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Inhibition of erythropoietin gene expression signaling involves the transcription factors GATA-2 and NF-B ¹

Institute of Physiology, University of Luebeck, Luebeck, Germany

²Correspondence: Institute of Physiology, University of Luebeck, Ratzeburger Allee 160, 23538 Luebeck, Germany. E-mail: jelkmann{at}physio.uni-luebeck.de

SPECIFIC AIMS

The proinflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor (TNF-) contribute to the anemia of chronic disease by decreasing erythropoietin (Epo) gene expression. The molecular mechanisms of this inhibition are not understood. We investigated the roles of transcription factors NF-B and GATA-2 in the negative action of IL-1ß and TNF- on Epo gene expression in the human hepatoma cell line HepG2.

PRINCIPAL FINDINGS

1. IL-1ß and TNF- induce NF-B DNA binding
Normoxic (20% O₂) and hypoxic (3% O₂) HepG2 cells stimulated with IL-1ß (300 pg/mL) or TNF- (10 ng/mL) for 1 h exhibited NF-B DNA binding. Hypoxia alone did not induce p50/p65 heterodimer DNA binding. A time course with IL-1ß showed that p50/p65 heterodimer formation was maximal after 30 min to 1 h, whereas signals decreased after 2 and 4 h of incubation. Binding assays were performed with extracts from cells stably transfected with pCMV-IB-M, a dominant-negative form of IB. pCMV-IB-M transfected cells showed a marked decrease in NF-B DNA binding compared with pCMV-IB transfected cells or nontransfected HepG2 cells. Preincubation of the reaction mixture with a polyclonal anti-p50 antibody induced supershifts of the specific signals in extracts from IL-1ß-treated nontransfected cells, whereas preincubation with a polyclonal anti-p65 antibody prevented p50/p65 and p65/p65 DNA binding.

2. Inhibition of NF-B DNA binding prevents Epo repression by IL-1ß and TNF-
Northern blots revealed a decrease of Epo mRNA in hypoxic HepG2 cells on treatment with IL-1ß or TNF- for 24 h. Cytokines also had decreased Epo mRNA levels in HepG2 cells transfected with pCMV-IB. However, Epo mRNA levels were higher in IL-1ß- or TNF--treated cells transfected with pCMV-IB-M than in pCMV-IB transfected cells treated with cytokines (Fig. 1 ).

View larger version (57K): [in this window] [in a new window]   Figure 1. 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-1ß (300 pg/mL) or TNF- (10 ng/mL). 15 µg total RNA from HepG2 cells (lanes 1–3), from HepG2 cells transfected with pCMV-IB (lanes 4–6), and from HepG2 cells transfected with pCMV-IB-M (lanes 7–9) were analyzed. The membrane was rehybridized to a 18 S rRNA probe. B) Mean values and SD of 3 separate experiments. Epo mRNA was related to 18 S rRNA. Hypoxic samples were set 100%. *P < 0.05 (Dunnett’s test).

Epo levels were twofold increased in culture supernatants of pCMV-IB-M vs. pCMV-IB transfected HepG2 cells after 24 h incubation at 3% O₂. This finding was true in both the presence and absence of the cytokines.

Oligo decoy experiments showed that IL-1ß failed to reduce Epo production when NF-B function was blocked by treating cells with an NF-B binding oligonucleotide (NF-B-wt). In contrast, the NF-B decoy oligonucleotide with the mutated NF-B binding site (NF-B-mut) did not prevent inhibition of Epo production by IL-1ß.

3. IL-1ß and TNF- increase GATA-2 DNA binding in hypoxia
HepG2 cells showed strong GATA-2 DNA binding in normoxia (Fig. 2 A) that was reduced on exposure of the cells to hypoxia. Stimulation with IL-1ß or TNF- for 4 h led to an increase of GATA-2 DNA binding in hypoxia. The specificity of signals seen in electrophoretic mobility shift assays (EMSAs) was demonstrated by competition experiments and by preincubation of the binding reaction mixtures with a specific antibody (Fig. 2B ).

View larger version (23K): [in this window] [in a new window]   Figure 2. GATA-2 DNA binding in HepG2 cells. A) EMSAs performed with 5 µg protein from untreated normoxic cells (lane 1), normoxic cells stimulated with IL-1ß (300 pg/mL, lane 2) or TNF- (10 ng/mL, lane 3), hypoxic cells (lane 4), and hypoxic cells stimulated with IL-1ß or TNF- (lanes 5 and 6) for 4 h (similar results were obtained in 3 independent experiments). B) EMSAs performed with 5 µg protein from untreated normoxic cells (lane 1), normoxic cells treated with IL-1ß (300 pg/mL, lane 2), 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 tables below.

4. A GATA site is necessary for the inhibition of the Epo promoter by IL-1ß and TNF-
HepG2 cells were transiently transfected with a construct spanning a region of the human Epo promoter containing either a GATA element (pGATA-wt) or this element mutated into TATA (pGATA-mut). Luciferase activity of normoxic cells transfected with pGATA-mut was 1.7-fold greater than in pGATA-wt transfected cells. In hypoxia luciferase activity was 1.8-fold increased compared to normoxia in both pGATA-wt and pGATA-mut cells. Stimulation of hypoxic pGATA-mut transfected cells with IL-1ß induced a 2.6-fold increase in luciferase activity compared with pGATA-wt IL-1ß-treated cells. A similar difference was seen when the effect of TNF- was studied in pGATA-mut vs. pGATA-wt cells.

Oligo decoy experiments showed that IL-1ß induced only a moderate decrease in Epo production 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 on IL-1ß treatment.

CONCLUSIONS

Impaired Epo synthesis in inflammatory diseases is of major clinical importance. Several groups of investigators have demonstrated that IL-1 and TNF- suppress Epo gene expression. However, the molecular mechanisms of this suppression have not been known. Our study suggests that the transcription factors NF-B and GATA-2 mediate the inhibitory effects of IL-1ß and TNF- on Epo production in HepG2 cells.

The Epo promoter and the 5' flanking region contain binding sites for NF-B and GATA-2. IL-1ß and TNF- induced NF-B activation in normoxic and hypoxic cells, whereas hypoxia did not activate NF-B. To investigate a possible involvement of NF-B activation in the regulation of Epo production, we inhibited NF-B function by stable transfection of HepG2 cells with a dominant-negative form of the inhibitory protein IB. Cells expressing the mutant form of IB showed higher rates of Epo production when treated with cytokines compared with cells expressing the normal form of IB, suggesting that NF-B activation is involved in the pathway leading to down-regulation of Epo production by IL-1ß and TNF-. These results were confirmed by RNA hybridization experiments showing decreased Epo mRNA levels in native HepG2 cells treated with IL-1ß or TNF- and in cells expressing the normal form of IB. In contrast, increased Epo mRNA levels were detected on cytokine treatment in cells expressing the mutant form of IB. Oligo decoy experiments revealed an increase in Epo production in cells with impaired NF-B function. Irrespective of Epo synthesis, these data add further evidence that NF-B can be involved in the repression of genes in contrast to most reports describing activation of gene transcription by NF-B.

GATA-2 is known as an inhibitory regulating factor of Epo gene expression, but its involvement in the repression of Epo production by cytokines had not been investigated. 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 modulates signal transduction pathways of the cytokines reducing Epo production. 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 the same sequence containing a TATA element instead of GATA. Cytokines decreased Epo promoter activity in cells transfected with the GATA plasmid compared to control cells, whereas Epo promoter activity was increased 2.5-fold when the TATA element was transfected, suggesting that an intact GATA element is necessary for the cytokine repression of the Epo promoter. In addition, oligo decoy experiments showed an increase in Epo production in cells with impaired GATA-2 function.

These findings suggest an important role of NF-B and GATA-2 in suppression of the Epo gene by proinflammatory cytokines. Previous studies have shown that the most important transcription factor for hypoxic stimulation of Epo transcription is the hypoxia-inducible factor 1 (HIF-1). However, HIF-1 is not involved in the inhibition of Epo gene expression by IL-1ß and TNF-, since recent studies from our laboratory have shown that these cytokines increase HIF-1 DNA binding. If anything, increased HIF-1 DNA binding should induce Epo gene expression.

IL-1ß and TNF- play a role in the development of the so-called anemia of chronic diseases (ACD). Based on our cell culture studies, we assume that NF-B and GATA-2 may be involved in the pathogenesis of ACD in vivo. A better understanding of the molecular mechanisms controlling the repression of the Epo gene might lead to new therapeutic agents to treat patients with anemia.

View larger version (33K): [in this window] [in a new window]   Figure 3. Schematic drawing of the erythropoietin gene and alterations of GATA-2 and NF-B during hypoxia and cytokine stimulation. Relevant promoter sequences with binding sites for GATA-2 and NF-B are indicated.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0168fje; to cite this article, use FASEB J. (September 5, 2002) 10.1096/fj.02-0168fje

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