Received for publication July 28, 2004.
Revised October 28, 2004.
Accepted for publication October 28, 2004.

Steroidogenic Factor-1 Interacts with CREB to Mediate cAMP Stimulation of CYP1B1 via a Far Upstream Enhancer

Abstract

CYP1B1 activates PAH carcinogens in cAMP-regulated tissues like the adrenal, ovary and testis. A 27-fold cAMP stimulation of the CYP1B1-luciferase reporter in Y-1 adrenal cells depends entirely on a far upstream enhancer region (FUER, -5298 to -5110). Cooperative participation of multiple SF-1 elements with the downstream CRE in FUER is essential for both basal and cAMP-stimulated activities of FUER. Basal and induced activities were similarly lowered by DAX-1, an SF-1 suppressor, and raised by SRC-1, an SF-1 coactivator. CBP that interacts preferentially with the phosphorylated-CREB increased the cAMP-induced FUER. 10T1/2 cells and HEK293 cells do not express SF-1. Introduction of exogenous SF-1 generated cAMP stimulation of the FUER in 10T1/2 fibroblasts. The same transfection only increased basal activity of FUER in HEK293 cells, despite presence of active CREB in cells. HEK293 cells therefore remain deficient in additional factor(s) critical to the cAMP stimulation of CYP1B1. Mutations of the PKA and the MAPK phosphorylation sites (Ser-430 and Ser-203) on SF-1 had no effect on the SF-1-dependent FUER stimulation in Y-1 and 10T1/2 cells. This contrasts with loss of activity with mutation of CREB at PKA phosphorylation site (Ser-133). SF-1 phosphorylation at these sites is therefore not essential for the cAMP stimulation and the cooperation with CREB. cAMP-enhanced AP-1 and Sp1 complexes in the proximal promoter region contributed substantially to both basal and cAMP-stimulated FUER activity. Chromatin immunoprecipitation from primary rat adrenal cells demonstrated cAMP stimulation of histone acetylation proximal to, respectively, the FUER and AP-1 sites of CYP1B1.

Key words: cAMP, Protein Kinase A, AP-1, CREB, Regulation of gene expression, Cytochrome P450, Regulation - transcriptional