Graham F. Wagner

Human stanniocalcin inhibits renal phosphate excretion in the rat

Stanniocalcin (STC) is a glycoprotein hormone first identified in bony fishes where it counteracts hypercalcemia by inhibiting gill calcium uptake and stimulating renal inorganic phosphate (Pi) reabsorption. Human STC (hSTC) has recently been cloned and sequenced and is highly homologous to the fish hormone at the amino acid level. The objective of this study was to examine the possible effects of hSTC on electrolyte homeostasis and renal function in the rat. Recombinant hSTC was expressed in bacteria and purified by metal‐ion affinity chromatography and reverse‐phase high performance liquid chromatography. Anesthetized animals were given bolus infusions of 1, 5, or 10 nmol hSTC per kilogram of body weight. Control animals received solvent alone. The most effective dosage was 5 nmol/kg, which caused significant reductions in both absolute and fractional phosphate excretion in comparison with control rats. The hSTC had no effect on the renal excretion of other ions, the glomerular filtration rate, renal blood flow, blood pressure, or plasma electrolytes (Na+, K+, Ca2+, Pi, Mg2+). The maximum effect of hSTC on phosphate excretion was observed 60–80 minutes postinjection. Lesser effects were obtained with higher and lower dosages of hormone. When renal cortical brush‐border membrane vesicles were isolated from control and hormone‐treated animals 80 minutes postinjection, the rate of Na+/Pi cotransport was found to be 40% higher in vesicles from hormone‐treated animals (p < 0.01; 5 nmol hSTC/kg). Together, the renal clearance and membrane vesicle data indicate that hSTC participates in the renal regulation of Pi homeostasis in mammals.

Comparative analysis of mammalian stanniocalcin genes.

The recent discovery of mammalian stanniocalcin (STC) prompted an investigation of its gene structure and expression pattern to study its function and regulation. We show that both the human and mouse genes are composed of four exons spanning about 13 kb, with 85% nucleotide sequence identity in coding regions. Remarkably high sequence conservation between species also exists in the approximately 3-kb 3′-untranslated region. Comparative analysis of the 5′-untranslated region and flanking DNA from the rat and human STC genes showed long stretches of CAG trinucleotide repeats and an additional (CA)25 dinucleotide repeat unique to the rat promoter. An analysis of STC expression in the mouse showed that ovary contained the highest level of messenger RNA, with lower, but detectable, levels in most tissues. In situ hybridization revealed strong, specific hybridization over the thecal-interstitial cells of the ovarian stroma, whereas immunohistochemical analysis indicated that STC was present not only in the str...

Development of a human stanniocalcin radioimmunoassay: serum and tissue hormone levels and pharmacokinetics in the rat.

Stanniocalcin (STC) is a polypeptide hormone that was first discovered in fish and recently identified in humans and other mammals. In fish STC is produced by one gland, circulates freely in the blood and plays an integral role in mineral homeostasis. In mammals, STC is produced in a number of different tissues and serves a variety of different functions. In kidney, STC regulates phosphate reabsorption by proximal tubule cells, whereas in ovary it appears to be involved in steroid hormone synthesis. However there is no information on circulating levels of STC in mammals or the regulation of its secretion. In this report we have developed a radioimmunoassay (RIA) for human STC. The RIA was validated for measuring tissue hormone levels. However human and other mammalian sera were completely devoid of immunoreactive STC (irSTC). To explore the possibility that mammalian STC might have a short half-life pharmacokinetic analysis was carried out in rats. STC pharmacokinetics were best described by a two compartment model where the distribution phase (t1/2(alpha)) equaled 1 min and the elimination phase (t1/2(beta)) was 60 min. However the STC in the elimination phase no longer crossreacted in the RIA indicating it had undergone substantial chemical modification, which could explain our inability to detect irSTC in mammalian sera. When we compared the pharmacokinetics of human and fish STC in mammalian and fish models the human hormone was always eliminated faster, indicating that human STC has unique structural properties. There also appears to be a unique clearance mechanism for STC in mammals. Hence there are major differences in the delivery and biology of mammalian STC. Unlike fishes, mammalian STC does not normally circulate in the blood and functions instead as a local mediator of cell function. Future studies will no doubt show that this has had important ramifications on function as well.

Studies on the regulation and characterization of plasma stanniocalcin in rainbow trout

Stanniocalcin (STC) is a hormone that is synthesized and secreted by the corpuscles of stannius (CS), endocrine glands that are unique to the bony fishes. The hormone inhibits Ca2+ transport from the aquatic environment into the bloodstream by way of the gills. Previous in vitro studies by our laboratory have shown that STC secretion is positively regulated by Ca2+ in a time- and dose-dependent fashion. In this report, we have examined circulating levels of STC in free-swimming, cannulated rainbow trout and how hormone levels are affected by surgical stress and intra-arterial infusions of mono and divalent cations. In addition, the plasma hormone has been concentrated by immunoaffinity chromatography and characterized by Western blot analysis. The results suggest that the in vivo response of the CS is extremely rapid and Ca(2+)-specific and that STC circulates in multiple molecular weight forms.

Evidence for calcium-sensing receptor mediated stanniocalcin secretion in fish.

The secretion of parathyroid hormone (PTH) and calcitonin (CT) in mammals are both tightly regulated by the prevailing levels of extracellular ionic calcium (Ca(2+)). And, it is now widely recognized that both of these Ca(2+) effects are mediated exclusively through a seven transmembrane calcium sensing receptor or CaR. As in the case of PTH and CT, the secretion of stanniocalcin (STC) in fish is tightly regulated by the levels of extracellular Ca(2+). Fish STC functions as an anti-hypercalcemic hormone such that a rise in extracellular Ca(2+) above the physiological set-point of approximately 1.2 mM provokes an immediate secretory response. Whether or not Ca(2+)-regulated STC secretion in fishes is mediated by similar type of receptor has never been addressed. Here, we have found that Ca(2+)-stimulated STC secretion in salmon is mimicked by CaR mimetics, pharmacological agents that increase the sensitivity of the CaR to calcium. NPS 467, a small organic molecule that acts as a positive allosteric modulator of the CaR and alters calciotropic hormone secretion in mammals, was examined for effects on serum levels of STC in trout. The IP administration of NPS R-467 had time and dose-dependent stimulatory effects on STC secretion that were indistinguishable from those of Ca(2+) loading. The effects of NPS 467 were stereospecific and had no effects on serum CT. NPS 467 induced STC release was also manifested by a downstream physiological response; the inhibition of gill calcium transport. A cDNA clone was amplified from a fish corpuscle of Stannius cDNA library with high homology to the human CaR. RT-PCR revealed that this transcript was also present in gill, kidney, pancreas, brain, muscle and spleen. These findings suggest that Ca(2+)-stimulated STC secretion in fishes is mediated by a calcium ion-sensing receptor similar to that in mammals.

Molecular cloning and cDNA sequence analysis of coho salmon stanniocalcin

Stanniocalcin (STC) (formerly known as both teleocalcin and hypocalcin) is an anti-hypercalcemic, glycoprotein hormone that is produced by the corpuscles of Stannius (CS), endocrine glands that are confined to bony fishes. The hormone has a unique amino acid sequence and exists as a disulfide-linked homodimer in the native state. In previous studies, we have described the purification and characterization of two salmon STCs, and examined the regulation of hormone secretion in response to calcium using both in vitro and in vivo model systems. This report describes the molecular cloning and cDNA sequence analysis of a coho salmon STC messenger RNA (mRNA) from a salmon CS lambda gt10 cDNA library. The STC mRNA in salmon is approximately 2 kilobases in length and encodes a primary translation product of 256 amino acids. The first 33 residues comprise the prepro region of the hormone, whereas the remaining 223 residues make up the mature form of the hormone. One N-linked, glycosylation consensus sequence was identified in the protein coding region as well as an odd number of half cysteine residues, the latter of which would allow for interchain bonding or dimerization of monomeric subunits. In addition, three sites were identified in the mature protein core of STC (two dibasic, one tribasic) that may be acted upon by endopeptidases to produce truncated forms of the hormone. In support of this latter possibility, Western blot analysis revealed multiple molecular weight forms of sTC within salmon glands.

The respiratory effects of stanniocalcin-1 (STC-1) on intact mitochondria and cells : STC-1 uncouples oxidative phosphorylation and its actions are modulated by nucleotide triphosphates

Stanniocalcin-1 (STC-1) is one of only a handful of hormones that are targeted to mitochondria. High affinity receptors for STC-1 are present on cytoplasmic membranes and both the outer and inner mitochondrial membranes of nephron cells and hepatocytes. In both cell types, STC-1 is also present within the mitochondrial matrix and receptors presumably enable its sequestration. Furthermore, studies in bovine heart sub-mitochondrial particles have shown that STC-1 has concentration-dependent stimulatory effects on electron transport chain activity. The aim of the present study was to determine if the same effects could be demonstrated in intact, respiring mitochondria. At the same time, we also sought to demonstrate the functionality, if any, of an ATP binding cassette that has only recently been identified within the N-terminus of STC-1 by Prosite analysis. Intact, respiring mitochondria were isolated from rat muscle and liver and exposed to increasing concentrations of recombinant human STC-1 (STC-1). Following a 1h exposure to 500 nM STC-1, mitochondria from both organs displayed significant increases in respiration rate as compared to controls. Moreover, STC-1 uncoupled oxidative phosphorylation as ADP:O ratios were significantly reduced in mitochondria from both tissues. The resulting uncoupling was correlated with enhanced mitochondrial (45)Ca uptake in the presence of hormone. Respiratory studies were also conducted on a mouse inner medullary collecting cell line, where STC-1 had time and concentration-dependent stimulatory effects within the physiological range. In the presence of nucleotide triphosphates such as ATP and GTP (5mM) the respiratory effects of STC-1 were attenuated or abolished. Receptor binding studies revealed that this was due to a four-fold decrease in binding affinity (KD) between ligand and receptor. The results suggest that STC-1 stimulates mitochondrial electron transport chain activity and calcium transport, and that these effects are negatively modulated by nucleotide triphosphates.

Stanniocalcin gene expression during mouse urogenital development: A possible role in mesenchymal-epithelial signalling

Stanniocalcin (STC) is a polypeptide hormone first discovered in fish and more recently in mammals. In mammals, the STC gene is widely expressed and the hormone is involved in a variety of functions, but STC does not normally circulate in the blood. In both kidney and gut, STC regulates phosphate fluxes across the transporting epithelia, whereas in brain it protects neurons against cerebral ischemia and promotes neuronal cell differentiation. However, the gene is most highly expressed in ovary and expression is dramatically up‐regulated by both pregnancy and nursing. STC mRNA levels are also high in the developing mouse embryo, but literally nothing is known of the tissue pattern of gene expression. Therefore, the aim of this study was to map the temporal and spatial patterns of gene expression during mouse embryologic development, starting with the urogenital system where the gene is so highly expressed in adults. STC mRNA was evident as early as E10.5 in both the mesonephros and genital ridge. Between E10.5 and 14.5 in developing kidney, STC was produced in undifferentiated mesenchyme cells and sequestered by ureteric bud epithelial cells that did not express the gene but nonetheless contained high levels of STC protein. Thereafter, the distribution pattern resembled that in adults such that gene expression predominated in collecting duct cells, whereas protein was present in most nephron segments. The pattern of gene expression during gonadal development was sexually dimorphic. In males, expression was first evident on E12.5 in interstitial mesenchyme cells surrounding the developing sex cords, whereas the protein accumulated in developing gonocytes within the sex cords that did not express the gene. This pattern became more pronounced over the course of gestation. In contrast, ovarian gene expression was only weakly evident during development. Collectively, the evidence suggests that in addition to its regulatory effects in adults, STC has novel and distinctive roles in the mesenchymal‐epithelial interactions that are vital to normal organogenesis.

Molecular cloning and characterization of stanniocalcin-related protein.

Stanniocalcin (STC) is a glycoprotein hormone first discovered in fish and recently identified in humans and mice. In this report we have described the cloning of an STC-like cDNA, designated here as STC related protein (STCrP). Human STCrP (hSTCrP) cDNAs were isolated as expressed sequence tags (ESTs) and predicted a polypeptide of 302 amino acids, with 58%, homology to human STC (hSTC). Ten of the eleven 1/2 cysteine residues that in STC allow for dimerization of monomeric subunits were conserved in hSTCrP. By Northern analysis, three hSTCrP mRNAs were detected and were most abundant in pancreas, spleen and kidney as well as a variety of different transformed cell lines. The high degree of sequence homology suggests that STC and STCrP may have been derived from a common ancestral gene.

Stanniocalcin (STC) in the Endometrial Glands of the Ovine Uterus: Regulation by Progesterone and Placental Hormones

Abstract Stanniocalcin (STC) is a hormone in fish that regulates calcium levels. Mammals have two orthologs of STC with roles in calcium and phosphate metabolism and perhaps cell differentiation. In the kidney and gut, STC regulates calcium and phosphate homeostasis. In the mouse uterus, Stc1 increases in the mesometrial decidua during implantation. These studies determined the effects of pregnancy and related hormones on STC expression in the ovine uterus. In Days 10–16 cyclic and pregnant ewes, STC1 mRNA was not detected in the uterus. Intriguingly, STC1 mRNA appeared on Day 18 of pregnancy, specifically in the endometrial glands, increased from Day 18 to Day 80, and remained abundant to Day 120 of gestation. STC1 mRNA was not detected in the placenta, whereas STC2 mRNA was detected at low abundance in conceptus trophectoderm and endometrial glands during later pregnancy. Immunoreactive STC1 protein was detected predominantly in the endometrial glands after Day 16 of pregnancy and in areolae that transport uterine gland secretions across the placenta. In ovariectomized ewes, long-term progesterone therapy induced STC1 mRNA. Although interferon tau had no effect on endometrial STC1, intrauterine infusions of ovine placental lactogen (PL) increased endometrial gland STC1 mRNA abundance in progestinized ewes. These studies demonstrate that STC1 is induced by progesterone and increased by a placental hormone (PL) in endometrial glands of the ovine uterus during conceptus (embryo/fetus and extraembryonic membranes) implantation and placentation. Western blot analyses revealed the presence of a 25-kDa STC1 protein in the endometrium, uterine luminal fluid, and allantoic fluid. The data suggest that STC1 secreted by the endometrial glands is transported into the fetal circulation and allantoic fluid, where it is hypothesized to regulate growth and differentiation of the fetus and placenta, by placental areolae.