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 1a and 2a (HIF-1a and HIF-2a) mRNA were analyzed by RT-PCR and the protein levels were analyzed by western blot and electrophoretic mobility shift assay. HIF-1a and HIF-2a 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-1a and HIF-2a was detectable until 6 h after the insults, but only HIF-1a upregulation was reduced in IL-6KO mice. In isolated hepatocytes, stimulation with a single dose of IL-6 induced a nuclear accumulation of HIF-1a, in parallel with an increase of EPO mRNA. No effect on HIF-2a expression was found. IL-6 appears to be the main regulator of EPO gene expression and a major contributor for HIF-1a induction in hepatocytes and Kupffer cells during acute-phase response. The increase of HIF-2a, predominantly expressed in endothelial cells and fibroblast-like cells, seems not to be affected by the lack of IL-6.

Ferroportin-1 is a ‘nuclear'-negative acute-phase protein in rat liver: a comparison with other iron-transport proteins

“…52,54 It should be noted that an induction of hepatic IRP - 25 and HIF-1a 46 gene expression has been reported in the APR model utilized in the present experiments. The upregulation of these iron transport molecules may also be due to an action of the major acute-phase cytokine (IL-6) as shown previously in vitro.…”

“…Ferroportin: a negative acute phase protein N Naz et al injury. 46 In fact, the upregulation of IL-1b and TNF-a gene expression is more intense in the injured muscle of IL-6 knock-out mice than in their wild-type littermate. 46 Therefore, we hypothesized that Fpn-1 is mainly controlled by IL-6 and partially by other acute-phase cytokines (IL-1b and TNF-a), which are released at the site of injury during APR.…”

“…46 In fact, the upregulation of IL-1b and TNF-a gene expression is more intense in the injured muscle of IL-6 knock-out mice than in their wild-type littermate. 46 Therefore, we hypothesized that Fpn-1 is mainly controlled by IL-6 and partially by other acute-phase cytokines (IL-1b and TNF-a), which are released at the site of injury during APR. 25,27 Moreover, these cytokines could also modulate the gene expression of other iron transport proteins.…”

Spontaneous onset of nonalcoholic steatohepatitis and hepatocellular carcinoma in a mouse model of metabolic syndrome

“…The primer sequence details are presented in Supplementary Table 1. [16][17][18] The amount of mRNA was calculated using glyceraldehyde-3-phosphate dehydrogenese as an endogenous control.…”

IL-36–Induced Toxicity in Neonatal Mice Involves TNF-α Production by Liver Myeloid Cells

“…After 6 h of stimulation, cells were collected and immunostained for flow cytometric analysis. AD mouse hepatocytes were isolated using a two-step enzymatic dissociation as previously described (27).…”