title: Cellular and molecular mechanisms regulating the hepatic erythropoietin expression during acute-phase response: a role for IL-6.
abstract: The source of circulating erythropoietin (EPO), the mediators and the mechanisms involved in the upregulation of EPO gene expression during acute-phase reaction are still poorly understood. Acute-phase reaction was induced by either intramuscular turpentine oil (TO) or intraperitoneal lipopolysaccharide (LPS) administration into wild-type and interleukin (IL)-6 knockout (KO) mice. Animals were killed at different time points and blood, liver and muscle tissue were collected. Serum levels of EPO were measured by enzyme-linked immunoadsorbent assay; liver and injured muscle samples were processed for RNA isolation and for protein analysis. EPO, hypoxia-inducible factors 1alpha and 2alpha (HIF-1alpha and HIF-2alpha) mRNA were analyzed by RT-PCR and the protein levels were analyzed by western blot and electrophoretic mobility shift assay. HIF-1alpha and HIF-2alpha localization was performed through immunofluorescence staining. EPO, HIF-1 and HIF-2 gene and protein expression levels were also analyzed in isolated mouse hepatocytes after stimulation with IL-6. In the wild-type animals, EPO serum levels increased dramatically at 12 h after the insults together with the hepatic gene expression. In TO-treated animals, the EPO gene expression reached an 8.2-fold increase at 12 h, and in LPS-treated mice a similar induction was recorded at 6 h (about 4.5-fold increase). In the IL-6KO strain, the upregulation after the inflammatory stimuli was much lower (only 2.0-fold increase). A progressive upregulation of HIF-1alpha and HIF-2alpha was detectable until 6 h after the insults, but only HIF-1alpha upregulation was reduced in IL-6KO mice. In isolated hepatocytes, stimulation with a single dose of IL-6 induced a nuclear accumulation of HIF-1alpha, in parallel with an increase of EPO mRNA. No effect on HIF-2alpha expression was found. IL-6 appears to be the main regulator of EPO gene expression and a major contributor for HIF-1alpha induction in hepatocytes and Kupffer cells during acute-phase response. The increase of HIF-2alpha, predominantly expressed in endothelial cells and fibroblast-like cells, seems not to be affected by the lack of IL-6.
authors.affiliation: Department of Gastroenterology and Endocrinology, Center of Internal Medicine, University of Göttingen, Göttingen, Germany.
authors.forename: Pierluigi
authors.initials: P
authors.lastname: Ramadori
authors.forename: Ghayyor
authors.initials: G
authors.lastname: Ahmad
authors.forename: Giuliano
authors.initials: G
authors.lastname: Ramadori
mesh_terms: D000210:Acute-Phase Reaction / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D057075:Enzyme Assays; D004921:Erythropoietin / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D005796:Genes; D022781:Hepatocytes / Q000378:metabolism; D000860:Hypoxia / Q000235:genetics / Q000378:metabolism; D051793:Hypoxia-Inducible Factor 1 / Q000235:genetics / Q000378:metabolism; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism*; D007728:Kupffer Cells / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014425:Turpentine / Q000378:metabolism; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism
chemical_list: D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D015850:Interleukin-6; D012333:RNA, Messenger; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D004921:Erythropoietin; D014425:Turpentine