Identification of galectin-3-binding protein as a factor secreted by tumor cells that stimulates interleukin-6 expression in the bone marrow stroma

We have previously demonstrated that neuroblastoma cells increase the expression of interleukin-6 by bone marrow stromal cells and that stimulation does not require cell-cell contact. In this study we report the purification and identification of a protein secreted by neuroblastoma cells that stimulates interleukin-6 production by stromal cells. Using a series of chromatographic purification steps including heparin-affinity, ion exchange, and molecular sieve chromatography followed by trypsin digestion and liquid chromatography tandem mass spectrometry, we identified in serum-free conditioned medium of neuroblastoma cells several secreted peptides including galectin-3-binding protein, also known as 90-kDa Mac-2-binding protein. We demonstrated the presence of the galectin-3-binding protein in the conditioned medium of several neuroblastoma cell lines and in chromatographic fractions with interleukin-6 stimulatory activity. Consistently, bone marrow stromal cells express galectin-3, the receptor for galectin-3-binding protein. Supporting a role for galectin-3-binding protein in stimulating interleukin-6 expression in bone marrow stromal cells, we observed that recombinant galectin-3-binding protein stimulated interleukin-6 expression in these cells and that interleukin-6 stimulation by neuroblastoma-conditioned medium was inhibited in the presence of lactose or a neutralizing anti-galectin-3 antibody. Down-regulation of galectin-3-binding protein expression in neuroblastoma cells also decreased the interleukin-6 stimulatory activity of the conditioned medium on bone marrow stromal cells. We also provide evidence that stimulation of interleukin-6 by galectin-3-binding protein involves activation of the Erk1/2 pathway. The data, thus, identifies galectin-3-binding protein as a factor secreted by neuroblastoma cells that stimulates the expression of interleukin-6 in bone marrow stromal cells and provides a novel function for this protein in cancer progression.     

A sialic acid-binding lectin from ovine placenta: purification,specificity and interaction with actin

A sialic-acid-specific lectin from ovine placental cotyledons was purified by affinity chromatography on bovine submaxillary mucin-agarose followed by gel filtration, and it showed a molecular weight of 65 000 by sodium dodecylsulfate-polyacrylamide gel electrophoresis. This lectin has the capacity to interact with actin, since in binds to actin-F in a cosedimentation assay and it acts as a mediator in the binding of action to the affinity column. The lectin agglutinated rabbit and rat erythrocytes, but not human A, B or O erythrocytes. Haemagglutination inhibition assays of different saccharides, glycoproteins and glycolipids indicate that this lectin has affinity for sialic acid, which is enhanced by its O-acetylation. The N-terminal sequence of the protein shows 92% identity with rabbit and porcine uterine calreticulin.     

Detection and characterization of an ovine placental lactogen stable intermediate in the urea-induced unfolding process.

The urea-induced equilibrium unfolding of ovine placental lactogen, purified from ovine placenta, was followed by size-exclusion chromatography, far-UV CD, and intrinsic tryptophan fluorescence. The data obtained by each of these methods showed a poor fit to a two-state model involving only a native and an unfolded form. A satisfactory fit required, instead, a model that involved a stable, partially folded form in addition to the native and unfolded ones. The results obtained from the best-fitting theoretical curves for the three-state model indicated that this intermediate state, which is the predominant species in solution at 3.6 M of urea activity, is compact, largely alpha-helical, and changes considerably the native-like tertiary packing around its tryptophan residues. These findings suggest that this stable intermediate exhibits properties similar to those that characterize the molten globule state.     

Ontogeny and regulation of ovine placental prostaglandin E2 synthase

Recent evidence suggests that ovine placental output of prostaglandin (PG) E2 rises through late gestation partly because of a direct effect of cortisol on PGH2 synthase 2 (PGHS-2) expression and activity within trophoblast tissue. Synthesis of PGE2 is also dependent, however, on PGE2 synthase (PGES), which converts PGH2 to PGE2. We hypothesized that PGES is expressed in the ovine placenta, and that, similar to PGHS-2, expression increases through gestation and is regulated positively by cortisol. Placental tissues from pregnant ewes in mid and late gestation, at term, and during early and active labor were analyzed to determine the gestational profile of PGES. The regulation of PGES expression was assessed in placental tissues from pregnant ewes in which intrafetal cortisol infusion was administered in late gestation, in the presence or absence of an aromatase inhibitor, to block the cortisol-stimulated rise in estradiol. Expression of PGES was analyzed by in situ hybridization, Western blot analysis, and immunohistochemistry. In the placentome, PGES localized to fetal trophoblast cells and endothelial cells in maternal blood vessels, consistent with its contribution to the rise in placental PGE2 output toward the onset of labor and with a role of PGE2 in the local regulation of uteroplacental blood flow, respectively. Expression of PGES mRNA and protein increased with gestation. However, there was no significant further change with labor or during cortisol infusion in the presence or absence of a rise in fetal plasma estradiol, in contrast to reported changes in PGHS-2. These results suggest that PGES is not coregulated with PGHS-2 in the sheep placenta at term. The progressive increase in PGES, however, likely contributes to the rise in circulating PGE2 in the fetus in late pregnancy.     

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.     

Plasma clearance of radiolabelled IGF-1 in the late gestation ovine fetus

We investigated the distribution of radiolabelled IGF-1 in the late gestation ovine fetus by exclusion gel chromatography following intravenous injection of 125I rh (recombinant human) met-IGF-1 into the chronically instrumented fetal lamb (120-130 days, n = 7). One minute after injection of 125I rh met-IGF-1 into the fetal femoral vein, 20.9 +/- 3.1% of the counts circulated in the 150K binding protein region, 55.0 +/- 3.7% in the 50K binding protein region and 18.7 +/- 0.6% in the free or 7K region. The chromatographic profiles obtained in the fetus were in general similar to those previously seen in the adult sheep. After an initial equilibration phase the half life of IGF-1 associated with the 150K binding fractions were 412.1 +/- 103.6 min. Two phases of clearance were observed for IGF-1 in association with the 50K binding fractions, an initial phase with a half life of 30.6 +/- 4.5 min followed by a second phase with a half life of 202.3 +/- 10.3 min. The 7K or 'free' form of IGF-1 had an initial half life of 12.6 +/- 5.1 min. Chromatography of samples of fetal tracheal fluid, fetal urine, amniotic fluid, maternal uterine venous plasma and maternal systemic plasma showed no movement of intact IGF-1 out of the fetal circulation into the fetal fluids or into the maternal circulation. However, when simultaneous samples were obtained from the fetal femoral artery and umbilical vein, higher radioactivity was consistently observed in the fetal femoral artery raising the possibility of placental uptake of IGF-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and immunolocalization of ovine placental retinol-binding protein.

A retinol-binding protein (RBP), synthesized and secreted by ovine allantois in vitro, was purified from culture medium. The protein consisted of three isoelectric variants (pI 5.3-6.1) of identical molecular masses of about 23,000 Da as determined by two-dimensional PAGE under reducing conditions. Thirty-one of the first 34 N-terminal amino acids of the purified protein were sequenced and shown to have complete homology with bovine placental and bovine plasma RBP. The ultraviolet absorption spectrum and fluorescence excitation and emission spectra of the purified ovine placental RBP indicated the presence of bound retinol. Metabolic labeling studies demonstrated that the protein was synthesized by placental membranes. Using antiserum to bovine placental RBP, ovine placental RBP was immunolocalized in trophectoderm of 13-day-old blastocysts and trophectodermal cells of the chorion, endodermal cells lining the allantois, and ectodermal cells lining the amnion of 23-, 45-, and 53-day-old conceptuses. Results from this study suggest that ovine placental membrane epithelia synthesize and secrete RBP. Transport, storage, and metabolism of retinol mediated by placental RBP may be essential for normal embryonic development during pregnancy.     

Ontogeny of insulin receptors in the rat hemochorial placenta

Binding of 125I-insulin to rat placental membranes was time and protein concentration dependent, reversible, and specific. Unlabeled porcine insulin competed for 125I-insulin binding with an IC50 of 65 nM, while IGF-I was much less potent with an IC50 of 2.12 mM. Specific binding of 125I-insulin decreased during the second half of gestation from Days 11 to 19. Scatchard analysis of the binding data for membranes prepared from Gestation Days 11 and 19 yielded typical curvilinear plots which showed a marked decrease in the number of binding sites in late gestation placenta. Beginning on Day 14, insulin binding was characterized with isolated labyrinth and basal zone portions of the hemochorial placenta. There was no evidence for differences in Kd values or the number of binding sites in these two functionally distinct portions of the rat placenta. Crosslinking of 125I-insulin followed by SDS-PAGE showed a single protein with a molecular weight of 130,000 from placental tissues on Gestation Days 11 and 19 and confirmed a gestational decrease in the number of insulin receptors. In solubilized, lectin-purified preparations from placenta and liver membranes, insulin stimulated the phosphorylation of a Mr 95,000 protein. 32P-incorporation into this 95,000 protein was stimulated fivefold by insulin in Day 11 placenta receptor, whereas no detectable 32P-incorporation was found in Day 19 placenta. Thus, while the alpha- and beta-subunits of insulin receptors in mid and late gestation placenta have molecular weights which are similar to receptors in maternal liver, data indicate the presence of a functional difference in insulin-stimulated kinase activities.     

Placental TonEBP/NFAT5 osmolyte regulation in an ovine model of intrauterine growth restriction

TonEBP/NFAT5 (the tonicity-responsive enhancer binding protein/nuclear factor of activated T cells) modulates cellular response to osmotic changes by accumulating inositol and sorbitol inside the cells. Our objective was to assess placental osmolytes, TonEBP/NFAT5 RNA and protein expression, and signaling molecules across gestation between control and intrauterine growth restriction (IUGR) ovine pregnancies. Pregnant sheep were placed in hyperthermic conditions to induce IUGR. Placental tissues were collected at 55, 95, and 130 days gestational age (dGA) to measure inositol, sorbitol, TonEBP/NFAT5 (NFAT5), sodium-dependent myo-inositol transporter (SMIT; official symbol SLC5A3), aldose reductase (AR), and NADPH (official symbol DE-CR1). Placental weight was reduced in IUGR compared to controls at 95 and 130 dGA. Osmolyte concentrations were similar between control and IUGR placentas, but both groups demonstrated a significant decrease in inositol concentration and an increase in sorbitol concentration with advancing gestation. Cytosolic NFAT5 protein decreased significantly from 55 to 95 dGA in both groups, and nuclear NFAT5 protein increased only at 130 dGA in the IUGR group, but no differences were seen between groups for either cytosolic or nuclear NFAT5 protein concentrations. DE-CR1 concentrations were similar between groups and increased significantly with advancing gestational age. AR was lowest at 55dGA, and SLC5A3 increased with advancing gestational age. We conclude that both placental osmolytes inositol and sorbitol (and their corresponding proteins SLC5A3 and AR) change with gestational age and are regulated, at least in part, by NFAT5 and DE-CR1 (NADPH). The inverse relationship between each osmolyte across gestation (e.g., inositol higher in early gestation and sorbitol higher in late gestation) may reflect nutritional needs that change across gestation.     

Purification and Characterization of a Human Brain Galectin-1 Ligand

Abstract: Our previous studies have characterized an endogenous lectin from human brain identified as galectin-1. A soluble ligand of galectin-1 was purified from human brain by affinity chromatography and preparative electrophoresis. The purified ligand (termed HBGp82, for human brain galectin-1-binding polypeptide of 82,000 daltons) has an apparent molecular mass of 82 kDa and is glycosylated by N -linked biantennary complex structures. HBGp82 was partially characterized by microsequencing of peptide fragments. Similar peptides were found in a heat shock of protein of 90,000 daltons, hsp90. However, comparison of apparent molecular weights and matrix-assisted laser desorption mass spectrometry clearly showed that HBGp82 differs to some degree from hsp90.     

Large-Scale Preparation of Recombinant Ovine Prolactin and Determination of Its in Vitro and in Vivo Activity

Recombinant bovine Ala-prolactin (PRL) (GenBank Accession No. V00112) in prokaryotic expression plasmid pMON3401 was mutated using a mutagenesis kit, to prepare plasmid encoding ovine PRL (oPRL) (GenBank Accession No. M27057) Escherichia coli cells transformed with this latter plasmid overexpressed large amounts of oPRL upon induction with nalidixic acid. The expressed protein, found in inclusion bodies, was refolded and purified to homogeneity on a Q-Sepharose column, yielding an electrophoretically pure fraction composed of over 98% monomeric protein of the expected molecular mass of 23 kDa. The biological activity of the recombinant oPRL after proper renaturation was evidenced in vitro by its ability to stimulate proliferation of rat lymphoma Nb₂ cells possessing PRL receptors, to stimulate luciferase activity in HEK 293 cells transiently transfected with oPRL receptors, and to induce progesterone secretion in primary cultures of luteal cells obtained from midpregnant ewes. In contrast to ovine growth hormone or ovine placental lactogen, recombinant oPRL had no galactopoietic effect in lactating ewes.     

Osteopontin is synthesized by uterine glands and a 45-kDa cleavage fragment is localized at the uterine-placental interface throughout ovine pregnancy

Osteopontin (OPN) is a phosphorylated and glycosylated, secreted protein that is present in various epithelial cells and biological fluids. On freezing and thawing or treatment with proteases, the native 70-kDa protein gives rise to 45- and 24-kDa fragments. Secreted OPN functions as an extracellular matrix (ECM) protein that binds cell surface receptors to mediate cell-cell adhesion, cell-ECM communication, and cell migration. In sheep and humans, OPN is proposed to be a secretory product of uterine glandular epithelium (GE) that binds to uterine luminal epithelium (LE) and conceptus trophectoderm to mediate conceptus attachment, which is essential to maintain pregnancy through the peri-implantation period. Cell-cell adhesion, communication, and migration likely are important at the interface between uterus and placenta throughout pregnancy, but to our knowledge, endometrial and/or placental expression of OPN beyond the peri-implantation period has not been documented in sheep. Therefore, the present study determined temporal and spatial alterations in OPN mRNA and protein expression in the ovine uterus between Days 25 and 120 of pregnancy. The OPN mRNA in total ovine endometrium increased 30-fold between Days 40 and 80 of gestation. In situ hybridization and immunofluorescence analyses revealed that the predominant source of OPN mRNA and protein throughout pregnancy was the uterine GE. Interestingly, the 45-kDa form of OPN was detected exclusively, continuously, and abundantly along the apical surface of LE, on conceptus trophectoderm, and along the uterine-placental interface of both interplacentomal and placentomal regions through Day 120 of pregnancy. The 45-kDa OPN is a proteolytic cleavage fragment of the native 70-kDa OPN, and it is the most abundant form in uterine flushes during early pregnancy. The 45-kDa OPN is more stimulatory to cell attachment and cell migration than the native 70-kDa protein. Collectively, the present results support the hypothesis that ovine OPN is a component of histotroph secreted by the uterine GE that accumulates at the uterine-placental interface to influence maternal-fetal interactions throughout gestation in sheep.     

Galectin-3 expression in macrophages is signaled by Ras/MAP kinase pathway and up-regulated by modified lipoproteins

To study the signaling pathway involved in the regulation of galectin-3 expression we used phorbol ester to stimulate macrophage differentiation of THP-1 cells. Treatment with phorbol 12-myristate 13-acetate (PMA) increased significantly the level of expression of galectin-3 in THP-1 cells. PMA-induced galectin-3 overexpression was blocked by: protein kinase C inhibitors staurosporine, calphostin C, and apigenin; tyrosine-specific protein kinase inhibitors genistein and tyrphostin A25; PD 98059, a selective inhibitor of mitogen-activated protein kinase (MAPK) kinase 1 (MEK1 or MKK1); and SB 203580, a specific inhibitor of p38 MAPK. Galectin-3 up-regulation was not affected by exposure to two inhibitors of cAMP-dependent protein kinase (PKA), H-89 and KT5720. Co-transfection of pPG3.5, a plasmid vector containing the rabbit galectin-3 promoter and the constructs pMCL-MKK1 N3 or pRC-RSV-MKK3Glu that constitutively express MKK1 and MKK3, raised the activity of galectin-3 promoter by 185% and 110%, respectively. Co-transfection with a Ha-Ras expression vector stimulated galectin-3 promoter activity approximately 10-fold. Expression of c-Jun or v-Jun raised the level of galectin-3 promoter activity more the three- and fourfold, respectively. Co-transfection of c-Jun and pPG3.5 5'-upstream deletion mutants resulted in a reduction of the galectin-3 promoter activity by 50% to 80%. Transfection of c-Jun, v-Jun or Ha-Ras increased significantly galectin-3 protein in THP-1 cells. These findings indicated that Ras/MEKK1/MKK1-dependent/AP-1 signal transduction pathway plays an important role in the expression of galectin-3 in PMA-stimulated macrophages. We further investigated the effect of modified lipoproteins on galectin-3 expression in macrophages. Murine resident peritoneal macrophages loaded with acetylated low-density lipoprotein (AcLDL) or oxidized LDL (OxLDL) showed increased galectin-3 protein and mRNA. These results showed that treatment of macrophages with PMA or modified lipoproteins results in galectin-3 overexpression. These findings may explain the enhanced expression of galectin-3 in atherosclerotic foam cells and suggest that Ras/MAPK signal transduction pathway is involved in controlling this gene.     

The heparin-binding lectin from ovine placenta: Purification and identification as histone H4

The heparin-binding lectin complex from ovine placental cotyledons was purified by affinity chromatography on heparin-agarose column. It showed three protein bands, which had molecular weights of 13 000, 15 000 and 17 000 by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and the presence of DNA by agarose gel electrophoresis. The protein components of the complex were separated by reverse-phase HPLC. The minimum inhibitory concentrations of glycosaminoglycans were significantly different for the lectin complex and the separated proteins, suggesting affinity changes upon DNA binding. The haemagglutinating activity specificity allowed the characterization of the fraction with a molecular weight of 13 000 as the heparin-binding lectin. This protein was identified as histone H4 by internal sequencing, thus showing that this is the histone responsible for the heparin-binding property of the complex. The accompanying proteins were tentatively identified as histones H2A and H2B. This revised version was published online in November 2006 with corrections to the Cover Date.     

Expression of an isoform of the testis-specific estrogen sulfotransferase in the murine placenta during the late gestational period

Cytosolic sulfotransferases play essential roles in regulating the activities and transfer of steroids. To evaluate their biological significance in the murine uterus and placenta during the course of gestation, we determined their activities with several steroids as substrates. Activated estrogen sulfotransferase (EST) was found in the placenta and uterus during the late gestational period. Reverse-transcribed cDNA of murine placental EST (mpEST) was isolated from mouse placenta at 18 days of gestation and its expression in the tissue coincided with a change in its enzyme activity. The open-reading frame of mpEST encodes a protein composed of 296 amino acids with a predicted molecular mass of 35.5 kDa and was revealed to be an isoform of the murine testis-specific EST gene (99.7%). Also, the amino acid sequence of mpEST showed 49.6 and 77.9% homology with human placental and endometrial EST, respectively, showing that it corresponds to human endometrial EST. COS-7 cells transfected with mpEST exhibited sulfotransferase activity with the phenolic hydroxy groups of steroids and artificial substrates. The best acceptor substrate was estrogen.     

Endothelium-derived nitric oxide synthase protein expression in ovine placental arteries

During the third trimester, fetoplacental and uterine blood flows increase dramatically to meet the high metabolic demands of the growing fetus. We hypothesized that the expression of endothelial nitric oxide synthase (eNOS) in fetoplacental artery endothelium and the concentrations of nitric oxide (NO) and cyclic GMP (cGMP) in amniotic fluid (AF) are increased during the third trimester of ovine gestation. Placental arteries and AF were collected from ewes at 110, 120, 130, and 142 days of gestation (n = 24; mean +/- SEM term = 145 +/- 3 days). Expression of eNOS protein was measured in intact and denuded placental arteries and in endothelium-derived protein by Western analysis and confirmed by immunohistochemistry. Concentrations of NO (nitrates plus nitrites) and cGMP were determined in AF. Placental artery eNOS protein expression was localized to the endothelium, where it was markedly greater than in vascular smooth muscle. Placental artery endothelium-derived eNOS expression and AF cGMP concentrations were similar at 110 and 120 days of gestation; however, both peaked at 130 days at levels two- to threefold above baseline (P < 0.05) before returning to baseline at 142 days of pregnancy. The AF NO (nitrates plus nitrites) levels, however, increased progressively between 120 days of gestation and term (P < 0.05). We concluded that endothelium-derived placental artery eNOS levels, AF NO (nitrates plus nitrites), and AF cGMP were markedly increased during the third trimester, thus supporting a role for NO-mediated elevations in cGMP in the control of fetoplacental blood flow.     

Large-scale preparation of recombinant ovine prolactin and determination of its in vitro and in vivo activity

Recombinant bovine Ala-prolactin (PRL) (GenBank Accession No. V00112) in prokaryotic expression plasmid pMON3401 was mutated using a mutagenesis kit, to prepare plasmid encoding ovine PRL (oPRL) (GenBank Accession No. M27057) Escherichia coli cells transformed with this latter plasmid overexpressed large amounts of oPRL upon induction with nalidixic acid. The expressed protein, found in inclusion bodies, was refolded and purified to homogeneity on a Q-Sepharose column, yielding an electrophoretically pure fraction composed of over 98% monomeric protein of the expected molecular mass of approximately 23 kDa. The biological activity of the recombinant oPRL after proper renaturation was evidenced in vitro by its ability to stimulate proliferation of rat lymphoma Nb(2) cells possessing PRL receptors, to stimulate luciferase activity in HEK 293 cells transiently transfected with oPRL receptors, and to induce progesterone secretion in primary cultures of luteal cells obtained from midpregnant ewes. In contrast to ovine growth hormone or ovine placental lactogen, recombinant oPRL had no galactopoietic effect in lactating ewes.     

Galectin-3 expression in response to LPS, immunomodulatory drugs and exogenously added galectin-3 in monocyte-like THP-1 cells

Galectin-3, a structurally unique beta-galactoside-binding lectin, through the specific protein?Cprotein and protein?Ccarbohydrate interactions participates in numerous biological processes, such as cell proliferation and apoptosis, adhesion and activation. Its expression and secretion by until now an unknown mechanism are modulated by diverse molecules and are dependent on different physiological and pathophysiological conditions. By autocrine and paracrine actions, galectin-3 modulates many immune reactions and affects various immune cells, particularly those of monocyte?Cmacrophage lineage. This is why galectin-3 has recently become an attractive therapeutic target. However, molecular mechanisms of its actions as well as regulatory mechanism of its expression and activation are still largely unknown. In this study, we show that lipopolysaccharide (LPS) provokes upregulation of galectin-3 expression on both gene and protein level in monocyte-like THP-1 cells, which can be inhibited by dexamethasone, but not with non-steroidal anti-inflammatory drugs aspirin and indomethacin. Resting and LPS-challenged monocyte-like THP-1 cells do not have detectable amount of surface-bound galectin-3, but are able to bind exogenously added galectin-3 with the same capacity. Although galectin-3 is generally considered to be a pro-inflammatory molecule, here we show that the exogenously added galectin-3 does not affect interleukin (IL)-1??, IL-6, IL-8, IL-10, IL-12p70 and TNF-?? production in resting and LPS-activated monocyte-like THP-1 cells nor influences its own gene expression level in those cells.