THE MAPK AND NRF2 PATHWAYS ACT IN CONCERT TO CONTROL IRON-INDUCED BMP6 EXPRESSION IN LIVER SINUSOIDA

Ruiyue QIU1, Stefania CUCINELLI1,2, Silvia COLUCCI1,2,3, Martina U. MUCKENTHALER1,2

1Department of Pediatric Oncology, Hematology and Immunology and Hopp Children Cancer Center (KiTZ), University Hospital Heidelberg, Heidelberg, Germany
2Molecular Medicine Partnership Unit (MMPU), EMBL and Heidelberg University, Heidelberg, Germany
3European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

Liver sinusoidal endothelial cells (LSECs) play a critical role in sensing systemic and/or liver iron levels and release bone morphogenetic proteins (BMPs) that control hepcidin levels in hepatocytes. Our previous results demonstrated that iron-dependent Bmp6 induction in LSECs requires component(s) of the hepatocyte secretome (PMID: 36187873), but the exact mechanism(s) of how iron controls BMP6 levels remains unclear.

Here we show that LSECs in iron-loaded Fpn(C326S) mice show activated PI3K-AKT and MAPK-ERK1/2 signaling pathways. Likewise, primary LSECs cultured in the presence of hepatocyte-conditioned medium with iron show increased phospho-ERK and phospho-AKT levels. Importantly, pretreatment of LSECs with MAPK signaling inhibitors targeting RAF, MEK1/2 and ERK1/2 (Raf265, U0126 and Ulixertinib, respectively), but not with the PI3K inhibitor Wortmannin, attenuated iron-induced Bmp6 expression under hepatocyte-conditioned medium, suggesting the involvement of the ERK1/2 pathway in iron-mediated Bmp6 control. Interestingly, the iron-induced Bmp6 response was only completely abolished when the ERK1/2 pathway was inhibited together with the Nrf2 pathway, suggesting a collaborative function of the ERK1/2 and Nrf2 pathways in controlling BMP6 levels.

Nrf2 is activated by pro-oxidant cellular states. Consistently, we show that H2O2 treatment of LSECs induces Bmp6 mRNA expression, a response that was fully prevented by the ROS scavenger N-Acetyl-L-Cystein (NAC) in the presence of hepatocyte-conditioned medium. By contrast, NAC only attenuated iron-induced Bmp6 mRNA levels, suggesting that iron in LSECs only partially activates Bmp6 by generating oxidative stress. Additional experiments are ongoing to evaluate how the ERK1/2 and Nrf2 pathways interact in controlling the iron sensing process in LSECs.