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. Author manuscript; available in PMC: 2018 Feb 6.
Published in final edited form as: Exp Clin Endocrinol Diabetes. 2010 Sep 8;119(2):81–85. doi: 10.1055/s-0030-1262860

Stanniocalcin 1 Induction by Thyroid Hormone Depends on Thyroid Hormone Receptor β and Phosphatidylinositol 3-kinase Activation

L C Moeller 1, N E Haselhorst 1, A M Dumitrescu 2, X Cao 4, H Seo 4, S Refetoff 2,3, K Mann 1, O E Janssen 1,5
PMCID: PMC5800749  NIHMSID: NIHMS937655  PMID: 20827662

Abstract

Context

Thyroid hormone (TH) mediated changes in gene expression were thought to be primarily initiated by the nuclear TH receptor (TR) binding to a thyroid hormone response element in the promoter of target genes. A recently described extranuclear mechanism of TH action consists of the association of TH-liganded TRβ with phosphatidylinositol 3-kinase (PI3K) in the cytosol and subsequent activation of the PI3K pathway.

Objective

The aim of this study was to examine the effect of TH, TRβ and PI3K on stanniocalcin 1 (STC1) expression in human cells.

Design

We treated human skin fibroblasts with triiodothyronine (T3) in the absence or presence of the PI3K inhibitor LY294002, a dominant negative PI3K subunit, Δp85α, and the protein synthesis inhibitor cycloheximide (CHX). The role of the TRβ was studied in cells from patients with resistance to thyroid hormone (RTH). STC-1 mRNA expression was measured by real-time PCR.

Results

We found an induction of STC1 by T3 in normal cells, but less in cells from subjects with RTH (2.7 ± 0.2 vs. 1.6 ± 0.04, P < 0.01). The effect of T3 was completely abrogated by blocking PI3K with LY294002 (3.9 ± 0.5 vs. 0.85 ± 0.5; P < 0.05) and greatly reduced after transfection of a dominant negative PI3K subunit, demonstrating dependency on the PI3K pathway.

Conclusion

These results establish STC1 as a TH target gene in humans. Furthermore, we show that STC1 induction by TH depends on both TRβ and PI3K activation.

Keywords: nongenomic action, hypoxia inducible factor 1, HIF-1

Introduction

Thyroid hormones (TH) are essential for normal development, growth and metabolism (Yen, 2001). Their action is principally mediated through triiodothyronine (T3), acting as a ligand for the TH receptors (TRs) α and β (Cheng et al., 2010, Yen, 2001). In the classical mode of TH action, the TRs are bound to TH response elements (TREs) in the promoter of target genes as homodimers or heterodimers with the retinoid X receptor (RXR). In the case of positively regulated genes, T3 binding then leads preferentially to a heterodimer formation with RXR, exchange of corepressors for coactivators, recruitment of basal transcription factors such as the RNA polymerase II and, subsequently, increased transcription of the TH target gene.

In addition to this classical mode of TH action, TH effects mediated by TRs but independent of TREs have recently been recognized (Moeller et al., 2006). Cao and others described a new mechanism of TH action in which the liganded TRβ interacts with the regulatory subunit of phosphatidylinositol 3-kinase (PI3K), p85α (Cao et al., 2005, Lei et al., 2004). This leads to activation of the PI3K signal transduction pathway with subsequent phosphorylation and activation of protein kinase B (PKB) and mammalian target of rapamycin (mTOR). It could be demonstrated that this initially non-genomic action of TH ultimately has genomic consequences. PI3K activation leads to induction of ZAKI4α, known to be strongly induced by TH (Cao et al., 2005). Furthermore, this mechanism of T3/TRβ action leads to induction of hypoxia-inducible factor (HIF-)1α, the tightly regulated subunit of the transcription factor HIF-1, and the HIF-1 target genes glucose transporter 1 (GLUT1) and phosphofructokinase (PFKP) (Moeller et al., 2005a, Moeller et al., 2005b). We also found an induction of the lactate transporter MCT4, which has recently been described as a HIF-1 target gene as well (Ullah et al., 2006).

A microarray study found stanniocalcin (STC)1 to be positively regulated by TH (Moeller et al., 2005b). STC1 is a polypeptide hormone that was originally identified as a regulator of calcium/phosphate homeostasis in fish (Wagner and Dimattia, 2006). During evolution from fish to mammals, STC1 maintained a function in calcium homeostasis, regulating mammalian intestinal calcium and phosphate transport (Madsen et al., 1998), but in evolutionary advanced animals, its role seems to have broadened to be involved in cellular metabolism, muscle and bone development and endothelial gene expression (Chakraborty et al., 2006, Filvaroff et al., 2002, McCudden et al., 2002). In humans, STC1 is expressed in heart, kidney, liver, ovary, prostate, thyroid c-cells and neurons (Chang et al., 2003, Sheikh-Hamad et al., 2003). Its physiological roles in humans as well as its regulation are not fully understood yet. STC1 has recently gained interest due to its involvement in cancer biology. Human STC1 was originally cloned in a screen for cancer-related genes and enhanced STC1 expression has been found in hepatocellular, colorectal and breast carcinoma as well as in medullary thyroid cancer (Chang et al., 1995, Chang et al., 2003).

In this communication we show that TH stimulate STC1 mRNA expression dependent on TRβ and PI3K activation.

Material and Methods

Cell culture and treatments

Human skin fibroblasts were obtained by punch biopsy from 2 normal individuals and 2 patients with RTH due to TRβ gene mutations, one heterozygous for a dominant negative TRβ mutation A317T (TRβmut) and the other homozygous for a TRβ gene deletion (TRβ0). Their use was approved by the Institutional Review Board of the University of Chicago. Primary cultures of fibroblasts were grown in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal bovine serum (FBS) as previously described in detail (Murata et al., 1987). At confluency, the medium was replaced with one containing thyroid hormone depleted bovine serum (T × BS), produced by treatment of FBS with anion exchange resin (Samuels et al., 1979). 48 h later, T3 was added to a final concentration of 0.1, 0.5 or 2 nM. Based on the free T3 concentration in the medium containing 10% FBS, measured by equilibrium dialysis, these 3 T3 doses correspond to a third to half, 1.5–2 times and 3–5 times physiological serum levels in humans. The inhibitors LY294002 (50 µM, Sigma-Aldrich GmbH, Taufkirchen, Germany) and cycloheximide, CHX (10 µg/ml, Sigma-Aldrich) were added to the medium 1 h before T3 treatment. Infections with recombinant adenovirus either expressing green fluorescent protein (Ad-GFP) or a dominant negative form of the p85α-regulatory subunit of PI3K (Ad-Δp85α) prior to T3 treatment were carried out as described previously (Cao et al., 2005). The experiments were terminated 24 h after T3 treatment by RNA extraction for real-time PCR. All experiments were carried out at early cell passages (< 10 passages).

Isolation and reverse transcription of RNA

The medium was removed and the dish was washed twice with ice cold Hank’s Buffered Saline Solution (HBSS, Invitrogen, Karlsruhe, Germany). Total RNA was extracted using phenol/guanidine isothiocyanate (TRIZOL, Invitrogen). RNA was reverse transcribed with the Superscript™ III RNase H Reverse Transcriptase Kit (Invitrogen) using 2 µg of total RNA and 100 ng of random hexamers.

Quantification of mRNA

Quantification of mRNAs was performed in an ABI Prism 7 700 Sequence Detection System (Applied Biosystems, Foster City, CA), using SYBR® Green I as detector dye. The reaction mixtures contained 25 µl QuantiTect™ SYBR® Green PCR Kit (QIAGEN, Hilden, Germany), 0.3 µMol/L of each primer, 10 ng template cDNA and nuclease-free water to a final volume of 50 µl. The oligonucleotide primers were designed to cross introns, based on a Human BLAST Search result (http://genome.ucsc.edu). STC1 (NCBI Entrez Nucleotide mRNA access number NM_003155): forward primer 5′-TGTGAGCCCCAGGAAATCC-3′(exon 1), reverse primer 5′-TTCCTGCACCTCAGCAATCA-3′ (exon 3); BTEB1 (NM_001206): forward primer 5′-CTC CCA TCT CAA AGC CCA TTA C-3′ (exon 2), reverse primer 5′-TGA GCG GGA GAA CTT TTT AAG G-3′ (exon 3). The primers were purchased from Operon Biotechnologies (Cologne, Germany). The reaction conditions were 95 C for 2 min followed by 40 cycles of 95 C for 15 s and 60 C for 45 s. To ensure specificity of the amplification, the PCR products were run on a gel and the single bands were of the expected size. STC1 expression was calculated relative to that in untreated cells and normalized for the housekeeping gene ubiquitin-conjugating enzyme E2D 2 (UBE2D2), using the 2−ΔΔCT method (Livak and Schmittgen, 2001). Quantification of TH dependent genes by microarray was carried out as described previously (Moeller et al., 2005b).

Data analysis

Real-time PCR results are expressed as mean ± SE and statistical analysis was done by ANOVA.

Results

STC1 expression is induced by T3 via the TRβ

STC1 was represented on the microarray chips that were used to study TH dependent gene expression in human fibroblasts as described previously (Moeller et al., 2005b). Primary cultures of human skin fibroblasts were treated for 24 h with increasing doses of T3, ranging from 0.1 to 2 nM following 48 h of TH-depleted medium to test for a dose response. In addition to cells from 2 normal individuals, fibroblasts from 2 patients with resistance to thyroid hormones were used. One patient has a heterozygous mutation (A317T, TRβmut) and the other a homozygous deletion of the TRβ gene (TRβ0). STC1 mRNA abundance after T3 treatment is expressed relative to that in fibroblasts cultured for 72 h in TH depleted medium. A dose dependent increase in STC1 mRNA was observed in cells from 2 normal individuals: 1.4- and 1.3-fold increase after 0.1 nM T3 and 1.8- and 2.1-fold increase after 0.5 nM T3, and 2.7 and 3.1-fold increase after 2 nM T3 (Fig. 1a). In the fibroblasts from subjects with RTH, no such effect of TH was observed, as 1.3-and 1.0-fold changes after 0.1 nM T3, 1.4- and 0.9-fold changes after 0.5 nM T3 and 1.3- and 0.7-fold changes after 2 nM T3 were found for the TRβmut and TRβ0 fibroblasts, respectively (Fig. 1a). These results demonstrate that STC1 is induced by TH. This effect is dose dependent and requires an intact TRβ.

Fig. 1.

Fig. 1

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Induction of STC1 mRNA expression by T3 in cultured human fibroblasts. Human skin fibroblasts were cultured in TH-depleted medium (TxBS) for 48 h and then treated with 3 different doses of T3 for 24 h prior to submission to microarray analysis. a, T3-dose dependent response of STC1 occurred in fibroblasts from normal individuals (left panels), but not in fibroblasts from 2 patients with RTH due to the dominant negative TRβ gene mutation, A317T (TRβmut) and homozygous deletion (TRβ0) (right panels). b, The effect of 0.5 and 2 nM T3 on STC1 mRNA expression was measured by real time PCR in fibroblasts from a normal individual and a patient with RTH (TRβ mutation A317T). Shown is the mean ± SE (n = 3 for each treatment).

These results were confirmed by real-time PCR in an independent series of experiments. A similar dose dependent increase in STC1 mRNA was observed in normal fibroblasts 24 h after addition of 0.5 and 2 nM T3 compared to untreated fibroblasts and expressed as fold-change (2.0 ± 0.2; P < 0.01 after 0.5 nM T3 and 2.7 ± 0.2; P < 0.005 after 2 nM T3) (Fig. 1b). This increase was greatly reduced in the RTH fibroblasts (1.3 ± 0.3; n.s. after 0.5 nM T3 and 1.6 ± 0.4; P < 0.005 after 2 nM T3) (Fig. 1b). The difference between the STC1 mRNA levels in normal and RTH fibroblasts was significant for the 2 nM T3 treatment (2.7 ± 0.2 vs. 1.6 ± 0.4; P < 0.01). These results confirm both the stimulatory effect of TH on STC1 expression as well as the crucial role of the TRβ.

T3-induced increase in STC1 mRNA requires de novo protein synthesis

Next, we investigated whether STC1 induction is direct or indirect, requiring de novo protein synthesis. Fibroblasts from a normal individual were cultured in T3-depleted medium for 48 h, followed by treatment with 2 nM T3 for 24 h in the absence or presence of the translation inhibitor CHX (10 µg/ml), added 1 h before T3. Again, an increase in STC1 mRNA after T3 treatment was observed. This increase was significantly diminished by pretreatment with CHX (2.7 ± 0.3 vs. 1.3 ± 0.1, P < 0.0005) (Fig. 2). These results indicate that STC1 is indirectly induced by TH.

Fig. 2.

Fig. 2

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Effect of T3 on STC1 mRNA in the absence and presence of the protein synthesis inhibitor CHX. Normal fibroblasts were cultured in DMEM with 10% TH-depleted bovine serum for 48 h and then treated with 2 nM T3 without or with pretreatment with CHX (10 µg/ml). Cells were harvested 24 h after T3 treatment. The relative amount of STC1 mRNA, as measured by real-time PCR and compared to fibroblasts not treated with T3, is plotted on the ordinate as fold increase (mean ± SE, n = 8).

T3-induced increase in STC1 mRNA is dependent on PI3K activation

To test whether the PI3K pathway is involved in mediating the T3 effect on STC1 expression, we treated normal fibroblasts with T3 in the absence and presence of the PI3K inhibitor LY294002. STC1 mRNA levels, measured by real-time PCR and expressed as fold-change relative to those in cells grown in T3-depleted serum, are shown in Fig. 3. Consistent with the previous results shown in Fig. 1, T3 treatment increased STC1 mRNA levels (3.9 ± 0.5; P < 0.005). This effect of T3 was completely abrogated by LY294002 (0.85 ± 0.5; n. s.). These results demonstrate that the stimulatory effect of TH on STC1 mRNA requires PI3K activation.

Fig. 3.

Fig. 3

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Effect of T3 and the PI3K inhibitor LY294002 on STC1 mRNA levels. Normal fibroblasts were cultured for 48 h in DMEM supplemented with 10% TH-depleted bovine serum (TxBS) and then treated for 24 h with 2 nM T3 in the absence or presence of LY294002 (LY, 50 µM), added 1 h before the addition of T3. The relative amount of mRNAs, as measured by real-time PCR, and compared to that in fibroblasts cultured in TH-depleted medium is plotted on the ordinates as fold increase. Data are expressed as mean ± SE (n = 3 for each treatment).

This was confirmed by a different approach. PI3K consist of the catalytic p110 subunit and the regulatory p85α subunit. Transfection of a dominant negative p85α mutant, Δp85α, can inhibit PI3K. Fibroblasts cultured in T3-depleted medium were incubated with T3 for 12 h after infection with either a green fluorescent protein expressing adenovirus (Ad-GFP) or an adenovirus expressing Δp85α (Ad-Δp85α). As shown in Fig. 4a, the infection with Δp85α reduced T3-induced STC1 expression to half compared to cells transfected with Ad-GFP, further demonstrating the involvement of PI3K. Basic transcription element binding protein 1 (BTEB1) mRNA induction by T3 in human cells on the other hand is independent of PI3K (Moeller et al., 2005a) and could therefore serve as control. BTEB1 expression was not affected by infection with Ad-Δp85α (Fig. 4b), suggesting that reduction of STC1 expression is a specific effect of PI3K inhibition.

Fig. 4.

Fig. 4

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Inhibition of T3-dependent STC1 expression by Δp85α. Adenovirus expressing either green fluorescent protein (Ad-GFP) or Δp85α (Ad-Δp85α) was infected at a M.O.I of 200 for 1 h. Fibroblasts were then incubated in DMEM with 5% T3-depleted FBS for 48 h and treated with T3 (10 nM) for 12 h. The relative amount of a, STC1 and b, BTEB1 mRNAs, measured by real-time PCR and compared to that in fibroblasts cultured in TH-depleted medium, is plotted on the ordinates as fold increase (mean of 2 experiments).

Discussion

A wide range of TH effects are mediated by its influence on gene expression. In recent years, several new mechanisms of TH effects on gene expression were described in addition to the classic nuclear mechanism of a TR/RXR heterodimer bound to a TRE in the promoter of a target gene. For example, TH can induce genes independent of TRs, as shown for basic fibroblast growth factor 2 (FGF2). TH binds to a membrane bound integrin, αVβ3, which subsequently leads to activation of the MAPK pathway and FGF2 mRNA increase (Davis et al., 2004).

Another mechanism of TH action leading to increased gene expression is the TR-mediated activation of the PI3K pathway by protein-protein interaction of the liganded TRβ with the p85α subunit of PI3K. The definition of the TRβ therefore has broadened beyond being merely a ligand-dependent transcription factor, acting on a TRE in the promoter of target genes. It can also induce gene expression through TRE-independent activation of signal transduction pathways such as the PI3K pathway (Cheng et al., 2010, Moeller et al., 2006).

We show here that STC1 is a TH inducible gene with a dose dependent mRNA response for physiological TH concentrations. The action of TH is TRβ mediated, because cells from patients with RTH showed a significantly reduced STC1 response to TH compared to cells from normal individuals.

In addition to TRβ, STC1 induction by TH is dependent on PI3K activation, since inhibiting PI3K with either LY294002 or Δp85α reduced the TH effect on STC mRNA expression. We observed the same pattern of TRβ- and PI3K-dependent induction for HIF-1α and subsequently its then known target genes PFKP and GLUT1 as well as for MCT4, which was later found to be a HIF-1 target gene, too (Moeller et al., 2005a, Ullah et al., 2006). Because STC1 was recently also reported to be a target gene of HIF-1 (Law et al., 2010, Yeung et al., 2005), it is reasonable to assume that TH exerts its effect on STC1 through the same TRβ/PI3K mediated mechanism.

There was no effect of T3 on STC1 expression in cells with homozygous deletion of the TRβ. As the resistance to TH in the case of heterozygous TRβ mutations is partial, a minimal response of STC-1 expression to T3 could be observed in these cells. Due to the variable degree of resistance among different mutations, there may be differences in the degree of STC1 expression, dependent on the mutation. In contrast, higher STC1 levels seem possible in the case of the TRβPV mutant, because in TRβPV/PV knock in mice, the TRβPV mutant bound significantly more to the PI3K-regulatory subunit p85α, resulting in a greater increase in the kinase activity than did TRβ1 in wild-type mice (Furuya et al., 2006).

To which physiological or pathophysiological effects could this mechanism of TRβ/PI3K mediated TH action contribute? A potential role of thyroid hormone in tumor progression was recently supported by the findings of Davis et al. They could demonstrate in glioblastoma cells that TH induced cell proliferation, which was mediated by the integrin αVβ3 and the MAPK pathway (Davis et al., 2006). Interestingly, STC1 was linked to angiogenesis in a glioblastoma cell line (Lal et al., 2001) and STC1 expression was enhanced in several types of cancer (colon, breast, hepatocellular and medullary thyroid cancer) (Chang et al., 2003). Further studies are needed to determine whether PI3K activation and STC1 induction by TH contribute to tumor progression.

Acknowledgments

This work was supported in part by grants DK 15070 from the National Institutes of Health and RR 00055 from the US public Health Service (to S. R.); Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (Monbukagakusho) of Japan (nos. 13470217 and 16390269) (to H.S.); by the 21st Century COE program “Integrated Molecular Medicine for Neuronal and Neoplastic Disorders” of Monbukagakusho (to H.S. and X.C.), the Howard Hughes Medical Institute (Predoctoral Fellowship to A.M.D.) and the Deutsche Forschungsgemeinschaft DFG (Mo 1018/1-1 to L.C.M.).

Footnotes

Conflict of Interest: None.

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