Hypoxia inhibits hepcidin expression in HuH7 hepatoma cells via decreased SMAD4 signaling

Abstract

Hepcidin negatively regulates systemic iron homeostasis in response to inflammation and elevated serum iron. Conversely, hepcidin expression is diminished in response to hypoxia, oxidative stress, and increased erythropoietic demand, though the molecular intermediates involved are incompletely understood. To address this, we have investigated hypoxic hepcidin regulation in HuH7 hepatoma cells either cultured alone or cocultured with activated THP-1 macrophages. HuH7 hepcidin mRNA expression was determined using quantitative polymerase chain reaction (Q-PCR). Hepcidin promoter activity was measured using luciferase reporter constructs containing a 0.9 kb fragment of the wild-type human hepcidin promoter, and constructs containing mutations in bone morphogenetic protein (BMP)/SMAD4, signal transducer and activator of transcription 3 (STAT3), CCAAT/enhancer-binding protein (C/EBP), and E-box-responsive elements. Hepatic expression of bone morphogenetic proteins BMP2 and BMP6 and the BMP inhibitor noggin was determined using Q-PCR, and the protein expression of hemojuvelin (HJV), pSMAD 1/5/8, and SMAD4 was determined by western blotting. Following exposure to hypoxia or H2O2, hepcidin mRNA expression and promoter activity increased in HuH7 cells monocultures but were decreased in HuH7 cells cocultured with THP-1 macrophages. This repression was attenuated by mutation of the BMP/SMAD4-response element, suggesting that modulation of SMAD signaling mediated the response to hypoxia. No changes in hepatocyte BMP2, BMP6 or noggin mRNA, or protein expression of HJV or pSMAD 1/5/8 were detected. However, treatment with hypoxia caused a marked decrease in nuclear and cytosolic SMAD4 protein and SMAD4 mRNA expression in cocultured HuH7 cells. Together these data indicate that hypoxia represses hepcidin expression through inhibition of BMP/ SMAD signaling.hepatocytes; macrophages; oxidative stress THE LIVER-EXPRESSED PEPTIDE hepcidin (23) has emerged as a major regulator of systemic iron homeostasis. During inflammation, hepcidin is secreted into the serum, where it blocks iron release from duodenal enterocytes and reticuloendothelial macrophages, causing hypoferremia (8,10,12,20,36). Hepcidin transcription is also sensitive to systemic iron levels, body iron stores, erythropoietic factors, and hypoxia in vivo (14,29,33), and the regulated production and release of the peptide plays a central role in the maintenance of iron homeostasis. This is supported by the inappropriate regulation of hepcidin expression seen in transgenic animal models of iron overload that lack functional genes for HFE, hemojuvelin (HJV), or transferrin receptor 2 (1, 6, 28, 31).The systemic and intracellular mechanisms that underlie negative regulation of hepcidin by hypoxia and oxidative stress are incompletely understood. Studies in rodent models have demonstrated dramatic effects of hypoxia on iron homeostasis: increased intestinal iron absorption (24, 25, 30, 37) and decreased expression of hepcidin (24,29,32). The molecular intermediates that signa...

Hepatic hypoxia-inducible factor-2 down-regulates hepcidin expression in mice through an erythropoietin-mediated increase in erythropoiesis

“…A decrease in BMP signaling could also be considered, as previously suggested. 27 Interestingly, it was recently proposed that hypoxia inhibits hepcidin expression in hepatoma cells via a marked decrease in SMAD4 mRNA expression, 37 indicating that the involvement of the BMP/SMAD signaling in hypoxia deserves further investigations. In vivo, hypoxia has been shown to decrease hepcidin expression in both acute and chronic experimental models 21,38,39 as well as in humans.…”

BMPS and Liver: More Questions than Answers

“…Hepcidin expression is not only regulated by BMPs, but also by HFE (high iron Fe) and/or TFR2 (transferrin receptor 2), but the nature of the interplay between these molecules and the BMP/HJV/hepcidin pathway is not known. It is also known that inflammatory cytokines, anemia and hypoxia regulate the expression levels of hepcidin, but how those pathways fit into the BMP pathway is just starting to be elucidated [101][102][103][104]. Although most evidence suggests that BMP6 produced and released by enterocytes is the master regulator of liver hepcidin levels, BMP6 is also expressed in liver cells [54].…”

Hypoxia and cytokines regulate carbonic anhydrase 9 expression in hepatocellular carcinoma cellsin vitro

“…On the other hand, IL-6 regulates other HIF-1α regulated genes such as vascular endothelial growth factor [24,25] , while IL-6 itself is regulated via the signal transducers and activators of transcription-Janus kinase pathway, and TNF-α induces HIF-1α expression through 3-phosphoinositide-dependent protein kinase-1-mediated I kappa B kinase beta [26,27] and nuclear factor "kappa-lightchain-enhancer" of the activated B-cells pathway [28] and has been shown to play a positive role in the induction of the HIF-1α regulated genes in human glioma [29,30] . In addition, enhanced levels of TGF-β are a common feature in human tumour cells [31] , and TGF-β is also released by infiltrating leukocytes and induces up-regulation of HIF-1α regulated genes [18,32] most probably without the involvement of the bone morphogenetic protein family [33,34] or the Smad family [35][36][37][38][39] pathways, despite the fact that TGF-β uses the Smad pathway which transcriptionally represses (inhibitor of differentiation 1) Ids proteins in epithelial cells. Also, CA9 causes a reduction in extracellular pH, thereby facilitating the breakdown of the extracellular matrix together with up-regulation of the genes involved in invasion and migration [40][41][42][43] .…”