Fig 1: Role of TLR4 in LPS-induced hepcidin expression by hepatocytes. (a–c) LPS-mediated hepcidin expression in the livers of mice. Eight-week-old male C57BL/6 mice (n=4) received an intravenous injection of LPS (500 µg kg-1). Six hours later, expression of Hamp1 mRNA from livers was measured by qRT-PCR (a), and the hepcidin concentration in the serum was measured by ELISA (b). The effect of LPS-induced hepcidin on ferroportin degradation in the spleen was verified by western blot analysis using an anti-ferroportin antibody. All the samples (n=4) were combined for western blot analyses (c). (d) Genotyping of genomic DNA isolated from the peripheral blood of bone marrow-transplanted chimeric mice. LPS-induced expression of hepcidin mRNA in mice receiving a bone marrow transplant (n=3–4). (e) Eight weeks after bone marrow transplantation, chimeric mice were challenged with LPS for 6 h, and expression of hepcidin mRNA in the liver was measured. (f) Concentration of LPS-induced hepcidin in serum from chimeric mice as measured by ELISA. (g) Proposed signaling pathway for LPS-induced hepcidin expression in hepatocytes.
Fig 2: LPS induces expression of hepcidin by hepatocytes via the TLR4 pathway. (a) Expression of Hamp1 mRNA in AML12 cells treated with LPS (1 µg ml-1) in the presence or absence of the TLR4 inhibitors polymyxin B (100 µg ml-1) or CLI095 (3 µM) for 24 h. Each inhibitor was used at the manufacturer’s recommended working concentration. (b–d) Expression of Hamp1 mRNA in WT versus TLR4 KO mice (b), MyD88 KO mice (c) and TRIF KO mice (d). Isolated mouse primary hepatocytes were treated with LPS (1 µg ml-1) for 24 h.
Fig 3: The TLR4 signaling pathway regulates the hepcidin promoter. (a) Effect of TLR4 downstream signaling molecules on hepcidin promoter activity. AML12 cells were co-transfected with a hepcidin promoter luciferase reporter (Hamp1-Luc; 200 ng) and each of the indicated signaling molecules (MyD88, IRAK1 and TRAF6; 400 ng). LPS (1 µg ml-1) treatment for 24 h before cell harvest was used as a positive control. (b) Effect of JNK–AP-1 signaling on hepcidin promoter activity. AML12 cells were co-transfected with Hamp1-Luc (200 ng) and JNK (1 or 2) or AP-1 (c-jun+c-fos) or NF-?B; p65; all at 400 ng). (c) Regulation of hepcidin promoter activity by activator protein-1 (AP-1). 293T cells were co-transfected with the wild-type hepcidin promoter, Hamp1-Luc (200 ng), or with an AP-1-binding site-specific mutated promoter, Hamp1 (AP-1 mt)-Luc (200 ng) and AP-1 (c-jun+c-fos; 400 ng). (d) Binding of AP-1 to the hepcidin promoter. Chromatin immunoprecipitation assay using an anti-c-jun antibody and the primers harboring the AP-1-binding site in LPS-treated (for 24 h) AML12 cells.
Fig 4: LPS induces hepcidin expression by hepatocytes. (a) AML12 cells were treated with different concentrations of LPS for 24 h, and the expression of mouse hepcidin (Hamp1) mRNA was measured by quantitative real time polymerase chain reaction (qRT-PCR). (b) AML12 cells were treated with LPS (1 µg ml-1) for the designated times. (c) Expression of mRNA encoding inducible nitric oxide synthase (iNOS) and Hamp1 in AML12 cells treated with 1 µg ml-1 LPS for 24 h. (d) AML12 cells were treated for 12 h with BMP6 (20 ng ml-1), IL-6 (20 ng ml-1) or LPS (1 µg ml-1), and the expression of Hamp1 mRNA was measured by qRT-PCR. (e) Hepcidin concentration in cell culture medium from LPS (1 µg ml-1)-treated AML12 cells. (f) Iron concentration in AML12 cells treated with LPS (1 µg ml-1) for 24 h.
Fig 5: LPS induces the expression of hepcidin in hepatocytes by activating JNK and activator protein-1 (AP-1). (a) Expression of Hamp1 mRNA in AML12 cells treated with PD98059 (a MEK inhibitor), LY294002 (a PI3K inhibitor), SP600125 (a JNK inhibitor), SB203580 (a p38 MAP kinase inhibitor) or Bay11-7082 (a NF-?B) inhibitor), all at 5 µM. Cells were exposed to each inhibitor for 1 h before treatment with LPS (1 µg ml-1) for 4 h. (b) LPS activates JNK and AP-1 in hepatocytes. AML12 cells were treated with LPS (1 µg ml-1) for the designated times. Cell lysates were prepared and examined by western blotting with anti-phospho-JNK, anti-phospho-c-jun, anti-JNK and anti-c-jun antibodies. An anti-alpha tubulin antibody was used as an internal control. The blots were prepared in duplicate for each independent western blot for JNK and c-Jun.
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