Stanniocalcin-1 Gene Research Articles

Synergistic effect of p53 on TSA-induced stanniocalcin 1 expression in human nasopharyngeal carcinoma cells, CNE2

Human stanniocalcin 1 (STC1) has recently been identified as a putative protein factor involved in cellular apoptosis. The use of histone deacetylase inhibitor (i.e. trichostatin A (TSA)) and doxorubicin (Dox) is one of the common treatment methods to induce apoptosis in human cancer cells. A study on TSA and Dox-mediated apoptosis may shed light on the regulation and function of STC1 in cancer treatment. In this study, TSA and Dox cotreatment in human nasopharyngeal carcinoma cells (CNE2) elicited synergistic effects on STC1 gene expression and cellular apoptosis. An activation of p53 (TP53) transcriptional activity in Dox- or Dox+TSA-treated cells was revealed by the increased expression levels of p53 mRNA/protein as well as p53-driven luciferase activities. To elucidate the possible involvement of p53 in STC1 gene transcription, a vector expressing wild-type or dominant negative (DN) p53 was transiently transfected into the cells. Both STC1 promoter luciferase constructs and chromatin immunoprecipitation assays did not support the direct role of p53 in STC1 gene transactivation. However, the synergistic effects of p53 on the induction of NF-κB phosphorylation and the recruitment of acetylated histone H3 in STC1 promoter were observed in TSA-cotreated cells. The overexpression of exogenous STC1 sensitized apoptosis in Dox-treated cells. Taken together, this study provides data to show the cross talk of NF-κB, p53, and histone protein in the regulation of STC1 expression and function.

Effects of the calcium-regulating glycoprotein hormone stanniocalcin-1 within the nucleus of the solitary tract on arterial pressure and the baroreceptor reflex

Receptors for the calcium-regulating glycoprotein hormone stanniocalcin-1 (STC-1) have been found within the CNS and whether these receptors exist within the nucleus of the solitary tract (NTS), and their possible role in the regulation of arterial pressure (AP) is unknown. Experiments were done in the rat to: (1) map the distribution of STC-1 receptors throughout NTS using in situ ligand binding that uses a stanniocalcin-alkaline phosphatase (STC-AP) fusion protein; (2) determine whether protein and gene expression for STC-1 exists within NTS using immunohistochemistry, Western blot and real time qPCR; (3) determine the effect of microinjection of STC-1 into NTS on AP and the baroreflex. Cells exhibiting STC-1 binding sites were found mainly within the caudal medial (Sm), gelantinous and commissural subnuclei of NTS. Cells containing STC-1 immunoreactivity were found to overlap those regions of NTS that contained STC-1 receptors. STC-1 protein and gene expression were also found within caudal NTS. In chloralose-urethane–anesthetized rats, microinjections of STC-1 (1.76–176 nM; 20 nl) into the caudal Sm elicited a dose-related decrease in AP. In contrast, injections of a nonbioactive form of STC-1 (STC-1+guanosine 5′-triphosphate [GTP]), the vehicle (0.9% saline), or GTP alone did not elicit cardiovascular responses. Additionally, injection of STC-1 into Sm potentiated the AP responses to electrical stimulation of the ipsilateral aortic depressor nerve. Finally, bilateral injection of STC-1 primary antiserum (1:1000; 100 nl) into Sm elicited a long lasting increase in AP, whereas microinjection of heat inactivated STC-1 antiserum did not alter AP. Taken together these data suggest that endogenous STC-1 signaling in NTS is involved in regulating the excitability of neurons that normally function as components of the baroreceptor reflex controlling AP.

Stanniocalcin-1 Regulates Re-Epithelialization in Human Keratinocytes

Stanniocalcin-1 (STC1), a glycoprotein hormone, is believed to be involved in various biological processes such as inflammation, oxidative responses and cell migration. Riding on these emerging evidences, we hypothesized that STC1 may participate in the re-epithelialization during wound healing. Re-epithelialization is a critical step that involves keratinocyte lamellipodia (e-lam) formation, followed by cell migration. In this study, staurosporine (STS) treatment induced human keratinocyte (HaCaT) e-lam formation on fibronectin matrix and migration via the activation of focal adhesion kinase (FAK), the surge of intracellular calcium level [Ca2+]i and the inactivation of Akt. In accompanied with these migratory features, a time- and dose-dependent increase in STC1 expression was detected. STC1 gene expression was found not the downstream target of FAK-signaling as illustrated by FAK inhibition using PF573228. The reduction of [Ca2+]i by BAPTA/AM blocked the STS-mediated keratinocyte migration and STC1 gene expression. Alternatively the increase of [Ca2+]i by ionomycin exerted promotional effect on STS-induced STC1 gene expression. The inhibition of Akt by SH6 and GSK3β by lithium chloride (LiCl) could respectively induce and inhibit the STS-mediated e-lam formation, cell migration and STC1 gene expression. The STS-mediated e-lam formation and cell migration were notably hindered or induced respectively by STC1 knockdown or overexpression. This notion was further supported by the scratched wound assay. Collectively the findings provide the first evidence that STC1 promotes re-epithelialization in wound healing.

STC1 Induction by PACAP is Mediated Through cAMP and ERK1/2 but not PKA in Cultured Cortical Neurons

The neuroprotective actions of PACAP (pituitary adenylate cyclase-activating polypeptide) in vitro and in vivo suggest that activation of its cognate G protein coupled receptor PAC1 or downstream signaling molecules,and thus activation of PACAP target genes, could be of therapeutic benefit. Here, we show that cultured rat cortical neurons predominantly expressed the PAC1hop and null variants. PACAP receptor activation resulted in the elevation of the two second messengers cAMP and Ca(2+) and expression of the putative neuroprotectant stanniocalcin 1(STC1). PACAP signaling to the STC1 gene proceeded through the extracellular signal-regulated kinases 1 and 2(ERK1/2), but not through the cAMP-dependent protein kinase (PKA), and was mimicked by the adenylate cyclase activator forskolin. PACAP- and forskolin-mediated activation of ERK1/2 occurred through cAMP, but not PKA.These results suggest that STC1 gene induction proceeds through cAMP and ERK1/2, independently of PKA, the canonical cAMP effector. In contrast, PACAP signaling to the BDNF gene proceeded through PKA, suggesting that two different neuroprotective cAMP pathways co-exist in differentiated cortical neurons. The selective activation of a potentially neuroprotective cAMP-dependent pathway different from the canonical cAMP pathway used in many physiological processes, such as memory storage, has implications for pharmacological activation of neuroprotection in vivo.

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.

Sp1 is a transcription repressor to stanniocalcin-1 expression in TSA-treated human colon cancer cells, HT29

Our previous study demonstrated that, stanniocalcin-1 (STC1) was a target of histone deacetylase (HDAC) inhibitors and was involved in trichostatin A (TSA) induced apoptosis in the human colon cancer cells, HT29. In this study, we reported that the transcriptional factor, specificity protein 1 (Sp1) in association with retinoblastoma (Rb) repressed STC1 gene transcription in TSA-treated HT29 cells. Our data demonstrated that, a co-treatment of the cells with TSA and Sp1 inhibitor, mithramycin A (MTM) led to a marked synergistic induction of STC1 transcript levels, STC1 promoter (1 kb)-driven luciferase activity and an increase of apoptotic cell population. The knockdown of Sp1 gene expression in TSA treated cells, revealed the repressor role of Sp1 in STC1 transcription. Using a protein phosphatase inhibitor okadaic acid (OKA), an increase of Sp1 hyperphosphorylation and so a reduction of its transcriptional activity, led to a significant induction of STC1 gene expression. Chromatin immunoprecipitation (ChIP) assay revealed that Sp1 binding on STC1 proximal promoter in TSA treated cells. The binding of Sp1 to STC1 promoter was abolished by the co-treatment of MTM or OKA in TSA-treated cells. Re-ChIP assay illustrated that Sp1-mediated inhibition of STC1 transcription was associated with the recruitment of another repressor molecule, Rb. Collectively our findings identify STC1 is a downstream target of Sp1.

PAC1hop, null and hip receptors mediate differential signaling through cyclic AMP and calcium leading to splice variant-specific gene induction in neural cells

Pituitary adenylate cyclase-activating polypeptide (PACAP)-mediated activation of its G protein-coupled receptor PAC1 results in activation of the two G proteins Gs and Gq to alter second messenger generation and gene transcription in the nervous system, important for homeostatic responses to stress and injury. Heterologous expression of the three major splice variants of the rat PAC1 receptor, PAC1hop, null and hip, in neural NG108-15 cells conferred PACAP-mediated intracellular cAMP generation, while elevation of [Ca2+]i occurred only in PAC1hop-, and to a lesser extent in PAC1null-expressing cells. Induction of vasoactive intestinal polypeptide (VIP) and stanniocalcin 1 (STC1), two genes potentially involved in PACAP's homeostatic responses, was examined as a function of the expressed PAC1 variant. VIP induction was greatest in PAC1hop-expressing cells, suggesting that a maximal transcriptional response requires combinatorial signaling through both cAMP and Ca2+. STC1 induction was similar for all three receptor splice variants and was mimicked by the adenylate cyclase activator forskolin, indicating that cAMP elevation is sufficient to induce STC1. The degree of activation of two different second messenger pathways appears to determine the transcriptional response, suggesting that cellular responses to stressors are fine-tuned through differential receptor isoform expression. Signaling to the VIP gene proceeded through cAMP and protein kinase A (PKA) in these cells, independently of the MAP kinase ERK1/2. STC1 gene induction by PACAP was dependent on cAMP and ERK1/2, independently of PKA. Differential gene induction via different cAMP dependent signaling pathways potentially provides further targets for the design of treatments for stress-associated disorders.

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 72 h 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 72 h. Medullary gene activity was significantly increased as well, but muted in comparison (∼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.

Stanniocalcin-2 is a HIF-1 target gene that promotes cell proliferation in hypoxia

Stanniocalcin-2 (STC2), the paralog of STC1, has been suggested as a novel target of oxidative stress response to protect cells from apoptosis. The expression of STC2 has been reported to be highly correlated with human cancer development. In this study, we reported that STC2 is a HIF-1 target gene and is involved in the regulation of cell proliferation. STC2 was shown to be up-regulated in different breast and ovarian cancer cells, following exposure to hypoxia. Using ovarian cancer cells (SKOV3), the underlying mechanism of HIF-1 mediated STC2 gene transactivation was characterized. Hypoxia-induced STC2 expression was found to be HIF-1α dependent and required the recruitment of p300 and HDAC7. Using STC2 promoter deletion constructs and site-directed mutagenesis, two authentic consensus HIF-1 binding sites were identified. Under hypoxic condition, the silencing of STC2 reduced while the overexpression of STC2 increased the levels of phosphorylated retinoblastoma and cyclin D in both SKOV3 and MCF7 cells. The change in cell cycle proteins correlated with the data of the serial cell counts. The results indicated that cell proliferation was reduced in STC2-silenced cells but was increased in STC2-overexpressing hypoxic cells. Solid tumor progression is usually associated with hypoxia. The identification and functional analysis of STC2 up-regulation by hypoxia, a feature of the tumor microenvironment, sheds light on a possible role for STC2 in tumors.

Identification and characterization of the hypoxia-responsive element in human stanniocalcin-1 gene

In this study, we aimed to identify the hypoxia-inducible factor-1 (HIF-1) binding motif in human STC1 gene promoter and to characterize the associated gene transactivation mechanism. Using normoxic human nasopharyngeal cancer cells (CNE2), we manipulated the stability of HIF-1α protein by overexpressing HIF-1α or the silencing of prolyl hydroxylase-2 (PHD2), to illustrate HIF-1 activation of STC1 promoter-driven luciferase activity. Subsequently luciferase activities of the deletion and mutated STC1 promoter constructs were investigated in HIF-1 overexpressed cells. The data revealed the presence of an authentic HRE motif in STC1 gene. This result was further supported by the chromatin immunoprecipitation (ChIP) assay. Using a similar experimental treatment, however, had no significant effect on the expression level of STC1 mRNA and protein. Moreover the activation of STC1 expression can be restored by the silencing of “factor inhibiting HIF-1” (FIH-1) in either HIF-1 overexpressed or PHD2 silenced cells. The data implied that the HIF-1-mediated STC1 gene expression required the recruitment of p300. This presumption was confirmed by the use of p300 inhibitor, chetomin and HIF-1α/p300 re-ChIP assay. Collectively our data provide the first evidence to show that STC1 is a FIH-inhibited gene with a functional HRE motif located at the upstream region between −2322/−2335. The data support the need for further investigation to reveal if STC1 can be used as a novel tumor marker for HIF-1 induction and for the monitoring of anti-angiogenic therapy.

Clinicopathological significance of stanniocalcin 2 gene expression in colorectal cancer

Laser microdissection (LMD) and microarray were used to identify genes associated with colorectal cancer. Stanniocalcin 2 (STC2) expression and clinicopathological significance in 139 clinical colorectal cancer samples were specifically investigated using real-time quantitative reverse transcription-polymerase chain reaction. A number of genes upregulated in colorectal cancer cells compared to normal colorectal epithelial cells were identified including STC2. STC2 gene expression in cancer tissue was higher than in corresponding normal colorectal epithelial tissue in 124 of 139 cases (89.2%, p < 0.01). Tumors with high STC2 expression showed higher frequencies of lymph node metastasis, lymphatic invasion, tumor depth, tumor size and AJCC Stage classification (p < 0.01). Patients with high STC2 expression also showed significantly worse overall survival rates than those with low STC2 expression (p < 0.01). Furthermore, STC2 gene appeared to be associated with colorectal cancer progression and may be a useful prognostic indicator for colorectal cancer.

Derlin-1 and Stanniocalcin-1 are Differentially Regulated in Metastasizing Canine Mammary Adenocarcinomas

Derlin-1, stanniocalcin-1, epithelial glycoprotein-2 (EGP-2) and maspin are associated with the metastasis of human breast cancer cells. This study reports the potential role of these molecules in metastasis of canine mammary tumours. Laser microdissected tissue samples were prepared from normal canine mammary gland and from simple adenomas, adenocarcinomas and their lymph node metastases. The expression of genes encoding the molecules of interest in these tissues was determined by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Most adenomas displayed reduced expression of derlin-1 mRNA. Few adenocarcinomas overexpressed derlin-1 mRNA, but all lymph node metastases overexpressed this gene product. Stanniocalcin-1 mRNA was not expressed within adenomas and was reduced in adenocarcinomas and their lymph node metastases. EGP-2 gene expression did not differ between normal, benign and malignant neoplastic tissues. Maspin gene expression varied markedly among the tumours with reduced or increased expression compared with normal mammary gland. Taken together, these results suggest that malignant behaviour of canine mammary adenocarcinoma is associated with reduced transcription of the stanniocalcin-1 gene and overexpression of the derlin-1 gene.

Cloning and expression of Stanniocalcin-1 gene in tissues of toad, Bufo bufo gargarizans

Cloning and expression of Stanniocalcin-1 gene in tissues of toad, Bufo bufo gargarizans

Histone deacetylase inhibitor-induced cellular apoptosis involves stanniocalcin-1 activation

Our previous studies have demonstrated the involvement of HIF-1 and p53 in the regulation of stanniocalcin-1 (STC1) gene transcription in human cancer cells. In this study, we reported that the treatment of human colon adenoma HT29 cells with a histone deacetylase (HDAC) inhibitor (i.e. trichostatin A, TSA) induced both cellular apoptosis and STC1 expression. The activation of STC1 expression was also observed in other TSA-treated human cancer cells (i.e. SKOV3, CaCo-2, Jurkat and CNE-2 cells). STC1 mRNA was rapidly induced within 4 h in TSA-treated HT29 cells, and was found to be transcriptionally regulated and was independent of new protein synthesis as revealed by ActD and CHX treatment respectively. The induction was correlated with increased cellular levels of acetyl histone H3 and H4 and acetyl NFκB. Chromatin immunoprecipitation (ChIP) assay showed the increased binding of acetyl histone H3 and H4 to STC1 promoter in the TSA-treated cells. A cotreatment of HT29 cells with a NFκB inhibitor (parthenolide) significantly inhibited the TSA-induced cellular levels of acetyl NFκB p65 and abolished the stimulation of STC1 gene expression. ChIP assay also demonstrated that TSA treatment increased while TSA/parthenolide cotreatment decreased NFκB p65 binding to STC1 gene promoter. In the STC1-luciferase promoter construct (1 kb) study, the data implied that the promoter can be activated by TSA treatment. Interestingly, the promoter region contains 2 putative NFκB binding sites. Consistent with the STC1mRNA expression data, TSA/parthenolide cotreatment also significantly inhibited the TSA-induced STC1 promoter-driven luciferase activity. Importantly, TSA-induced apoptotic process was found to be significantly reduced by the silencing of STC1 expression. This is the first study to show that histone hyper-acetylation and the recruitment of activated NFκB stimulated STC1 gene expression. In addition, our results support the notion that STC1 is a pro-apoptotic factor.

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-1α 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-1α 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.

Vascular endothelial growth factor regulates Stanniocalcin-1 expression via Neuropilin-1-dependent regulation of KDR and synergism with fibroblast growth Factor-2

Stanniocalcin-1 (STC-1) is a glycoprotein hormone originally identified as a regulator of calcium and phosphate homeostasis in bony fish. Up-regulation of the mammalian homolog in numerous gene profiling studies of angiogenesis and Vascular Endothelial Growth Factor-A (VEGF-A 165)-regulated gene expression, suggests that regulation of this factor may be a key feature of the angiogenic response. Here we investigated the mechanisms mediating VEGF-A 165-induced STC-1 gene expression in human endothelial cells. VEGF-A 165, acting via VEGFR2/KDR, induced STC-1 through de novo transcription, mediated primarily via intracellular protein kinase C (PKC)- and extracellular signal-regulated protein kinase (ERK)-dependent pathways. VEGF-A 165-induced STC-1 mRNA expression was synergistically enhanced up to 2-fold by co-treatment with FGF-2, in a mechanism dependent on VEGFR2/KDR and FGFR1. Production of STC-1 protein by endothelial cells was also induced by VEGF-A 165 and synergistically enhanced by co-treatment with FGF-2. Synergism between VEGF-A 165 and FGF-2 was mediated via a novel neuropilin-1 (NP-1)-dependent mechanism, as indicated by the complete inhibition of synergism with either EG3287, a specific neuropilin antagonist, or siRNA-mediated NP-1 knockdown, and by the inability of the VEGF-A 121 isoform to synergise with FGF-2. Surprisingly, we found that NP-1 knockdown also markedly reduced KDR expression in HUVECs, and enhanced the VEGF-A 165-induced reduction in KDR expression resulting from receptor-mediated endocytosis. These findings support a role for NP-1 in mediating synergistic effects between VEGF-A 165 and FGF-2, which may occur in part through a contribution of NP-1 to KDR stability.

Impact of gestation and labor on the expression of the polypeptide hormone, stanniocalcin‐1, in guinea pig and human myometrium

Stanniocalcin-1 (STC1) is a small polypeptide hormone which plays a major role in calcium homeostasis. It is involved in physiological processes such as ion transport and reproduction and development. During pregnancy, STC1 production by the ovaries is upregulated and released into circulation. We found that the myometrium is one target of this systemically delivered hormone. The expression of STC1 in the myometrium and its role in labor and dysfunctional labor are unclear. Here we investigate the expression and regulation of STC1 in guinea pig and human myometrium during pregnancy under various physiological conditions. Gene profiling was performed with myometrial RNA obtained from nonpregnant, mid-pregnant (MP), and term-pregnant (TP) guinea pigs and at primary cesarean of human preterm no labor; preterm labor (PTL); term no labor; and term labor (TL). Fold-changes in gene expression were verified by real time pcr. Western blots were used to further determined STC1 protein levels in each myometrial condition. We found a decrease (−9.2 fold) in the gene expression of STC1 in guinea pig myometrium during MP and a contrasting rise (1.7 fold) closer to term. STC1 gene expression increased in human myometrium during normal TL but no change was observed at PTL. STC1 protein levels were greater at term than preterm. These findings suggest that STC1 may be gestationally regulated and important for normal pregnancy outcome. Supported by: HL49041-12 (CPW) and HD049185-01 (CPW)

Molecular characterization and expression analysis of stanniocalcin-1 in turbot ( Scophthalmus maximus)

Stanniocalcin-1 (STC-1) is an anti-hypercalcemic hormone that is produced by the corpuscles of Stannius (CS) in the teleost. The hormone is a homodimeric glycoprotein involved in the calcium and phosphate regulation in both teleost fish and mammals. In the present study, we have cloned and characterized a full-length cDNA of STC-1 from the turbot ( Scophthalmus maximus) CS and examined its expression pattern in various tissues. The turbot STC cDNA (1246 nucleotides) encodes a putative preprohormone of 248 amino acids (aa) with a signal peptide of 18 aa and a pro-sequence peptide of 14 aa followed by a mature protein of 216 aa. The deduced aa sequence of turbot STC-1 shows the highest sequence identity (86.3%) with that of European flounder STC-1 among the fish and mammalian STC-1. The aa sequence of turbot STC-1 showed moderate sequence identity with those of salmoniforms (chum salmon and rainbow trout), cypriniforms (zebrafish), ancient fish (gar and bowfin), and humans (43.7–66.9%), respectively, whereas it showed less identity with those of zebrafish and human STC-2 (23.8–25.4%). RT-PCR analysis revealed that the turbot STC-1 gene is expressed in the CS, pituitary, brain, kidney, liver, heart, muscle, and gonad. In a transient transfection of turbot STC-1 cDNA construct into human embryonic kidney 293T cells followed by Western blot analysis, a recombinant STC-1 secreted into a medium was detected as a single band of 28 kDa under reducing conditions and a 50 kDa protein under non-reducing conditions. Deglycosylation of the recombinant STC-1 led to a decrease in the molecular mass. Our data indicate that the turbot STC-1 gene is widely expressed and the secreted protein exists as a homodimeric glycoprotein.

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.