S1627 THE HEMOCHROMATOSIS PROTEINS HJV AND TFR2 ARE DISPENSABLE FOR ACUTE BMP2‐MEDIATED HEPCIDIN UPREGULATION IN MICE
Authors: Pagani A. , Pettinato M. , Colucci S. , Dulja A. , Rauner M. , Nai A. , Camaschella C. , Altamura S. , Muckenthaler M. , Silvestri L. 
Published: June 2019

Background:

Hepcidin is the liver hormone that maintains body iron homeostasis. In hemochromatosis (HH), a hereditary disease due to mutations in HFE, TFR2, HJV and hepcidin itself, iron accumulates in several tissues and organs because levels of hepcidin are inappropriately low. Hepcidin is regulated by the liver BMP‐SMAD pathway, whose activation requires BMP type I (ALK2 and ALK3) and type II receptors (the redundant BMPR2 and ACVR2A), hemochromatosis proteins (HFE, TFR2 and the BMP‐coreceptor HJV), and BMP ligands (BMP2 and BMP6). BMP2 signals preferentially through ALK3 for basal hepcidin activation, whereas BMP6, increased by body iron, signals via ALK2. The current model considers that the HH proteins functionally interact with ALK3 and are part of ALK3‐BMP2 branch of the pathway, thus forming a multiprotein complex important for basal hepcidin modulation. Moreover the characterization of Bmp2

LSECKO
, Bmp6

KO
, HH proteins KO models and double knock out animals (Bmp6

KO

‐HH

KO
with worsen phenotypes), indicates that the two branches of BMP‐SMAD signaling do not compensate each other and require different ligands.

Aims:

Since HH proteins interact mainly with ALK3, we investigated the effect of BMP2 on hepcidin regulation in murine models of hereditary hemochromatosis (Hjv KO and Tfr2 KO).

Methods:

In vivo studies were performed on Hjv KO and Tfr2 KO mice, compared to wild type controls, treated with a single BMP2 injection (24 mg/mouse) and sacrificed 4 hrs later. We analyzed: iron parameters (TS%, LIC, SIC, serum hepcidin); BMP‐SMAD target genes (Hamp, Id1), ligands (Bmp6, Bmp2) and inflammation target genes (Saa1, Il6, Il1β, Tnfα, Ifnγ, Tgfβ) expression with qRT‐PCR; SMAD1/5/8 and STAT3 phosphorylation with standard western blot technique. We studied the effect of BMP2 treatment ex vivo on cultured primary hepatocytes isolated from wt, Hjv KO, and Tfr2 KO.

Results:

The treatment protocol did not change iron parameters of wild type, Hjv KO and Tfr2 KO mice. Hamp mRNA and serum hepcidin were both increased by BMP2 in HH mice and the BMP‐SMAD pathway was activated as in WT mice. Interestingly, although Bmp6 levels were increased in iron loaded HH mice, Bmp2 levels were not, and the expression of both ligands was not affected by BMP2 injection. Systemic administration of BMP2 activated an inflammatory response hallmarked by increased expression of the liver acute phase protein Saa1 in WT and HH mice through STAT3 phosphorylation. Serum IL‐6 as well as spleen Il‐6, Tnfα, IfnY and Tgfβ mRNA levels were not affected by BMP2. In contrast, Il1β was significantly upregulated in BMP2‐treated mice. To evaluate if an inflammatory signal contributed to hepcidin upregulation, we analyzed wt, Hjv KO and Tfr2 KO primary hepatocytes. Hamp and the BMP‐SMAD target gene Id1 were strongly increased in a BMP2 dose‐dependent manner both in WT and HH hepatocytes in the absence of Saa1 changes, excluding a direct effect of BMP2 on STAT3 in these cells.

Summary/Conclusion:

Overall our results demonstrate that:

TFR2 and HJV are dispensable for BMP2‐dependent hepcidin activation in vivo in an acute setting

The BMP2‐induced proinflammatory response is mediated by its action on non parenchymal cells, likely macrophages

Bmp2