding to its receptor could be instrumental in diagnosing the gonadotropin resistance ovary syndrome.