M1994 Molecular Factors Involved in Determining Genetic Susceptibility to Mallory-Denk Body Formation

Abstract

Background:Mallory-Denk bodies (MDBs) are hepatocyte inclusions associated with alcoholic and nonalcoholic steatohepatitis, and some forms of drug-induced liver injury. Understanding the biological significance of MDBs and devising means to alter their formation has clinical relevance to protein aggregation diseases beyond the liver. Feeding mice 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) produces, in a strain-selective manner, inclusions resembling human MDBs. Aims: We hypothesized that proteomic comparison of livers from MDBprone C57BL/6 mice and MDB-resistant C3H mice, before and after DDC treatment, will illuminate the molecular mechanisms regulating susceptibility of C57BL/6 mice to MDB formation. Methods: 2-dimensional (2D) differential-in-gel electrophoresis and mass spectrometry of liver protein homogenates resulted in the identification of >200 charged isoforms with >2 fold differences between the two strains. The results for some of the proteins were validated in control and DDC-treated livers, and isolated hepatocytes using 1D/2D gel analysis followed by immunoblotting. Enzymatic activities were also measured. Results: Proteins involved in cellular homeostasis and survival, such as carbonic anhydrase 3 (CA3) and GAPDH are expressed at lower levels in C57BL/6 livers. Whereas DDC treatment strongly induces CA3 and GAPDH expression in isolated C3H hepatocytes, it has no effect in C57BL/ 6 hepatocytes. There is significantly higher expression of reactive oxygen species-generating cytochrome P450 and detoxifying glutathione-S-transferase enzymes, and lower expression of the antioxidant enzyme peroxiredoxin 6 in the C57BL/6 livers, indicating a state of elevated oxidative stress. Furthermore, the livers, but not other organs, of untreated and DDC-fed C3H mice have a 2-fold higher expression of nucleoside diphosphate kinase B (NDPK-B), a multifunctional protein involved in regulating intracellular energy pools and protection from oxidative stress. Additionally, isolated C3H hepatocytes have 6-fold higher NDPK expression and activity relative to C57BL/6 hepatocytes, indicating that differences in total liver NDPK levels are accounted for by differences in hepatocytes. Conclusions: Genetic predisposition to MDB formation is likely related, at least in part, to deficiencies in systems involved in regulating intracellular homeostasis, energy generation and protection from oxidative stress. NDPK may be an upstream effector functioning in a strain-dependent and tissue-selective manner to limit MDB formation. Similar molecular determinants might contribute to the genetic differences in human liver diseases that are associated with MDB formation.