A Novel Crosstalk Between Iron Homeostasis and mTOR Mediated By the Immunophilin FKBP12

Introduction

Hepcidin, the liver hormone that regulates iron homeostasis, is mainly activated via the BMP/SMAD pathway. Among other stimuli, as erythropoiesis expansion, inflammation and hypoxia, hepcidin expression is influenced by drugs, including the mTOR inhibitor rapamycin (RAPA) (Mleczko-Sanecka et al., Blood 2014). In the liver, BMP type I receptors, ALK2 and ALK3, are both essential for hepcidin regulation (Steinbiecker et al., Blood 2012), whereas BMP type II receptors, BMPR2 and ACVR2A, have a redundant role (Mayeur et al., Blood 2014). The signaling is activated when BMP type II receptors phosphorylate residues of the glycine/serine rich (GS) domain of BMP type I receptors, which then activate SMAD1/5/8. Treatment with RAPA increases hepcidin expression in murine hepatocytes and may cause microcytic anemia in patients (Przybylowski P. et al., Transplant Proc. 2013). However, the mechanisms involved in hepcidin and mTOR crosstalk are unknown.

Methods

Hepcidin, BMP-SMAD and mTOR target genes were analyzed in human hepatoma cell lines and murine primary hepatocytes (HCs), treated with RAPA (100 nM), Torin1 (100 nM), tacrolimus (TAC, 1 μg/ml) in the presence or absence of the BMP pathway inhibitor DMH1 (0.5 μg/ml) or of the ligands BMP6, BMP2, Activin B (ActB) and Activin A (ActA) (1-100 ng/ml). ALK2^wt was mutagenized in the GS domain (R206H, Q207E) or close to the GS domain (R258S) to generate ALK2^mut with reduce binding to FKBP12 (Taylor et al., Cancer Res. 2014). SMAD1/5/8 phosphorylation was analyzed by Western Blotting in cells transfected with SMAD1 and ALK2^wt or ALK2^mut and treated or not with TAC, BMP6 and ActA. Binding of FKBP12 to ALK2^wt and ALK2^mut was assessed by coimmunoprecipitation of tagged proteins in transfected cells treated as above. Eight weeks old C57BL/6 wild type male mice were treated with a single dose of TAC (10 mg/kg) or vehicle and sacrificed at different time points (3-18 hrs). Hepcidin expression, LIC, SIC, serum iron and hematological parameters were analyzed by standard methods.

Results

We analyzed hepcidin expression in hepatoma cells and primary HCs treated with RAPA, an inhibitor of mTORC1, and Torin1, an ATP-competitive inhibitor of mTORC1 and 2. Hepcidin is increased by RAPA, but not Torin1, in a SMAD1/5/8 dependent way since DMH1 abrogates the effect. RAPA inhibits mTORC1 when complexed with FKBP12, an immunophilin that binds BMP receptors to avoid leakage activation of the pathway. To investigate whether hepcidin upregulation by RAPA is mediated by FKBP12 sequestration, we used genetic and pharmacologic approaches. First, we confirmed by coimmunoprecipitation that ALK2^mut have a reduced ability to bind FKBP12. Then we transfected hepatoma cells with ALK2^wt and ALK2^mut and analyzed hepcidin and BMP pathway activation. Overexpression of ALK2^mut increases hepcidin through SMAD1/5/8 as shown by high levels of SMAD1 phosphorylation, an effect abrogated by DMH1. Second, we treated hepatoma cells and primary HCs with TAC, a calcineurin inhibitor that binds FKBP12. This treatment increases hepcidin through SMAD1/5/8, suggesting a mechanism shared with RAPA. The same effect is observed in vivo since hepcidin is increased at 6 hrs post-injection in TAC-treated wt mice. Our results identify FKBP12 as a novel regulator of hepcidin. In addition, FKBP12 displacement alters the BMP receptor selectivity to ligands. Despite ALK2^wt preferentially binds BMP6, ALK2^mut become responsive to ActA, a TGF-β ligand that signals through SMAD2/3. Hepcidin activation by BMP6, BMP2 and ActB is comparable in ALK2^wt and ALK2^mut expressing cells. However, ActA upregulates hepcidin (through SMAD1/5/8) only in ALK2^mut transfected cells. This effect is due to the impaired ability of ALK2^mut to bind FKBP12, since it is observed even in ALK2^wt transfected cells pretreated with TAC.

Conclusions

FKBP12 contributes to hepcidin regulation both in vitro and in vivo, thus adding a new player to the BMP-dependent hepcidin activation and a potential pharmacologic target for disorders characterized by low hepcidin and iron overload. Furthermore the ability of ALK2 to respond to Activin A, which is released in inflammation, links the BMP pathway-hepcidin activation to the inflammatory response.