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Letters to the Editor
Endoplasmic reticulum stress controls iron metabolism through TMPRSS6 repression and hepcidin mRNA stabilization by RNA-binding protein HuR
The liver hormone hepcidin controls the main inflows of iron into plasma by binding to and inducing the degra- dation or the occlusion of the iron export activity of fer- roportin, the only known cellular exporter of iron.1,2 When hepcidin concentrations are high, iron is trapped in enterocytes of the duodenum, hepatocytes, and macrophages. Hepcidin production by the hepatocytes is induced by a number of stimuli, most notably iron, through the BMP-SMAD signaling pathway,3 and inflam- matory signals, through the IL-6/ STAT3 signaling axis.4 In addition, hepcidin has also been reported to respond to intracellular stress, namely endoplasmic reticulum (ER) stress which is involved in a number of pathophysiologi- cal states, including the inflammatory response, nutrient disorders and viral infection. A previous study has sug- gested that hepcidin induction by ER stress is controlled by the BMP-SMAD pathway,5 but the exact mechanism is still uncertain.
ER stress may have an important role in the develop- ment of nonalcoholic fatty liver disease (NAFLD).6 Indeed, hepatic lipid accumulation induces ER stress, and, in turn, the ER stress response promotes hepatic lipogenesis, thus creating a positive-feedback loop, which may contribute to the development of hepatic steatosis.6 ER stress has also been implicated in the development of hepatocellular injury and fibrosis and the progression of simple steatosis to nonalcoholic steatohepatitis (NASH). Interestingly, approximately one third of patients with NAFLD show signs of disturbed iron homeostasis as indi- cated by elevated serum ferritin with normal or mildly elevated transferrin saturation, mild hepatic iron deposi- tion7 and increased hepcidin production.8 Excess iron is proposed to aggravate the natural course of NAFLD because of its capability to catalyze the formation of toxic hydroxyl radicals that cause cellular damage. Iron accu- mulation in NAFLD is mainly due to impaired iron export from hepatocytes and Kupffer cells7 which might well be the consequence of hepcidin induction by ER stress.
Given the potential impact of ER stress-induced hep- cidin on hepatic iron deposition in NAFLD patients, it is essential to better understand the molecular mechanisms leading to the induction of hepcidin in this context. In order to definitely elucidate these mechanisms, we used a model of acute ER stress induced in mice by tuni- camycin. We demonstrated that induction of hepcidin by ER stress requires repression of TMPRSS6, the gene cod- ing for the inhibitor of BMP-SMAD1/5/8 signaling, matriptase-2, and stabilization of hepcidin mRNA by the RNA-binding protein, HuR.
In order to investigate the kinetics of hepcidin induc- tion by ER stress over time, wild-type (WT) mice received one intraperitoneal (IP) injection of tunicamycin (Tm) (2 mg/kg), a well-known ER stress inducer, and were sacri- ficed at time points ranging from 3 to 24 hours after injec- tion. As expected, Tm injection triggered ER stress in the liver (Online Supplementary Figures S1A-B, S2A-B). As shown in Online Supplementary Figure S1C, hepcidin gene expression progressively increased and reached a maxi- mum 6 hours after Tm injection. Therefore, this time point was chosen for performing all the following exper- iments.
As expected, we show that hepcidin induction coin- cides with an activation of the BMP-SMAD pathway
(Figure 1A-C; Online Supplementary Figure S1D) which is dependent on the Bmp type 1 receptor Bmpr1a (Online Supplementary Figure S3) 6 hours after Tm injection. In this study, our objective was to find out the mechanisms that activate BMP-SMAD signaling during ER stress, leading to excessive hepcidin production. As shown in the Online Supplementary Figure S4A-H, Tm did not increase the expression of the ligands known to activate Bmp-Smad signaling and hepcidin gene expression in cir- cumstances other than ER stress, i.e., Bmp63 and Bmp29 and does not modulate the expression of other genes belonging to this pathway. Another ligand of the TGF-β family, activin B, was suggested to induce hepcidin in ER stress5 but, as shown in the Online Supplementary Figure S4I-J, Tm similarly induced Id1 and hepcidin gene expression in Inhbb-/- mice, lacking activin B, and in WT controls. A role for activin B in this process is thus unlikely.
Matriptase-2, encoded by the TMPRSS6 gene, is a strong inhibitor of the BMP-SMAD signaling pathway and of hepcidin expression.10 Interestingly, Tm injection significantly suppressed matriptase-2 at mRNA (Figure 1D; Online Supplementary Figure S1E) and protein (Online Supplementary Figure S5) levels, which could explain the observed activation of BMP-SMAD signaling and induc- tion of hepcidin expression in these mice. In order to con- firm this hypothesis, we assessed the response of Tmprss6-/- mice to Tm. Notably, in the absence of stimula- tion, Smad5 phosphorylation and Id1 expression are, as expected, constitutively high in Tmprss6-/- mice, but they have not reached their peak and can still be further induced by iron dextran (Online Supplementary Figure S6F- G). This demonstrates that Tmprss6-/- mice have the abil- ity to activate the BMP-SMAD signaling in response to external stimuli. However, although ER stress was simi- larly induced in both Tmprss6-/- and WT mice (Online Supplementary Figure S6A-D), Smad5 phosphorylation (Figure 1E) and Id1 expression (Figure 1F) were not fur- ther increased by Tm in Tmprss6-/- mice. In order to deter- mine if the loss of BMP-SMAD activation was not blunt- ed by the iron deficiency anemia of Tmprss6-/- mice, we used Bmp6-/- - Tmprss6-/- mice which have a BMP signaling similar to WT mice and no iron deficiency anemia.11 In this mouse model, BMP signaling is not induced by Tm injection either (Online Supplementary Figure S7A-B) con- firming that repression of matriptase-2 is required for activation of BMP-SMAD signaling by ER stress. Of note, lack of Bmp6 only does not prevent the induction of BMP signaling and hepcidin expression in response to ER stress (Online Supplementary Figure S8).
Quite surprisingly though, and despite the lack of fur- ther Smad5 activation, hepcidin induction was not totally abolished in mice lacking matriptase-2 (Figure 1G; Online Supplementary Figure S7C), suggesting that a second mechanism contributes to the whole magnitude of hep- cidin upregulation in ER stress.
In order to characterize this additional mechanism observed in Tmprss6-/- mice, we used the HepG2 hepatoma cell line that expresses TMPRSS6 mRNA at a level so low that a siRNA directed against it is unable to promote any activation of BMP-SMAD signaling (data not shown). The HepG2 cell line is thus a good model to characterize hepcidin regulation by ER stress independ- ently of matriptase-2 and BMP-SMAD signaling. Treatment of HepG2 cells with Tm induces ER stress (Figure 2A) and hepcidin (Figure 2B; Online Supplementary Figure S9A) even in the absence of Smad5 activation or ID1 mRNA induction (Figures 5C-D; Online Supplementary Figure S9B).
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haematologica | 2021; 106(4)