NAG-1/GDF15 modulates hepcidin expression through STAT3 and SMAD pathways

Hepcidin, a liver-derived hormone, is a central regulator of iron homeostasis and a key contributor to anemia of inflammation (AI). This study investigates the regulatory role of NAG-1 (Nonsteroidal Anti-Inflammatory Drug-Activated Gene-1; also known as GDF15) in modulating hepcidin expression. In NAG-1 transgenic mice, elevated circulating NAG-1 was associated with markedly reduced hepatic hepcidin expression, demonstrating an in vivo role for NAG-1 in hepcidin suppression. To further dissect the mechanism, we established HepG2 cell lines stably overexpressing either NAG-1 WT (wild type), which produces both pro- and mature forms, or the R193A mutant, which secretes only the pro-form. Both WT and R193A significantly suppressed IL-6- and BMP6-induced hepcidin expression, with the mutant showing slightly stronger inhibition in some assays. Mechanistically, NAG-1 inhibited both the JAK/STAT3 and BMP6/SMAD pathways by reducing STAT3 activation and Smad1/5/9 phosphorylation, thereby limiting their recruitment to the HAMP promoter. Furthermore, co-expression of NAG-1 with the transcriptional corepressor SMILE produced an additive suppression of hepcidin, through enhanced inhibition of STAT3 signaling, despite only weak physical interaction between the two proteins. Together, these findings establish NAG-1 as a negative regulator of hepcidin transcription and demonstrate that the pro-form retains substantial biological activity within cells, providing new therapeutic insights into the management of anemia of inflammation through modulation of iron metabolism.