Cyclooxygenase-2 mediates induction of the renal stanniocalcin-1 gene by arginine vasopressin.

In rats and mice, the renal stanniocalcin-1 (STC-1) gene is expressed in most nephron segments, but is differentially induced in response to dehydration. In cortical segments STC-1 mRNA levels are upregulated by the hypertonicity of dehydration, while hypovolemia causes gene induction in the inner medulla (papilla). In both cases induction is mediated by arginine vasopressin (AVP) acting via the V2 receptor (V2R). The intent of STC-1 gene upregulation during dehydration has yet to be established. Therefore, to narrow down the range of possible actions, we mapped out the pathway by which V2R occupancy upregulates the gene. V2R occupancy activates two different renal pathways in response to dehydration. The first is antidiuretic in nature and is mediated by direct V2R occupancy. The second pathway is indirect and counter-regulates AVP-mediated antidiuresis. It involves COX-2 (cyclooxygenase-2) and the prostanoids, and is activated by the V2R-mediated rise in medullary interstitial osmolality. The resulting prostanoids counter-regulate AVP-mediated antidiuresis. They also upregulate renal cytoprotective mechanisms. The present studies employed models of COX inhibition and COX gene deletion to address the possible involvement of the COX pathway. The results showed that both general and specific inhibitors of COX-2 blocked STC-1 gene induction in response to dehydration. Gene induction in response to dehydration was also abolished in COX-2 null mice (cortex and papilla), but not in COX-1 null mice. STC-1 gene induction in response to V2R occupancy was also uniquely abolished in COX-2 nulls (both regions). These findings therefore collectively suggest that AVP-mediated elevations in STC-1 gene expression are wholly dependent on functional COX-2 activity. As such, a permissive role for STC-1 in AVP-mediated antidiuresis can be ruled out, and its range of possible actions has been narrowed down to AVP counter-regulation and renal cytoprotection.

Stanniocalcin-1 co-localizes with insulin in the pancreatic islets.

The polypeptide hormone stanniocalcin-1 (STC-1) is widely expressed in mammals and signals both locally and systemically. In many tissues STC-1 ligand is sequestered by target cell organelles (mitochondria, nuclei, and cholesterol lipid droplets) to exert diverse biological effects. Most notably, STC-1 serves as an uncoupler of oxidative phosphorylation in liver, muscle, and kidney mitochondria. The present paper describes the identification of STC-1 receptors in mouse pancreatic ß cells and the discovery that the ligand co-localizes with insulin in pancreatic ß cells. In situ hybridization (ISH) analysis subsequently revealed that pancreatic ß cells were the source of the ligand. Intriguingly however, all ISH signal was localized over putative islet cell nuclei as opposed to the cell cytoplasm. Real-time qPCR and agarose gel electrophoresis revealed that the STC-1 amplicon generated from islet cell total RNA was the same size as that from kidney. However, relative levels of STC-1 gene expression were >100-fold lower in islets than those in kidney tissue. Collectively, these findings are indicative of a local STC-1 signalling pathway in pancreatic ß cells. The role of STC-1 in this context remains to be established, but it could very well entail the regulation of ß cell mitochondria membrane potential which is an integral aspect of regulated insulin release. Interestingly, STC-1 immunoreactivity was not evident in embryonic pancreatic islets, suggesting that ligand synthesis may only commence postnatally.

Stanniocalcin-1 in the subfornical organ inhibits the dipsogenic response to angiotensin II.

Recently, receptors for the calcium-regulating glycoprotein hormone stanniocalcin-1 (STC-1) have been found within subfornical organ (SFO), a central structure involved in the regulation of electrolyte and body fluid homeostasis. However, whether SFO neurons produce STC-1 and how STC-1 may function in fluid homeostasis are not known. Two series of experiments were done in Sprague-Dawley rats to investigate whether STC-1 is expressed within SFO and whether it exerts an effect on water intake. In the first series, experiments were done to determine whether STC-1 was expressed within cells in SFO using immunohistochemistry, and whether protein and gene expression for STC-1 existed in SFO using Western blot and quantitative RT-PCR, respectively. Cells containing STC-1 immunoreactivity were found throughout the rostrocaudal extent of SFO. STC-1 protein expression within SFO was confirmed with Western blot, and SFO was also found to express STC-1 mRNA. In the second series, microinjections (200 nl) of STC-1, ANG II, a combination of the two or the vehicle were made into SFO in conscious, unrestrained rats. Water intake was measured at 0700 for a 1-h period after each injection in animals. Microinjections of STC-1 (17.6 or 176 nM) alone had no effect on water intake compared with controls. However, STC-1 not only attenuated the drinking responses to ANG II for about 30 min, but also decreased the total water intake over the 1-h period. These data suggest that STC-1 within the SFO may act in a paracrine/autocrine manner to modulate the neuronal responses to blood-borne ANG II. These findings also provide the first direct evidence of a physiological role for STC-1 in central regulation of body fluid homeostasis.

Characterization of stanniocalcin-1 receptors in the rainbow trout.

Mammalian stanniocalcin-1 (STC-1) is one of several ligands targeted to mitochondria. High affinity STC-1 receptors are present on the mitochondrial membranes of nephron cells, myocytes, and hepatocytes, to enable ligand sequestration within the matrix. However, STC-1 receptors have not been characterized in fish. Nor is it known if mitochondrial targeting occurs in fish. The aim of the study was to address these questions. Saturation binding assays were carried out to obtain estimates of K(D) and B(max). They revealed the presence of saturable, high-affinity receptors on both membranes and mitochondria of liver, muscle, and gill filament. In situ ligand binding (ISLB) was used to localize receptors at the histological level and revealed some unexpected findings. In cranium, for instance, receptors were found mainly in the cartilage matrix, as opposed to the chondrocytes. In brain, the majority of receptors were located on neuropil areas as opposed to neuronal cell bodies. In skeletal muscle, receptors were confined to periodic striations, tentatively identified as the Z lines. Receptors were even found on STC-1 producing corpuscles of Stannius cells, raising the possibility of there being an autocrine feedback loop or, perhaps, a soluble binding protein that is released with the ligand to regulate its bioavailability.

Vasopressin controls stanniocalcin-1 gene expression in rat and mouse kidney.

Renal stanniocalcin-1 (STC-1) is made by collecting duct principal cells for autocrine and paracrine targeting of the distal nephron. While the underlying purpose of this targeting is poorly understood, increased targeting is tied to changes in extracellular fluid (ECF) balance. For example, water deprivation is a potent stimulator of renal STC-1 gene activity in both rats and mice. The effects are most evident in cortical kidney where transcript levels are increased as much as 8-fold, as compared to 2-fold in the papilla. As is now known, this gene upregulation occurs in response to the dual consequences of water deprivation; hypertonicity followed by hypovolemia. The cortical gene has proven to be uniquely responsive to hypertonicity and that in papilla to hypovolemia; the implication being that STC-1 has different roles in the two zones, both of which are somehow related to ECF balance. The role of arginine vasopressin (AVP) in maintaining ECF balance is well established. Moreover, hypertonicity and hypovolemia are, respectively, the primary and secondary stimulators of AVP release. Therefore the present study explored the hypothesis that AVP was responsible for inducing the STC-1 gene in one or both zones. The results showed that this was indeed the case. AVP had time and dose-dependent stimulatory effects on the gene in both rat and mouse cortical kidney. In the papilla, however, gene regulation was more complex, as AVP was inhibitory in rats but stimulatory in mice. Further studies on papilla revealed that angiotensin II (ANG II) was stimulatory in rats, but inhibitory in mice. Moreover, ANG II attenuated the stimulatory effects of AVP in mouse cortex and papilla. Receptor agonist studies revealed that the effects of AVP in both zones were mediated exclusively through the V2 receptor (V1a, V1b and oxytocin-specific agonists had no effect). The findings serve to further implicate STC-1 in the renal control of ECF balance.

The renal stanniocalcin-1 gene is differentially regulated by hypertonicity and hypovolemia in the rat.

Stanniocalcin-1 (STC-1) is made by kidney collecting duct cells for autocrine and paracrine targeting of nephron cell mitochondria. Here, the ligand stimulates respiratory uncoupling and calcium uniport activity. However, the underlying purpose of these actions and how the renal gene is regulated are poorly understood. In a previous study, we described the time-dependent, stimulatory effects of water deprivation on renal STC-1 mRNA levels in both rats and mice. In cortical kidney, STC-1 mRNA levels were increased 8-fold by 72h of water deprivation, whereas the gene response in outer and inner medulla was less pronounced (2-4 fold). Gene induction occurred equally in males and females and was accompanied by increased mitochondrial STC-1 protein levels. As water deprivation increases extracellular fluid (ECF) tonicity and at the same time reduces ECF volume, the present study examined the individual effects of hypertonicity and hypovolemia on renal gene activity in rats. Hypertonicity, whether induced by mannitol, glucose or NaCl, uniquely stimulated the cortical gene, to the extent that transcript levels were positively correlated with serum osmolality. This was in contrast to high dietary sodium, which had no bearing on cortical or medullary transcript levels. The situation was reversed in the case of hypovolemia. Inner medullary gene expression was uniquely induced by hypovolemia (low sodium diet or polyethylene glycol) such that transcript levels were positively correlated with hematocrit, while cortical gene activity was unaffected or reduced. Hence, the cortical and medullary genes proved to be differentially regulated by changing ECF tonicity and volume, respectively. The findings are therefore indicative of cortical and medullary STC-1 having separate roles in the renal control of ECF balance.

Induction of the renal stanniocalcin-1 gene in rodents by water deprivation.

Stanniocalcin-1 (STC-1) is made by kidney collecting duct cells for targeting of nephron mitochondria to promote respiratory uncoupling and calcium uniport activity. However, the purpose of these actions and how the renal gene is regulated are poorly understood. This study has addressed the latter issue by monitoring renal STC-1 gene expression in different models of kidney function. Unilateral nephrectomy and over-hydration had no bearing on renal gene activity in adult Wistar rats. Dehydration, on the other hand, had time-dependent stimulatory effects in male and female kidney cortex, where STC-1 mRNA levels increased 8-fold by 72h. Medullary gene activity was significantly increased as well, but muted in comparison ( approximately 2-fold). Gene induction was accompanied by an increase in mitochondrial sequestration of STC-1 protein. Aldosterone and angiotensin II had no bearing on STC-1 gene induction, although there was evidence of a role for arginine vasopressin. Gene induction was unaltered in integrin alpha1 knockout mice, which have an impaired tonicity enhancer binding protein (TonEBP) response to dehydration. The STC-1 gene response could be cytoprotective in intent, as dehydration entails a fall in renal blood flow and a rise in medullary interstitial osmolality. Alternatively, STC-1 could have a role in salt and water balance as dehydration necessitates water conservation as well as controlled natriuresis and kaliuresis.

The corpuscles of Stannius, calcium-sensing receptor, and stanniocalcin: responses to calcimimetics and physiological challenges.

This study has examined whether the calcium-sensing receptor (CaSR) plays a role in control of stanniocalcin-1 (STC-1), the dominant calcium regulatory hormone of fish, comparable with that demonstrated for CaSR in the mediation of ionized calcium regulation of PTH secretion in mammals. In a previous study, we have cloned flounder STC-1 from the corpuscles of Stannius (CS). Here, we report the cloning and characterization of the CS CaSR, and the in vivo responses of this system to altered salinity, EGTA induced hypocalcemia, and calcimimetic administration. Quantitative PCR analysis demonstrated, for the first time, that the CS are major sites of CaSR expression in flounder. Immunoblot analysis of CS proteins with CaSR-specific antibodies revealed a broad band of approximately 215-300 kDa under nonreducing conditions, and bands of approximately 215-300 kDa and approximately 120-150 kDa under reducing conditions. There were no differences in CS CaSR mRNA expression or plasma STC-1 levels between seawater and freshwater (FW)-adapted fish, although CS STC-1 mRNA expression was lower in FW animals. Immunoblots showed that glycosylated monomeric forms of the CaSR migrated at a lower molecular mass in CS samples from FW animals. The ip administration of EGTA rapidly induced hypocalcemia, and a concomitant lowering of plasma STC-1. Calcimimetic administration (1 mg/kg R-568) rapidly increased plasma STC-1 levels, and reduced plasma concentrations of calcium, phosphate, and magnesium when compared with S-568-treated controls. Together, these findings support an evolutionary conserved role for the CaSR in the endocrine regulation of calcium before the appearance of parathyroid glands in tetrapods.

Stanniocalcin 1 is a luminal epithelial marker for implantation in pigs regulated by progesterone and estradiol.

Stanniocalcin 1 (STC1) is a glycoprotein that decreases calcium and increases phosphate in cells/tissues. This investigation examined endocrine regulation of STC1 in endometria of pigs during the estrous cycle and pregnancy. STC1 mRNA was present exclusively in luminal epithelium (LE) between d 12 and 15 of the estrous cycle, increased between d 12 and d 20, and was not detectable by d 30 of pregnancy. STC1 protein was also detected in uterine flushings. To determine effects of estrogen and progesterone, pigs were ovariectomized and treated with these hormones alone or together. Progesterone, but not estrogen, induced STC1 in LE. Cotreatment with progesterone and estrogen further stimulated STC1 over progesterone alone. To determine effects of pseudopregnancy, nonpregnant gilts were given daily injections of estradiol benzoate from d 11 to d 14. STC1 was not expressed in LE on d 90 of pseudopregnancy, suggesting that the estradiol given to induce pseudopregnancy and/or long-term exposure to progesterone are required for down-regulation of STC1. To determine effects of long-term progesterone, without effects of estradiol, pigs were ovariectomized on d 12, given daily injections of progesterone through d 39, and hysterectomized on d 40 after estrus. STC1 was expressed in LE of progesterone-treated pigs, suggesting that estrogen is involved in down-regulation of STC1. We conclude that STC1 is induced in LE by progesterone and further stimulated by estrogen, and its down-regulation in LE by d 25 likely requires exposure of the progestinized uterus to estrogen. The temporal and cell type-specific expression of STC1 makes this gene a unique marker for implantation in pigs.

Distribution of stanniocalcin binding sites in the lamina terminalis of the rat.

Stanniocalcin (STC-1), a 50 kDa glycoprotein hormone that regulates calcium/phosphate homeostasis in bony fish and mammals, has been shown to be expressed in central neurons and choroid plexus, and to exert a protective effect against hypercalcemic and hypoxic damage to neurons. Circumventricular organs are known to function in the regulation of ion and body fluid balance. Therefore, the possibility exists that STC-1 may be involved in the regulation of calcium/phosphate and fluid homeostasis through its actions on these central sites. In the present study, the distribution of STC-1 binding sites in forebrain circumventricular organs of the rat were investigated by in situ ligand binding using a stanniocalcin-alkaline phosphatase (STC-AP) fusion protein. Cells exhibiting STC-1 binding sites were found throughout the lamina terminalis. Dense cytoplasmic staining was observed predominantly within ependymal cells lining the anterior third ventricle region (AV3V), as well as cells of the choroid plexus. Additionally, neurons of the organum vasculosum of the lamina terminalis, the dorsal and ventral components of the median preoptic nucleus and the rostral aspects of the subfornical organ exhibited dense STC-1 cytoplasmic staining. STC-1 binding sites were also found in cells of the supraoptic nucleus, suprachiasmatic nucleus and anteroventral preoptic nucleus. These data suggest that STC-1 binding sites localized on the ependymal cells of the AV3V region and neurons of circumventricular organs may be associated with neuronal pathways involved in calcium/phosphate and fluid homeostasis.

Epigenetic and HIF-1 regulation of stanniocalcin-2 expression in human cancer cells.

Mammalian stanniocalcin-2 (STC2) is a secreted glycoprotein hormone with a putative role in unfolded protein response and apoptosis. Here we reported that STC2 expression was sporadically abrogated in human cancer cells by transcriptional silencing associated with CpG island promoter hypermethylation. Direct sequencing of bisulfite-modified DNA from a panel of seven human cancer cell lines revealed that CpG dinucleotides in STC2 promoter was methylated in human ovarian epithelial cancer (SKOV3, OVCAR3 and CaOV3), pancreatic cancer (BxP3), colon adenoma (HT29), and leukemia (Jurkat cells). STC2 CpG island hypermethylation was accompanied with a low basal STC2 expression level. Treatment of these cancer cells with 5-aza-2'-deoxycytidine (5-aza-CdR), an inhibitor of DNA methylation significantly induced STC2 expression. Using SKOV3 cells as a model, the link between DNA demethylation and STC2 expression was consistently demonstrated with hydralazine treatment, which was shown to reduce the protein level of DNA methyltransferase 1 (DNMT1) but stimulated STC2 expression. Two human normal surface ovarian cell-lines (i.e. IOSE 29 and 398) showed no methylation at CpG dinucleotides in the examined promoter region and were accompanied with high basal STC2 levels. Hypoxia stimulated STC2 expression in SKOV3 cells was markedly increased in 5-aza-CdR pretreated cells, showing that DNA methylation may hinder the HIF-1 mediated activation. To elucidate this possibility, RNA interference studies confirmed that endogenous HIF-1 alpha was a key factor for STC2 gene activation as well as in the synergistic induction of STC2 expression in 5-aza-CdR pretreated cells. Chromatin immunoprecipitation (ChIP) assay demonstrated the binding of HIF-1 alpha to STC2 promoter. The binding was increased in 5-aza-CdR pretreated cells. Collectively, this is the first report to show that STC2 was aberrantly hypermethylated in human cancer cells. The findings demonstrated that STC2 epigenetic inactivation may interfere with HIF-1 mediated activation of STC2 expression.

Stanniocalcin-1 secretion and receptor regulation in kidney cells.

Kidney collecting duct principal cells are the main source of stanniocalcin-1 (STC-1) production and secretion. From there, the hormone targets thick ascending limb and distal convoluted tubule cells, as well as collecting duct cells. More specifically, STC-1 targets their mitochondria to exert putative antiapoptotic effects. Two distal tubule cell lines serve as models of STC-1 production and/or mechanism of action. Madin-Darby canine kidney-1 (MDCK-1) cells mimic collecting duct cells in their synthesis of STC-1 ligand and receptor, whereas inner medullary collecting duct-3 (IMCD-3) cells respond to additions of STC-1 by increasing their respiration rate. In the present study, MDCK cell STC-1 secretion was examined under normal and hypertonic conditions, vectorally, and in response to hormones and signal transduction pathway activators/inhibitors. STC-1 receptor regulation was monitored in both cell lines in response to changing ligand concentration. The results showed that NaCl-induced hypertonicity had concentration-dependent stimulatory effects on STC-1 secretion, as did the PKC activator TPA. Calcium and ionomycin were inhibitory, whereas calcium receptor agonists had no effect. Angiotensin II, aldosterone, atrial natriuretic factor, antidiuretic hormone, and forskolin also had no effects. Moreover, STC-1 secretion exhibited no vectoral preference. STC-1 receptors were insensitive to homologous downregulation in both cell lines. In contrast, they were upregulated when STC-1 secretion was inhibited by calcium. The findings suggest that hypertonicity-induced STC-1 secretion is regulated through PKC activation and that high intracellular calcium levels are a potent inhibitor of release. More intriguingly, the results suggest that the receptor may not accompany STC-1 in its passage to the mitochondria.

Induction of stanniocalcin-1 expression in apoptotic human nasopharyngeal cancer cells by p53.

There is growing evidence to suggest that altered patterns of STC1 gene expression relate to the process of human cancer development. Our previous study has demonstrated the involvement of HIF-1 in the regulation of STC1 expression in human cancer cells. Recently, STC1 has been implicated as a putative pro-apoptotic factor in regulating the cell-death mechanism. Thus it would be of interest to know if STC1 is regulated by a tumor suppressor protein, p53. In this study, we provide evidence to demonstrate that the induction of STC1 expression in apoptotic human nasopharyngeal cancer cells (CNE2) is mediated by the activation of p53. Our study indicated that the activation of STC1 and heat-shock protein (hsp70) accompanied iodoacetamide (IDAM)-induced apoptosis in CNE-2. In addition, cellular events such as GSH depletion, mitochondrial membrane depolarization, reduction of pAkt and procaspase-3, and the induction of total p53 protein, acetylated p53, and annexin V positive cells were observed. The activation of STC1 was found to be at the transcriptional level and was independent of prior protein synthesis. Co-treatment of IDAM exposed cells with N-acetyl cysteine (NAC) prevented cell death by restoring mitochondrial membrane potential and cellular levels of GSH. NAC co-treatment also suppressed STC1 expression but had no effect on IDAM-induced hsp70 expression. RNA interference studies demonstrated that endogenous p53 was involved in activating STC1 gene expression. Collectively, the present findings provide the first evidence of p53 regulation of STC1 expression in human cancer cells.

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.

The stanniocalcin family of proteins.

Stannniocalcin (STC) is a polypeptide hormone that was originally identified in bony fishes as a systemic regulator of mineral metabolism, and is best known for its regulatory effects on calcium/phosphate transport by the gills, gut and kidneys. The mammalian homolog to fish STC was discovered in 1995 and has resulted in progressively growing interest ever since as to its possible role in humans. Moreover, new discoveries in the mammalian STC field are resulting in significant reappraisals as to its role in fishes. Perhaps the most significant of these has been the discovery of a second gene encoding stanniocalcin-related protein, or STC-2, first in mammals and subsequently in fish. This review covers the comparative endocrinology of the STCs in fishes and mammals from the perspectives of structure, function and regulation. It then delves into some of the newer aspects of STC-1/STC-2 biology that have been uncovered using both classical and transgenic approaches. Of these, one of the most intriguing discoveries relates to the receptor-mediated sequestration of STC by target cell organelles. The functions of other newly discovered mammalian and fish STC variants are also discussed, as is the recent discovery of STC-related homologs in invertebrates. Based on our current state of knowledge, it is apparent that STC has an ancient lineage and that the STC family of proteins is proving to have significant roles in metabolism, reproduction and development.

Passive immunization of lactating mice with stanniocalcin-1 antiserum reduces mammary gland development, milk fat content, and postnatal pup growth.

During pregnancy and lactation in rodents, stanniocalcin-1 (STC-1) production by the ovaries is upregulated markedly and released into the circulation. The mammary glands are one target of this systemically delivered hormone. The purpose of this study was to lower serum levels of STC-1 in lactating mice through passive immunization so as to monitor the effects on mammary gland function and postnatal pup growth. Passive immunization significantly reduced circulating hormone levels, and pup growth was significantly compromised (30%), even though control and experimental litters had ingested equal amounts of milk. When mammary glands were analyzed, the alveolar area was significantly reduced in antibody-treated mothers. An analysis of milk composition revealed no changes in lactose, protein, or electrolyte levels but an approximately 40% reduction in triglyceride levels. The latter was due to a significant reduction in mammary gland lipoprotein lipase activity and led to a significant buildup of triglycerides in the serum. Body fat content was also significantly reduced in pups from antibody-treated mothers, whereas pup fecal fat content was increased. In mothers, passive immunization also caused significant behavioral effects, in particular, increased locomotor and hindleg rearing activities. Collectively, the results suggest that systemically derived STC-1 has important effects on mammary gland development and the transfer of serum-based triglycerides into milk. Locomotor effects suggest that STC-1 also has a role in maternal behavior.

Stanniocalcin (STC) in the endometrial glands of the ovine uterus: regulation by progesterone and placental hormones.

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.

Nuclear targeting of stanniocalcin to mammary gland alveolar cells during pregnancy and lactation.

In most mammalian tissues, the stanniocalcin-1 gene (STC-1) produces a 50-kDa polypeptide hormone known as STC50. Within the ovaries, however, the STC-1 gene generates three higher-molecular-mass variants known as big STC. Big STC is targeted locally to corpus luteal cells to block progesterone release. During pregnancy and lactation, however, ovarian big STC production increases markedly, and the hormone is released into the serum. During lactation, this increase in hormone production is dependent on a suckling stimulus, suggesting that ovarian big STC may have regulatory effects on the lactating mammary gland. In this report, we have addressed this possibility. Our results revealed that virgin mammary tissue contained large numbers of membrane- and mitochondrial-associated STC receptors. However, as pregnancy progressed into lactation, there was a decline in receptor densities on both organelles and a corresponding rise in nuclear receptor density, most of which were on milk-producing, alveolar cells. This was accompanied by nuclear sequestration of the ligand. Sequestered STC resolved as one approximately 135-kDa band in the native state and therefore had the appearance of a big STC variant. However, chemical reduction collapsed this one band into six closely spaced, lower-molecular-mass species (28-41 kDa). Mammary gland STC production also underwent a dramatic shift during pregnancy and lactation. High levels of STC gene expression were observed in mammary tissue from virgin and pregnant rats. However, gene expression then fell to nearly undetectable levels during lactation, coinciding with the rise in nuclear targeting. These findings have thus shown that the mammary glands are indeed targeted by STC, even in the virgin state. They have further shown that there are marked changes in this targeting pathway during pregnancy and lactation, accompanied by a switch in ligand source (endogenous to exogenous). They also represent the first example of nuclear targeting by STC.

Hypoxia-inducible factor-1-mediated activation of stanniocalcin-1 in human cancer cells.

Stanniocalcin-1 (STC1) is an endocrine hormone originally discovered in the corpuscles of Stannius, endocrine glands on kidneys of bony fishes, and also has been identified in mammals. The mammalian STC1 gene is widely expressed in various tissues and appears to be involved in diverse biological processes. There is growing evidence to suggest that altered patterns of gene expression have a role in human cancer development. Recently STC1 has been identified as a stimulator of mitochondrial respiration and has been hypothesized to be functionally related to the Warburg effect, of which hypoxia-inducible factor (HIF)-1 plays a key role in reprogramming tumor metabolism. This prompted us to examine the involvement of HIF-1 in the regulation of STC1 expression in tumor hypoxia. Our data reveal that hypoxia can stimulate STC1 gene expression in various human cancer cell lines, including those derived from colon carcinomas, nasopharyngeal cancer (CNE-2, HONE-1, HK-1), and ovarian cancer (CaOV3, OVCAR3, SKOV3). By far, the greatest response was observed in CNE-2 cells. In further studies on CNE-2 cells, desferrioxamine, cobalt chloride, and O(2) depletion all increased HIF-1alpha protein and STC1 mRNA levels. Desferrioxamine treatment, when coupled with Fe replenishment, abolished these effects. RNA interference studies further confirmed that endogenous HIF-1alpha was a key factor in hypoxia-induced STC1 expression. The ability of vascular endothelial growth factor to stimulate STC1 expression in CNE-2 cells was comparatively low. Collectively, the present findings provide the first evidence of HIF-1 regulation of STC1 expression in human cancer cells. The studies have implications as to the role of STC1 in hypoxia induced adaptive responses in tumor cells.

Characterization of big stanniocalcin variants in mammalian adipocytes and adrenocortical cells.

The hormone stanniocalcin (STC) is widely distributed, and in rodents the highest levels of expression are in the ovaries. In both cows and rodents, ovarian STC consists of three high-molecular-weight variants collectively known as big STC. In the ovary, big STC is made by theca cells and interstitial cells and is targeted to lipid storage droplets of nearby luteal cells to inhibit progesterone release. An endocrine pathway is operative during pregnancy and lactation. Whether or not big STC is made by tissues other than ovary has never been addressed. Therefore, the purpose of this study was to determine via a detailed characterization of adrenal glands and adipocytes whether big STC is present in other cells that store and release lipids. The results showed that STC was made in bovine and mouse adrenals, mainly in steroidogenic, adrenocortical cells. The majority of ligand and receptor were likewise confined to cortical zone cells. As in luteal cells, high levels of ligand and receptor were found in the adrenocortical cell lipid droplet fraction. However, adrenals made only the largest (135 kDa) of the three big STC variants. Nonetheless, adrenal STC had much greater receptor affinity than a mixture of the three big STC variants. Adipocytes contained all three big STC variants, and both ligand and receptor were heavily concentrated on the lipid droplets. Moreover, adipocyte lipid storage droplets had 50-fold more receptors than those in steroidogenic cells, indicating that big STC is heavily targeted to adipose cells. The findings collectively support the hypothesis that big STC is not unique to ovarian steroidogenic cells but is in fact common to cells with a role in lipid storage and release.