Evidence for a lack of a direct transcriptional suppression of the iron regulatory peptide hepcidin by hypoxia-inducible factors.

BACKGROUND: Hepcidin is a major regulator of iron metabolism and plays a key role in anemia of chronic disease, reducing intestinal iron uptake and release from body iron stores. Hypoxia and chemical stabilizers of the hypoxia-inducible transcription factor (HIF) have been shown to suppress hepcidin expression. We therefore investigated the role of HIF in hepcidin regulation. METHODOLOGY/PRINCIPAL FINDINGS: Hepcidin mRNA was down-regulated in hepatoma cells by chemical HIF stabilizers and iron chelators, respectively. In contrast, the response to hypoxia was variable. The decrease in hepcidin mRNA was not reversed by HIF-1alpha or HIF-2alpha knock-down or by depletion of the HIF and iron regulatory protein (IRP) target transferrin receptor 1 (TfR1). However, the response of hepcidin to hypoxia and chemical HIF inducers paralleled the regulation of transferrin receptor 2 (TfR2), one of the genes critical to hepcidin expression. Hepcidin expression was also markedly and rapidly decreased by serum deprivation, independent of transferrin-bound iron, and by the phosphatidylinositol 3 (PI3) kinase inhibitor LY294002, indicating that growth factors are required for hepcidin expression in vitro. Hepcidin promoter constructs mirrored the response of mRNA levels to interleukin-6 and bone morphogenetic proteins, but not consistently to hypoxia or HIF stabilizers, and deletion of the putative HIF binding motifs did not alter the response to different hypoxic stimuli. In mice exposed to carbon monoxide, hypoxia or the chemical HIF inducer N-oxalylglycine, liver hepcidin 1 mRNA was elevated rather than decreased. CONCLUSIONS/SIGNIFICANCE: Taken together, these data indicate that hepcidin is neither a direct target of HIF, nor indirectly regulated by HIF through induction of TfR1 expression. Hepcidin mRNA expression in vitro is highly sensitive to the presence of serum factors and PI3 kinase inhibition and parallels TfR2 expression

Effects of hypoxia, DMOG and DP on TfR2 protein expression in HepG2 cells.

<p>(<b>A</b>) FACS analysis of HepG2 cells with a TfR2 mouse monoclonal antibody demonstrating TfR2 expression on HepG2 cells under baseline conditions in comparison with an isotype-matched negative control antibody (co - TfR2). (<b>B</b>) After 16 h exposure to hypoxia a moderate induction was detected. After 16 h exposure to DMOG (<b>C</b>) or DP (<b>D</b>) TfR2 protein expression was reduced. C–D, representative of three independent experiments; B, representative of two out of three experiments.</p

The hypoxic response of the <i>hepcidin</i> promoter is cell type-specific and independent of HIF.

<p>(<b>A</b>) Sequence of the human <i>hepcidin</i> promoter (acc. no. AD000684.1) with two putative HIF binding motifs which also conform to E-box/USF binding sites (yellow boxes, red letters) and one additional HIF binding site (yellow); binding sites for p53 (light blue), AP1 (blue-green), C/EBPα (green), STAT3 (pink), SMAD (grey) are also marked; mRNA sequence (bold), translation start codon (ATG); underlined sequence represents region highly conserved between human and murine <i>hepcidin 1</i> gene. Lower sequence, two putative HREs were identified in the mouse <i>hepcidin 1</i> gene about 2.1 kbp upstream to the transcription start. These HIF binding sites do not conform to E-box/USF binding site consensus sequence. (<b>B</b>) A 617-bp human <i>hepcidin</i> promoter construct (HAMP.prom) responded differently to hypoxia (hyp), DMOG and DP in HepG2 and Huh7 cells after 16 h of stimulation (co = control). Data are means±SD of five (HepG2 cells) or three (Huh7 cells) independent experiments. (<b>C</b>) SiRNA knock-down of HIF-1α (1α) or HIF-2α (2α) did not reverse the down-regulation of promoter activity by DMOG in Huh7 cells; a 6xHRE luciferase reporter served as control. 3 luc siRNAs served as negative control for the pGL2-based <i>hepcidin</i> promoter constructs and 2 luc siRNA as negative control for the pGL3 6xHRE; deletion of the putative HREs in the <i>HAMP</i> promoter (HAMP.promΔHRE) did not alter the response of the luciferase construct to DMOG nor did HIF-α knock-down. (<b>D</b>) Deletion of putative HREs (HAMP.promΔHRE) did not alter the response of the <i>hepcidin</i> promoter to DMOG, DP or hypoxia in Huh7 cells. (<b>E</b>) Overexpression of a stable HIF-1α triple mutant (HIF-1αTM) tended to increase <i>hepcidin</i> promoter activity in comparison with the empty vector control (pcDNA3). The 6xHRE reporter served as positive and the promoter-less pGL2basic vector as negative control, respectively; <i>HAMP</i> promoter activities given in B–D are means of three independent experiments±SD. *<i>p</i><0.05; **<i>p</i><0.01 <i>vs.</i> unstimulated control (co).</p

Response of hepcidin transcript levels to hypoxia and chemical HIF stabilization in human hepatoma cells.

<p>(<b>A</b>) RNase protection assays (RPA) demonstrating hepcidin (HAMP) mRNA regulation and HIF target gene (IGFBP1 and ANGPTL4) induction in HepG2 cells after 16 h exposure to hypoxia (1% O₂), dimethyloxalylglycine (<i>abbreviated</i> DG <i>or</i> DMOG) or 2,2′dipyridyl (DP). (<b>B</b>) Hepcidin (HAMP), IGFBP1 and ANGPTL4 mRNA regulation in Huh7 cells. U6sn RNA served as loading control. Quantification was performed by phosphoimaging. Data are expressed as means±standard deviation (SD) of the indicated number of experiments; *<i>p</i><0.05, **<i>p</i><0.01 <i>vs.</i> unstimulated controls.</p

Hypoxia and hypoxia-Inducible Factor-1\alpha Modulate Lipopolysaccharide-Induced Dendritic Cell Activation and Function

Dendritic cells (DC) play a key role in linking innate and adaptive immunity. In inflamed tissues, where DC become activated, oxygen tensions are usually low. Although hypoxia is increasingly recognized as an important determinant of cellular functions, the consequences of hypoxia and the role of one of the key players in hypoxic gene regulation, the transcription factor hypoxia inducible factor 1\alpha (HIF-1\alpha), are largely unknown. Thus, we investigated the effects of hypoxia and HIF-1alpha on murine DC activation and function in the presence or absence of an exogenous inflammatory stimulus. Hypoxia alone did not activate murine DC, but hypoxia combined with LPS led to marked increases in expression of costimulatory molecules, proinflammatory cytokine synthesis, and induction of allogeneic lymphocyte proliferation compared with LPS alone. This DC activation was accompanied by accumulation of HIF-1\alpha protein levels, induction of glycolytic HIF target genes, and enhanced glycolytic activity. Using RNA interference techniques, knockdown of HIF-1alpha significantly reduced glucose use in DC, inhibited maturation, and led to an impaired capability to stimulate allogeneic T cells. Alltogether, our data indicate that HIF-1\alpha and hypoxia play a crucial role for DC activation in inflammatory states, which is highly dependent on glycolysis even in the presence of oxygen

Transcriptional induction of hepcidin expression by IL-6 and BMP-2.

<p>(<b>A</b>) RNase protection assays demonstrating dose-dependent induction of hepcidin mRNA by IL-6 in HepG2 and Huh7 cells. U6sn RNA served as loading control. (<b>B</b>) Quantification of hepcidin mRNA induction; means±SD of three independent experiments; *<i>p</i><0.05, **<i>p</i><0.01 <i>vs.</i> unstimulated cells. (<b>C</b>) IL-6 blunted, but did not abrogate the down-regulation of hepcidin mRNA levels by DMOG. IGFBP1 mRNA served as control for the hypoxic stimulation. (<b>D</b>) IL-6 activated the human <i>hepcidin</i> promoter (HAMP.prom) in HepG2 and Huh7 cells. (<b>E</b>) Activation of the <i>hepcidin</i> promoter by BMP-2 (100 ng/ml) was mediated primarily by the proximal promoter, since fusion of the 5′adjacent CpG island to the proximal promoter (HAMP.prom.CpG) did not significantly alter the response to BMP-2. The activation was more pronounced under serum-reduced conditions (0.4% FCS). D–E, data are means±SD of three independent experiments; *<i>p</i><0.05, **<i>p</i><0.01 <i>vs.</i> unstimulated controls.</p

Response of hepcidin transcript levels to serum deprivation and protein kinase inhibition.

<p>(<b>A</b>) Serum withdrawal rapidly decreased hepcidin transcript levels in Huh7 cells. (<b>B</b>) After 40 h of FCS reduction from 10% to 0.4%, hepcidin transcripts were hardly detectable by RPA in HepG2 cells, whereas hypoxic IGFBP1 induction was not affected. Representative of three independent experiments. U6sn RNA served as loading control. (<b>C</b>) Exposure of Huh7 cells to protein kinase inhibitors revealed that the pan kinase inhibitor staurosporine (stauro; 0.5 µM) and the PI3 kinase inhibitor LY294002 (LY; 10 µM) reduced hepcidin expression similar to serum deprivation, whereas the p38 SAP kinase inhibitor SB202190 (SB; 10 µM) had no effect and the MEK1/2 inhibitor UO126 (1 µM) even increased HAMP/18S ratios. Data are results of qRT PCR analyses and given as means of three independent experiments±SEM; *p<0.05; **p<0.01.</p

Suppression of hepcidin transcript levels is not mediated by HIF-1α or HIF-2α.

<p>(<b>A</b>) SiRNA-mediated knock-down of HIF-1α (1α) or HIF-2α (2α) did not attenuate the down-regulation of hepcidin mRNA levels in HepG2 cells after 16 h of stimulation; luciferase (luc) siRNA served as negative control. (<b>B</b>) Immunoblots demonstrating the efficiency of HIF-1α and HIF-2α knock-down in HepG2 and Huh7 cells. (<b>C</b>) mRNA expression of the established HIF target genes ANGPTL4 (HIF-1α target) and IGFBP1 (HIF-2α target in hepatoma cells) after HIF-α knock-down in HepG2 cells. (<b>D</b>) Two independent HIF-1α siRNAs (1α, 1α*) reversed the hypoxic down-regulation of the negatively regulated HIF-1 target NUP98. (<b>E</b>) In Huh7 cells hepcidin mRNA down-regulation by DMOG was not affected by HIF-α knock-down, whereas the decrease of NUP98 mRNA was reversed by HIF-1α knock-down. Results shown are representative of at least three independent experiments.</p