The calcium-sensing receptor is silenced by genetic and epigenetic mechanisms in unfavorable neuroblastomas and its reactivation induces ERK1/2-dependent apoptosis.

Neuroblastic tumors (NTs) include the neuroblastomas, ganglioneuroblastomas and ganglioneuromas. We have reported previously that the calcium-sensing receptor is expressed in differentiated, favorable NTs but almost undetectable in unfavorable neuroblastomas. We have now detected hypermethylation of a particular region within the CpG island encompassing the CaSR gene promoter 2 in neuroblastoma cell lines and 25% primary neuroblastomas. Hypermethylation of this region was associated with reduced CaSR messenger RNA expression and several predictors of poor outcome in neuroblastomas, including MYCN amplification. Treatment with 5'aza-2-deoxycitidine and/or trichostatin A restored CaSR expression in MYCN-amplified cell lines. Following 5'aza-2-deoxycitidine exposure, decreased percentages of methylated CpG sites were observed at the above-mentioned region. By interphase fluorescence in situ hybridization, variable percentages of nuclei with monosomy of chromosome 3, where the human CaSR gene resides, were observed in more than 90% of primary NTs of all subgroups. Nuclei harboring this alteration were heterogeneously distributed among tumor cells. Ectopic overexpression of the calcium-sensing receptor in two MYCN-amplified neuroblastoma cell lines in which this gene is silenced by promoter hypermethylation significantly reduced their in vitro proliferation rates and almost abolished their capacity to generate xenografts in immunocompromised mice. Finally, upon acute exposure to calcium, the primary activator of this receptor, calcium-sensing receptor-overexpressing neuroblastoma cells underwent apoptosis, a process dependent on sustained activation of ERK1/2. These data would support the hypothesis that epigenetic silencing of the CaSR gene is neither an in vitro artefact in neuroblastoma cell lines nor an irrelevant, secondary event in primary NTs, but a significant mechanism for neuroblastoma survival.

Characterization and measurement of the plasma alpha- and beta-sex hormone-binding globulin paralogs in salmon.

When the biochemical characteristics of coho salmon SHBG (csSHBG) plasma were examined, two different steroid-binding profiles were obtained corresponding to recombinant csSHBG-alpha and csSHBG-beta. These SHBG paralogs share only 24% sequence identity, and this explains their unique steroid-binding properties. Both proteins bind testosterone, but csSHBG-alpha also binds androstenedione (Kd = 2.8 nm) and ethinylestradiol with high affinity, whereas csSHBG-beta binds estradiol (Kd = 0.8 nm) preferentially. When analyzed by gel filtration, csSHBG-alpha displays the properties of a 153-kDa homodimer, whereas csSHBG-beta elutes as a 68-kDa monomer. The unique steroid-binding properties of csSHBG-alpha and csSHBG-beta allowed us to develop an assay for their measurements in immature (pre-smolt) and mature coho salmon blood. Plasma csSHBG-alpha levels were 3- to 4-fold higher than those of csSHBG-beta irrespective of developmental stage or sex and correlate with each other. The major site of csSHBG-alpha expression in pre-smolts and mature fish is the liver, but low levels of csSHBG-alpha mRNA are present in stomach/intestine of mature fish. In pre-smolts, high levels of csSHBG-beta mRNA are present in gills and ovary, whereas csSHBG-beta mRNA is most abundant in muscle and stomach/intestine of mature fish. Based on the differences in csSHBG-alpha and csSHBG-beta plasma levels and their tissue expression profiles, we conclude that gills and/or muscle contribute mainly to plasma SHBG-beta in coho salmon. The assays we have developed will enable studies of how SHBG-alpha/SHBG-beta biosynthesis is regulated throughout the salmonid life cycle and how they influence steroid hormone action in these fish.

In silico identification of anthropogenic chemicals as ligands of zebrafish sex hormone binding globulin.

Anthropogenic compounds with the capacity to interact with the steroid-binding site of sex hormone binding globulin (SHBG) pose health risks to humans and other vertebrates including fish. Building on studies of human SHBG, we have applied in silico drug discovery methods to identify potential binders for SHBG in zebrafish (Danio rerio) as a model aquatic organism. Computational methods, including; homology modeling, molecular dynamics simulations, virtual screening, and 3D QSAR analysis, successfully identified 6 non-steroidal substances from the ZINC chemical database that bind to zebrafish SHBG (zfSHBG) with low-micromolar to nanomolar affinities, as determined by a competitive ligand-binding assay. We also screened 80,000 commercial substances listed by the European Chemicals Bureau and Environment Canada, and 6 non-steroidal hits from this in silico screen were tested experimentally for zfSHBG binding. All 6 of these compounds displaced the [(3)H]5alpha-dihydrotestosterone used as labeled ligand in the zfSHBG screening assay when tested at a 33 microM concentration, and 3 of them (hexestrol, 4-tert-octylcatechol, and dihydrobenzo(a)pyren-7(8H)-one) bind to zfSHBG in the micromolar range. The study demonstrates the feasibility of large-scale in silico screening of anthropogenic compounds that may disrupt or highjack functionally important protein:ligand interactions. Such studies could increase the awareness of hazards posed by existing commercial chemicals at relatively low cost.

Sex hormone-binding globulin in fish gills is a portal for sex steroids breached by xenobiotics.

As in most vertebrates, plasma sex hormone-binding globulin (SHBG) is produced in fish liver and regulates sex steroid access to target tissues. Low levels of SHBG mRNA are present in zebra fish gills but are unlikely to account for the high amounts of immunoreactive SHBG in filaments and lamellae. Although the uptake of steroids by fish from water has been reported to correlate with their affinity for SHBG, it is not known how this occurs. Our studies of zebra fish SHBG have revealed its preference for biological active androgen (testosterone), as well as for androstenedione, a sex steroid precursor that also acts as a pheromone in some fish. In addition to natural steroids, zebra fish SHBG has a high affinity for synthetic steroids, such as ethinylestradiol and progestins (levonorgestrel and norethindrone), that are present in waste water systems. Because steroids can pass across fish gills, we examined whether SHBG serves as a portal for natural and synthetic steroids controlling their flux between the blood and aquatic environment. The results indicate that SHBG ligands are rapidly and specifically removed from water by the fish through their gills, whereas the accumulated steroids are released slowly. The capacity of fish to sequester SHBG ligands from water is similar between sexes, independent of size, and characterized by a wide dynamic range. We conclude that SHBG controls the flux of sex steroids across fish gills and that this highly specialized function can be hijacked by xenobiotic ligands of fish SHBGs.

Sex hormone-binding globulin expression in sea bass (Dicentrarchus labrax L.) throughout development and the reproductive season.

Sex hormone-binding globulin (SHBG) transports androgens and estrogens in the blood of vertebrate species, including fish, and regulates the bioavailability and metabolic clearance of these steroids. Liver is the major site of plasma SHBG synthesis, while an SHBG homologue, known as the androgen-binding protein, is produced in testes. When shbg gene expression was examined throughout European sea bass (Dicentrarchus labrax L.) development, SHBG mRNA was clearly detectable at 7 days post-fertilization and persisted throughout embryonic development. In male and female sea bass, the liver is the principal site of shbg gene expression, as determined by SHBG mRNA analyses. Immunoreactive SHBG is present in the liver and villous stroma of the intestine in both sexes. It is also present in the interstitial space between testicular lobules, and the connective tissue surrounding the ovary in the non-reproductive season and around post-vitellogenic oocytes. Plasma SHBG levels were measured over a 10-month period as male sea bass undergo sexual maturation. Immature females of the same age were also studied over the same time interval. The mean+/-S.E.M. plasma SHBG levels in 2-year-old males and females are lower (80+/-15nM and 82+/-16nM, respectively) during the winter reproductive season (December-March) than the spring (April-June) months (144+/-32nM and 193+/-18nM, respectively). In both sexes, plasma SHBG levels start to decline 1-2 months before the reproductive season, coincident with a period of rapid weight gain, while increases after the reproductive season are not accompanied by significant changes in body weight. In addition, plasma SHBG in triploid (sterile) and diploid (fertile) male sea bass do not differ during the first spawning season. These data suggest that the decrease in plasma SHBG levels during sexual maturation in sea bass is related to nutritional or metabolic effects in relation to water temperatures and food intake, rather than changes in gonadal sex steroid production.

Sea bass (Dicentrarchus labrax) sex hormone binding globulin: molecular and biochemical properties and phylogenetic comparison of its orthologues in multiple fish species.

Sex hormone binding globulin (SHBG) binds and transports androgens and estrogens in the blood of vertebrate species including fish. We have used oligonucleotide primers corresponding to highly conserved regions of the SHBG coding sequences within the zebrafish and fugufish genomes to obtain a 1528 bp cDNA encoding SHBG from tissue RNA extracts from the European sea bass. Amino-terminal sequence analysis of recombinant sea bass SHBG indicated that its deduced precursor polypeptide includes a 35-residue secretion signal polypeptide, and the 361-residue mature sea bass SHBG sequence exhibits 45-67% sequence identity with SHBGs from other fish species that have been determined directly (for zebrafish) or deduced (for rainbow trout, medaka and fugufish) from sequences within public databases. The sea bass SHBG (39,894 Da) comprises a tandem repeat of laminin G-like domains typical of SHBG sequences; contains three N-glycosylation sites, and exists as a 118,300 +/- 11,500 Da homodimer. Sea bass SHBG exhibits a high affinity (K(d) = 8.8 nM for 17beta-estradiol) and specificity for gonadal steroids and their precursors (e.g., 17beta-estradiol > testosterone > dehydroepiandrosterone > 5alpha-dihydrotestosterone > androstenedione >11-ketotesterone). Interestingly, the affinity of sea bass SHBG for the synthetic estrogen, 17alpha-ethynylestradiol was found to be essentially identical to that for 17beta-estradiol. The availability of SHBG sequences in sea bass and other fish set the stage for detailed studies of SHBG function in fish reproductive physiology, as well as its potential role as a target of endocrine disruptors.

Molecular and functional characterization of sex hormone binding globulin in zebrafish.

SHBG (sex hormone binding globulin) transports androgens and estrogens in the blood of vertebrates including fish. Orthologs of SHBG in fish are poorly defined, and we have now obtained a zebrafish SHBG cDNA and characterized the zebrafish SHBG gene and protein through molecular biological, biochemical, and informatics approaches. Amino-terminal analysis of zebrafish SHBG indicated that its deduced precursor sequence includes a 25-residue secretion polypeptide and exhibits 22-27% homology with mammalian SHBG sequences and 41% with a deduced fugufish SHBG sequence. The 356-residue mature zebrafish SHBG (39,243 Da) sequence comprises a tandem repeat of laminin G-like domains typical of SHBG sequences; contains three N-glycosylation sites; and exists as a 105,000 +/- 8700 Da homodimer. Zebrafish SHBG exhibits a high affinity and specificity for sex steroids. An RT-PCR indicated that SHBG mRNA first appears in zebrafish larva, and SHBG mRNA was localized within the liver and gut at this stage of development by whole-mount in situ hybridization. In adult fish, SHBG mRNA was found in liver, testis, and gut. In the liver, immunoreactive SHBG was present in hepatocytes and concentrated in intrahepatic bile duct cells, whereas in the testis it was confined to cells surrounding the seminiferous tubule cysts. In the intestine, immunoreactive SHBG was present in the stroma and epithelial cells of the villous projections and the surrounding muscle. The production and presence of SHBG in the gut of developing and adult zebrafish suggests a novel role for this protein in regulating sex steroid action at this site.

Phylogenetic comparisons implicate sex hormone-binding globulin in "masculinization" of the female spotted hyena (Crocuta crocuta).

Exposures to sex steroids during fetal development are thought to contribute to the unique urogenital anatomy and social dominance of the female spotted hyena: overt phenotypes not shared by other hyenids (i.e. striped hyena, brown hyena, and aardwolf). Because both androgens and estrogens influence development of genitalia and behavior, and because plasma SHBG regulates their access to tissues, we compared the Shbg gene sequences, structures, and steroid-binding properties in the four extant hyenids. We found the hyenid Shbg genes (>95% identical) and mature protein sequences (98% identical) are highly conserved. As in other mammals, the hyenid SHBG all bind 5α-dihydrotestosterone with high affinity (K(d) = 0.62-1.47 nm), but they also bind estrone and dehydroepiandrosterone with similarly high affinity, and this unusual property was attributed to specific amino acids within their SHBG steroid-binding sites. Phylogenetic comparisons also indicated that the spotted hyena SHBG precursor uniquely lacks two leucine residues and has a L15W substitution within its secretion signal polypeptide, the reduced size and hydrophobicity of which markedly decreases the production of SHBG and may therefore explain why serum SHBG concentrations in male and female spotted hyenas are approximately five times lower than in other hyenids. This is important because low plasma SHBG concentrations in spotted hyenas will increase exposure to biologically active androgens and estrogen as well as to their precursors (dehydroepiandrosterone and estrone), which may contribute to the masculinized external genitalia of female spotted hyenas and to female social dominance over males.