Abstract
The nature of host-virus interactions in hepatitis B virus infection is incompletely understood. Since soluble factors, e.g., cytokines and metals, may exacerbate liver injury in chronic hepatitis, we considered that defining the effects of receptor-mediated signaling upon viral replication will be significant. Consequently, we studied effects of iron or TGF-β-induced TGF-β/BMP signaling in the HepG2 2.2.15 cell model of hepatitis B virus replication. We found iron and TGF-β increased hepcidin mRNA expression or TGF-β receptor kinase activity, respectively, which indicated that 2.2.15 cells responded appropriately to these substances. However, iron increased but TGF-β decreased hepatitis B virus mRNA and DNA expression. TGF-β induced expression at the mRNA level of multiple TGF-β/BMP pathway genes. This change was not observed in iron-treated cells. On the other hand, presence of SMAD proteins in iron or TGF-β-treated cells, including of SMAD4, did confirm convergence of TGF-β/BMP signaling pathways under these conditions. Since transcription factors in TGF-β/BMP signaling pathways could not have directly targeted hepatitis B virus itself, we studied whether iron or TGF-β exerted their effects through alternative mechanisms, such as by involvement of antiviral cellular microRNAs. We discovered cellular microRNA expression profiles were significantly different in iron or TGF-β-treated cells compared with untreated control cells. In many cases, exposure to iron or TGF-β changed microRNA expression in opposite directions. Introduction in cells of sequences representing such differentially expressed microRNAs, e.g., hsa-miR-125a-5p and -151-5p, even reproduced effects on virus replication of iron- or TGF-β. We surmised that TGF-β/BMP pathway members, i.e., SMADs, likely governed iron or TGF-β-induced microRNA expression. Iron may have mediated Drosha/DGCR8/heme-mediated processing of microRNAs. In turn, cellular microRNAs regulated replication of hepatitis B virus in iron or TGF-β-treated cells. This knowledge should advance studies of mechanisms in viral-host interactions, hepatic injury, and therapeutic developments for hepatitis B.
Highlights
Intra- and extracellular soluble signaling molecules are involved in hepatitis virus replication but these interactions are not well understood
Iron increased SMAD-4 expression, which was partly blocked by TGF-b1 receptor kinase inhibitor (TKI). These results indicated differences in the nature of intracellular signaling in cells treated with iron or transforming growth factor (TGF)-b activation of SMAD-4 by both iron and TGF-b indicated confluence of TGF-b/bone morphogenetic protein (BMP) signaling in these conditions
This confirmed that individual miRNAs were capable of altering hepatitis B virus (HBV) replication. These studies established 2.2.15 cells were appropriately responsive to iron and TGF-b. This was evidenced by greater hepcidin expression, which is transcriptionally regulated via BMP signaling [20], and requires the cofactors, hemojuvelin and neogenin [21]
Summary
Intra- and extracellular soluble signaling molecules are involved in hepatitis virus replication but these interactions are not well understood. Interferon-a has widely been used for treating HBV with JAK/STAT signaling serving intermediary roles [3] The role of these intracellular signaling pathways in transducing antiviral effects of interferon is far from complete and new information is still emerging [4]. After activation of TGF-b- or BMP receptors leads to heteromeric complexing between SMADs, followed by engagement with the common-mediator, SMAD-4, which is required and sufficient for regulation of nuclear transcription, and in this way, brings together TGF-b/BMP signaling pathways. How these diverse intracellular signaling pathways may regulate replication of HBV (or other viruses) is yet to be clarified
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