The FASEB Journal express article 10.1096/fj.02-0168fje. Published online September 5, 2002. Inhibition of erythropoietin gene expression signaling involves the transcription factors GATA-2 and NF-κB Katia La Ferla, Christian Reimann, Wolfgang Jelkmann, and Thomas Hellwig-Bürgel Institute of Physiology, University of Luebeck, Luebeck, Germany Corresponding author: Wolfgang Jelkmann, Institute of Physiology, University of Luebeck, Ratzeburger Allee 160, 23538 Luebeck, Germany. E-mail: firstname.lastname@example.org ABSTRACT The anemia of chronic inflammatory and malignant diseases is partly due to impaired synthesis of the hormone erythropoietin (Epo). The proinflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α) suppress in vitro Epo gene expression and Epo protein secretion. However, the molecular mechanisms of this inhibition are poorly understood. The human Epo promoter and the 5' flanking region contain several recognition sequences for transcription factors acting either positively or negatively. Herein, we investigated the roles of the transcription factors GATA-2 and NF-κB in the modulation of Epo gene expression by IL-1β and TNF-α in the human hepatoma cell line HepG2. Electrophoretic mobility shift assays revealed increased GATA-2 and NF-κB DNA binding in cells treated with IL-1β or TNF-α. Reporter gene assays with a sequence from the Epo promoter in front of the firefly luciferase gene showed that the cytokines reduced Epo reporter gene activity. Functional inactivation of GATA-2 and NF-κB by oligo-decoy techniques prevented the inhibition of Epo production by IL-1β and TNF-α. In HepG2 cells stably transfected with a dominant-negative form of IκBα, the activation of NF-κB was inhibited, while Epo mRNA levels and Epo secretion increased. Thus, both GATA-2 and NF-κB seem to be involved in the suppression of Epo gene expression by IL- 1β and TNF-α in vitro and may be responsible for impaired Epo synthesis in inflammatory diseases in vivo. Key words: cytokines • anemia • interleukin 1 • tumor necrosis factor α • promoter T he hormone erythropoietin (Epo) is essential in the proliferation and differentiation of erythrocytic progenitor cells in bone marrow and in the maintenance of a steady supply of red blood cells. Epo is produced mainly in the kidney and the liver. Epo synthesis is increased in response to tissue hypoxia, being regulated by a negative feedback mechanism involving oxygen tension. Abnormally low plasma Epo concentrations are measured in patients with malignant, infectious, or autoimmune diseases (1, 2). The pathogenesis of their anemia, known as anemia of chronic disease (ACD), involves several factors, including impaired iron turnover and, most importantly, reduced erythropoiesis. Administration of recombinant human Epo can improve ACD, whereas iron administration is much less effective. Epo mRNA expression and Epo protein secretion are induced by hypoxia in the human hepatoma cell lines HepG2 and Hep3B (3). In both cell lines, tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) cause a dose-dependent decrease of hypoxia-induced Epo gene expression and Epo secretion (4–6). Furthermore, IL-1 has been shown to inhibit murine erythropoiesis in vitro and in vivo (7, 8) and to suppress renal Epo gene expression in rats (9). The most important transcription factor for hypoxic stimulation of Epo production is the hypoxia-inducible factor 1 (HIF-1). Because IL-1β and TNF-α activate HIF-1 and increase HIF-1 DNA binding, HIF-1 cannot be the mediator of the cytokine-induced Epo repression (10). Apart from the hypoxic response element, the Epo promoter and the 5' flanking region contain other potentially regulatory elements. The minimal DNA sequence required for hypoxic basal transcription, a region from bp -32 to -1 in respective to the transcriptional startpoint, includes a binding site for the GATA transcription factors (11). The GATA motif is highly conserved among species and located in a region where a TATA motif is present in many other genes (12). GATA-2 has been reported to inhibit Epo gene expression (12–14). In addition, the 5' flanking region of the Epo gene contains several binding sites for NF-κB (15). One of these is adjacent to the minimal hypoxia-responsive element of the Epo promoter. NF-κB transcription factors are common mediators of proinflammatory cytokines and can modulate the expression of many genes involved in inflammation. This study was intended to investigate in vitro the influence of proinflammatory cytokines on GATA-2 and NF-κB activation in relation to Epo synthesis. Effects of TNF-α and IL-1β on GATA-2 and NF-κB DNA binding were studied in HepG2 cells. Reporter gene assays and oligo-decoy experiments (16, 17) were carried out as additional approaches to study the involvement of GATA-2 and NF-κB in Epo gene expression. MATERIALS AND METHODS Cell cultures The human hepatoma cell line HepG2 was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ Braunschweig). Cells were grown in RPMI 1640 medium (Invitrogen, Carlsbad, CA), supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml,) and 10% fetal calf serum (FCS) in a humidified incubator at 37°C. For preparation of nuclear extracts, we cultured cells with serum- and antibiotic-free medium for 24 h. For hypoxia experiments, we placed cells in an incubator at 3% O2 (Heraeus incubators, Kendro, Newtown, CT). Recombinant human TNF-α was a gift from BASF/Knoll (Ludwigshafen, Germany) and recombinant human IL-1β from Ciba-Geigy (Basel, Switzerland). Nuclear protein extraction Nuclear extracts were prepared according to established protocols (18, 19) with minor modifications. In brief, at the end of the incubation period, cells were kept on ice, washed once with ice-cold phosphate-buffered saline (PBS), scraped off, and collected. The cell suspensions were centrifuged at 1000g for 5 min at 4°C. The cell pellets were resuspended in 300 µl of low- salt buffer (10 mmol/l HEPES, pH 7.9; 1.5 mmol/l MgCl2; 10 mmol/l KCl). After incubation on ice for 10–15 min, cell membranes were destroyed by adding 25 µl of a 10% solution of NP-40 and vigorously mixing for 30 s. Nuclei were collected by centrifugation and resuspended in 100 µl high-salt buffer (20 mmol/l HEPES, pH 7.9; 420 mmol/l NaCl; 1.5 mmol/l MgCl2; 0.2 mmol/l EDTA; 25% glycerol) and incubated on ice for 20 min on a shaking platform. Buffers were supplemented with 2 µg/ml aprotinin, 10 µg/ml leupeptin, 20 µg/ml pepstatin, 1 mmol/l sodium- ortho-vanadate, 0.5 mmol/l benzamidine, 2 mmol/l levamisole, 10 mmol/l β-glycerophosphate, 0.5 mmol/l DTT, and 0.4 mmol/l PMSF just before use. Nuclei were centrifuged at 12500g at 4°C for 10 min. Supernatants were shock-frozen and stored at –80°C. Protein concentrations were determined by the Lowry method with the commercially available DC protein assay kit (BioRad Laboratories, Hercules, CA). Electrophoretic mobility shift assay (EMSA) For EMSAs, the following synthetic oligonucleotides were used: for NF-κB 5'- AGTTGAGGGGACTTTCCCAGGC-3' and the complementary strand 5´- GCCTGGGAAAGTCCCCTCAACT-3', for GATA-2 5'- CACACATGCAGATAACACCCCCGACC-3' and the complementary strand 5'- GGTCGGGGCTGTTATCTGCATGTGTG-3'. Single-stranded oligonucleotides were labeled with T4-polynucleotide kinase (MBI Fermentas) and γ [32P]-ATP. A twofold molar excess of unlabeled complementary oligonucleotides was annealed, and double-stranded oligonucleotides were purified on spin columns (Micro Bio-Spin P30, BioRad). Binding reactions were performed for 30 min on ice in 20 µl buffer (1 mmol/l MgCl2; 0.5 mmol/l M EDTA; 0.5 mmol/l DTT; 50 mmol/l NaCl; 10 mmol/l Tris-HCl, pH 7.5; 4% glycerol) containing 5 µg protein, 1 µg poly(dI:dC), and 15,000 cpm labeled oligonucleotide. Binding complexes were resolved by electrophoresis in vertical nondenaturing 6% polyacrylamide gels, using 0.3 × TBE as running buffer. Gels were dried, and binding complexes were quantitated by phosphoimager analysis. For supershift experiments, 2 µg of anti-NF-κB p-50 H-119 (Santa Cruz Biotechnology, Santa Cruz, CA), 5 µg of anti-NF-κB p-65 (Rel A) (Biomol, Stressgen Biotechnologies, San Diego, CA), and 1.6 µg of anti-GATA-2 H116 (Santa Cruz Biotechnology) were incubated with binding reaction mixtures for 1 h on ice before the labeled probe was added. Plasmid construction Epo reporter plasmids were constructed using the Kpn I and Sac I restriction enzyme sites within the PGL3-prom luciferase plasmid (Promega, Madison, WI). DNA inserts were synthetic oligonucleotides spanning the sequence from bp -38 to -14 of the Epo promoter (CACACATGCAGATAACAGCCCCGAC) containing either the GATA element (pGATAwt) or the GATA element mutated to TATA (pGATAmut). Constructs were verified by sequence analysis. Transfection HepG2 cells (5 × 106) in 400 µl RPMI 1640 medium were transiently transfected with 50 µg of the previously mentioned constructs by electroporation (250 V, 950 µF) with a Gene Pulser 2 (BioRad). The transfected cells were allowed to grow for 24 h. After medium renewal, cells were stimulated with cytokines. Luciferase activity was determined after 24 h. All results obtained for firefly luciferase were normalized to total cellular protein. NF-κB-responsive and -nonresponsive HepG2 cells were generated by transfection with either pCMV-IκBα or pCMV- IκBα-M (Clontech, Palo Alto, CA) by electroporation. pCMV- IκBα- transfected cells overexpress IκBα, whereas pCMV-IκBαM-transfected cells overexpress a mutated form of IκBα, with a serine-alanine mutation at residues 32 and 36. This form is not phosphorylated by Iκ-kinases and remains bound to NF-κB, leading to inhibition of NF-κB activation. After electroporation, cells were allowed to grow for 48 h and selected in medium containing 0.5 mg/ml of G418. Oligo-decoy experiments HepG2 cells were seeded at subconfluence in 96-well plates in medium with FCS. Double- stranded phosphorothioate backbone oligonucleotides with binding sites for either NF-κB (NF- κB-wt) or GATA-2 (GATA-2-wt) or with no-functional binding sites (NF-κB-mut, GATA-2- mut), respectively, were added to cell cultures to a final concentration of 3 mmol/l for 24 h. The sequences of oligonucleotides were NF-κB-wt 5'-AGTTGAGGGGACTTTCCCAGGC-3', NF- κB-mut 5'-AGTTGAGCCTGAACCCCCAGGC-3'; GATA-wt 5'- ACGCACACATGCAGATAACAGCCCCGACCC-3', GATA-mut 5´- ACGCACCACTGCAGGGATCAGCCCCGACCC-3' and the respective complementary strands. After medium renewal, cells were exposed to hypoxia alone or in combination with IL-1β. After 12 h of hypoxic incubation, medium was collected and assayed for secreted Epo. The efficiency of oligonucleotide uptake was proven with FITC-labeled oligonucleotides and fluorescence microscopy. About 30–40% of cells showed FITC fluorescence after the 24 h uptake period (data not shown). Epo concentrations were related to total cellular protein determined by the Bradford method. Assay of Epo Cells were seeded at a density of 5 × 106/well in 6-well plates or 1 × 105/well in 96-well plates and incubated at 3% O2 for 12 h or 24 h as specified. We determined Epo concentrations in cell culture supernatants by ELISA, using a commercial kit (Medac). RNA isolation and Northern blot Total RNA was isolated with the guanidinium thiocyanate-phenol-chloroform method (20). Total RNA (15 µg) was separated on a 0.7 mol/l formaldehyde/1% agarose gel and vacuum transferred onto a nylon membrane (Biodyne A membrane, Pall Corporation, East Hills, NY). Epo probe was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) of RNA from hypoxic HepG2 cells with Epo-specific primers as previously described (10). The probe was labeled with α [32P]-dCTP and a random prime DNA labeling kit (MBI Fermentas). Hybridization was carried out in 50% formamide, 1× Denhardt’s, and 0.2% sodium dodecyl sulfate (SDS) at 42°C for 24 h. The membrane was washed with 0.2× SSC/0.1% SDS at 60°C for 1 h, sealed in a plastic bag, and analyzed by phosphoimaging. The membrane was rehybridized with a 18 S rRNA probe for normalization of the Epo signals. Statistics Results are given as the mean and SD. Statistical analyses were performed using the methods of Student, Dunnett, or Bonferroni as appropriate. P was considered significant at the 5% level. RESULTS Induction of NF-κB DNA binding by IL-1β and TNF-α Normoxic (20% O2) and hypoxic (3% O2) HepG2 cells stimulated with IL-1β (300 pg/ml) or TNF-α (10 ng/ml) for 1 h showed increased NF-κB DNA-binding (Fig. 1A). Hypoxia alone did not induce p50/p65 heterodimer DNA binding. A time-course study with IL-1β showed that p50/p65 heterodimer formation was maximal between 30 min and 1 h, whereas the signals decreased after 2 and 4 h of incubation (Fig. 1B). Binding assays were also performed with extracts from cells stably transfected with pCMV- IκBα-M, a dominant-negative form of IκBα. pCMV-IκBα-M-transfected cells showed a marked decrease in NF-κB DNA binding compared with pCMV-IκBα-transfected cells or to nontransfected HepG2 cells (Fig. 1C). Preincubation of the reaction mixture with a polyclonal anti-p50 antibody induced supershifts of the specific signals in extracts from IL-1β-treated nontransfected cells, whereas preincubation with a polyclonal anti-p65 antibody prevented p50/p65 and p65/p65 DNA binding. Effects of NF-κB inhibition on Epo production Northern blots confirmed a decrease of Epo mRNA in hypoxic HepG2 cells upon treatment with IL-1β or TNF-α for 24 h. The cytokines also decreased Epo mRNA levels in HepG2 cells transfected with pCMV-IκBα. However, Epo mRNA levels were higher in IL-1β- or TNF-α- treated cells transfected with pCMV-IκBα-M than in pCMV-IκBα-transfected cells treated with the cytokines (Fig. 2). Epo levels increased about twofold in culture supernatants of pCMV-IκBα-M compared with pCMV-IκBα-transfected HepG2 cells following 24 h incubation at 3% O2, in both the presence and the absence of cytokines (Fig. 3). Oligo-decoy experiments showed that IL-1β failed to reduce Epo production when NF-κB function was blocked by treating cells with a NF-κB binding oligonucleotide (NF-κB-wt). In contrast, the NF-κB decoy-oligonucleotide with the mutated NF-κB binding site (NF-κB-mut) did not prevent the inhibition of Epo production by IL-1β (Fig. 4). Induction of GATA-2 DNA binding by IL-1β and TNF-α HepG2 cells showed strong GATA-2 DNA binding in normoxia (Fig. 5A), which was reduced upon exposure of the cells to hypoxia. Stimulation with IL-1β or TNF-α for 4 h led to an increase in GATA-2 DNA binding in hypoxia. The specificity of signals seen in EMSAs was demonstrated by competition experiments and by preincubation of the binding reaction mixtures with a specific antibody (Fig. 5B). Influence of IL-1β and TNF-α on Epo promoter activity Luciferase activity of normoxic cells transfected with pGATAmut was 1.7-fold greater than in pGATAwt-transfected cells. In hypoxia, luciferase activity was increased compared with normoxia in both pGATAwt and pGATAmut cells. Stimulation of hypoxic pGATAmut- transfected cells with IL-1β induced a 2.6-fold increase in luciferase activity compared with pGATAwt IL-1β-treated cells. A similar difference was seen when the effect of TNF-α was studied in pGATA-mut vs. pGATA-wt cells (Fig. 6). Oligo-decoy experiments showed that with respect to Epo production, IL-1β induced only a moderate decrease when GATA-2 function was blocked by treating cells with a GATA binding oligonucleotide (GATA-2-wt). In cells treated with an ineffective mutated oligonucleotide (GATA-2-mut), GATA-2 function was maintained and HepG2 cells produced little Epo upon IL- 1β treatment (Fig. 7). DISCUSSION The proinflammatory cytokines IL-1β and TNF-α are central mediators of immune responses. They have several biological activities in common, including the ability to induce anemia by inhibiting erythropoiesis. IL-1β and TNF-α suppress Epo production in vitro (4–6, 21). Our study suggests that this effect is mediated by the transcription factors NF-κB and GATA-2 in HepG2 cells. IL-1β and TNF-α induced NF-κB activation in normoxia and hypoxia, whereas hypoxia alone did not activate NF-κB. To investigate a possible involvement of NF-κB activation in the control of Epo production, we inhibited NF-κB function by stable transfection of HepG2 cells with a dominant-negative form of the inhibitory protein IκBα, which is usually present in HepG2 cells (22). Cells expressing the mutant form of IκBα showed higher rates of Epo production when treated with cytokines compared with cells expressing the normal form of IκBα, suggesting that NF-κB activation is involved in the pathway leading to the down- regulation of Epo production by IL-1β and TNF-α. These results were confirmed by RNA hybridization experiments showing decreased mRNA levels in native HepG2 cells treated with IL-1β or TNF-α and in cells expressing the normal form of IκBα. In comparison, Epo mRNA levels in cells expressing the mutant form of IκBα were higher upon cytokine treatment. Furthermore, oligo-decoy experiments revealed an increase in Epo production in cells with impaired NF-κB function. These data give additional evidence that NF- κB can be involved in the repression of certain genes, whereas most reports describe activation of gene transcription by NF-κB. Earlier studies have shown that NF-κB mediates TNF-α- induced suppression of the genes encoding cytochrome P-450 1A1 (23) and the carcinoma- associated epithelial cell adhesion molecule (24). This gene suppression has been explained by competition of NF-κB with other transcription factors for the cofactor p 300/CREB-binding protein (CBP) (23, 24). The transcription cofactor P300/CBP is also involved in hypoxia- induced Epo gene expression (25). In addition, we have shown previously that exogenous cAMP- analogs partly reverse the inhibition of Epo mRNA expression by IL-1β and TNF-α (6). GATA-2 binds to a specific motif present in the hypoxia-inducible promoter of the Epo gene (12–14). In addition, nitric oxide is thought to stimulate Epo gene expression by reducing GATA-2 availability (26), whereas hydrogen peroxide exerts the opposite effect (27). The role of GATA-2 in the inhibition of Epo production by cytokines has not been investigated previously. Our results show that GATA-2 DNA binding was decreased in hypoxic vs. normoxic cells, whereas Epo production increased in hypoxia. Treatment with cytokines restored signals in EMSAs to a level comparable to those observed under normoxic conditions, suggesting that GATA-2 may modulate signal transduction pathways of the cytokines reducing Epo production. To further prove that GATA-2 is involved in the cytokine-induced reduction of Epo synthesis, we performed reporter gene assays with a construct spanning a sequence from the Epo promoter, including the GATA element, and investigated the influence of cytokines on luciferase activity. We also measured luciferase activity in cells transfected with a sequence containing TATA instead of the GATA element. Cytokines decreased Epo promoter activity in cells transfected with pGATAwt compared with control cells, whereas Epo promoter activity was increased 2.5- fold when the TATA element was transfected. Others (12) have shown that a point mutation of the GATA sequence into TATA enhances transcriptional activity of the Epo promoter even in normoxia. Thus, the GATA motif in the Epo promoter can avoid binding and activation by TATA binding proteins, probably explaining the low basal expression of the Epo gene in normoxia. Our results confirm that the GATA element is a repressor element for the Epo gene. The observation that its activity is enhanced by IL-1β and TNF-α is a novel finding. Moreover, oligo-decoy experiments showed an increase in Epo production by cells with impaired GATA-2 function. It is generally assumed that HIF-1 is the primary transcription factor in the hypoxic induction of Epo gene expression. HIF-1 binds to the Epo enhancer as a dimer composed of an α- and a β- subunit (28). In the presence of O2, the α-subunit is rapidly prolyl- and asparaginyl- hydroxylated, targeted by the von Hippel-Lindau protein/E3 ubiquitation ligase complex, and degraded by the ubiquitin-proteasome system (29–32). In hypoxia, HIF-1α is enabled to enter the nucleus and to form the active heterodimer with HIF-1β. We have recently shown that neither IL-1β nor TNF-α suppresses HIF-1 DNA binding in HepG2 cells. 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W., and Schofield, C. J. (2002) Hypoxia inducible factor (HIF) asparagine hydroxylase is identical to factor inhibiting HIF (FIH) and is related to the cupin structural family. J Biol Chem 277, 26351–26355 33. Stiehl, D. P., Jelkmann, W., Wenger, R. H., and Hellwig-Bürgel, T. (2002) Normoxic induction of the hypoxia-inducible factor-1α by insulin and interleukin-1β involves the phosphatidylinositol 3-kinase pathway. FEBS Letters 512, 157–162 Received April 17, 2002; accepted July 15, 2002. Fig. 1 Figure 1. NF-κB DNA binding in HepG2 cells. A) Electrophoretic mobility shift assays (EMSAs) performed with 5 µg protein from untreated normoxic cells (lane 1), normoxic cells stimulated with IL-1β (300 pg/ml) or tumor necrosis factor (TNF)-α (10 ng/ml) (lanes 2 and 3), hypoxic control cells (lane 4), and hypoxic cells stimulated with IL-1β or TNF-α (lanes 5 and 6) for 1 h (similar results were obtained in three independent experiments). B) Time course of NF-κB DNA binding in normoxic HepG2 cells stimulated with IL-1β. C) EMSAs performed with 5 µg protein from control and IL-1β- treated normoxic cells: nontransfected cells (G2, lanes 1 and 2), with pCMV-IκBα (IκBα, lanes 3 and 4) and pCMV- IκBα-M (M, lanes 5 and 6) transfected HepG2 cells stimulated with IL-1β. Specificity of signals was shown by incubation of binding reactions for 1 h on ice with polyclonal antibodies against the p50 (lane 7) or p65 (lane 8) subunit of NF-κB in supershift assays (G2 [S]). Induction ratios are given in the tables below the figure. Fig. 2 Figure 2. Epo mRNA expression. A) Representative Northern blot of cells incubated under hypoxic conditions (3% O2) for 24 h in the absence or presence of IL-1β (300 pg/ml) or TNF-α (10 ng/ml). Total RNA (15 µg) from HepG2 cells (lanes 1–3), from HepG2 cells transfected with pCMV-IκBα (lanes 4–6), and from HepG2 cells transfected with pCMV- IκBα-M were analyzed. The membrane was rehybridized to a 18 S rRNA probe. B) Mean values and SD of three separate experiments. Epo mRNA was related to 18 S rRNA. Hypoxic samples were set 100%. *P < 0.05 (Dunnett’s test). Fig. 3 Figure 3. Epo production in IκBα-transfected cells. HepG2 cells transfected with pCMV-IκBα or with pCMV-IκBα- M were incubated under hypoxic conditions (3% O2) with or without cytokines (IL-1β, 300 pg/ml; TNF-α, 10 ng/ml) for 24 h. Epo concentrations were related to total cellular protein (mean ±SD of four separate experiments).*P < 0.05 (Student’s t test). Fig. 4 Figure 4. Effects of NF-κB decoy on Epo production. Double-stranded oligonucleotides (3 mmol/l) with functional (NF-κB-wt) or mutated binding site (NF-κB-mut) for NF-κB were added to cell cultures. Epo concentrations in culture supernatants were assessed after 12 h in hypoxia (3% O2) with or without IL-1β (300 pg/ml). Results are means ±SD of three experiments. *P < 0.05 (Bonferroni’s test). Fig. 5 Figure 5. GATA-2 DNA binding in HepG2 cells. A) Electrophoretic mobility shift assays (EMSAs) performed with 5 µg protein from untreated normoxic cells (lane 1), normoxic cells stimulated with IL-1β (300 pg/ml, lane 2) or TNF-α (10 ng/ml, lane 3), hypoxic cells (lane 4), and hypoxic cells stimulated with IL-1β or TNF-α (lanes 5 and 6) for 4 h (similar results were obtained in three independent experiments). B) EMSAs performed with 5 µg protein from untreated normoxic cells (lane 1), normoxic cells treated with IL-1β (300 pg/ml, lane 2), and normoxic cells preincubated with a 100-fold excess of an AP-1 consensus oligonucleotide (1 pmol) as unspecific competitor (u.c., lane 3) or with the GATA oligonucleotide (1 pmol) as specific competitor (s.c., lane 4). Preincubation of the binding reaction with an anti-GATA-2 polyclonal antibody (lane 5) prevented GATA-2 DNA-binding. Induction ratios are given in the tables below the figure. Fig. 6 Figure 6. Reporter gene assay of HepG2 cells transfected with pGATA-wt and pGATA-mut. Cells were stimulated with IL-1β (300 pg/ml) or TNF-α (10 ng/ml) for 24 h. Luciferase activity was compared with untreated normoxic cells set 100% and normalized to total cellular protein (mean ±SD of three separate experiments). Fig. 7 Figure 7. Effects of GATA-2 decoy on Epo production. Cells were treated with double-stranded oligonucleotides (3 mmol/l) with a functional binding site (GATA-wt) and a mutated binding site (GATA-mut) for GATA. Epo concentrations in culture supernatants were assessed after 12 h in hypoxia (3% O2) with or without IL-1β (300 pg/ml).