Human fibroblast-derived growth factor is a mitogen and chemoattractant for endothelial cells

Human fibroblasts were found to produce a potent mitogen and chemoattractant for fetal bovine aortic endothelial cells. Homogenates from AG1523 and AG1518 foreskin, CCD18Lu lung, and CCD18Co colon fibroblasts produced half-maximal stimulation of endothelial cell growth at concentrations of 1-7 micrograms/ml. The factor was purified from large-scale cultures of the CCD18Co fibroblasts using cation exchange chromatography and heparin-Sepharose chromatography. Such preparations were mitogenic for endothelial cells in vitro at concentrations of about 5-10 ng/ml, and promoted chemotaxis at 0.1-1 ng/ml. Heparinase treatment of the cells prevented the chemotactic response. These properties suggest that the factor may be related to fibroblast growth factor.     

Human sperm chemotaxis: is progesterone a chemoattractant?

Follicular fluid (FF) induces sperm chemotaxis in human spermatozoa. Progesterone also causes sperm accumulation. However, sperm accumulation can be caused by chemotaxis, chemokinesis, and trapping of various kinds. It has been suggested that progesterone also induces chemotaxis in human spermatozoa. In view of the physiological significance of sperm chemotaxis in human fertilization and its potential clinical implications, it is important to determine unequivocally whether chemotaxis is induced by progesterone and, if so, whether progesterone in FF is the chemoattractant. To resolve these questions we looked for characteristic changes in the direction of sperm swimming toward pure progesterone as well as toward FF before and after progesterone removal. Progesterone caused sperm accumulation and hyperactivation-like motility, but it caused very few changes in the direction of sperm swimming that are characteristic of chemotaxis. Removal of progesterone (and other steroids) from FF by charcoal treatment abolished the sperm hyperactivation-like motility but not sperm chemotaxis. These results suggest that while progesterone might be a weak chemoattractant, it is not the major chemoattractant in FF. Progesterone probably causes human sperm accumulation mainly by inducing hyperactivation-like motility and, as a consequence, sperm trapping.     

RGS1 is expressed in monocytes and acts as a GTPase-activating protein for G-protein-coupled chemoattractant receptors.

The leukocyte response to chemoattractants is transduced by the interaction of transmembrane receptors with GTP-binding regulatory proteins (G-proteins). RGS1 is a member of a protein family constituting a newly appreciated and large group of proteins that act as deactivators of G-protein signaling pathways by accelerating the GTPase activity of G-protein alpha subunits. We demonstrate here that RGS1 is expressed in human monocytes; by immunofluorescence and subcellular fractionation RGS1 was localized to the plasma membrane. By using a mixture of RGS1 and plasma membranes, we were able to demonstrate GAP activity of RGS1 on receptor-activated G-proteins; RGS1 did not affect ligand-stimulated GDP-GTP exchange. We found that RGS1 desensitizes a variety of chemotactic receptors including receptors for N-formyl-methionyl-leucyl-phenylalanine, leukotriene B4, and C5a. Interaction of RGS proteins and ligand-induced G-protein signaling can be demonstrated by determining GTPase activity using purified RGS proteins and plasma membranes.     

Bergenin: a computationally proven promising scaffold for novel galectin-3 inhibitors

Bergenin is a C-glycoside of 4-O-methylgallic acid that is isolated from medicinal plants such as Flueggea leucopyrus, Bergenia crassifolia, Mallotus philippensis, Corylopsis spicata, Caesalpinia digyna, Mallotus japonicus, and Sacoglottis gabonensis. Even though there appears to be ample evidence from South Asian traditional medicine that bergenin possesses strong anticancer activity, no comprehensive scientific study has been carried out to test its anticancer potency. Therefore, in this study, the potential mechanisms of action for bergenin’s postulated anticancer activity were examined using computational techniques. Firstly, bergenin was tested for its toxicity as a drug candidate using in silico toxicity analysis. It was found that bergenin is nontoxic according to modern toxicity measures. The optimized structure of bergenin was obtained at the DFT-B3LYP/6-31G(d) level of theory. Potential biological targets of bergenin were identified using reverse docking calculations. Reverse docking results suggested that galectin-3 is a potential target of bergenin. Gelectin-3 is an enzyme that plays a major role in cell–cell adhesion, cell-matrix interactions, macrophage activation, angiogenesis, metastasis, and apoptosis in cancer, making it a popular target in anticancer drug design. Among the many potential biological targets predicted by reverse docking calculations, galectin-3 was selected as it complies with the primary objective of this study. The binding of bergenin to galectin-3 was studied by conventional forward docking calculations. Classical molecular dynamics (MD) simulations were used to study the stability of the galectin-3:bergenin complex. Docking calculations indicated that bergenin has the potential to effectively bind to the carbohydrate recognition domain (CRD) of galectin-3. As well as electrostatic and van der Waals interactions, a few strong hydrogen bonds were found to be involved in the binding of bergenin to galectin-3. There is also a plausible π-stacking interaction between the aromatic moiety of bergenin and the His158 residue at the binding site. A 50-ns MD simulation was carried out for the bergenin:galectin-3 complex in a cubic water box with periodic boundary conditions. The MD results showed that the bergenin:galectin-3 complex is highly stable and confirmed the veracity of the docking results, which suggested that bergenin potentially exerts an inhibitory effect on galectin-3. This study therefore sheds new light on the anticancer activity of bergenin and demonstrates that bergenin could potentially be used to develop more potent galectin-3 inhibitors. The study also provides scientific evidence supporting the use of bergenin-containing plants in cancer treatments in Eastern traditional medicine.

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Graphical abstract Bergenin in the galectin-3 binding site

     

A role for galectin-3 in CD13-mediated homotypic aggregation of monocytes

We recently reported that anti-CD13 mAbs induce homotypic aggregation of monocytic cells. This phenomenon is signal transduction dependent and does not require CD13 aminopeptidase activity. Since CD13 is heavily glycosylated and a member of the galectin family (galectin-4) has been shown to associate with CD13 in the intestinal epithelium, we hypothesized that CD13-mediated aggregation might proceed through a carbohydrate-dependent mechanism involving galectin-3, the most highly expressed galectin on monocytes. We report here that lactose and anti-galectin-3 antibodies completely abrogate homotypic aggregation induced by anti-CD13 antibodies. Furthermore, galectin-3 co-immunoprecipitates with CD13 from resting U-937 cells and this association decreases during the aggregation process, a phenomenon that may have functional implications. Together, the results presented here point to a key role for galectin-3 in CD13-mediated homotypic aggregation of monocytic cells.     

Adiponectin downregulates galectin-3 whose cellular form is elevated whereas its soluble form is reduced in type 2 diabetic monocytes

Galectin-3 plays a role in atherosclerotic diseases, and the effect of adiponectin that protects from atherosclerotic diseases on monocytic galectin-3 was analysed. Adiponectin reduced galectin-3 mRNA, its cellular and soluble form, and this effect was impaired in T2D cells. Cellular galectin-3 was higher in monocytes of overweight than normal-weight donors and was highest in T2D cells. Cellular galectin-3 positively correlated with the BMI of the donors and negatively with soluble monocyte galectin-3. Circulating levels of total adiponectin did not correlate with cellular or soluble galectin-3 indicating that additional factors contribute to higher cellular monocytic galectin-3 in obesity and T2D.     

Regulatory role of thioredoxin in homocysteine-induced monocyte chemoattractant protein-1 secretion in monocytes/macrophages

We have previously shown that homocysteine (Hcy) can induce monocyte chemoattractant protein-1 (MCP-1) secretion via reactive oxygen species (ROS) in human monocytes. Here, we show that Hcy upregulates expression of an important antioxidative protein, thioredoxin (Trx), via NADPH oxidase in human monocytes in vitro. The increase of Trx expression and activity inhibited Hcy-induced ROS production and MCP-1 secretion. Of note, 2-week hyperhomocysteinemia (HHcy) ApoE^−/− mice showed accelerated lesion formation and parallel lower Trx expression in macrophages than ApoE^−/− mice, suggesting that HHcy-induced sustained oxidative stress in vivo might account for impaired Trx and hence increased ROS production and MCP-1 secretion from macrophages, and subsequently accelerated atherogenesis.     

Detection of galectin-3 by novel peptidic photoprobes

Photoprobes were prepared with specificity for binding, labeling, and visualizing galectin-3 in a mixture of proteins. The probes were derived from a galectin-3 binding 15-mer peptide sequence in which a benzophenone photolabel was incorporated at the N-terminus and in another case as a phenyl alanine replacement in the middle of the sequence. Detection of galectin-3 was possible in Escherichia coli lysates that were spiked with various amounts of galectin-3.     

GDNF is a chemoattractant for enteric neural cells

In situ hybridization revealed that GDNF mRNA in the mid- and hindgut mesenchyme of embryonic mice was minimal at E10.5 but was rapidly elevated at all gut regions after E11, but with a slight delay (0.5 days) in the hindgut. GDNF mRNA expression was minimal in the mesentery and in the pharyngeal and pelvic mesenchyme adjacent to the gut. To examine the effect of GDNF on enteric neural crest-derived cells, segments of E11.5 mouse hindgut containing crest-derived cells only at the rostral ends were attached to filter paper supports and grown in catenary organ culture. With GDNF (100 ng/ml) in the culture medium, threefold fewer neurons developed in the gut explants and fivefold more neurons were present on the filter paper outside the gut explants, compared to controls. Thus, in controls, crest-derived cells colonized the entire explant and differentiated into neurons, whereas in the presence of exogenous GDNF, most crest-derived cells migrated out of the gut explant. This is consistent with GDNF acting as a chemoattractant. To test this idea, explants of esophagus, midgut, superior cervical ganglia, paravertebral sympathetic chain ganglia, or dorsal root ganglia from E11.5-E12.5 mice were grown on collagen gels with a GDNF-impregnated agarose bead on one side and a control bead on the opposite side. Migrating neural cells and neurites from the esophagus and midgut accumulated around the GDNF-impregnated beads, but neural cells in other tissues showed little or no chemotactic response to GDNF, although all showed GDNF-receptor (Ret and GFRalpha1) immunoreactivity. We conclude that GDNF may promote the migration of crest cells throughout the gastrointestinal tract, prevent them from straying out of the gut (into the mesentery and pharyngeal and pelvic tissues), and promote directed axon outgrowth.     

半乳糖凝集素3在骨肉瘤患者血清及肉瘤组织中的表达研究

通过检测骨肉瘤患者血清中半乳糖凝集素3(galectin-3)的水平以及骨肉瘤组织标本中galectin-3的表达情况,探讨其在骨肉瘤患者血清及肿瘤组织中的表达情况并分析其与骨肉瘤临床特征的关系。实验结果显示galectin-3在骨肉瘤患者血清中的表达水平与Enncking分期相关,且随着骨肉瘤组织临床分期及肿瘤发展,galectin-3的表达呈上升趋势,这说明galectin-3与骨肉瘤的发生、发展密切相关,可能在其发生、发展过程中起着重要作用。     

Interaction of a novel cysteine and histidine-rich cytoplasmic protein with galectin-3 in a carbohydrate-independent manner

We have used the yeast two-hybrid system to search for cytoplasmic proteins that might assist in the intracellular trafficking of the soluble beta-galactoside-binding protein, galectin-3. We utilised as bait murine full-length galectin-3 to screen a murine 3T3 cDNA library. Several interacting clones were found to encode a partial open reading frame and a full-length clone was obtained by rapid amplification of cDNA ends methodology. In various assays in vitro the novel protein was shown to bind galectin-3 in a carbohydrate-independent manner. The novel protein contains an unusually high content of cysteine and histidine residues and shows significant sequence homologies with several metal ion-binding motifs present in known proteins. Confocal immunofluorescence microscopy of permeabilised 3T3 cells shows a prominent perinuclear, as well as cytoplasmic, localisation of the novel protein.     

Human galectin-8 isoforms and cancer

Galectins are animal lectins that can specifically bind β-galactosides. Thirteen galectins have already been described. This review focuses on a specific member of this family: galectin-8. This galectin was discovered in prostate cancer cells eight years ago and has been studied extensively in the last few years. The galectin-8 gene (LGALS8) encodes numerous mRNAs by alternate splicing and the presence of three unusual polyadenylation signals. These mRNAs encode six different isoforms of galectin-8: three belong to the tandem-repeat galectin group (with two CRDs linked by a hinge peptide) and three to the prototype group (with one CRD). Various studies showed that galectin-8 is widely expressed in tumor tissues as well as in normal tissues. The level of galectin-8 expression may correlate with the malignancy of human colon cancers and the degree of differentiation of lung squamous cell carcinomas and neuro-endocrine tumors. Recently, the differences in galectin-8 expression levels between normal and tumor tissues have been used as a guide for the selection of strategies for the prevention and treatment of lung squamous cell carcinoma. These experiments are still under investigation, but demonstrate the potential of galectin-8 research to enhance our understanding of, and possibly prevent, the process of neoplastic transformation. Published in 2004.     

Production and characterization of a monoclonal antibody able to discriminate galectin-1 from galectin-2 and galectin-3

Antisera raised against galectin-1 exhibit crossreactivities with other galectins or related molecules. In order to overcome this problem, a monoclonal antibody to human brain galectin-1 was obtained by selecting clones without reactivity toward galectin-3. This mAb specifically bound galectin-1 of various animal origins but neither galectin-2 nor galectin-3. Western-blotting analysis of soluble human brain extracts after 2D gel electrophoresis revealed only the two most acidic isoforms of galectin-1. The ability of this mAb to bind galectin-1/asialofetuin complexes indicates that its epitope is not localized in the carbohydrate recognition domain of galectin-1. This particularity induces with efficiency its monospecificity.      

Arachidonic acid is a chemoattractant for Dictyostelium discoideum cells

Cyclic AMP (cAMP)is a natural chemoattractant of the social amoeba Dictyostelium discoideum. It is detected by cell surface cAMP receptors. Besides a signalling cascade involving phosphatidylinositol 3,4,5-trisphosphate (PIP3), Ca2+ signalling has been shown to have a major role in chemotaxis. Previously, we have shown that arachidonic acid (AA) induces an increase in the cytosolic Ca2+ concentration by causing the release of Ca2+ from intracellular stores and activating influx of extracellular Ca2+. Here we report that AA is a chemoattractant for D. discoideum cells differentiated for 8-9 h. Motility towards a glass capillary filled with an AA solution was dose-dependent and qualitatively comparable to cAMP-induced chemotaxis. Ca2+ played an important role in AA chemotaxis of wild-type Ax2 as ethyleneglycol-bis(b-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA) added to the extracellular buffer strongly inhibited motility. In the HM1049 mutant whose iplA gene encoding a putative Ins(1,4,5)P3 -receptor had been knocked out, chemotaxis was only slightly affected by EGTA. Chemotaxis in the presence of extracellular Ca2+ was similar in both strains. Unlike cAMP, addition of AA to a cell suspension did not change cAMP or cGMP levels. A model for AA chemotaxis based on the findings in this and previous work is presented.     

Human galectin-8 isoforms and cancer

Galectins are animal lectins that can specifically bind beta-galactosides. Thirteen galectins have already been described. This review focuses on a specific member of this family: galectin-8. This galectin was discovered in prostate cancer cells eight years ago and has been studied extensively in the last few years. The galectin-8 gene ( LGALS8) encodes numerous mRNAs by alternate splicing and the presence of three unusual polyadenylation signals. These mRNAs encode six different isoforms of galectin-8: three belong to the tandem-repeat galectin group (with two CRDs linked by a hinge peptide) and three to the prototype group (with one CRD). Various studies showed that galectin-8 is widely expressed in tumor tissues as well as in normal tissues. The level of galectin-8 expression may correlate with the malignancy of human colon cancers and the degree of differentiation of lung squamous cell carcinomas and neuro-endocrine tumors. Recently, the differences in galectin-8 expression levels between normal and tumor tissues have been used as a guide for the selection of strategies for the prevention and treatment of lung squamous cell carcinoma. These experiments are still under investigation, but demonstrate the potential of galectin-8 research to enhance our understanding of, and possibly prevent, the process of neoplastic transformation.     

Brain-derived growth factor is a chemoattractant for fibroblasts and astroglial cells

Bovine brain-derived growth factor (BDGF), a 16-17 kDa protein with biochemical properties resembling brain-derived acidic fibroblast growth factor (acidic FGF) and endothelial cell growth factor, was found to have potent chemotactic activity for bovine ligament fibroblasts, human skin fibroblasts and rat astroglial cells, maximal at 100-200 pg/ml. The chemotactic activity was completely blocked by protamine sulfate (5 ug/ml), an inhibitor of receptor-binding and mitogenic activity of BDGF. BDGF did not stimulate migration of human monocytes. These results indicate that the effects of BDGF 'in vivo' might extend to mesenchymal cell recruitment.