INTRAHEPATIC STAT-3 DNA BINDING DOES NOT CORRELATE WITH INTRANUCLEAR PHOSPHO-STAT-3 LEVELS IN SEPTIC MICE

Abstract

Objective: Intrahepatic signal transducer and activator of transcription (STAT)-3 activation and acute phase gene expression of α2-macroglobulin (A2M) has shown to predict outcome after cecal ligation and double puncture (2CLP) in rats. Previous studies have shown that STAT-3 DNA binding is decreased dramatically in mice 16 and 24 hours following 2CLP and remains attenuated compared to single puncture (CLP). We hypothesize that this decrease is from a disruption of the intracellular STAT-3 signaling pathway. Methods: Liver homogenates were collected from 6-8 weeks old C57 B1/6 mice 3, 6, 16, 24, 48, and 72 hours after sham operation (SO) and 2CLP. Liver homogenates from unoperated control (T0) animals were also collected. Nuclear and cytosolic protein extracts were made from liver homogenates. Electrophoretic mobility shift assays (EMSA) were previously done on nuclear extracts detailing STAT-3 DNA binding activity following SO, and 2CLP. Western blotting was performed on hepatic nuclear protein extracts following SO and 2CLP for phosphorylated- STAT-3 (p-STAT-3). Results: Hepatic intranuclear p-STAT-3 levels were undetectable in T0 animals. However, significant levels were detected in samples taken 3 and 6 hours after 2CLP. Intranuclear p-STAT-3 levels were decreased at 16 hours and undetectable at 24 hours post 2CLP. However, levels increased again 48 and 72 hours post 2CLP. A similar pattern but markedly decreased intranuclear p-STAT-3 levels were observed after SO and there were no detectable levels 48 and 72 hours post SO. Conclusion: Intranuclear p-STAT-3 levels initially correlated with STAT-3 DNA binding, but did not last beyond 24 hours in 2CLP. Although there is signal attenuation 16 and 24 hours after 2CLP, the lack of STAT-3 DNA binding despite elevated intranuclear p-STAT-3 levels 48 and 72 hours post 2CLP suggests a defect with intranuclear STAT-3 regulation.