Mechanisms of mallory body formation induced by okadaic acid in drug-primed mice

Abstract

Drug-primed mice form Mallory bodies in their liver after various types of liver injury such as heat shock, drug refeeding, or ethanol ingestion. However, the mechanisms involved that lead to Mallory body formation after these different treatments are unknown. There may be a common pathway of Mallory body formation that is initiated by these different types of injuries. Recently it was shown that the phosphatase 1/2A inhibitor okadaic acid induced Mallory body formation, suggesting that the mechanism of formation involves hyperphosphorylation or oxidative stress-induced NFkappaB activation. To test this hypothesis we exposed drug-primed mice to okadaic acid and measured phosphorylation of Mallory body proteins immunohistochemically and by immunoblot chemiluminescence using an antibody specific for phosphothreonine. NFkappaB activation was measured by a gel shift retardation assay of nuclear lysates. Beginning 15 min after okadaic acid injection, complex changes were progressively seen in the liver cells focally including aggregation of cytokeratins 8 and 18 in hepatocytes which otherwise failed to stain normally with cytokeratin antibody. The aggregates stained positive with ubiquitin and phosphothreonine antibodies. Immunoblots showed a progressive increase in positive staining of the Mallory body band with the antibody to phosphothreonine. NFkappaB activation was progressive up to 2 h after okadaic acid treatment but was downregulated 7 days later. In summary we show for the first time the effect of okadaic acid on the liver cytokeratins in vivo. We conclude that hyperphosphorylation and NFkappaB activation may play a role in the early phases of Mallory body formation.