Abstract
In most organs, the first line of defense against invading viruses is the epithelial cell, which provides an initial innate response and can provide subsequent immune control. Hepatitis virus infection of human hepatocytes illicit a strong antiviral response involving the upregulation of interferon-stimulated gene 15 (ISG15) and subsequent antiviral mechanism, ISGylation. Chronic infection can lead to liver diseases such as hepatocellular carcinoma. As tumor cells are defined as de-differentiated cells with stem cell-like characteristics, we wanted to study the expression of ISG15 and ISGylation during hepatocyte differentiation from induced-pluripotent stem cells (iPSCs). In these cells, we measured mRNA and protein expression following treatment with RNA viral mimetic poly(I:C) and interferon (IFN). ISGylation was not detected in iPSCs despite increased expression of ISG15 following viral mimetic or IFN treatments. When compared to hepatocyte-like cells (HLCs), we observed an attenuation in IFN-induced expression of the ISGylation conjugating enzyme, UBE2L6 due to a lower activation of the Signal Transducer and Activator of Transcription 1 (STAT1), a major transcription factor involved in the antiviral response. Furthermore, we determined iPSCs constitutively express the Suppressor of Cytokine Signaling 1 (SOCS1), a negative regulator of STAT1 activation. Knockdown of SOCS1 mRNA expression promoted observable ISGylation in iPSCs with increased UBE2L6 expression and STAT1 activation following IFN treatment. Additionally, we determined that the lower baseline expression level of ISGylation activating enzyme, UBE1L in iPSCs is related to epigenetic regulation associated with transcriptional repression, such as methylated CpG sites at the gene promoter region. These data revealed that ISGylation is regulated at baseline (UBE1L), in a STAT1-independent (ISG15), and a STAT1-dependent (UBE2L6) manner in which iPSCs characteristically have an attenuated interferon response due to a sustained intrinsic expression of SOCS1. Thus, ISGylation is suppressed.