Identification of Ras/RAF MAPK and mTOR signaling as critical pathways for hepcidin suppression. (A) The interconnected signaling network, which involves Ras/MAPK, PI3K/Akt/mTOR, and β-catenin signaling, plays a crucial role in key cell processes such as proliferation, anabolic growth, nutrient homeostasis, survival, and motility. Small molecules used to modulate pathway activity are shown. (B,C,F) (B) Huh7 cells, (C) Hep3B cells, or (F) murine primary hepatocytes were treated with the indicated concentrations of small molecules: Wortmannin (4 hours), rapamycin (15 hours), sorafenib (9 hours), or metformin (15 hours). Levels of hepcidin mRNA expression were determined by real-time qPCR analysis and presented as fold change compared with untreated cells. (D-E) Huh7 cells were transfected with the wild-type and mutated hepcidin promoter reporter constructs. (D) The cells were cotransfected with expression vectors containing complementary DNA sequences of the indicated signaling genes. The clones were designed to encode wild-type (WT) and constitutively active (CA) protein variants (supplemental Table 2). Forced expression of HJV served as a positive control. (E) Cells were treated with rapamycin for 15 hours. The results are presented as fold change of luciferase activity (±SD of Firefly/Renilla) compared with overexpression of empty vector control (D) or untreated cells (E). (G) Shown are relative hepcidin mRNA, plasma, and splenic iron levels in mice 6 weeks after tamoxifen-induced systemic loss of Rasa1. The efficiency of Rasa1 deletion at the protein level in liver and spleen tissue is shown in supplemental Figure 6. Results represent a mean of at least 3 (B,F), 4 (C-D), or 5 (E) independent experiments. Statistically significant changes are marked by *(P < .05) or **(P < .001).