Am J Physiol Regul Integr Comp Physiol-2004-Ma-R600-7

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doi: 10.1152/ajpregu.00115.2004287:R600-R607, 2004. First published 20 May 2004; Am J Physiol Regul Integr Comp Physiol

FandreyYaluan Ma, Patricia Freitag, Jie Zhou, Bernhard Brüne, Stilla Frede and Joachimfactor-1through augmented accumulation of hypoxia-inducibleThyroid hormone induces erythropoietin gene expression

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Thyroid hormone induces erythropoietin gene expression through augmented

accumulation of hypoxia-inducible factor-1

Yaluan Ma,1 Patricia Freitag,1 Jie Zhou,2 Bernhard Brune,2 Stilla Frede,1 and Joachim Fandrey1

1University of Duisburg-Essen, Institute of Physiology, D-45147 Essen; and 2University of

Kaiserslautern, Faculty of Biology, Department of Cell Biology, D-67663 Kaiserslautern, Germany

Submitted 18 February 2004; accepted in final form 14 May 2004

Ma, Yaluan, Patricia Freitag, Jie Zhou, Bernhard Brune, Stilla

Frede, and Joachim Fandrey. Thyroid hormone induces erythropoi-etin gene expression through augmented accumulation of hypoxia-inducible factor-1. Am J Physiol Regul Integr Comp Physiol 287:R600–R607, 2004. First published May 20, 2004; 10.1152/ajpregu.00115.2004.—Oxygen is of vital importance for the metabolism andfunction of all cells in the human body. Hypoxia, the reduction of oxygen supply, results in adaptationally appropriate alterations ingene expression through the activation of hypoxia-inducible factor 1(HIF-1) to overcome any shortage of oxygen. Thyroid hormones arerequired for normal function of nearly all tissues, with major effectson oxygen consumption and metabolic rate. Thyroid hormones havebeen found to augment the oxygen capacity of the blood by increasingthe production of erythropoietin (EPO) and to improve perfusion byvasodilation through the augmented expression of adrenomedullin(ADM). Because the hypoxic expression of both genes depends onHIF-1, we studied the influence of thyroid hormone on HIF-1 activa-tion in the human hepatoma cell line HepG2 under normoxic andhypoxic conditions. We found that thyroid hormones increasedHIF-1␣ protein accumulation by increasing HIF-1␣ protein synthesisrather than attenuating its proteasomal degradation. HIF-1␣ expres-sion directly correlated with augmented HIF-1 DNA binding andtranscriptional activity of luciferase reporter plasmids, whereasHIF-1␤ levels remained unaffected. Knocking down HIF-1␣ by shortinterfering RNA (siRNA) clearly demonstrated that thyroid hormone-induced target gene expression required the presence of HIF-1. Al-though an increased association of the two known coactivators of HIF-1, p300 and SRC-1, was found, thyroid hormone did not affectthe activity of the isolated COOH-terminal transactivating domain of HIF-1␣. Increased synthesis of HIF-1␣ may contribute to the adaptiveresponse of increased oxygen demand under hyperthyroid conditions.

hypoxic gene expression; oxygen sensing

ERYTHROPOIETIN (EPO), a 30.4-kDa glycoprotein hormone, isthe major physiological stimulator of red blood cell formationin mammals (18). The main EPO production sites are thekidney in adults and the liver in fetuses (6). EPO production isinduced by hypoxia, a state when oxygen supply does not

cover the demand of the tissue. EPO mRNA levels are induced50- to 100-fold in vitro by physiologically relevant levels of hypoxia (9). In vivo under severe hypoxia, production of EPOcan be increased up to 1,000-fold (18, 29).

Oxygen-dependent EPO expression is regulated by hypoxia-inducible factor 1 (HIF-1), a heterodimer of the O2-labile120-kDa ␣-subunit, and the constitutive 91- to 94-kDa ␤-sub-unit (37). The cellular levels of HIF-1␣ are adjusted by theubiquitin-proteasome-dependent degradation of HIF-1␣ under

normoxic conditions, allowing to tightly couple the proteinappearance to the ambient oxygen tension (31). At high P O2,HIF-1␣ is posttranslationally hydroxylated at proline residues402 and 564 by O2-sensitive prolyl hydroxylases, termedPHD1, PHD2, and PHD3, which are recognized today as themost likely cellular O2 sensors (7, 17). Hydroxylated HIF-1␣ isrecognized by the tumor suppressor von Hippel-Lindau(pVHL) protein for ubiquitination by the E3 ubiquitin-proteinligase (23). Under hypoxia, HIF-1␣ evades proteasomal deg-radation because of the lack of proline hydroxylation and

concomitantly accumulates and is translocated into the nucleusto form the HIF-1 complex by dimerization with constitutivelyexpressed HIF-1␤ (identical to aryl hydrocarbon receptor nu-clear translocator 1, ARNT1) (14, 37). Binding of HIF-1 toDNA at HIF-binding site (HBS) within the hypoxia responseelement (HRE) in the 3Ј-flanking EPO enhancer increasesexpression of the gene under hypoxic conditions (33). Inaddition to hypoxic accumulation, the trans-activity of HIF-1␣is regulated by the O2-sensitive asparagyl-hydroxylase FIH-1(factor inhibiting HIF-1) (22). Under hypoxia the lack of hydroxylation of Asp803 allows binding of the adapter proteinp300 to recruit further coactivator proteins such as steroidreceptor coactivator-1 (SRC-1) (3, 20).

Like EPO, the expression of adrenomedullin (ADM), ahypotensive peptide originally isolated from a pheochromocy-toma, is hypoxia inducible (4). Promoter analysis revealed thatthis induction was primarily mediated by HIF-1 bound toregulatory DNA sequences within the promoter of rodent (4,25) and human cells (12, 16).

Thyroid hormone is required in nearly all tissues, with majoreffects on oxygen consumption and metabolic rate. Adaptationto this increased metabolic demand is partly achieved by potenteffects of thyroid hormone on erythropoiesis and thus bloodoxygen capacity. Thyroid hormones directly increase the pro-liferation of erythroid progenitors (5, 13) and thyroid hormonereceptors were identified on nucleated erythroid cells isolatedfrom hypoxic hamsters (1). Apart from the direct effect in

erythroid precursors, thyroid hormone directly enhanced hy-poxia-inducible EPO formation both in the isolated perfusedrat kidney and HepG2 cells (10). In addition, tissue perfusionmay be increased through the stimulated expression of thepotent vasodilator ADM by thyroid hormones (15, 25).

The molecular mechanisms of thyroid hormone-inducibleEPO and ADM expression have not yet been elucidated.Thyroid hormone binds to the intracellular thyroid hormonereceptor (TR), a member of the nuclear hormone receptor

Address for reprint requests and other correspondence: J. Fandrey, Institutfur Physiologie, Universitatsklinikum Essen, Universitat Duisburg-Essen,Hufelandstrasse 55, D-45122 Essen (E-mail: [email protected]).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement ”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Am J Physiol Regul Integr Comp Physiol 287: R600–R607, 2004.First published May 20, 2004; 10.1152/ajpregu.00115.2004.

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family, which acts as a transcription factor and regulates geneexpression (8). Moreover, it has been reported that steroidreceptor coactivator-1 (SRC-1) functions as a positive regula-tor of the TR-mediated transactivation pathway (19). Thusherein we studied whether thyroid hormones impinge on theHIF-1 activation pathway and by that means affect hypoxia-inducible expression of EPO and ADM.

MATERIALS AND METHODS

Cell culture and in vitro stimulation. The human hepatoma cellline, HepG2, obtained from the American Type Culture Collection(ATCC HB 8065) was grown in RPMI 1640 supplemented with 10%fetal calf serum (FCS), penicillin (100 U/ml), and streptomycin (100g/ml) in a humidified atmosphere (5% CO2 in air) at 37°C. Care wastaken to avoid formation of cell clumps that would result in aheterogeneous distribution of the pericellular oxygen tension. Toexamine the effects of thyroid hormone, cells were regularly culturedserum free in the RPMI 1640 medium containing 1% serum supple-ment (SS; transferrin, insulin, selenium, albumin; Sigma, Munich,Germany) for 24 h before the experiment to remove the effects of serum-derived hormones. Transiently transfected HepG2 cells were

kept in RPMI 1640 supplemented with 1% SS for 8 h before theexperiments.

At the beginning of an experiment, HepG2 cells received freshmedium (RPMI 1640 supplemented with 1% SS) containing therespective thyroid hormone concentration. Triiodothyronine (T3) andthyroxine (T4; Sigma, Munich, Germany) stock solutions (14.86 mMin 1:4 1 N HCl-ethanol for T3 and 56.2 mM in 4 N ammoniumhydroxide in methanol for T4) were stored at Ϫ80°C. To achievehypoxic conditions, culture dishes were placed in a Heraeus incubator(Hanau, Germany) with 5% CO2, and nitrogen (N2) to balance fordifferent O2 concentrations. Hypoxia was defined as 3% O2 if notindicated otherwise. Control normoxic cells were placed in an incu-bator (5% CO2 in air) for equivalent time periods. For reoxygenationexperiments, cells were exposed to hypoxia for 4 h and then trans-ferred to 21% O2 for different times. Nuclear extracts were prepared

using the method of Schreiber et al. (32) and subjected to Western blotanalysis and electrophoretic mobility shift assays (EMSAs).

Quantitative real-time RT-PCR analysis. Total RNA was extractedby guanidinium isothiocyanate extraction as described (34). Total RNA(1 g) was reverse transcribed with oligo(dT) and Moloney murineleukemia virus reverse transcriptase (Promega). Gene expression of human EPO was quantitated using the qPCR Mastermix for SYBR GreenI (Eurogentec, Belgium) and the GeneAmp 5700 sequence DetectionSystem (PE Biosystems). The PCR reactions were set up in a finalvolume of 25l with 0.5l cDNA, 1ϫ reaction buffer containing SYBRGreen I, 10 pmol forward (F), and 10 pmol reverse primer (R). Primersets used for EPO: (F) 5Ј-CTCCGAACAATCACTGCT-3Ј and (R)5Ј-GGTCATCTGTCCCCTGTCCT-3Ј; ADM: (F) 5Ј-GGATGCCGC-CCGCATCCGAG-3Ј and (R) 5Ј-GACACCAGAGTCCGACCCGG-3Ј;␤-actin: (F) 5Ј-TCACCCACACTGTGCCCATCTA CGA-3Ј and (R)

5Ј-CAGCGGAACCGCTCATTGCCAATGG-3 Ј; HIF-1␣: (F) 5Ј-GCT-GGC CCCAGCCGCTGGAG-3Ј and (R) 5Ј-GAGTGCAGGGTCAG-CACTAC-3Ј. Oligonucleotides were purchased from Invitrogen.

Agarose gel electrophoresis confirmed the specificity of the ampli-fication product. The resulting PCR fragments were visualized onethidium bromide-stained 1.5% agarose gels. Tenfold dilutions of purified PCR products starting at 1 pg to 0.1 fg were used asstandards. Amplification conditions were set to 10 min at 95°Cfollowed by 45 PCR cycles (15 s at 95°C, 1 min at 60°C). Thequantity of cDNA used in each reaction was normalized to the ␤-actincDNA and expressed as cDNA per microgram total RNA.

ELISA for EPO. EPO protein in the culture supernatant wasmeasured by ELISA (Quantikine IVD EPO; R&D System, Wiesba-den-Nordenstadt, Germany).

Protein extract preparation and Western immunoblotting. Nuclearextracts were prepared using the methods of Schreiber et al. (32). Allprocedures were performed at 4°C. Briefly, HepG2 cells (70 – 80%density of the cells in dishes with 10-cm diameter) were washed withcold PBS, drained, and scraped from plates with 150 l cold nuclearextract buffer A (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl,0.5 mM PMSF, 0.5 mM DTT, 0.4% NP-40, 1ϫ protease-inhibitor-cocktail, Roche), transferred into Eppendorf tubes, and incubated onice for 20 min. The cell lysate was centrifuged at 5,000 g for 5 min at4°C and the supernatant was discarded. The pellet was resolved in 80

l cold nuclear extract buffer B (20 mM HEPES pH 7.9, 420 mM

NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT,1ϫ protease-inhibitor-cocktail) and homogenized with a magneticstirrer on ice for 30 min. Cellular debris was removed by centrifuga-tion at 13,000 g for 15 min at 4°C. The supernatant was used as anuclear extract, and 5 l of supernatant was taken out for proteindetermination. The supernatant was stored at Ϫ80°C. For whole celllysates, cells were lysed on the plate with 100 l extract buffer (0.1%NP-40, 300 mM NaCl, 10 nM Tris pH 7.9, 1 mM EDTA, 1:7 dilutionprotein-inhibitor-cocktail) for 20 min on ice. Extracts were spun downin a microfuge, 3,600 g for 5 min at 4°C, quantitated using theBio-Rad protein assay reagent and stored at Ϫ80°C. After addition of 1

⁄ 4

volume of 4ϫ sample buffer (50 mM Tris pH 6.8, 2% SDS, 5%␤-mercaptoethanol, 0.0125% bromphenol blue, 1% glycerin), 75 gof total cell lysate or 20 g nuclear extract per lane were subjected to7.5% SDS-PAGE, separated, and transferred to a nitrocellulose mem-

brane (0.2 m pore size; Schleicher and Schuell). Blots were stainedwith Ponceau S solution to ensure equal protein loading and transfer.The membranes were blocked with 5% nonfat dry milk powder inTBS-T, incubated with anti-human HIF-1␣ mouse monoclonal(Transduction Laboratories, Heidelberg, Germany; 1:250 dilution inTBS-T containing 5% nonfat milk), anti-HIF-2␣ (Novus Biologicals),or anti-nuclear factor (NF)-B (Cell Signalling, Hamburg, Germany).Horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit an-tibodies were used as a secondary antibody at a 1:10,000 dilution inTBS-T containing 5% nonfat milk. Anti-␣-tubulin antibody (at a1:500 dilution, Santa Cruz) was used as a loading control. Immuno-

reactive proteins were visualized using the enhanced chemilumines-cence plus (ECL) detection system followed by exposure to X-ray

film (Agfa, Mortsel, Belgium).

Electrophoretic mobility shift assay. Double-stranded oligonucleo-tides containing the HIF binding site from the HRE (5Ј-GCC CTACGT GCT GTC TCA-3Ј) of the EPO enhancer were used as probes.The double-strand fragments were end labeled by filling in 5Ј over-hangs with 32P-labeled adenosine triphosphate ([32P]dATP) using T4

polynucleotide kinase. DNA-protein binding reactions were carriedout in a total volume of 20 l containing 5 g nuclear extract, 30 fmol32P-labeled oligonucleotides, and a nonspecific competitor (50 ng calf thymus DNA) in a binding buffer with a final concentration of 12 mMHEPES, 4 mM Tris (pH 7.9), 60 mM potassium chloride, 1 mMEDTA, and 1 mM dithiothreitol. After incubation for 30 min at roomtemperature, 1 g anti-HIF-1␣ antibody was added for supershift

detection of the HIF-1 complex and incubated overnight at 4°C. Theproducts were analyzed by electrophoresis in 5% non-denaturingpolyacrylamide gels. Electrophoresis was performed at 80 V in 0.25ϫTBE buffer at 4°C for 4 h. The dried gels were exposed to X-ray filmsand PhosphoImage sheets overnight.

Immunoprecipitation. One milligram of cell extract was incubated2 h at 4°C with 2.5 g of anti-Src-1 antibody (sc-6098, Santa Cruz)or anti-p300 (sc-584, Santa Cruz), followed by overnight incubationwith 20 l of protein A-Sepharose beads (Santa Cruz) on a rotator at4°C. Beads were washed five times with 1ϫ TBS denatured samplesand 4ϫ sample buffer at 95°C for 5 min. Beads were removed bycentrifugation, and supernatants were loaded on 7.5% SDS-PAGEfollowed by Western blotting and detection with the anti-HIF-1␣antibody as described above.

R601THYROID HORMONES ACTIVATE HIF-1

AJP-Regul Integr Comp Physiol • VOL 287 • SEPTEMBER 2004 • www.ajpregu.org



Transient transfection assays. For reporter gene assays, HepG2cells were transfected by electroporation. The luciferase reporter geneplasmid pH3SVL containing an SV40 promoter-luciferase unit down-stream of six HIF binding sites from the transferrin enhancer was akind gift of R. Wenger, Zurich (30). To measure the activation of HIF-1␣ by its COOH terminus, the Gal4 chimeric activator/reportersystem was used in which the HIF-1␣ 775– 826 COOH-terminaltransactivating domain (C-TAD) was fused to Gal4 DNA bindingdomain. The CMV promoter ensures expression of the fusion protein.The reporter plasmid contains the luciferase gene under the control of Gal4 binding sites. Both plasmids of the chimeric activator/reportersystem were generously provided by Peter Ratcliffe, Oxford, UK (28).HepG2 (1 ϫ 107) cells were electroporated with 10 g plasmid at 975F, 250 V in 0.4-mm-thick cuvettes in 400 l of RPMI 1640serum-free medium using a Gene Pulser and Capacitance Extenderapparatus (Bio-rad). After overnight incubation, cells were preincu-bated in 1% SS medium for 8 h, and then the medium was replacedby a thin layer of fresh 1% SS medium containing 50 nM T 3 or carrieralone. The plates were incubated for another 18 –24 h in 21% or 3%O2. Cells were lysed with 100 l 1ϫ reporter lysis buffer. Luciferaseactivity was measured with the luciferase assay system (Promega,Heidelberg, Germany). Luciferase activity was expressed in relativelight units (RLU) and normalized to total cellular protein measured byusing the Bio-Rad protein assay kit.

35S-radioisotopic labeling. Cells, starved for 1 h in serum- andmethionine-free medium (PromoCell, Heidelberg, Germany), werereplaced with methionine-free medium containing 1% SS and 100Ci/ml [35S]methionine (ICN Biomedicals) for 6 h in the absence orpresence of 50 nM T3. Cells were then washed with PBS, resuspendedin lysis buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, 0.5%NP-40, 5% glycerol, 1 mM PMSF, protease inhibitor cocktail, pH 7.5)followed by immediate vortexing (3 ϫ 15 s). After centrifugation(15,000 g for 30 min) supernatants were transferred into new tubes.The supernatant containing 1 mg total protein was supplied with 1 ganti-HIF-1␣ antibody and incubated at 4°C for 1 h. Thereafter, 50 lprotein G microbeads (Miltenyi Biotec, Bergisch Gladbach, Ger-many) was added and incubations continued at 4°C overnight. Beadswere magnetically collected following the manufacturer’s manual andwashed three times with 100 l lysis buffer. Coprecipitated proteinswere finally eluted by adding 95°C preheated SDS-PAGE samplebuffer according to the manufacturer’s manual. Protein samples wereseparated on 7.5% SDS-PAGE. The gel was dried for 2 h and exposedto X-ray films for 32 h.

Statistics. Statistical significance was calculated using the Graph-Pad Instat software applying the one-way ANOVA followed by theBonferroni multiple comparison post test.

RESULTS

Thyroid hormones enhance expression of the HIF-1 target genes EPO and ADM. Expression of EPO and ADM is undercontrol of the HIF-1 complex and was thus stimulated by

hypoxia (3% O2) in HepG2 cells. Quantitation of mRNA levelsby quantitative real-time PCR revealed that thyroid hormonessignificantly stimulated normoxic expression and augmentedhypoxic induction of EPO and ADM expression after 3 h of treatment (Fig. 1).

HepG2 cells, maintained in six-well culture plates for 24 h,produced 15.8 Ϯ 0.4 mIU EPO/ml supernatant in the absenceof thyroid hormone under normoxia. T3 provoked formation of 17.6 Ϯ 0.6 mIU EPO/ml (P Ͻ 0.05 vs. untreated controls; n ϭ4), whereas T4 led to the production of 18.7 Ϯ 0.7 mIUEPO/ml (P Ͻ 0.05 vs. untreated controls; n ϭ 4). Underhypoxia, HepG2 cells secreted 26.1 Ϯ 0.7 mIU EPO/ml EPOprotein in 24 h, which was further increased to 39.1 Ϯ 4.1 mIU

EPO/ml by T3 or 30.6 Ϯ 1.4 mIU EPO/ml by T4 (P Ͻ 0.05 forboth thyroid treated groups vs. untreated controls; each groupn ϭ 4). Thus HepG2 cells showed an oxygen-dependent EPOand ADM gene expression. Thyroid hormones significantlystimulate expression of both genes under normoxia and hyp-oxia.

HIF-1␣ protein accumulation in HepG2 cells is stimulated by thyroid hormone. To investigate whether stimulation of

HIF-1-dependent target gene expression by thyroid hormonesrequires HIF-1 signaling, we determined HIF-1␣ protein levelsby Western analysis performed with whole cell lysate. HepG2cells were treated with T3 or T4 under normoxia or hypoxia for6 h. T3, and to a lesser extent T4, induced HIF-1␣ protein levelsin HepG2 cells incubated under hypoxic (3% O2) conditionsfor 6 h (Fig. 2 A). Effective concentrations of T3 on HIF-1␣protein expression ranged from 2 to 500 nM (data not shown).With the use of very long exposure times (L) it was revealedthat T3 also enhanced HIF-1␣ protein levels under normoxicconditions (Fig. 2 A). In contrast, HIF-1␤ was constitutivelypresent and expression remained unchanged by thyroid hor-mone treatment (Fig. 2 A). In addition, 50 nM T3 had no effect

Fig. 1. Thyroid hormones stimulate the expression of erythropoietin (EPO)and adrenomedullin (ADM) mRNA. HepG2 cells were treated with 100 nMtriiodothyronine (T3) or 1 M thyroxine (T4) for 3 h in 21% O2 and 3% O2 and

mRNA for EPO ( A) and ADM ( B) was quantitated by real time PCR. Shownare representative data from 1 of 3 experiments. Each value is the mean withSD 3 separate culture dishes. Statistically significant difference from untreatedcontrols (#P Ͻ 0.05; ##P Ͻ 0.01); statistically significant differences fromrespective hypoxic controls without T3 (*P Ͻ 0.05; **P Ͻ 0.01).

R602 THYROID HORMONES ACTIVATE HIF-1




on NF-B accumulation, whereas HIF-1␣ levels were in-creased (Fig. 2 B). Interestingly, HepG2 cells displayed highlevels of HIF-2␣ under normoxia, which were only marginallyincreased by hypoxia. In the same cells, HIF-1␣ showed astrong hypoxic response. Nevertheless, 50 nM T3 increasedHIF-1␣ accumulation but did not affect HIF-2␣ levels(Fig. 2 B).

To test whether thyroid hormones affect transcription of thehif-1␣ gene, the effect of thyroid hormones on HIF-1␣ mRNAlevels was determined by RT-PCR. As shown in Fig. 2C ,HIF-1␣ mRNA levels remained constant, suggesting that thy-roid hormone most likely induced HIF-1␣ protein expressionvia posttranscriptional regulation.

Thyroid hormones do not stabilize HIF-1␣ but increase itssynthesis. To elucidate posttranscriptional mechanisms, weexamined whether T3 would stabilize HIF-1␣. Therefore,HepG2 cells were exposed to hypoxia for 4 h and then returnedto normoxia. Although T3 increased HIF-1␣ protein levelsunder hypoxia it did not affect HIF-1␣ degradation on reoxy-genation (Fig. 3 A). On the transition from hypoxia to reoxy-

genation, HIF-1␣ completely disappeared within 30 min incontrol and T3-treated cells. When the destruction of HIF-1␣was inhibited by the addition of MG132, an inhibitor of proteasomal degradation, T3 was still able to increase HIF-1␣protein levels (Fig. 3 B). The addition of the translationalinhibitor cycloheximide (CHX), however, prevented the T3-dependent increase of HIF-1␣ (Fig. 3C ). It is interesting to note

that under conditions of 3% O2 in the incubation gas and acorresponding pericellular PO2 of 5–7 mmHg, HIF-1␣ wascleared from the cells when new synthesis is prevented byCHX. Again, as shown above, T3 did not increase the half-lifeof HIF-1␣, but CHX prevented the T3-induced increase inHIF-1␣ levels. Because these data indicated that T3 might

Fig. 2. Thyroid hormones increase the accumulation of hypoxia-induciblefactor (HIF)-1␣. HepG2 cells were exposed to 100 nM T3 or 1 M T4 in 3%

O2 for 6 h and whole cell lysates (75 g/lane) were submitted to Western blotanalysis using anti-HIF-1␣ and anti-HIF-1␤ antibody. Short (S) and long (L)exposure times of the membrane to the film are shown ( A). T3 (50 nM) appliedfor 6 h increased HIF-1␣ but left HIF-2␣, nuclear factor (NF)-B, and␣-tubulin unchanged ( B). From parallel cultures, total RNA was prepared,reverse transcribed to cDNA, and submitted to HIF-1␣- and ␤-actin-PCR (C ).

Fig. 3. T3 does not inhibit the degradation but increases production of HIF-1␣protein. HepG2 cells were exposed to 50 nM T3 under 3% O2 for 4 h, thenreoxygenated for different time periods. Nuclear extracts (20 g/lane) weresubmitted to Western blot analysis ( A). HepG2 cells were exposed to 50 nM T3

under 3% oxygen for 4 h in the presence of proteasomal inhibitor MG132 ( B)and whole cell lysates (100 g/lane) were submitted to Western blot analysisusing anti-HIF-1␣ antibody and anti-␣-tubulin antibody. Cotreatment with thetranslational inhibitor cycloheximide (CHX) and T3 is shown in C . Cells weretreated with/without T3 under normoxic/hypoxic conditions as indicated for4 h. Thereafter CHX at 100 M was added for the time periods indicated, andwhole cell lysates were subjected to Western blot analyses; ␣-tubulin served asa loading control. In D, labeling of nascent HIF-1␣ protein by [35S]methioninewas performed to detect the increased synthesis of HIF-1␣ by treatment with50 nM of T3.





elevate HIF-1␣ levels by increasing the rate of translation,35S-radioisotopic labeling of nascent HIF-1␣ protein was per-formed. In these experiments, T3 clearly increased HIF-1␣protein synthesis (Fig. 3 D).

Thyroid hormone increases the formation of an HBS bound complex containing HIF-1␣. To test whether T3 also increasedHIF-1 DNA binding, nuclear extracts from HepG2 cells treated

with or without T3 in 3% O2 for 6 h were isolated for EMSAusing a double-stranded oligonucleotide containing the HBSfrom the EPO 3Ј-enhancer as a probe (Fig. 4).

Induction of HIF-1 DNA-binding activity was detected inthe nuclear extracts from hypoxic controls (lane 3). T3 in-creased HIF-1 DNA-binding activity under hypoxic (lane 4)conditions. To confirm the identity of the HIF-1 complex,supershift experiments were performed with the monoclonalanti-HIF-1␣ antibody, which shifted the complete DNA/pro-tein complex to lower mobility. This indicates that in HepG2cells, T3 induced HIF-1␣ is part of the HIF-1 complex.

Thyroid hormone stimulates the transcriptional activity of HIF-1. To investigate whether the T3-induced HIF-1 complexresulted in an increased transcriptional activity of HIF-1, lu-ciferase reporter gene assays were performed with the plasmidpH3SVL containing an SV40 promoter-luciferase unit down-stream of six HIF binding sites from the transferrin enhancer.Twenty-four hours after transient transfection, cells were in-cubated under normoxic and hypoxic conditions in the pres-

ence or absence of 50 nM T3 for another 18 h. T3 significantlyincreased the HIF-1-dependent activation of the reporter gene(Fig. 5 A).

In contrast, the biological inactive analog, reverse T3, waswithout any effect (data not shown). To further prove therequirement of HIF-1␣ for the T3 effect, HIF-1␣ was knockeddown by siRNA for HIF-1␣. HepG2 cells were ef ficiently

cleared from HIF-1␣ protein (Fig. 5 B) and HIF-1␣ mRNA(data not shown) although a faint band was still visible insiRNA and T3-treated cells (Fig. 5 B, lane 12). A subsequentlyperformed luciferase reporter gene assay revealed the completeloss of hypoxic and T3-dependent induction (Fig. 5 A).

Thyroid hormone does not stimulate the activity of theC-TAD of HIF-1␣ despite the increased formation of HIF-1␣- p300/SRC-1 complexes. To study whether T3, in addition toincreasing HIF-1␣ protein levels, also induces the activity of the C-TAD, coimmunoprecipitation for the known coactivatorsof HIF-1 p300 and SRC-1 was performed. For both coactiva-tors, increased amounts of complexes with HIF-1␣ were found(Fig. 6 A). However, when the activity of the C-TAD was

Fig. 4. Thyroid hormone increases HIF-1 DNA binding activity. Nuclearextracts (5 g/lane) prepared from HepG2 cells treated with 50 nM T3 undernormoxic or hypoxic conditions for 6 h were incubated with 32P-labeled probespanning the hypoxia response element (HRE) from the EPO enhancer. Arrowsindicate the inducible HIF-1 complex and the supershifted complex; a consti-tutive (C), an unspecific band (U) and the free probe are labeled accordingly.Supershift analysis was performed with anti-HIF-1␣ antibody (lane 5).

Fig. 5. Thyroid hormone stimulates HIF-1␣ transcriptional activity. HepG2cells transiently transfected with the luciferase reporter construct pH3SVLHRE-luciferase reporter construct were exposed to 50 nM T3 for 18 h innormoxia (NOX) or hypoxia (HOX). Cells were lysed and luciferase activitywas measured (solid bars in A). To prove the critical role of HIF-1␣ for theT3-dependent induction of the reporter gene, HIF-1␣ was knocked down withsiRNA for HIF-1␣. HIF-1␣ protein was ef ficiently reduced with sequencespecific siRNA, whereas siRNA with a scrambled sequence had no effect of HIF-1␣ levels ( B). T3-dependent induction of HIF-1 activity as determined by

the activation of the reporter gene was lost in HIF-1␣ siRNA-treated cells(shaded bars in A). Each bar represents the mean with SD in parentheses of atleast 3 separate experiments. *Significant difference (P Ͻ 0.05) betweenthyroid hormone-treated cultures and their respective nontreated control (NOXor HOX); #significant difference between normoxic and hypoxic cultures (P Ͻ0.001).





determined, T3 was without any effect although hypoxia in-creased the activity of the C-TAD significantly (Fig. 6 B). Thusincreased coimmunoprecipitation of HIF-1␣ with p300 andSRC-1 under T3 treatment probably reflects more input, i.e.,HIF-1␣ loading, rather than supporting the notion that theinteraction between HIF-1␣ and p300 or SRC-1 was increased.

DISCUSSION

Thyroid hormones exert a calorigenic effect on the tissuethat increases the demand for oxygen. Therefore erythropoie-sis, which provides the necessary oxygen capacity of the bloodand the control of erythropoiesis by EPO have always beenclosely linked with the effects of thyroid hormones (2). Mostinvestigators understood increased serum levels of EPO inhyperthyroidism to be caused by a greater demand for oxygenin the tissue under the action of thyroid hormones. Increasedoxygen consumption was thought to cause a suf ficiently lowPO2 in the tissue to trigger the production of EPO (26).However, there is no experimental evidence that the action of thyroid hormone on EPO production exclusively depends on an

increase in oxygen consumption. In fact, some groups found anoncalorigenic effect of thyroid hormones on erythropoiesiswhen oxygen consumption was not closely correlated with theincrease in radio-iron incorporation into red blood cells afterthe application of thyroid hormone (5, 27). A direct, noncal-origenic stimulation of EPO production in vitro was revealedby direct measurement of oxygen consumption in HepG2 cells

(10) and further supported by the experiment in which HepG2cells were maintained in hypoxia due to diffusion-limitedoxygen supply (24). In this setting, thyroid hormones exertedtheir full stimulatory effect on EPO production, although theculture conditions prevented oxygen consumption-dependentchanges in the PO2 (10).

The biological meaning of the effect of thyroid hormones onEPO production and erythropoiesis appears obvious due to thetight coupling of blood oxygen capacity and oxygen demand of the tissue. In contrast, the finding that T3 stimulates theexpression of the hypotensive hormone ADM in vitro and invivo (25) is less clear, but it has been hypothesized that a betterperfusion caused by vasodilation may be advantageous for thetissue under the influence of thyroid hormones (25). Bothgenes, EPO and ADM, have in common that they are hypoxiainducible and that the hypoxic induction depends on activationof HIF-1 (4). Herein, hypoxia-induced expression of bothgenes was significantly augmented by thyroid hormones (Fig.1) and we therefore aimed at studying potential effects of T3 onHIF-1 signaling. Indeed, T3 increased HIF-1␣ protein accumu-lation, HIF-1 DNA-binding activity, and HIF-1-mediated re-porter gene transcription, whereas the biologically inactive rT3

was without effect (data not shown). T3 stimulated HIF-1under normoxia but also augmented the hypoxic activation of HIF-1. This is in support of earlier findings where T3 stimu-lated EPO protein synthesis in HepG2 cells and isolated per-fused rat kidneys under hypoxic conditions (10).

One hypothesis to explain the increased HIF-1 activation bythyroid hormones might be that an increased O2 consumptioncould cause a decrease of the PO2 if O2 diffusion becomeslimiting. However, our setup for hypoxic exposure makes thishypothesis very unlikely. First, direct measurement of the O2

consumption of HepG2 cells under the influence of T3 revealedno increase (10). Second, a PO2 in the gas phase of ϳ20 mmHg(corresponding to 3% O2) results in a pericellular PO2 of Ͻ1mmHg (24), even with cells of an ϳ80% confluent cell mono-layer as used in this study. Finally, if aggravated hypoxiaaccounted for the increase in HIF-1␣ accumulation and acti-vation, T3 treatment should have delayed the degradation andincrease the stability of HIF-1␣, which was not the case (Fig.3 A). Considering that thyroid hormone might increase HIF-1␣

gene expression was rejected because HIF-1␣ mRNA levelswere not elevated by T3 treatment (Fig. 2C ). This findingindicated that the T3-inducible accumulation of HIF-1␣ proteinmight be due to an increase of HIF-1␣ protein synthesis. Totest this hypothesis we used MG132, a specific inhibitor of theubiquitin proteasome complex, to maintain ubiquitinatedHIF-1␣ and block HIF-1␣ degradation. By MG132 treatmentthe rate of HIF-1␣ accumulation is at large a function of therate of HIF-1␣ synthesis. T3 significantly enhanced HIF-1␣protein levels in the presence of MG132 under both normoxicand hypoxic conditions (Fig. 3 B). The effect of T3 was inhib-ited by cycloheximide, an inhibitor of translation (Fig. 3C ), anddirect labeling of nascent HIF-1␣ with [35S]methionine con-

Fig. 6. Thyroid hormones do not increase the activity of the COOH-terminaltransactivating domain (CTAD) of HIF-1␣. HepG2 cells were treated with T3

for 6 h under normoxic or hypoxic conditions. Immunoprecipitation wasperformed using anti-SRC-1 and anti-p300 antibodies (IP). Subsequently,precipitates were subjected to Western blot and HIF-1␣ was detected by amonoclonal antibody ( A). The two-hybrid GAL4-system was used to test forthe activity of the CTAD (amino acids 775 to 826 of HIF-1␣), which was fusedin frame to the DNA binding domain of GAL4. GAL4-luciferase was cotrans-fected to determine the activity of the fusion protein ( B). Shown are the meanswith SD of 3 to 4 separate experiments with or without stimulation by T3 undernormoxic and hypoxic conditions. #Significant difference between normoxicand hypoxic cultures (P Ͻ 0.001).





firmed the stimulation of translation of HIF-1␣ by T3 (Fig. 3 D).Moreover, HIF-1␣ was indispensable for T3-induced stimula-tion of HIF-1-dependent reporter and target gene expression asshown by knocking down HIF-1␣ by siRNA. Thus increasedsynthesis by T3 cooperates with hypoxic stabilization andaccumulation of HIF-1␣ that is due to inhibition of prolylhydroxylase activity (7) and ensures enhanced expression of

the HIF-1 target genes EPO and ADM.It appeared important to us to test whether thyroid hormones

would also specifically increase the activity of HIF-1␣. How-ever, although T3 caused an increased coimmunoprecipitationof the known HIF-1␣ coactivator p300 and SRC-1, the activityof the C-TAD of HIF-1␣ was not stimulated by T3, although asignificant hypoxic induction was observed (Fig. 6). Thusincreased capture of the HIF-1␣ binding protein p300 andSRC-1 is most likely due to the higher input of HIF-1␣ inT3-treated cells, but does not indicate a specific increase of HIF-1 activity by T3. Therefore the stimulation of HIF-1-dependent gene expression by T3 appears to be solely mediatedthrough increased HIF-1␣ synthesis and thus accumulation.

It is interesting that stimulation of target gene expressionwas first observed under hypoxic conditions when increasedlevels of HIF-1 are also activated by concomitant hypoxia (10).Thus the effect of thyroid hormone is similar to that observedwith insulin and a series of growth factors, including insulin-like growth factor (IGF)-1, IGF-2, EGF, basic fibroblastgrowth factor-2 (FGF-2), HGF, and HER2 (neu) receptortyrosine kinase (11, 21, 35, 36, 38). All of above hormones andgrowth factors do not stabilize HIF-1␣ but increase the rate of HIF-1␣ synthesis and would therefore act in concert withhypoxic accumulation and activation of HIF-1␣.

ACKNOWLEDGMENTS

We are grateful to P. J. Ratcliffe and R. Wenger for plasmids and T.

Kietzmann and K. Rutkowski for contribution in the very early phase of thisstudy.

GRANTS

This study was supported by a grant from the Deutsche Forschungsgemein-schaft (DFG Fa 225/18 –1).

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