Increased hepatic JNK activation by ethanol is mediated by transcriptional suppression of mitogen‐activated protein kinase phosphatase 1 (Mkp1): role of cAMP‐specific protein kinase A

Abstract

Background Increased and sustained JNK activation has been shown to be detrimental for hepatocyte survival and liver injury in various diseases, including alcohol-associated liver disease (ALD). Among three JNK isoforms, JNK1 and 2 are expressed in hepatocytes and both play a role in mouse models of ALD. Mkp1 phosphatase has been identified as a regulator of JNK activation. A previous study showed that Mkp1 degradation plays a critical role in sustained JNK activation and hepatocyte death from acute ethanol exposure. However, the effect of chronic ethanol exposure on hepatocyte Mkp1 expression is not known. Methods Male C57B/6 mice were subjected to intragastric (iG) feeding of ethanol for 8 weeks with weekly ethanol binge (5g/kg) starting in the second week of iG feeding. Pair-fed control mice were fed an isocaloric high-fat liquid diet. Plasma ALT, AST and histologic grading were performed to evaluate steatosis, liver inflammation and injury. Liver immunohistochemistry was performed to evaluate apoptosis (TUNEL staining). Myeloperoxidase (MPO) activity assay and chloroacetate esterase (CAE) staining for neutrophil infiltration were also performed. Liver RNA sequencing was performed on an Illumina NextSeq 500. Data analysis was done using the Tuxedo suite pipeline. Validation studies were carried out by performing real time qPCR and Western blot. Primary mouse hepatocytes were exposed to 50 mM ethanol for 48 hours followed by TNF stimulation. Statistical analysis was performed using ANOVA. Results Histological examination of liver sections confirmed the development of severe alcohol-associated steatohepatitis in ethanol fed mice via significant micro- and macro-vesicular steatosis, polymorphonuclear leukocyte (PMN) infiltration, CAE and TUNEL-positive cells. ALT and AST levels were significantly elevated in ethanol-fed mice. JNK activation was evident by Western blot analysis of phosphorylated JNK levels. RNA Seq analysis showed significant changes in expression of several Mkps; however, only Mkp1 levels were significantly downregulated, which was further validated by real time qPCR. Importantly, ethanol exposure of primary mouse hepatocytes led to a significant decrease in Mkp1 mRNA expression which further declined following TNF stimulation. These changes in Mkp1 were accompanied by sustained JNK activation and cell death. Moreover, ethanol exposure and TNF stimulation markedly increased cAMP specific PDE4 expression resulting in reduction in hepatocyte cAMP levels. Significantly, PDE4 inhibition and PKA specific agonists prevented the suppressive effect of ethanol and TNF on Mkp1 and resultant activation of JNK and cell death. Conclusion Our results strongly implicate ethanol-induced decline in hepatic Mkp1 as a significant mechanism regulating JNK activation and hepatocyte death in ALD. Further, these data strongly suggest that the ethanol induced decrease in cAMP/PKA signaling plays a causal role in regulating the Mkp1/JNK axis in hepatocytes and development of liver injury.