Novel PKA-associated pathways for the regulation of hepatocyte survival and pharmacological applications

Abstract

Hepatocytes are primary targets for toxic insults such as drugs, alcohol and viruses. Hepatocyte death is evident and a common feature in a variety of liver disease ranging from drug induced liver injury, viral/alcoholic/autoimmune hepatitis to metabolic disorders. Therefore, preserving functional hepatocytes is important for intervention and amelioration of liver injury. Currently, therapeutic approaches targeting hepatocyte death are challenging from the lower clinical outcomes. Thus, molecular targets and pathways related to hepatocyte survival need to be discovered. Protein kinase A (PKA) mediates the molecular signaling transduction between receptors/therapeutic drugs and cellular targets. The numerous targets associated with PKA determine its nature as an important regulator for energy metabolism. The aims of this study are to explore novel molecules associated with PKA pathway targeting hepatocyte survival and specify the molecules and underlying mechanisms. As an aim to find a pharmacological approach aiming at PKA-associated pathway, this study investigated the possibilities of methylene blue (MB), a mitochondria-targeting drug in hepatocyte protection. MB treatment resulted in a PKA-dependent activation of liver kinase B 1 (LKB1). LKB1 then induced activation of AMP-activated protein kinase (AMPK) that caused inhibitory phosphorylation of glycogen synthase kinase 3b (GSK3b). MB treatment also induced an AMPK-independent early inhibition of GSK3b. siRNA knockdown of PKA eliminated the inhibitory phosphorylation of GSK3b by MB, suggesting the early inhibition of GSK3b by MB is dependent on PKA. Together, the ability of MB in protecting hepatocytes and liver against toxicant induced injury was confirmed by both in vitro and in vivo experiments. These results support the notion that MB facilitates dual inhibition of GSK3b downstream of PKA, and protects hepatocyte. Liver X receptor a (LXRa) is a nuclear receptor and oppositely regulates lipid metabolism and inflammation. In the analysis of public gene expression omnibus (GEO) database, LXRa level significantly decreased in different injury conditions in hepatocytes, mice and human liver. LXRa agonist treatment attenuated CCl4 induced hepatocyte injury, whereas LXRa-/- mice had more hepatocyte death and liver injury in response to CCl4, indicating LXRas protective effects on hepatocytes. In an additional bioinformatics study, LXRa protection of hepatocytes may result from antagonizing transforming growth factor b (TGFb) signaling, which was confirmed by subsequent experiments using AML12 cell and mouse primary hepatocytes. Immunoblotting, quantitative real-time polymerase chain reaction (qRT-PCR), reporter gene assay and chromatin immunoprecipitation (ChIP) analysis were used to study molecular mechanism downstream of LXRa. Cannabinoid receptor 2 (CB2) was identified as a transcriptional target of LXRa. CB2 contributed to LXRas protective effects on hepatocytes via upregulation of microRNA-27b (miR-27b). MiR-27b was demonstrated here as a novel upstream inhibitory regulator of ubiquitin specific peptidase 4 (USP4), a deubiquitylating enzyme for TGFb receptor 1 (TbRI). In addition, the changes on USP4 and TbRI by CB2 agonist were attenuated by PKA inhibition. Finally, albumin promoter-driven lentiviral delivery of LXRa protected hepatocytes from injury. Together, LXRa transcriptionally upregulates CB2, leading to miR-27b upregulation and contributes to protecting hepatocytes via repression of USP4 and TbRI. Taken together, the outcomes of this study demonstrate not only PKA-dependent GSK3b inhibition for hepatocyte survival activated by pharmacological agent, but also CB2-mediated PKA pathway associated with LXRa for hepatocyte protection, providing novel exogenous and endogenous pathways and pharmacological insights for hepatocyte survival, and potentially for the cure of liver diseases.