Introduction: We have been interested in the regulation of early gene activation in monocytes as these are implicated in the pathogenesis of acute inflammatory states such as sepsis. A key early gene upregulated by endotoxin is c-jun. C-jun undergoes serine phosphorylation by c-jun N-terminal kinase (JNK) and associates with c-fos to form the transcriptional activation complex, AP-1, to direct transcription of inflammatory genes, such as IL-1β. With a growing understanding of the regulatory relationship between kinases and phosphatases involved in cell signaling pathways, we hypothesized that the endogenous serine/threonine phosphatase, PP2A, regulates JNK activity. Methods: The human acute monocytic leukemia cell line, THP-1, was used for all studies. Stimulation was performed with 1 μg/ml LPS (E. coli). Okadaic acid (OA, 1-500 nM, Sigma) pretreatment time was 30 minutes in all experiments. JNK activity was determined by standard kinase assay following immunoprecipitation of cell lysates with anti-JNK antibody. Presence of phosphorylated c-jun and PP2A subunits was determined by Western blot of whole cell extracts. Nuclear translocation of AP-1 complex was determined by ELISA. AP-1 transcriptional activity was measured by transient transfections with a luciferase-linked, tandem AP-1 (X 3) promoter. Supernatant levels of IL-1β were measured by ELISA. Results: Constitutive presence of PP2A was found. Inhibition of PP2-A with OA, following LPS stimulation resulted in a >20-fold increase in JNK activity, as well as a substantial increase in phosphorylated c-jun and increased nuclear translocation/binding of AP-1 (56%). These findings were associated with AP-1 transcriptional activity as supported by increased expression of AP-1-driven luciferase and increased AP-1-dependent, IL-1β expression in cell culture supernatants following OA treatment. Conclusions: PP2A appears to be a crucial regulator of JNK activity. The data support the hypothesis that phosphatases may be key endogenous regulators of cytokine signaling and thus valid therapeutic targets in the setting of inflammation.
ASJC Scopus subject areas
- Critical Care and Intensive Care Medicine