Hepatocyte GPCR signaling regulates IRF3 to control hepatic stellate cell transdifferentiation

Background Interferon Regulatory Factor 3 (IRF3) is a transcription factor that plays a crucial role in the innate immune response by recognizing and responding to foreign antigens. Recently, its roles in sterile conditions are being studied, as in metabolic and fibrotic diseases. However, the search on the upstream regulator for efficient pharmacological targeting is yet to be fully explored. Here, we show that G protein-coupled receptors (GPCRs) can regulate IRF3 phosphorylation through of GPCR-G alpha protein interaction.Results IRF3 and target genes were strongly associated with fibrosis markers in liver fibrosis patients and models. Conditioned media from MIHA hepatocytes overexpressing IRF3 induced fibrogenic activation of LX-2 hepatic stellate cells (HSCs). In an overexpression library screening using active mutant G alpha subunits and Phos-tag immunoblotting, G alpha s was found out to strongly phosphorylate IRF3. Stimulation of G alpha s by glucagon or epinephrine or by G alpha s-specific designed GPCR phosphorylated IRF3. Protein kinase A (PKA) signaling was primarily responsible for IRF3 phosphorylation and Interleukin 33 (IL-33) expression downstream of G alpha s. PKA phosphorylated IRF3 on a previously unrecognized residue and did not require reported upstream kinases such as TANK-binding kinase 1 (TBK1). Activation of G alpha s signaling by glucagon induced IL-33 production in hepatocytes. Conditioned media from the hepatocytes activated HSCs, as indicated by alpha-SMA and COL1A1 expression, and this was reversed by pre-treatment of the media with IL-33 neutralizing antibody.Conclusions G alpha s-coupled GPCR signaling increases IRF3 phosphorylation through cAMP-mediated activation of PKA. This leads to an increase of IL-33 expression, which further contributes to HSC activation. Our findings that hepatocyte GPCR signaling regulates IRF3 to control hepatic stellate cell transdifferentiation provides an insight for understanding the complex intercellular communication during liver fibrosis progression and suggests therapeutic opportunities for the disease.