Presence Of Gal-1 Research Articles

Galectin-1 modulation of neutrophil reactive oxygen species production depends on the cell activation state

Here we report the effects of exogenous and endogenous galectin-1 (Gal-1) in modulating the functional responses of human and murine neutrophils at different stages of activation, i.e. naive, primed, and activated. Exposure to Gal-1 did not induce ROS production in either naive or N-formyl-methionyl-leucyl-phenylalanine-primed (fMLP; 10−9 M) neutrophils. However, Gal-1 elicited a concentration-dependent ROS production in neutrophils activated with fMLP at concentrations ranging from 10-8 M to 10-6 M. Additional fMLP (10-7 M) stimulation of fMLP-activated neutrophils increased ROS production, whose intensity was inversely related to the fMLP concentration used in the first activation step (10-8 M to 10-6 M), and was not influenced by the presence of Gal-1. Naive neutrophils treated with Gal-1 and then exposed to fMLP (10-6 M) or phorbol-12-myristate-13-acetate (10-7 M) produced less ROS, as compared to naive neutrophils not treated with Gal-1. Interestingly, these in vitro Gal-1 effects were associated with Gal-1 carbohydrate-binding activity and the ability to decrease FPR-1 (formyl peptide receptor 1) expression in naive human neutrophils. Conversely, positive ROS modulation by Gal-1 in activated neutrophils was not associated with FPR-1 expression but it was related to its carbohydrate recognition. In vitro, fMLP stimulation of Gal-1-/- mouse neutrophils produced more ROS than fMLP stimulation of Gal-1+/+ neutrophils and this effect may be associated with increased FPR-1 expression. Exogenous Gal-1 induced ROS production in Gal-1-/- mouse neutrophils more effectively than in Gal-1+/+ mouse neutrophils. Compared to Gal-1+/+ mice, Gal-1-/- mice exhibited lower bacterial load in the peritoneal fluid and peripheral blood, thus indicating a greater bactericidal activity in vivo. These findings demonstrate that endogenous Gal-1 restricts ROS generation that correlates with bacterial killing capacity in inflammatory neutrophils. Thus, endogenous and exogenous Gal-1 may either positively or negatively modulate the effector functions of neutrophils according to the cell activation stage.

Genetic and Epigenetic Strategies Potentiate Gal4 Activation to Enhance Fitness in Recently Diverged Yeast Species

Certain genes show more rapid reactivation for several generations following repression, a conserved phenomenon called epigenetic transcriptional memory. Following previous growth in galactose, GAL gene transcriptional memory confers a strong fitness benefit in Saccharomyces cerevisiae adapting to growth in galactose for up to 8 generations. A genetic screen for mutants defective for GAL gene memory revealed new insights into the molecular mechanism, adaptive consequences, and evolutionary history of memory. A point mutation in the Gal1 co-activator that disrupts the interaction with the Gal80 inhibitor specifically and completely disrupted memory. This mutation confirms that cytoplasmically inherited Gal1 produced during previous growth in galactose directly interferes with Gal80 repression to promote faster induction of GAL genes. This mitotically heritable mode of regulation is recently evolved; in a diverged Saccharomyces species, GAL genes show constitutively faster activation due to genetically encoded basal expression of Gal1. Thus, recently diverged species utilize either epigenetic or genetic strategies to regulate the same molecular mechanism. The screen also revealed that the central domain of the Gal4 transcription factor both regulates the stochasticity of GAL gene expression and potentiates stronger GAL gene activation in the presence of Gal1. The central domain is critical for GAL gene transcriptional memory; Gal4 lacking the central domain fails to potentiate GAL gene expression and is unresponsive to previous Gal1 expression.

Abstract LB-180: Targeting galectin-1 in combination with radiation and immune checkpoint therapy in head and neck cancers

Abstract Head and neck cancers (HNC) development shows significant changes in the immune profile, with lower systemic lymphocyte counts and reduced tumor-infiltrating lymphocytes, which inversely correlate with survival. Galectin-1 (Gal-1) is secreted at high level by cancer cells, including HNC, and contributes to tumor-immune escape and disease progression. Previously, we have shown that both hypoxia and radiotherapy (RT) can stimulate Gal-1 secretion. Immune checkpoint therapies, specifically targeting the PD1 pathway, allows T cells to be effective in shrinking tumors and lengthen survival in patients with metastatic HNC. However, these therapies work only in a small number of patients with tumors showing significant T cell infiltration. Targeting Gal-1 in HNC therefore, could be a useful therapeutic approach, as it may synergize with radiation and immunotherapy. Using the CRISPR/Cas9 deletion approach, we developed an orthotopic model of HNC with/without Gal-1 expression. We found that in both subcutaneous and orthotopic model of oral cancer, Gal-1 KO (knock-out) tumors showed marked decrease in tumor growth and nodal metastases. These differences were attributed to enhanced infiltration of CD4+ and CD8+ T cells, in the absence of Gal-1. Using a series of transendothelial migration assays in vitro and tracking adoptively transferred T cells in vivo, we show that presence of Gal-1 in the tumor microenvironment strongly suppresses T cell infiltration into tumors. We found that Gal-1 secretion led to a systemic immune suppressive response not just intra-tumorally but also in the circulating blood compartment, resulting in reduction of both CD4+ and CD8+ T cells. Concurrently, we detected higher levels of IFN-γ secretion in mice with Gal-1 KO tumors. We also observed a higher expression of PD-L1 and Gal-9 on the endothelium of WT-Gal1 compared to Gal-1 KO tumors, leading to T cell exhaustion and ineffective systemic immune response. In addition, there was noteworthy increase in the PD1+ and Tim3+ T cells in Gal-1 WT tumors compared to Gal-1 KO tumors, suggesting a role of Gal-1 in T cell anergy. Finally, we show that knocking out Gal-1 either genetically or by a blocking antibody, we could convert an anti-PD1 Ab non-responsive tumor into a responsive tumor with a significant decrease in tumor burden. In summary, our study shows Gal-1 in the tumor microenvironment could hamper immune response by impeding transendothelial T cell migration into the tumor, blocking pro-inflammatory cytokine secretions and up-regulating inhibitory immune checkpoints ligands in both tumor endothelial and T cells. Our data suggests that Gal-1 may mediate poor-response or resistance to anti-PD1 therapy and combinatorial approaches of galectin-1 inhibition with PD-1 checkpoint inhibitors may enhance therapeutic efficacy in HNC. (Acknowledgements: BMS for Anti-PD1 and Anti-Gal1 Abs. Dr R. Uppaluri, for MOC-2 oral cancer cells). Citation Format: Dhanya K. Nambiar, Todd Aguilera, Joshua Daniel Bloomstein, Hongbin Cao, Albert Koong, Quynh Thu Le. Targeting galectin-1 in combination with radiation and immune checkpoint therapy in head and neck cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-180. doi:10.1158/1538-7445.AM2017-LB-180

Presence of galectin-1 in the epidermal and dermal thickening of keloid tissues

Background: Keloids are recognized as benign dermal fibroproliferative tumors that result from aberrant wound healing in susceptible individuals. Nevertheless, the etiology and pathophysiology of keloids is not fully elucidated yet. A protein that has been implicated in the wound healing process, conversion of fibroblasts into myofibroblasts and production of ECM components, inflammation, and generation of galectin-glycan complexes, is galectin-1 (Gal-1). However, its presence as well as its implications in benign dermal fibroproliferative tumors such as keloids, has not been considered. Therefore, the aim of this study is to ascertain whether Gal-1 is present in keloid tissues. Methods: Seven biopsies from patients diagnosed with keloid were selected. Four normal skin specimens were obtained from patients undergoing aesthetic surgery procedures. To identification of collagen bundles, some histological sections were stained following the Mason´s trichrome protocol. The presence of Gal-1 was assessed by immunofluorescence staining using confocal laser scanning microscopy. Results: Immunostaining revealed that Gal-1 was present in the uppermost cell layers of the thickened epidermis with a gradient of expression that seems to increase from basal layer to suprabasal layers. Surprisingly, Gal-1 was particularly found outlining the cellular margin and intercellular bridges of the upper-spinous and granular keratinocytes and was absent in the epidermal keratinocytes of normal adult human skin. Interestingly, in the reticular dermis the presence of Gal-1 was noticeable in the cytoplasm of elongated fibroblasts that were forming cellular bundles of collagen and along the thick and compact collagen bundles, whereas in the dermis of normal skin, Gal-1 immunoreactivity was weak in some fibroblasts. Conclusions: Our findings suggest that in keloid tissues Gal-1 would not only be regulating the dermal fibroblast proliferation, ECM production, particularly collagen, and abnormal vascularization, but would also be contributing to the altered stratification and terminal keratinocyte differentiation and importantly, to epidermal thickening.

Galectin-1 induces inflammation and cartilage degeneration in osteoarthritis through NF-κB-signaling

Purpose: Cartilage consists of numerous glycoproteins and proteoglycans, which are of significance for its functionality. In osteoarthritis (OA), we previously observed an altered glycophenotype of cartilage and chondrocytes. We also examined the presence of galectins and found that Galectin-1 (Gal-1) predominantly accumulated in regions of severely degenerated cartilage. The interaction of glycans and galectins is crucial in many physiological or pathological processes. While a protective role of Gal-1 is described in rheumatoid arthritis (RA), little is known about the role of galectins in onset and progression of OA. Therefore, the present study investigates in detail the presence and biological activity of Gal-1 in OA cartilage and chondrocytes. Methods: OA cartilage was obtained from patients who underwent total knee replacement surgery. The degeneration status of cartilage from 29 patients was assessed in a total of 231 regions of interest using the Mankin score (MS). Gal-1 presence was detected by immunohistochemical staining and the percentages of positive chondrocytes were assessed. OA chondrocytes were isolated and secretion of Gal-1 to the cell culture medium was determined by ELISA. Binding of Gal-1-FITC to the surface of chondrocytes was analyzed in cell suspension. Expression of cartilage matrix components and matrix metalloproteinases (MMPs) in Gal-1-treated chondrocytes was analyzed with RT-qPCR. Cell culture supernatants were subjected to ELISA for determination of MMP levels. For microarray analyses, RNA was isolated from chondrocytes cultured in absence or presence of Gal-1 (n=5). Bioinformatic analyses were performed using Gene Set Enrichment Analyses (GSEA) and subsequent NF-κB analyses were carried out using quantitative Western blot and RT-qPCR. Results: Immunohistochemical staining revealed increasing amounts of Gal-1-positive chondrocytes in regions of degenerated cartilage. Correlation analysis indicated a significant positive correlation between Gal-1 presence (% chondrocyte positivity) and cartilage degeneration (p<0.0001, Wilcoxon‘s signed rank test). In vitro, Gal-1 was secreted by chondrocytes, whereby this secretion was not inducible by pro-inflammatory cytokines. Binding of Gal-1 to chondrocyte surfaces and resulting effects were inhibitable by cognate sugar (lactose). Gal-1 treatment of chondrocytes increased the expression of matrix degrading enzymes (ADAMTS4, MMP1, MMP3, MMP13) and pro-inflammatory cytokines (IL1B, TNFA), while the expression of matrix components (AGC1, COL2A1) was decreased. In agreement, elevated secretion levels of pro-MMP-1, MMP-3 and pro-MMP-13 were detected. Analyses of the microarray data revealed an overexpression of chemokines and cytokines in Gal-1-treated chondrocytes. GSEA showed an overrepresentation of NF-κB DNA binding motifs in the promoters of most significantly induced genes prompting further investigation of the NF-κB signaling cascade. Western blot analyses showed that Gal-1 induced phosphorylation of IκBα and p65, and RT-qPCR experiments revealed an impairment of Gal-1-mediated IL1B transcription by inhibition of specific NF-κB pathway components. Conclusions: This study is first to suggest a functional link between Gal-1 and OA manifestation. In situ, Gal-1 presence correlates with cartilage degeneration, which points towards its qualification as novel tissue biomarker. In vitro, Gal-1 induces the production of pro-inflammatory cytokines and matrix degrading enzymes. Importantly, Gal-1 secretion is not induced by pro-inflammatory conditions, indicating its position as an upstream mediator acting as a trigger of inflammation and matrix breakdown in OA. The deleterious activity of Gal-1 in OA, which is mediated by the NF-κB pathway, stands in contrast to its protective role in RA. In summary, our results highlight the role of Gal-1 in onset and progression of OA and suggest that targeting Gal-1 signaling may be a promising therapeutic strategy.

Stimulation of Syncytium Formation in vitro in Human Trophoblast Cells by Galectin-1

Background Galectin-1 (gal-1), a member of the mammalian β-galactoside-binding proteins, exerts biological effects by recognition of glycan ligands, including those involved in cell adhesion and growth regulation. In trophoblast cells, gal-1 binds to cell surface glycoproteins (e.g., Mucin1). It has been demonstrated that gal-1 recognizes appropriate glycotopes on the syncytiotrophoblast and extravillous trophoblast from second trimester human placenta and choriocarcinoma cells BeWo, which reveal two coexisting phenotypes, the cytotrophoblast-like and the syncytiotrophoblast-like phenotype. So the aim of this study was to investigate the effect of gal-1 on syncytium formation in BeWo and human villous trophoblasts (HVT) cells. Materials and methods The effect of gal-1 on syncytium formation was investigated with immunocytochemical and double immunofluorescence stainings, cell-labelling and Real-time RT-PCR. BeWo choriocarcinoma and HVT cells were incubated in vitro for 24 and 48 h in the absence (controls) and presence of gal-1 and forskolin and stained with antibodies against Ki67, β-catenin, E-cadherin and syncytin. BeWo and HVT cells were incubated with 60 μg/ml gal-1 for 48 h (BeWo) or 96 h (HVT) and cell fusion was detected by fluorescent cell-labelling solution. Finally, BeWo cells were incubated for 1 h or 48 h in the absence and presence of 60 μg/ml gal-1 and Real-time RT-PCR was performed. Results We showed with immunocytochemical staining a downregulation of β-catenin expression in the 24 h BeWo cell culture and with double immunofluorescence staining an inhibition of the β-catenin and E-cadherin expression in the 48 h BeWo cell culture stimulated with gal-1 or forskolin. The inhibition of E-cadherin was demonstrated on mRNA level in the 1 h BeWo cell culture too. Increased cell fusion was also showed with DiO and DiI fluorescent cell-labelling solution in the 48 h BeWo cell culture. In addition, we demonstrated the downregulation of Ki67 protein expression in the 24 h BeWo cell culture and on mRNA level in the 1 h BeWo cell culture. We also showed the upregulation of syncytin protein and mRNA expression after incubation of the 48 h BeWo cell culture with gal-1 or forskolin. Similar results were obtained with HVT cells: the amount of cell fusion was significantly increased in the gal-1 treated 48 h HVT cell culture in vitro compared to untreated cells as demonstrated with β-catenin and E-cadherin double immunofluorescence staining. This increase was also shown by fluorescent cell-labelling with DiO and DiI in the 96 h HVT cell culture compared to untreated cells. Conclusion Our data suggest that gal-1 stimulates the syncytium formation in choriocarcinoma cells BeWo and HVT cells in vitro and inhibits the expression of β-catenin, E-cadherin and in addition Ki67 in BeWo cells. Therefore gal-1 may be a major trigger for the process of trophoblast cell fusion.

Effects of galectin-1 on regulation of progesterone production in granulosa cells from pig ovaries in vitro.

The detection of galectin-1 (gal-1) in pig granulosa cell lysates by immunoblotting and its cytosolic as well as membrane-associated localization prompted us to study its effects on cell proliferation and regulation of progesterone synthesis. The lectin stimulated the proliferation of granulosa cells from pig ovaries cultured in serum-free medium. Gal-1 inhibited the FSH-stimulated progesterone synthesis of granulosa cells. This inhibitory effect was strongly reduced by the disaccharidic competitor lactose at 30 mM. The absence of inhibitory effects on dibutyryl-cAMP (db-cAMP), forskolin, and pregnenolone-enhanced cellular progesterone synthesis suggests that gal-1interferes with the receptor-dependent mechanism of FSH-stimulated progesterone production. In FSH-stimulated granulosa cells, western blot analysis revealed the gal-1-mediated suppression of the cytochrome P450-dependent cholesterol side chain cleavage enzyme (P450(SCC)) that catalyzes the conversion of cholesterol to pregnenolone. In the presence of 30 mM lactose, the gal-1-reduced P450(SCC) expression was prevented. Strongly reduced mRNA levels were recorded for P450(SCC) and 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) when FSH-stimulated granulosa cells were cultured in the presence of gal-1. We conclude that gal-1 exerts its inhibitory effect on steroidogenic activity of granulosa cells by interfering the hormone-receptor interaction resulting in decreased responses to FSH stimulation.

Molecular mechanisms implicated in galectin-1-induced apoptosis: activation of the AP-1 transcription factor and downregulation of Bcl-2.

Galectins are emerging as a new class of bioactive molecules with specific immunomodulatory properties. Galectin-1 (Gal-1), a member of this family, has been shown to induce apoptosis of mature T cells and immature thymocytes. To gain insight into the intracellular signals transduced by Gal-1 upon binding to mature T cells, we investigated whether this protein triggered activation of the dimeric AP-1 transcription factor. A marked increase in the binding of nuclear extracts to synthetic oligonucleotides containing the AP-1 consensus sequence, could be detected by an electrophoretic mobility shift assay, when T cells were cultured for 30 min in the presence of Gal-1. This DNA-binding activity was preceded by a rapid increase in the levels of c-Jun mRNA, as determined by Northern blot analysis. Requirement of AP-1 for Gal-1-induced apoptosis was confirmed by the dose-dependent reduction on the level of DNA fragmentation observed when cells were pre-treated with curcumin (an inhibitor of AP-1 activation) before exposure to Gal-1. Finally, evidence is also provided by Western blot analysis, showing that Gal-1 inhibits Concanavalin A (Con A) induction of Bcl-2 protein. Results presented in this study provide the first experimental evidence regarding AP-1 and Bcl-2 as targets of the signal transduction pathway triggered by Gal-1 and set the basis for a more in depth understanding of the molecular mechanisms of T-cell death regulation.