Ceramide Induces Human Hepcidin Gene Transcription through JAK/STAT3 Pathway
Fig 5
Activation of STAT3 signaling was required for the induction of HAMP transcription by ceramide (A)
HAMP mRNA expression in HepG2 cells, treated with solvent (solv.) or C2 ceramide for 8 hours in the presence of either 5 μM JAK inhibitor I or DMSO (control), was determined by Taqman qPCR. HAMP expression in treated cells was calculated as fold change of that in respective control cells treated with solvent. (B) HepG2 cells, pre-transfected with STAT3 or control siRNA, were treated with C2 ceramide or solvent (solv.) in the presence of 5 μM JAK inhibitor I or DMSO. HAMP mRNA levels, determined by qPCR, were expressed as fold expression of that in respective controls treated with solvent. (C) HepG2 cells, transfected with pGL3-basic vector harboring 0.6 kb HAMP promoter, were treated with C2 ceramide or solvent (solv.) for 8 hours in the presence or absence of 5 μM JAK inhibitor I. Dual luciferase assays were performed and HAMP promoter activity, calculated as fold change of that in corresponding control cells treated with solvent, was expressed in relative luciferase units. (D) HepG2 cells, pre-transfected with STAT3 or control siRNA, were subsequently transfected with pGL3-basic vector harboring 0.6 kb wild-type HAMP promoter. After treatment with solvent (solv.) and C2 ceramide in the presence of either JAK inhibitor I or DMSO, dual luciferase reporter assays were performed. HAMP promoter activity, expressed as relative luciferase units, was expressed as fold change of that in respective control cells treated with solvent. Asterisks indicate statistical significance (P<0.05).