GSK3β/NF-κB -dependent transcriptional regulation of homeostatic hepatocyte Tnf production

Maintenance of hepatocyte homeostasis plays an important role in mediating the pathogenesis of many diseases. A growing body of literature has established a critical role played by tumor necrosis factor-alpha (TNF alpha) in maintaining hepatocyte homeostasis; however, the transcriptional mechanisms underlying constitutive Tnf expression are unknown. Whole liver fractions and primary hepatocytes from adult control C57BL/6 mice and the murine hepatocyte cell line AML12 were assessed for constitutive Tnf expression. Impacts of glycogen synthase kinase-3 beta (GSK3 beta) and nuclear factor kappa B (NF-kappa B) inhibition on constitutive Tnf expression were assessed in AML12 cells. Finally, AML12 cell proliferation following GSK3 beta and NF-kappa B inhibition was evaluated. Constitutive Tnf gene expression is present in whole liver, primary hepatocytes, and cultured AML12 hepatocytes. Cytokine-induced Tnf gene expression is regulated by NF-kappa B activation. Pharmacological inhibition of GSK3 beta resulted in a time- and dose-dependent inhibition of Tnf gene expression. GSK3 beta inhibition decreased nuclear levels of the NF-kappa B subunits p65 and p50. We determined that NF-kappa B transcription factor subunit p65 binds to consensus sequence elements present in the murine TNF alpha promoter and inhibition of GSK3 beta decreases binding and subsequent Tnf expression. Finally, AML12 cell growth was significantly reduced following GSK3 beta and NF-kappa B inhibition. These results demonstrate that GSK3 beta and NF-kappa B are essential for mediating Tnf expression and constitutive hepatocyte cell growth. These findings add to a growing body of literature on TNF alpha mediated hepatocyte homeostasis and identify novel molecular mechanisms involved in mediating response to various disease states in the liver. NEW & NOTEWORTHY Maintenance of hepatocyte homeostasis plays an important role in controlling the pathogenesis of many diseases. Our findings add to a growing body of literature on tumor necrosis factor-alpha (TNF alpha)-mediated hepatocyte homeostasis and identify novel molecular mechanisms involved in regulating this response.