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Endocrinology, doi:10.1210/en.2007-1238
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Endocrinology Vol. 149, No. 5 2241-2250
Copyright © 2008 by The Endocrine Society

Thyroid Hormone Induces Hypoxia-Inducible Factor 1{alpha} Gene Expression through Thyroid Hormone Receptor β/Retinoid X Receptor {alpha}-Dependent Activation of Hepatic Leukemia Factor

Teresa Otto and Joachim Fandrey

Institut für Physiologie, Universität Duisburg-Essen, D-45122 Essen, Germany

Address all correspondence and requests for reprints to: Joachim Fandrey, M.D., Institut für Physiologie, Universität Duisburg-Essen, Hufelandstrasse 55, D-45122 Essen, Germany. E-mail: joachim.fandrey{at}uni-due.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Thyroid hormones are important regulators of differentiation, growth, metabolism, and physiological function of virtually all tissues. Active thyroid hormone T3 affects expression of genes that encode for angiogenic proteins like adrenomedullin or vascular endothelial growth factor and erythropoietin, as well as for glucose transporters and phospho fructokinase that determine glucose use. Interestingly, those target genes are also hypoxia inducible and under the control of the oxygen-dependent transcription factor hypoxia-inducible factor (HIF)-1). We and others have reported that T3 stimulates HIF-1 activation, which intimately links T3 and HIF-1 induced gene expression. Here, we studied intracellular pathways that mediate HIF-1{alpha} regulation by T3. We found that T3-dependent HIF-1 activation is not limited to hepatoma cells but is also observed in primary human hepatocytes, kidney and lung carcinoma cells. T3 increased the HIF-1{alpha} subunit mRNA and protein within a few hours through activation of the thyroid hormone receptor β retinoid X receptor {alpha} heterodimer because knockdown of each of the partners abrogated the stimulation by T3. However, T3 had no direct effect on transcription of HIF-1{alpha}, but activation of the thyroid hormone receptor β/retinoid X receptor {alpha} heterodimer by T3 stimulated expression of the hepatic leukemia factor, which increases HIF-1{alpha} gene expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TO SUPPLY CELLS with sufficient oxygen and nutrients when they are scarce goods, the transcription factor complex hypoxia-induced factor (HIF)-1 coordinates the expression of genes that ensure proliferation and cell death, vascularization, and vascular tone, and finally respiration and glycolysis. HIF-1 consists of two subunits, the oxygen-sensitive {alpha}-subunit (120 kDa) and the constitutively expressed HIF-1β (91–94 kDa, arylhydrocarbon nuclear translocator). Both subunits belong to a subfamily of bHLH-PAS-transcription factors that also includes the HIF-1{alpha} orthologues HIF-2{alpha} and HIF-3{alpha} (1).

The activation of HIF-1 is a multistep process, including posttranslational stabilization, nuclear translocation, and transcriptional activation. Under normoxia HIF-1{alpha} is subjected to oxygen-dependent hydroxylation, ubiquitinylation, and degradation in proteasomes. Hydroxylated HIF-1{alpha} binds von Hippel-Lindau tumor suppressor protein, which acts as a recognition site for an E3-ubiquitin ligase that marks HIF-1{alpha} for degradation by polyubiquitinylation (2). The interaction between HIF-1{alpha} and von Hippel-Lindau tumor suppressor protein requires the O2- and iron-dependent hydroxylation of proline 402 and 564 in the central oxygen-dependent degradation domains of HIF-1{alpha} by prolyl hydroxylases (1, 2, 3). Because of the oxygen dependence of prolyl hydroxylase activity, these enzymes are classified as the cellular oxygen sensors, which regulate the amount of HIF-1{alpha}-subunits in the cell (3). Under hypoxia the hydroxylation and subsequent degradation of HIF-1{alpha} cease, and the HIF-1{alpha} subunits can accumulate.

Transactivation is dependent on an additional oxygen sensor, the HIF-asparaginyl-hydroxylase factor inhibiting HIF-1, which hydroxylates the asparagine (Asn803 in human HIF-1{alpha}) within the C-terminal transactivation domain. Hydroxy-Asn prevents the binding of p300/CBP to HIF-1{alpha} that serves as a scaffold for recruiting further transcription factors, and can facilitate the transcription by acetylation and relocation of histone proteins (4). As a consequence the HIF-1{alpha}-C-terminal transactivation domain remains inactive under normoxic conditions (5). Well-known HIF-1 target genes are erythropoietin (EPO) (5, 6), adrenomedullin (ADM) (7), vascular endothelial growth factor (VEGF) (8), glucose transporters (GLUTs) (9), or glycolytic enzymes (10) that control erythropoiesis, angiogenesis, and glucose use, demonstrating the central role of HIF-1 for hypoxic adaptation on systemic, local, and cellular levels (11).

Thyroid hormone T3 as a mediator of increased metabolic rate and oxygen consumption has regulated HIF-1{alpha} protein accumulation (12, 13). However, discrepancies with respect to the time course and mechanism of HIF-1{alpha} induction were observed. Short-term regulation of HIF-1{alpha} by T3 in human hepatoma cells HepG2 was attributed to increased translation of HIF-1{alpha} mRNA, but no evidence for the engaged signaling pathways was provided (12). In contrast, primary human fibroblasts reacted with late HIF-1{alpha} stimulation, i.e. after 24 h, by T3-mediated induced transcription using nonnuclear signaling via phosphatidylinositol 3 (PI3)-kinase (13).

Compared with nonnuclear signaling, T3 classically binds to specific nuclear receptors, the thyroid hormone receptors (TRs) (14). TRs are members of the nuclear receptor superfamily, which includes receptors for steroid hormones and retinoids and that act as transcriptions factors directly regulating target gene expression by binding to regulatory DNA elements and coactivator complexes (15). Two isoforms of TRs, TR{alpha} and TRβ, can either bind to DNA as monomers, or form homodimers or heterodimers (14) with proteins such as retinoid X receptors (RXRs) to bind to thyroid hormone response elements (TREs) (16). TRs can either repress or activate gene expression, depending on the presence of active T3. If T3 is not available, receptors interact with a transcriptional corepressor complex, including histone deacetylases, to form a compact, "turned-off" conformation of chromatin. T3 binding to receptors results in a conformational change. The corepressor complex dissociates and enables binding to the coactivator complex with histone transacetylase activity, which imposes an open configuration on adjacent chromatin. This molecular rearrangement alters repressed receptors into transcriptional activators (17).

Interestingly, functions and, therefore, target genes of T3 and HIF-1{alpha} are overlapping, such as expression of EPO (6, 18), ADM (19), GLUT, and glycolytic enzymes (13). Thus, here, we studied how thyroid hormones exert their effects on HIF-1 by classical signaling through the TRβ/RXR{alpha} nuclear receptor activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
The human hepatoma cell line, HepG2, was cultured in RPMI 1640 (BioWhittaker; Cambrex Karlskoga AB, Karlskoga, Sweden) supplemented with 10% fetal calf serum (FCS) (Life Technologies, Inc., Karlsruhe, Germany), 1% penicillin (100 U/ml), and 1% streptomycin (100 g/ml) (Life Technologies). The human primary liver cells were grown in Williams E supplemented with 10% FCS (Life Technologies), 1% penicillin/streptomycin (Life Technologies), 1% sodium pyruvate (Life Technologies), 1% L-glutamine (Life Technologies), 1.5% 1 M HEPES (Life Technologies), 0.08% insulin (Life Technologies), and 0.02% dexamethasone (Life Technologies). The human kidney carcinoma cell line KiKa was cultured in RPMI 1640 (BioWhittaker) supplemented with 10% 575H serum (Biochrom, Berlin, Germany), 1% penicillin (100 U/ml), and 1% streptomycin (100 g/ml) (Life Technologies). The lung carcinoma cell line A549 was cultured in Ham’s F12 (with glutamine and 25 mM HEPES; PAA, Cölbe, Germany) with 10% FCS (Life Technologies), 1% penicillin (100 U/ml), and 1% streptomycin (100 g/ml) (Life Technologies). All cells were cultured in a humidified atmosphere (5% CO2 in air) at 37 C. To exclude gene mutations, especially known for thyroid receptors in hepatocellular carcinoma cell lines (20), cells were used until they reached the 15th passage.

For experiments cells were maintained under serum free conditions with 1% serum supplement (Sigma-Aldrich, Seelze, Germany) 24 h before and during the experiments. To achieve hypoxic conditions, all cell lines were placed in a Heraeus incubator (Hanau, Germany) with 5% CO2 and nitrogen to balance for 3% O2 concentration.

Reagents
T3, tetraiodothyro-acetic acid (Tetrac), actinomycin D, and cycloheximide were obtained from Sigma-Aldrich. Small interfering RNAs (siRNAs) against TRβ (hp-validated siRNA Hs_THRB_7) were from by QIAGEN (Hilden, Germany), and siRNA against TR{alpha} (ON-TARGETplus SMARTpool human TR{alpha}), RXR{alpha} (ON-TARGETplus SMARTpool human RXR{alpha}), HIF-1{alpha} (siGENOME duplex human HIF-1{alpha} siRNA), and hepatic leukemia factor (HLF) (siGENOME SMARTpool human HLF), as well as nontarget siRNA (siCONTROL nontargeting siRNA no. 2) were from Dharmacon (Perbio, Bonn, Germany). Oligofectamine and all primers were purchased from Invitrogen (Karlsruhe, Germany). For plasmid transfection Fugene 6 (Roche GmbH, Mannheim, Germany) was used. The inhibitors SB203590, Wortmannin, LY294002, and Rapamycin were purchased from Calbiochem (Merck Chemicals Ltd., Darmstadt, Germany), and PD98059 and U0126 from Cell Signaling Technology, Inc. (Danvers, MA). HIF-1{alpha} antibody was from Biotransduction Laboratories (Lexington, KY) and {alpha}-tubulin from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany). Oligo(dT) and reverse transcriptase were obtained from Promega (Heidelberg, Germany). SYBR Green I was from Eurogentec (Köln, Germany).

Quantitative real-time PCR
RNA was isolated by the guanidinium isothiocyanate extraction as described (21). Total RNA (1 µg) was reverse transcribed with oligo(dT) and Moloney murine leukemia virus reverse transcriptase (Promega). Gene expression was quantitated using the qPCR Mastermix for SYBR Green I (Eurogentec) and the GeneAmp 5700 sequence Detection System (PE Biosystems, Applied Biosystems, Oxford, UK) or iCycler (Bio-Rad, Munich, Germany). The real-time PCR were set up in a final volume of 25 µl with 1 µl cDNA and a primer concentration of 10 pmol each. Used forward (F) and reverse (R) primers: ADM, (F) 5'-GGATGCCGCCCGCATCCGAG-3' and (R) 5'-GACACCAGAGTCCGACCCGG-3'; β-actin, (F) 5'-TCACCCACACTGTGCCCATCTACGA-3' and (R) 5'-CAGCGGAACCGCTCATTGCCAATGG-3'; HIF-1{alpha}, (F) 5'-GCTGGCCCCAGCCGCTGGAG-3' and (R) 5'-GAGTGCAGGGTCAGCACTAC-3'; TR{alpha}, (F) 5'-ATCTATCCACATTGCCACA-3' and (R) 5'-TGGTAAACTCGCTGAAGGCT-3'; TRβ, (F) 5'-GGAGAACCGGGAGAAAAGAC-3' and (R) 5'-GGGCATTGACTATTGGTGCT-3'; RXR{alpha}, (F) 5'-TTCTCCACCCAGGTGAACTC-3' and (R) 5'-GAGCTGATGACCGAGAAAGG-3'; and HLF, (F) 5'-TCAGACCAGGTCAGCTGTTG-3' and (R) 5'-GCTGTGGCTTCAGTTCTTCC-3'. Dilutions of purified PCR products starting at 1 pg to 0.001 fg were used as standards. Amplification conditions were set to 10 min at 95 C, followed by 40 PCR cycles for β-actin and 45 PCR cycles for the other genes (15 sec at 95 C, 1 min at 60 C). The quantity of any cDNA was normalized to β-actin cDNA and expressed as fold induction.

Protein extract preparation and Western immunoblotting
For whole cell lysates, primary liver cells were lysed with 100 µl and all other cell lines with 50 µl extract buffer [0.1% NP-40, 300 mM NaCl, 10 nM Tris (pH 7.9), 1 mM EDTA, 1:10 dilution protein-inhibitor-cocktail] for 20 min on ice. Extracts were centrifuged (3600 x g for 5 min at 4 C), and the supernatant was quantitated using the Bio-Rad protein assay reagent and stored at –20 C. After addition of one fourth volume of 4x sample buffer [50 mM Tris (pH 6.8), 2% sodium dodecyl sulfate, 5% β-mercaptoethanol, 0.0125% bromphenol blue, and 1% glycerin], samples were subjected to 7.5% SDS-PAGE, separated, and transferred to a nitrocellulose membrane (0.2-µm pore size; Whatman, Schleicher & Schuell, Dassel, Germany). Blots were stained with Ponceau S solution (Bio-Rad) to ensure equal protein loading and transfer. The membranes were blocked with blocking solution (5% nonfat dry milk powder in Tris-buffered saline with Tween 20) overnight and afterwards were incubated with primary antibodies diluted in blocking solution (HIF-1{alpha} and HIF-1β 1:500). After washing with Tris-buffered saline with Tween 20 [20 mM Tris-Base, 140 mM NaCl (pH 7.6) with 1:2000 Tween 20], horseradish peroxidase-conjugated antimouse IgG or antirabbit antibodies were used as a secondary antibody at a 1:1000 dilution in blocking solution. Anti-{alpha}-tubulin antibody (at a 1:750 dilution; Santa Cruz Biotechnology) was used as a loading control. Immunoreactive proteins were visualized using the luminol coumarin acid H2O2 system [per membrane: 12.5 µl 90 mM coumarin acid, 25 µl 250 nM luminol, 1.5 µl 30% H2O2 in 5 ml 100 mM Tris/Cl (pH 8.5)] for hepatic cells and ECL Advanced System (GE Healthcare, formerly Amersham Biosciences, Munich, Germany) for all other cells, followed by exposure to x-ray film (Agfa, Cologne, Germany).

Immunohistochemistry
To ensure the stabile adhesion to glass, sterile coverslips were incubated with poly-D-lysin for 30 min before adding cells. After the experiments cells were fixed with a 1:1 acetone-methanol mixture and finally blocked with 3% BSA diluted in PBS for 30 min. Including three washing steps with PBS between both steps, the first antibody (1:50 in blocking solution) was incubated for 2 h, and then the second antibody (Alexa 568 antimouse, 1:400 in PBS) was incubated dry and lightproof for 1 h. The coverslips were fixed on the slides using Moviol and were dried overnight in the dark. The immunostained cells were visualized by fluorescence microscopy on the next day.

Transient transfection assays
For reporter gene assays, cells were transfected with Fugene 6 (Roche GmbH). The luciferase reporter gene plasmid pH3SVL containing an SV40 promoter-luciferase unit downstream of six HIF binding sites from the transferrin enhancer was a kind gift of Professor R. Wenger (Zurich, Switzerland). The promoter plasmid pGL3-Prom800 containing the 5'-untranslated region (UTR) and the promoter of HIF-1{alpha} (–541 to –1 bp) was a kind gift of Professor C. Michiels (Namur, Belgium). The bicistronic vector RhifF contains the complete 5'-UTR of HIF-1{alpha}, which includes an internal ribosomal entry site (IRES). It was a kind gift of Professor B. Brune (Frankfurt, Germany).

Cells were transfected with 0.25 µg plasmid and 0.75 µl Fugene six per 24-well. After overnight incubation in serum free media, experiments were started. Afterwards, cells were lysed with 100 µl 1x lysis buffer. After 24 h at –20 C, luciferase activity was measured with the Firefly Luciferase assay kit (Biotium, Biotrend Chemikalien GmbH, Cologne, Germany) or Dual-Luciferase reporter assay system (Promega) regarding the description and was expressed in relative light units (RLUs) normalized to total cellular protein.

siRNA transfection assays
Different end concentrations of 20 µM stocks of siRNAs were transfected with 10 µl Oligofectamine (Invitrogen) per six-well dish. After 24-h incubation without serum and antibiotics, the medium was changed, and experiments were started.

Statistics
Statistical significance was calculated using the GraphPad Prism 5.0 software (GraphPad Software Inc., San Diego, CA) applying the one-way ANOVA, followed by the Bonferroni test. An asterisk indicates a P value less than 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Active thyroid hormone T3 enhances HIF-1{alpha} accumulation, nuclear translocation, and HIF-1 target gene expression
In contrast to our previous study (12), the concentration of T3 was reduced to 10 nM, reflecting the upper range of physiological T3 levels measured in FCS (data not shown). HepG2 hepatoma cells treated with 10 nM T3 under normoxia or hypoxia showed maximum HIF-1{alpha} protein levels after 5 h hypoxic incubation, whereas an increase under normoxia was almost undetectable by Western blot analysis (Fig. 1AGo). HIF-1β protein was constitutively present and was not influenced by thyroid hormone treatment (data not shown). Likewise, HIF-2{alpha} was not affected by T3 (data not shown), confirming our earlier data (12) and excluding aggravated hypoxia due to T3-stimulated O2 consumption as a cause for increased HIF-1{alpha} levels. Immunohistochemistry for HIF-1{alpha} revealed a remarkable increase of nuclear HIF-1{alpha} after 5-h incubation with T3, even under normoxia, whereas constitutively expressed HIF-1β was not changed (Fig. 1BGo).


Figure 1
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  		FIG. 1. Stimulation of HIF-1 accumulation, transactivation, and HIF-1-dependent ADM expression in HepG2 cells by T3. A, Time course of HIF-1{alpha} accumulation. HepG2 cells were treated with 10 nM T3 or carrier dimethyl sulfoxide under normoxic (NOX) (21% O2) or hypoxic (HOX) (3% O2) conditions. One hundred micrograms of whole cell lysates were submitted to Western blot analysis using anti-HIF-1{alpha}. {alpha}-Tubulin served as the loading control (n = 9). T3-induced HIF-1{alpha} accumulation reached its maximum after 5 h incubation. B, Immunofluorescence of HIF-1{alpha} (red) and HIF-1β (green). HepG2 cells were treated with 10 nM T3 or carrier dimethyl sulfoxide for 5 h under normoxic (21% O2) or hypoxic (3% O2) conditions. Acetone/methanol-fixed cells were submitted to immunofluorescence analysis using HIF-1{alpha} and HIF-1β, detected by Alexa 568 (red, for HIF-1{alpha}) and Cy2 (green, for HIF-1β) (n = 8). HIF-1{alpha} accumulation and translocation into the nucleus were induced by T3, whereas HIF-1β was not changed. C, Activity of HIF-1 complex. HepG2 cells were transfected with reporter gene vector pH3SVL containing three hypoxia response elements (HRE) from the transferrin 5'-enhancer. After transfection, cells were exposed to 10 nM T3 or carrier dimethyl sulfoxide under hypoxic (3% O2) conditions (n = 16). T3 significantly induced the activity of HIF-1 after 5-h incubation. D, Expression of ADM. HepG2 cells were treated with 10 nM T3 or carrier dimethyl sulfoxide under hypoxic (3% O2) conditions. After isolation of RNA and RT into cDNA, samples were quantitated using real-time PCR. Femtograms of ADM cDNA is shown, normalized to 1 pg β-actin (n = 12). T3-induced HIF-dependent ADM induction was observed after 5-h incubation. *, P < 0.05.

 
To determine whether the T3-regulated HIF-1{alpha} exhibits transcriptional activity by forming a heterodimeric complex with HIF-1β that binds to HIF-1 binding sites, we transfected HepG2 cells with a reporter construct containing six HIF-1 binding sites. T3 increased HIF-1 activity after 5 h compared with hypoxia alone, which corresponds well to the highest levels of HIF-1{alpha} protein (Fig. 1CGo). Control vector pGL3 was induced by T3 about 9.3 ± 0.12% after 5 h, which was significantly less than T3-mediated induction of the pH3SVL vector (20.5 ± 0.5%). Likewise, the HIF-1 target gene ADM was induced by T3 (Fig. 1DGo).

T3-dependent HIF-1{alpha} induction is not mediated by kinase pathways
T3 may exert its function by activation of nonnuclear second messenger pathways or acting via nuclear receptors. Incubation with T3 for 24 h has recently been reported to induce HIF-1{alpha} by nonnuclear pathways (22). Therefore, we started to inhibit MAPKs p38 with SB203590 and p42/44 with PD98059, and the more specific U0126. None of the MAPK-inhibitors had an effect on T3-mediated HIF-1{alpha} accumulation but, depending on concentration, reduced hypoxic HIF-1{alpha} (Fig. 2Go). Likewise, the inhibition of phosphatidylinositol-3-kinase by Wortmannin and LY294002, as well as the inhibition of P70S6-kinase/mammalian target of Rapamycin (mTor) by Rapamycin had no effect on T3-induced HIF-1{alpha} protein.


Figure 2
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  		FIG. 2. Nonnuclear regulation of T3-induced HIF-1{alpha} accumulation. T3-mediated HIF-1{alpha} accumulation is not regulated by MAPK, PI3-kinase, or p70S6 kinase/mTOR. HepG2 cells were incubated with 10 nM T3 or carrier dimethyl sulfoxide under hypoxic (3% O2) conditions. In addition, inhibitors of p38 [SB203590 (SB)], p42/44 [PD98059 (PD) and U0126 (U0)], PI3-kinase [Wortmannin (WM) and LY294002 (LY)], p70S6 kinase/mTOR [Rapamycin (RM)], or carrier were added before starting the experiments. After the experiments, cells were lysed and used for Western blot analysis (n = 6). {alpha}-Tubulin served as a loading control. Although basic HIF-1{alpha} accumulation was reduced by all MAPK-inhibitors, the T3-induced HIF-1{alpha} accumulation remained unaffected. T3-dependent HIF-1{alpha} accumulation also remained unaffected by all other inhibitors.

 
T3-dependent HIF-1{alpha} induction is mediated by TRβ and RXR{alpha} in hepatoma cells and primary human hepatocytes
To determine whether T3 regulates HIF-1{alpha} transcription via nuclear receptors, we transfected HepG2 cells with siRNA against the two isoforms of TR and the heterodimerization partner retinoid receptor RXR{alpha}. Although a knockdown of TR{alpha} (87% efficiency) did not reduce the T3-mediated HIF-1{alpha} accumulation, knockdown of TRβ (86% efficiency) as well as knockdown of RXR{alpha} (78% efficiency) abrogated the T3 effect on HIF-1{alpha} (Fig. 3Go). Treatment with Tetrac, a product of deamination and oxidative decarboxylation of T4, showed no effect on HIF-1{alpha} accumulation.


Figure 3
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  		FIG. 3. Nuclear signaling by thyroid hormones induced HIF-1{alpha} accumulation. A, T3-mediated HIF-1{alpha} accumulation is regulated by TRβ/RXR{alpha} heterodimer. HepG2 cells were transfected with no siRNA (Mock), nontarget siRNA (siLuc) or 50 nM siRNA against HIF-1{alpha} (siHIF), TR{alpha} (siTR{alpha}), TRβ (siTRβ), and RXR{alpha} (siRXR{alpha}). Transfection with nontarget siRNA served as a control. Cells were incubated with 10 nM T3 or carrier dimethyl sulfoxide under hypoxic (3% O2) conditions. At the end of the experiments, cells were lysed and used for Western blot analysis. {alpha}-Tubulin served as a loading control (n = 8). Although the knockdown of TR{alpha} did not reduce the T3 effect on HIF-1{alpha} protein, a knockdown of HIF-1{alpha} produced a reduction of accumulation. The knockdown of TRβ and RXR{alpha} caused a distinct loss of T3-induced HIF-1{alpha}, whereas the basic hypoxic level remained unaffected. B, HIF-1{alpha} accumulation is induced by T3 and T4, but not by Tetrac. HepG2 cells were treated with 10 nM T3, T4, Tetrac, or carrier dimethyl sulfoxide for 5 h under hypoxic (3% O2) conditions. One hundred micrograms of whole cell lysates were submitted to Western blot analysis using anti-HIF-1{alpha}. {alpha}-Tubulin served as a loading control (n = 3). Tetrac had no effect on HIF-1{alpha} accumulation, whereas both thyroid hormones induced HIF-1{alpha}.

 
Primary human hepatocytes were used to test for a similar induction of HIF-1{alpha} in nonmalignant cells. As in HepG2 cells, a maximum of HIF-1{alpha} induction by T3 was observed after 5 h incubation (Fig. 4AGo), whereas hypoxic HIF-1{alpha} accumulation was faster than in HepG2 cells. Knockdown of TRβ (70% efficiency) and RXR{alpha} (70% efficiency) in primary hepatocytes abolished T3-stimulated HIF-1{alpha} accumulation, whereas the knockdown of TR{alpha} (73% efficiency) had no effect (Fig. 4BGo). Consequently, T3-induced ADM expression in primary hepatocytes was suppressed when TRβ was blocked (Fig. 4CGo). In comparison to knockdown of HIF-1{alpha}, ADM expression was even more reduced by TRβ knockdown, indicating that T3 may directly induce ADM in addition to the effects through HIF-1{alpha}.


Figure 4
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  		FIG. 4. HIF-1{alpha} induction in primary human hepatocytes. A, Time course of T3-regulated HIF-1{alpha} accumulation. Primary human hepatocytes were treated with 10 nM T3 or carrier dimethyl sulfoxide as a control under hypoxic (3% O2) conditions. One hundred fifty micrograms of whole cell lysate were submitted to Western blot analysis using anti-HIF-1{alpha}. {alpha}-Tubulin served as a loading control (n = 6). Primary hepatocytes respond to T3 with a maximally induced HIF-1{alpha} accumulation after 5 h. B, Regulation by TRβ/RXR{alpha} heterodimer. Primary hepatocytes were transfected with 100 nM siRNA against HIF-1{alpha} (siHIF1{alpha}), TRβ (siTRβ), HLF (siHLF), siTR{alpha} (siTR{alpha}), and RXR{alpha} (siRXR{alpha}). The transfection with no siRNA (Mock) or nontarget siRNA (siLuc) served as controls. Afterwards, the cells were incubated for 5 h with 10 nM T3 or carrier dimethyl sulfoxide under hypoxic (3% O2) conditions. After experiments, cells were lysed and used for Western blot analysis. {alpha}-Tubulin served as a loading control (n = 6). The knockdown of TRβ, RXR{alpha}, and HLF caused a loss in HIF-1{alpha} induction by T3, whereas hypoxic accumulation remained unaffected. C, Regulation of ADM expression after HIF-1{alpha} and TRβ knockdown. Primary human hepatocytes were transfected with 100 nM siRNA against HIF-1{alpha} and TRβ. The transfection with nontarget siRNA served as a control. Cells were incubated for 5 h with 10 nM T3 or carrier dimethyl sulfoxide under hypoxic (3% O2) conditions. RNA was isolated, transcribed into cDNA, and measured with quantitative real-time PCR. Femtograms of ADM cDNA are shown, normalized to 1 pg β-actin (n = 3). Although a knockdown of TRβ completely abrogated the T3-induced ADM expression, the knockdown of HIF-1{alpha} left a minimal induction of ADM by T3, which may be HIF-1 independent.

 
Active T3 does not influence translation but transcription
To examine further the mechanisms by which T3 increases HIF-1{alpha} protein levels, we studied whether T3 affects HIF-1{alpha} mRNA translation. HepG2 cells were treated with 10 µg/ml cycloheximide to inhibit translation that had, however, no effect on T3-induced HIF-1{alpha} levels but decreased hypoxic HIF-1{alpha} accumulation (Fig. 5AGo). Analysis of cap-independent translation using a bicistronic reporter gene vector that included the 5'-UTR of HIF-1{alpha} with an IRES exhibited a strong increase in translational efficiency under hypoxia, but no additional induction by T3 (Fig. 5BGo).


Figure 5
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  		FIG. 5. T3-mediated HIF-1{alpha} induction does not depend on increased translation. A, Inhibition with cycloheximide does not diminish T3-induced HIF-1{alpha} accumulation. HepG2 cells were incubated with 10 nM T3 or carrier dimethyl sulfoxide for 5-h hypoxia (3% O2). Ten, 20, and 30 min before the end of the experiments, 10 µg/ml cycloheximide (CHX) or carrier dimethyl sulfoxide was added. Seventy-five micrograms of whole cell lysate were submitted to Western blot analysis using anti-HIF-1{alpha}. {alpha}-Tubulin served as a loading control (n = 6). The inhibition of translation did not influence T3-induced HIF-1{alpha} accumulation. B, Cap-independent translation by IRES is not responsible for T3-induced HIF-1{alpha} accumulation. HepG2 cells were transfected with bicistronic vector pRF [empty control (RF)] of pRhifF (RhifF) containing the complete 5'-UTR of HIF-1{alpha}, including an IRES. Cells were exposed to 10 nM T3 or carrier dimethyl sulfoxide under hypoxic (3% O2) conditions and lysed. RLUs were determined and normalized to µg total protein (n = 12). No T3-induced activity of reporter gene was observed. C, Actinomycin D abrogated the T3-induced HIF-1{alpha} accumulation. HepG2 cells were incubated with 10 nM T3 or carrier dimethyl sulfoxide under hypoxia (3% O2). At the beginning of the experiment, cells were treated with 1 µg/ml actinomycin D or carrier dimethyl sulfoxide. One hundred micrograms of whole cell lysate were submitted to Western blot analysis using anti-HIF-1{alpha}. {alpha}-Tubulin served as a loading control (n = 9). Inhibition of transcription completely prevented T3-induced HIF-1{alpha} accumulation.

 
In contrast, inhibition of transcription with 1 µg/ml actinomycin D completely abrogated the T3-induced HIF-1{alpha} accumulation (Fig. 5CGo).

Active T3 does not induce HIF-1{alpha} expression through the –541 to –1 bp HIF-1{alpha} promoter but a further upstream HLF binding site
To determine effects of T3 on hif-1{alpha} gene expression, we quantitated hif-1{alpha} mRNA isolated from HepG2 cells at different time points. Maximum induction by T3 was found after 4 h incubation (Fig. 6AGo), which matches with T3-induced HIF-1{alpha} protein after 5 h incubation. To examine whether this effect is mediated by activation of the hif-1{alpha} promoter, HepG2 cells were transfected with reporter gene construct in which luciferase was under control of the previously published promoter sequences (–541 to –1) of hif-1{alpha}. This vector did not contain sequences responsible for the induction of HIF-1{alpha} expression by T3 (Fig. 6BGo). However, extension of the promoter by 120 bp revealed a potential binding site for HLF (Fig. 6CGo).


Figure 6
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  		FIG. 6. T3 mediates HIF-1{alpha} mRNA induction. A, Time course of T3-mediated HIF-1{alpha} expression. HepG2 cells were incubated with 10 nM T3 or carrier dimethyl sulfoxide under hypoxic (HOX) (3% O2) conditions. RNA was isolated, transcribed into cDNA, and measured by quantitative real-time PCR. Femtograms of HIF-1{alpha} cDNA are shown, normalized to 1 pg β-actin (n = 12). Quantitative PCR showed a significant T3-mediated increase in HIF-1{alpha} mRNA after 4 h. B, T3 has no influence on hif-1{alpha} promoter (Pro) (–541 to –1 bp) activity. HepG2 cells were transfected with reporter gene vector pGL3-Prom800 in comparison to cell transfected with pGL3 as control (C) containing most of the hif-1{alpha} promoter (–541 to –1 bp). After transfection, cells were exposed to 10 nM T3 or carrier dimethyl sulfoxide as a control under hypoxic (3% O2) conditions. Afterwards, cells were lysed, and RLUs were determined. RLUs were normalized to µg total protein (n = 16). No T3-induced activity of promoter was observed. C, Transcription factor binding sites within remaining 120 bp hif-1{alpha} promoter. Using TFsearch we identified HLF (score > 90), which is able to bind at the beginning of the hif-1{alpha} promoter.

 
HLF only occurs in the liver, kidney, and lung. Therefore, we tested ovarian carcinoma cells OVCAR3 and osteosarcoma cells U2OS, which both did not react to T3 with enhanced HIF-1{alpha} transcription (data not shown). In contrast, in kidney carcinoma cells, KiKa and lung cancer cells A549 T3 increased HIF-1{alpha} protein after 4–5 h (Fig. 7AGo). Treatment with T3 increased HLF expression after 3 h in both KiKa and HepG2 cells (Fig. 7Go, B and C). Finally, when HLF was knocked down (98% efficiency), T3-dependent induction of HIF-1{alpha} protein accumulation was almost completely abrogated (Fig. 7DGo).


Figure 7
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  		FIG. 7. Kidney carcinoma cells KiKa and lung carcinoma cells A549: indication for importance of HLF. A, Time course of HIF-1{alpha} accumulation. KiKa and A549 cells were treated with 10 nM T3 or carrier dimethyl sulfoxide under hypoxic (HOX) (3% O2) conditions. Seventy-five micrograms of whole cell lysate were submitted to Western blot analysis using anti-HIF-1{alpha}. {alpha}-Tubulin served as a loading control (n = 4). Both cell types responded to T3 with an induced HIF-1{alpha} accumulation. B, Time course of T3-regulated HLF expression in KiKa cells. KiKa cells were incubated with 10 nM T3 or carrier dimethyl sulfoxide under hypoxic (3% O2) conditions. RNA was isolated, transcribed into cDNA, and measured by quantitative real-time PCR. Femtograms of HLF cDNA are shown, normalized to 1 pg β-actin (n = 9). After 3-h incubation, HLF expression was stimulated by T3. C, Time course of T3-regulated HLF expression in HepG2 cells. HepG2 cells were incubated with 10 nM T3 or carrier dimethyl sulfoxide as a control under hypoxic (3% O2) conditions. RNA was isolated, transcribed into cDNA, and measured by quantitative real-time PCR. Femtograms of HLF cDNA are shown, normalized to 1 pg β-actin (n = 9). After 3 h incubation, HLF expression was stimulated by T3. D, T3-mediated HIF-1{alpha} accumulation depends on the presence of HLF. HepG2 cells were transfected with 50 nM siRNA against HLF (siHLF). The transfection with no siRNA (Mock) and nontarget siRNA (sint) served as a control. Cells were incubated with 10 nM T3 or carrier dimethyl sulfoxide for 5 h under hypoxic (3% O2) conditions. After the experiments, cells were lysed and used for Western blot analysis. {alpha}-Tubulin served as a loading control (n = 6). The knockdown of HLF resulted in reduction of T3-mediated HIF-1{alpha} accumulation, whereas the basic hypoxic level remained unaffected. *, P < 0.05.

 
Our data strongly suggest that T3 mediates its effects through TRβ/RXR{alpha}-dependent expression of HLF, which bind to upstream promoter sequences of hif-1{alpha}, thus increasing hif-1{alpha} gene expression.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is well known that many HIF-1 regulated genes are implied with T3-mediated effects on the regulation of glucose homeostasis, vascularization, apoptosis, and proliferation. A comparison between HIF-1- and T3-dependent genes demonstrated an overlap of many target genes, e.g. ADM (23, 24), GLUT 1 (25, 26), TGFβ1 (27, 28), transferrin (29, 30), endothelial nitric oxide synthase (31, 32), and enolase 1 (10, 13). Previous studies revealed that T3 induced HIF-1{alpha} accumulation in human hepatocellular carcinoma cells (HepG2) and primary fibroblasts (12, 13). However, whereas T3 increased HIF-1{alpha} after 5-h incubation in HepG2 cells, human primary fibroblasts only responded after 24 h. Because in our previous study higher T3 concentrations (100 nM) were used (12), here, we reduced T3 to a high (patho) physiological concentration that we also measured in FCS used for culturing the cells (data not shown; measured by Biofocus, Recklinghausen, Germany). T3 still induced HIF-1{alpha} accumulation (Fig. 1AGo), translocation (Fig. 1BGo), as well as HIF-1 activity (Fig. 1CGo) after 5 h-incubation, also demonstrated by HIF-1 target gene expression of ADM.

PI3-kinase dependent signaling had been implied for long-term stimulation of HIF-1{alpha} protein by T3 in primary fibroblasts (22). However, our data (Fig. 2Go) exclude a significant contribution of the major protein kinase pathways to T3 signaling during the early effects on HIF-1{alpha}. We confirmed that hypoxic HIF-1{alpha} accumulation is influenced by MAPK p38 (33, 34) as well as MAPK p42/44 (35, 36). Therefore, we focused on the meaning of nuclear TRs. Indeed, the targeted knockdown of TRβ and RXR{alpha} resulted in the loss of T3-induced HIF-1{alpha} accumulation (Fig. 3Go). By contrast, T3-induced HIF-1{alpha} protein remained unaffected when TR{alpha} was inhibited. Although HIF-1β is able to interact with the corepressor silencing mediator of retinoid acid thyroid hormone (23) and might play an important role in activating the TRs, HIF-1β was not influenced by blocking any TRs (Fig. 3Go). Therefore, we conclude that T3 effects on HIF-1{alpha} depend on the TRβ/RXR{alpha} heterodimer.

To address the concern that the effect of T3-induced HIF-1{alpha} accumulation is only observed in a tumor-cell line, we repeated the critical previous experiment with primary human hepatocytes. Administration of T3 induced HIF-1{alpha} protein after 4–5 h (Fig. 4AGo) and exhibited the same dependence of TRβ (Fig. 4BGo) as in HepG2 cells. Additional experiments showed that T3-induced ADM expression was blocked by the knockdown of HIF-1{alpha} as well as TRβ (Fig 4CGo). Thus, our data from the permanent tumor cell line HepG2 were fully confirmed in primary human hepatocytes.

Because Ma et al. (12) had suggested an induction of HIF-1{alpha} mRNA translation by T3, we examined HIF-1{alpha} accumulation in cells treated with the inhibitor of translation cycloheximide (Fig. 5AGo). Although general hypoxic HIF-1{alpha} levels were reduced by cycloheximide, no specific reduction of the T3 was achieved. Cap-independent translation via an IRES in the HIF-1{alpha}-5'-UTR (37) was studied by a bicistronic vector. We confirmed that hypoxia-mediated cap-independent translation for HIF-1{alpha} was effective (38), but we did not observe any inducibility by T3 (Fig. 5BGo), confirming our data on the lack of mTOR involvement in the T3 effect (Fig. 2BGo). However, inhibition of transcription by actinomycin D completely abrogated T3-mediated HIF-1{alpha} accumulation. Because the increase in HIF-1{alpha} mRNA was not mediated through regulatory DNA elements in the so far known HIF-1{alpha} promoter (–541 to –1 to the start of the 5'-UTR), we searched for transcription factor binding sites using the program TFsearch (Computational Biology Research Center; www.cbrc.ip). TFsearch revealed no TRE but indicated high-score binding sites (score > 90) for three proteins potentially involved in T3-regulated HIF-1{alpha} transcription: c-Rel, a member of the nuclear factor-{kappa}B pathway; CAATT/enhancer binding protein {alpha}; and HLF (Fig. 6CGo). In contrast to the ubiquitous abundance of c-Rel and thyroid hormone-regulated CAATT/enhancer binding protein {alpha} (39), HLF is limited to the liver, kidney, lung, and adult nervous system (40). Additional experiments had shown no effect of T3 on HIF-1{alpha} in ovarian carcinoma (OVCAR3) and osteosarcoma (U2OS) cells (data not shown), but a T3-dependent increase in kidney and lung carcinoma cells (Fig. 7AGo). This cell-specific HIF-1{alpha} induction by T3 very much reflects the expression pattern of HLF, which is induced by T3 after 3 h in KiKa cells (Fig. 7BGo) and HepG2 (Fig. 7CGo).

The human HLF gene was originally isolated from leukemic blasts of patients with pro-B cell acute lymphoblastic leukemia. HLF belongs such as the thyrotroph embryonic factor and albumin promoter D-box binding protein to the proline and acidic amino acid-rich protein subfamily of basic leucine-zipper transcription factors. Subfamily members are able to form heterodimers among each other and bind to similar DNA consensus sequences. These members have important roles in the control of normal growth and differentiation (40, 41). Despite of the potential role of HLF in regulation of programmed cell death and synaptic transmission, HLF-mediated functions remain vague, especially in mammals (42). To establish HLF as the connecting factor between TRs and HIF-1 expression, we searched for possible TREs in regulatory domains of HLF. Indeed, we identified two potential TREs (consensus sequence in boldface letters): a composite element 5'-ACGAGGTCAAACTGGCAGAGGGACACGC-3' (at –834 bp upstream of transcriptional start site; ER-9 element), and a single TRE 5'-AGAGGTCACTA-3' (at –1304 bp), which might serve as binding sites for the heterodimer TRβ/RXR{alpha} to control HLF expression by T3. Finally, when we knocked down HLF, T3-induced HIF-1{alpha} accumulation was abrogated.

In summary, our results provide evidence that T3-induced HIF-1{alpha} accumulation depends on increased expression of the hif-1 gene mediated by HLF. HLF activation results from classical nuclear signaling of the TRβ/RXR{alpha} receptor heterodimer. Our results do not appear to be limited to tumor cells but were confirmed in primary human hepatocytes. Thus, T3-induced HIF-1{alpha} expression may cooperate with hypoxic HIF-1 activation and expression of HIF-1 target genes like ADM and EPO in a tissue-specific manner that depends on the presence of HLF.


   Acknowledgments
 
We thank Professor Roland Wenger (Zuerich, Switzerland) for the pH3SVL vector, Professor Bernhard Brune (Frankfurt, Germany) for the bicistronic pRhifF vector, Dr. Carine Michiels (Namur, Belgium) for the Prom800 vector, and Dr. Igor M. Sauer (Berlin, Germany) for primary human hepatocytes.


   Footnotes
 
The study was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG Fa 225/18-2).

Disclosure Statement: T.O. has nothing to declare. J.F. has served as an expert witness for Johnson & Johnson and Ortho Biotech.

First Published Online January 31, 2008

Abbreviations: ADM, Adrenomedullin; EPO, erythropoietin; FCS, fetal calf serum; GLUT, glucose transporter; HIF, hypoxia-inducible factor; HLF, hepatic leukemia factor; IRES, internal ribosomal entry site; mTOR, mammalian target of rapamycin; PI3, phosphatidylinositol 3; RLU, relative light unit; RXR, retinoid X receptor; siRNA, small interfering RNA; Tetrac, tetraiodothyro-acetic acid; TR, thyroid hormone receptor; TRE, thyroid hormone response element; UTR, untranslated region; VEGF, vascular endothelial growth factor.

Received September 7, 2007.

Accepted for publication January 18, 2008.


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 Materials and Methods
 Results
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