Basic Study
Copyright ©The Author(s) 2021. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Hepatol. Oct 27, 2021; 13(10): 1378-1393
Published online Oct 27, 2021. doi: 10.4254/wjh.v13.i10.1378
Direct modulation of hepatocyte hepcidin signaling by iron
Lin-Na Yu, Shi-Jin Wang, Cheng Chen, Vanessa Rausch, Omar Elshaarawy, Sebastian Mueller
Lin-Na Yu, Shi-Jin Wang, Cheng Chen, Vanessa Rausch, Sebastian Mueller, Center for Alcohol Research and Salem Medical Center, University of Heidelberg, Heidelberg 69121, Germany
Omar Elshaarawy, Department of Hepatology, Gastroenterology and Liver Transplantation, National Liver Institute, Menoufia University, Shebine Elkom 35121, El Salvador
Omar Elshaarawy, Department of Gastroenterology, Royal Liverpool University Hospital, Liverpool L7 8XP, United Kingdom
Author contributions: Yu LN and Wang SJ equally contributed to the manuscript and share co-first-authorship; Yu LN, Mueller S and Rausch V conceived the study; Yu LN, Wang SJ and Mueller S designed the experiments and analyzed the data; Yu LN, Wang SJ and Chen C performed the experiments; Mueller S wrote and critically discussed and revised the manuscript; All authors approved the final version of the article.
Supported by the Deutche Forschungsgemeinschaft, No. RA 2677/1-2 (to Mueller S).
Conflict-of-interest statement: The authors declare no potential conflicts of interest regarding this article.
Data sharing statement: All data including Supplementary Figureary materials are contained within the manuscript.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Sebastian Mueller, PhD, Professor, Center for Alcohol Research and Salem Medical Center, University of Heidelberg, Zeppelinstraße 11-33, Heidelberg 69121, Germany. sebastian.mueller@urz.uni-heidelberg.de
Received: February 27, 2021
Peer-review started: February 27, 2021
First decision: April 18, 2021
Revised: May 4, 2021
Accepted: August 27, 2021
Article in press: August 27, 2021
Published online: October 27, 2021
Processing time: 236 Days and 18.6 Hours
Abstract
BACKGROUND

Liver-secreted hepcidin is the systemic master switch of iron homeostasis and decreased levels of hepcidin are considered to cause iron overload not only in hereditary hemochromatosis but also in hemolytic anemia and chronic liver diseases. The regulation of hepcidin is complex and its response to iron is still not completely understood.

AIM

To study the direct effect of iron on various established hepcidin signaling pathways in hepatoma cells or primary hepatocytes.

METHODS

Hepcidin mRNA expression was studied by quantitative real-time (qRT)-PCR in the presence of various forms of iron including ferric ammonium citrate (FAC) in hepatoma cells (Huh7), murine primary hepatocytes and an established co-culture model of phorbol myristate acetate-differentiated THP-1 monocytes and Huh7 cells. To analyze hepcidin signaling, the response to bone morphogenetic protein 6 (BMP6), interleukin (IL)-6, IL-1β, hypoxia and lipopolysaccharide (LPS) were studied. Hepcidin and small mothers against decapentaplegic 6 (SMAD6) mRNA levels were assessed by qRT-PCR and the expression of phosphorylated signal transducer and activator of transcription 3 (phospho-STAT3), STAT3, phospho-SMAD1/5/8 and SMAD1 proteins were analyzed by western blot.

RESULTS

All iron III forms including FAC efficiently blocked hepcidin mRNA expression at non-toxic dosages in Huh7 cells or primary hepatocytes in a time and dose-dependent manner (P < 0.001; P < 0.05). Hepcidin blockage could be efficiently blunted by iron chelators salicylaldehyde isonicotinoyl hydrazone (SIH) and Desferal (P < 0.001). FAC also inhibited BMP6, hypoxia, IL-1β and IL-6-mediated hepcidin induction (P < 0.001; P < 0.001; P < 0.05; P < 0.001), and FAC also inhibited LPS-mediated hepatic hepcidin induction in co-culture model (P < 0.001). Moreover, FAC reduced SMAD6 mRNA and p-SMAD1/5/8 protein expression at basal or upon stimulation by BMP6 (P < 0.05; P < 0.01), and FAC also reduced SMAD6 and p-SMAD1/5/8 expression under hypoxia (P < 0.01; P < 0.05). However, FAC has no significant effect on p-STAT3 protein expression at basal or upon stimulation by various stimuli. Notably, in the presence of the BMP/SMAD signaling pathway inhibitor LDN193189 Hydrochloride (LDN), FAC was unable to further decrease hepcidin, SMAD6 and p-SMAD1/5/8 expression compared with LDN alone.

CONCLUSION

Iron directly blocks hepatocellular hepcidin signaling through the BMP/SMAD pathway but independent of STAT3. This mechanism may contribute to continued iron overload in many pathophysiological conditions ultimately causing a vicious cycle of continued hepcidin suppression.

Keywords: Hepcidin/iron metabolism, Iron overload, Inflammation, Hypoxia, BMP/SMAD, STAT3

Core Tip: Hepcidin is paradoxically and strongly suppressed during hemolytic iron overload. Although various upstream regulators of hepcidin have been discovered, the direct iron sensing mechanisms by hepcidin remain obscure. This study investigated the direct effect of iron on hepcidin signaling and for the first time to show that iron directly blocks hepcidin transcription via bone morphogenetic protein/small mothers against decapentaplegic but not the STAT3 signaling in various established in vitro models of hepcidin signaling.