Hepatocyte toll-like receptor 4 mediates lipopolysaccharide-induced hepcidin expression

Hepcidin expression is induced by inflammatory molecules such as lipopolysaccharide (LPS) via a macrophage-mediated pathway. Although hepatocytes directly respond to LPS, the molecular mechanism underlying toll-like receptor (TLR)-dependent hepcidin expression by hepatocytes is mostly unknown. Here we show that LPS can directly induce the mRNA expression and secretion of hepcidin by hepatocytes via TLR4 activation. Using hepatocytes deficient in TLR4, myeloid differentiation factor 88 (MyD88) and TIR domain-containing adaptor inducing interferon-β (TRIF), we demonstrated that LPS-induced hepcidin expression by hepatocytes is regulated by its specific receptor, TLR4, via a MyD88-dependent signaling pathway. Hepcidin promoter activity was significantly increased by MyD88-dependent downstream signaling molecules (interleukin-1 receptor-associated kinase (IRAK) and tumor necrosis factor receptor-associated factor 6 (TRAF6), which activate c-Jun N-terminal kinase (JNK) and activator protein-1 (AP-1). We then confirmed that LPS stimulation induced the phosphorylation of JNK and c-Jun, and observed strong occupancy of the hepcidin promoter by c-Jun. Promoter mutation analysis also identified the AP-1-binding site on the hepcidin promoter. Finally, bone marrow transplantation between wild-type and TLR4 knockout mice revealed that hepatic TLR4-dependent hepcidin expression was comparable to macrophage TLR4-dependent hepcidin expression induced by LPS. Taken together, these results suggest that TLR4 expressed by hepatocytes regulates hepcidin expression via the IRAK-TRAF6-JNK-AP-1 axis.