Cyclic AMP mediated GSTP1 gene activation in tumor cells involves the interaction of activated CREB-1 with the GSTP1 CRE: a novel mechanism of cellular GSTP1 gene regulation.

Abstract

The human GSTP1 gene is frequently over-expressed in many human cancers and the expression increases with tumor progression and is associated with a more aggressive biology, poor patient survival, and resistance to therapy. The molecular regulation of the human GSTP1 gene during malignancy is, however, still not well understood. Recently, we reported the presence of a cAMP response element (CRE) in the 5'-region of the human GSTP1 gene, raising the possibility that the cAMP signaling pathway, frequently aberrant in human cancers, may play an important role in the transcriptional activation of the GSTP1 gene in human tumors. In this study, we report that the GSTP1 gene is an early cAMP response gene. Treatment of cells of the human lung carcinoma cell line, Calu-6, with 25 microM forskolin to activate the cAMP pathway resulted in a rapid and significant (sevenfold after 30 min) increase in GSTP1 gene transcripts, which peaked at 12-fold after 4 h. The forskolin-activated GSTP1 transcription in Calu-6 cells was suppressed dose-dependently by a 2-h pre-treatment with 0.1, 1.0, and 10 microM of the adenylate cyclase inhibitor, 2', 5'-dideoxyadenosine. Western blot analysis showed a rapid, fivefold increase, in GSTP1 protein levels after treatment with 25 microM forskolin, with a peak at 2 h post-treatment. The levels of phosphorylated CRE (Ser133) binding protein-1 (CREB-1) increased rapidly, sevenfold at 30 min, and reached 10-fold at 4 h following forskolin treatment. Intracellular cAMP levels also increased rapidly reaching 12-fold at 30 min. Gel mobility shift and supershift assays and DNase/footprinting analyses demonstrated that CREB-1 bZIP and CREB-containing nuclear extracts recognized the GSTP1 CRE with high affinity and specificity. Binding of CREB-1 bZIP to the GSTP1 CRE was abolished when the GSTP1 CRE sequence 5'-CGTCA-3', was mutated at the core nucleotides. Finally, transfection studies using luciferase plasmid constructs showed the GSTP1 CRE to be required for the cAMP-activated gene expression. Together, these findings describe a novel cAMP- and CREB-1-mediated mechanism of transcriptional regulation of the GSTP1 gene and suggest that this may be an important mechanism underlying the increased GSTP1 expression observed in tumors with an aberrant cAMP signaling pathway and in normal cells under conditions of stress, associated with increased intracellular cAMP.