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Letters to the Editor
cidin 3’UTR sequence or a non-relevant sequence in the BMPR1A gene. In cells treated with Tm, the level of hep- cidin 3’UTR measured in the immunoprecipitate collect- ed with HuR antibody was increased 20 to 6,700-fold rel- ative to that in the immunoprecipitate collected with control IgG (Figure 2H; Online Supplementary Figure S10A- B). No such enrichment of hepcidin 3’UTR is seen in cells treated with mock. Furthermore, there was no enrich- ment of the non-relevant sequence in the immunoprecip- itate collected with HuR antibody (Figure 2I; Online Supplementary Figure S10C-D), indicating that Tm treat- ment specifically promotes binding of the RNA stabilizer HuR to hepcidin 3’UTR.
Interestingly, while HuR mRNA expression is stimulat- ed in response to ER stress in WT and Tmprss6-/- mice (Online Supplementary Figures S1F, S6E), it is blunted when Bmpr1A is missing (Online Supplementary Figure S3H). This is consistent with the previously reported transcriptional control of HuR by BMP-SMAD signal- ing.13 Accordingly, hepcidin mRNA expression is not sta- bilized in Bmpr1Ahep/hep mice.
In the present study, we showed that ER stress up-reg- ulates the iron-regulatory hormone hepcidin through two complementary mechanisms. The first involves the inhi- bition of matriptase-2, which activates BMP-SMAD sig- naling without the induction of Bmp2 and Bmp6 or the requirement for activin B. The second is the stabilization of hepcidin mRNA by the RNA-binding protein, HuR. These two mechanisms appear to act sequentially. Indeed, whereas the induction of another target of BMP-SMAD signaling, Id1, reached a plateau 3 hours after Tm injection (Online Supplementary Figure S1D), hepcidin mRNA did not achieve its maximal level at this time point and continued to rise at least 2-fold between 3 and 6 hours (Online Supplementary Figure S1C). This coincided with the induction of Hur expression in the liver (Online Supplementary Figure S1F). Thus, the effect of HuR on hepcidin mRNA stabilization is very likely sec- ondary to the activation of BMP-SMAD signaling. Moreover, in WT mice, in which both mechanisms are functional, hepcidin is increased 5.3 times whereas in Bmp6-/--Tmprss6-/- mice in which the BMP signaling is not activated, hepcidin expression is only induced 2.8 times. This suggests that both mechanisms each contribute to half of the whole magnitude of hepcidin induction in response to ER stress. Interestingly, HuR mRNA is not increased in mice lacking Bmpr1A specifically in hepato- cytes. A functional BMP-SMAD signaling pathway is thus necessary for the induction of hepcidin by ER stress.
Chronic liver ER stress, promoted at least in part by liver fatty acid accumulation,6 contributes to the progres- sion of NAFLD towards NASH.14-16 Mild to moderate hepatic iron accumulation, seen in hepatocytes and/or in cells of the reticuloendothelial system of one third of the patients with NAFLD,7 is also a factor of poor prognosis. Finally, NAFLD patients often have high levels of hep- cidin.8 In vitro, HuR stabilizes hepcidin mRNA in response not only to ER stress as demonstrated here but also to saturated fatty acids.12 As saturated fatty acids accumulate in the liver in NAFLD and induce ER stress, it is highly likely that ER stress is the intermediary between fatty acids and activation of HuR and that HuR also partici- pates in hepcidin regulation in patients with NAFLD. Nonetheless, the role of HuR on the stabilization of hep- cidin in vivo will need to be confirmed by further studies.
Audrey Belot,1 Ophélie Gourbeyre,1 Anais Palin,1
Aude Rubio,1 Amélie Largounez,1 Céline Besson-Fournier,1 Chloé Latour,1 Megane Lorgouilloux,1 Inka Gallitz,2
Alexandra Montagner,3 Arnaud Polizzi,3 Marion Régnier,3 Sarra Smati,3 An-Sheng Zhang,4 Manuel D. Diaz-Munoz,5 Andrea U. Steinbicker,2 Hervé Guillou,3 Marie-Paule Roth,1 Hélène Coppin1 and Delphine Meynard1
1IRSD, Université de Toulouse, INSERM, INRA, ENVT, UPS, Toulouse, France; 2Department of Anesthesiology, Intensive Care and Pain Medicine, University Hospital Muenster, University of Muenster, Muenster, Germany; 3Institut National de La Recherche Agronomique (INRA), UMR1331 ToxAlim, Toulouse, France and
4Cell, Developmental, and Cancer Biology, Oregon Health & Science University, Portland, OR, USA and 5CPTP, INSERM UMR1043/CNRS U5282, Toulouse, France
Correspondence:
DELPHINE MEYNARD - delphine.meynard@inserm.fr
doi:10.3324/haematol.2019.237321
Disclosures: no conflicts of interest to disclose.
Contributions: AB, OG, AP, AR, and AL performed experiments, analyzed, and discussed results; CB-F, CL, ML, IG, AM, AP, MR, SS and AUS performed experiments; MD-M, AUS, HG analyzed and discussed results; A-SZ provided antibody against matriptase-2, DM, HC, and M-PR discussed data and wrote the manuscript; DM designed research, performed experiments, analyzed and discussed data, and wrote the manuscript; and all authors reviewed and approved the final manuscript.
Acknowledgments: the authors are grateful to Carlos Lopez Otin (University of Oviedo, Oviedo, Spain) for kindly providing the original Tmprss6−/− mice on a mixed genetic background. They also thank Florence Capilla (Experimental Histopathology Platform, Toulouse Purpan), and members of the INSERM US006 facility (Toulouse)
for their technical assistance and help in the mouse breeding..
Funding: DM was supported by the Cooley’s Anemia Foundation, the French Foundation for Rare Diseases, the Région Midi-Pyrénées and ANR (ANR-17-CE14-0036-01 and ANR-17-CE14-0031-01). AUS was supported by a research grant of the German Research Foundation (DFG, STE-1985/4-1). HG was supported by grants from Région Occitanie and ANR (ANR-15-CE14-0026-Hepatokind). MDD-M was supported by ATIP-AVENIR (INSERM/CNRS) program and by Plan-Cancer (C18003BS). A-SZ was supported
by a grant from NIH (R01DK102791). This work was also support- ed by the “Programme des Investissements d’Avenir” ANINFIMIP (ANR-11EQPX-0003).
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