A complex signaling network involving protein kinase CK2 is required for hepatitis C virus core protein-mediated modulation of the iron-regulatory hepcidin gene expression

Abstract

Hepatitis C virus (HCV) infection is associated with hepatic iron overload and elevated serum iron that correlate to poor antiviral responses. Hepcidin (HAMP), a 25-aa cysteine-rich liver-specific peptide, controls iron homeostasis. Its expression is up-regulated in inflammation and iron excess. HCV-mediated hepcidin regulation remains controversial. Chronic HCV patients possess relatively low hepcidin levels; however, elevated HAMP mRNA has been reported in HCV core transgenic mice and HCV replicon-expressing cells. We investigated the effect of HCV core protein on HAMP gene expression and delineated the complex interplay of molecular mechanisms involved. HCV core protein up-regulated HAMP promoter activity, mRNA, and secreted protein levels. Enhanced promoter activity was abolished by co-transfections of core with HAMP promoter constructs containing mutated/deleted BMP and STAT binding sites. Dominant negative constructs, pharmacological inhibitors, and silencing experiments against STAT3 and SMAD4 confirmed the participation of both pathways in HAMP gene regulation by core protein. STAT3 and SMAD4 expression levels were found increased in the presence of HCV core, which orchestrated SMAD4 translocation into the nucleus and STAT3 phosphorylation. To further understand the mechanisms governing the core effect, the role of the JAK/STAT-activating kinase CK2 was investigated. A CK2-dominant negative construct, a CK2-specific inhibitor, and RNAi interference abrogated the core-induced increase on HAMP promoter activity, mRNA, and protein levels, while CK2 acted in synergy with core to significantly enhance HAMP gene expression. Therefore, HCV core up-regulates HAMP gene transcription via a complex signaling network that requires both SMAD/BMP and STAT3 pathways and CK2 involvement.

Alterations in the iron homeostasis network: A driving force for macrophage-mediated hepatitis C virus persistency

“…We have previously showed that HCV core induces HAMP gene expression in hepatoma cells 23 and this may also apply to early HCV infection. Conversely, upon established infection, hepcidin expression dropped in our in vitro model and in patient sera, 12,13 in step with studies from chronic HCV patients with low intrahepatic HAMP mRNA 24 and decreased serum hepcidin, 12,13 and earlier in vitro RNA data.…”

“…Then, the RNA fraction was subjected to qRT-PCR, essentially as described in ref. 23 The gene specific primers used for HAMP, 18S rRNA, IL-6 and NS3-2A have been published elsewhere. 23,59 Results were analyzed with the internal standard-curve method and normalized to 18S rRNA to provide the relative mRNA expression.…”

“…23 The gene specific primers used for HAMP, 18S rRNA, IL-6 and NS3-2A have been published elsewhere. 23,59 Results were analyzed with the internal standard-curve method and normalized to 18S rRNA to provide the relative mRNA expression. In all PCR experiments, unless otherwise stated, the relative mRNA expression of mock (mean § SD) was set as 100% or fold-change and all other values were depicted as a ratio of this.…”

“…40 mg of cell lysates were used in Western blotting, as published before. 23,59 Gel photographs presented in each figure panel are representative results from samples that were derived from the same experiment and processed in parallel. All experiments were performed at least in triplicate.…”

“…23,59 The percentage change in protein expression of the control cells was set as 100% and all other values were a percentage of the control, except when absolute peptide concentration values were measured in ng/ml. The assays were repeated at least 3 times in quadruplicates.…”