Coordinated Induction of GST and MRP2 by cAMP in Caco-2 Cells. Role of protein kinase A signaling pathway and toxicological relevance

Abstract

The cAMP pathway is a universal signaling pathway regulating many cellular processes including metabolic routes, growth and differentiation. However, its effects on xenobiotic biotransformation and transport systems are poorly characterized. The effect of cAMP on expression and activity of GST and MRP2 was evaluated in Caco-2 cells, a model of intestinal epithelium. Cells incubated with the cAMP permeable analog dibutyryl cyclic AMP (db-cAMP: 1,10,100 μM) for 48 h exhibited a dose–response increase in GST class α and MRP2 protein expression. Incubation with forskolin, an activator of adenylyl cyclase, confirmed the association between intracellular cAMP and upregulation of MRP2. Consistent with increased expression of GSTα and MRP2, db-cAMP enhanced their activities, as well as cytoprotection against the common substrate 1-chloro-2,4-dinitrobenzene. Pretreatment with protein kinase A (PKA) inhibitors totally abolished upregulation of MRP2 and GSTα induced by db-cAMP. In silico analysis together with experiments consisting of treatment with db-cAMP of Caco-2 cells transfected with a reporter construct containing CRE and AP-1 sites evidenced participation of these sites in MRP2 upregulation. Further studies involving the transcription factors CREB and AP-1 (c-JUN, c-FOS and ATF2) demonstrated increased levels of total c-JUN and phosphorylation of c-JUN and ATF2 by db-cAMP, which were suppressed by a PKA inhibitor. Co-immunoprecipitation and ChIP assay studies demonstrated that db-cAMP increased c-JUN/ATF2 interaction, with further recruitment to the region of the MRP2 promoter containing CRE and AP-1 sites. We conclude that cAMP induces GSTα and MRP2 expression and activity in Caco-2 cells via the PKA pathway, thus regulating detoxification of specific xenobiotics.