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

Iron-Induced Expression of Bone Morphogenic Protein 6 in Intestinal Cells Is the Main Regulator of Hepatic Hepcidin Expression In Vivo

BACKGROUND & AIMS: Recent studies identified bone morphogenic protein 6 (BMP6) as a key regulator of hepatic hepcidin expression and iron metabolism, but the cellular source of BMP6 and the reason for its specific effect on hepatocytes are unknown. METHODS: BMP and hepcidin expression upon iron sensing were analyzed in vivo in BMP6(-/-) and BMP6(+/+) mice and ex vivo in tissue and in vitro in cells of the liver and the small intestine. RESULTS: BMP6(-/-) mice developed severe hepatic iron accumulation and reduced hepcidin expression with increasing age. This phenotype could be triggered in younger BMP6(-/-) mice by dietary or parenteral iron application. Furthermore, both treatments induced a marked up-regulation of BMP6 expression in the small intestine of BMP6(+/+) mice. Ex vivo treatment of intestinal tissue of BMP6(+/+) mice with iron sulfate or holo-transferrin confirmed epithelial cells as an inducible source of BMP6. In contrast, iron overload did not promote a striking induction of BMP6 expression in hepatocytes or macrophages. Furthermore, iron-supplemented diet induced a compensatory up-regulation of BMP2, BMP4, and BMP9 in the small intestine of BMP6(-/-) mice that was apparently not sufficient to assure iron homeostasis. As a potential explanation, analysis of hepatocytes revealed an expression pattern of BMP receptor subunits preferentially used by BMP6, and treatment of hepatocytes with different recombinant BMPs identified BMP6 as the most potent stimulator of hepcidin expression. CONCLUSIONS: Epithelial cells of the small intestine are the predominant cellular source of BMP6 upon iron sensing. Our findings reveal a previously unknown mechanism in which the small intestine controls iron homeostasis

GLUT1 expression is increased in hepatocellular carcinoma and promotes tumorigenesis

Accelerated glycolysis is one of the biochemical characteristics of cancer cells. The glucose transporter isoform 1 (GLUT1) gene encodes a key rate-limiting factor in glucose transport into cancer cells. However, its expression level and functional significance in hepatocellular cancer (HCC) are still disputed. Therefore, we aimed to analyze the expression and function of the GLUT1 gene in cases of HCC. We found significantly higher GLUT1 mRNA expression levels in HCC tissues and cell lines compared with primary human hepatocytes and matched nontumor tissue. Immunohistochemical analysis of a tissue microarray of 152 HCC cases revealed a significant correlation between Glut1 protein expression levels and a higher Ki-67 labeling index, advanced tumor stages, and poor differentiation. Accordingly, suppression of GLUT1 expression by siRNA significantly impaired both the growth and migratory potential of HCC cells. Furthermore, inhibition of GLUT1 expression reduced both glucose uptake and lactate secretion. Hypoxic conditions further increased GLUT1 expression levels in HCC cells, and this induction was dependent on the activation of the transcription factor hypoxia-inducible factor-1alpha. In summary, our findings suggest that increased GLUT1 expression levels in HCC cells functionally affect tumorigenicity, and thus, we propose GLUT1 as an innovative therapeutic target for this highly aggressive tumor

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

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

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

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

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