A SEQUENCE IN THE RAT PIT-1 GENE PROMOTER CONFERS SYNERGISTIC ACTIVATION BY GLUCOCORTICOIDS AND PROTEIN KINASES-C

The 5'-flanking region of the gene for Pit-1, a pituitary-specific transcription factor, was isolated from a rat liver genomic library and sequenced. Expression of a reporter construct containing Pit-1 promoter sequences linked to the bacterial chloramphenicol acetyltransferase (CAT) gene was assessed by transient transfection in rat pituitary GH(4)C(1) cells. Treatment of transfected cells with either dexamethasone (DEX) for 48 h or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) for the final 20 h of the 48-h posttransfection period had minimal effects on CAT expression. However, CAT activity was elevated about 20-fold when transfected cells were treated with both DEX and TPA. This apparent synergistic activation was lost when DEX treatment was also limited to the final 20 h of the 48-h posttransfection period, suggesting that a time-dependent accumulation of a DEX-induced gene product might be involved. This putative DEX-induced product appeared to be relatively stable, because synergistic activation was observed in cells treated with DEX alone for 36 h, followed by a 10-h incubation without DEX before the addition of TPA. The Pit-1 gene promoter region between -210 and -142 from the transcription start site conferred synergistic regulation by DEX and TPA when placed upstream of position -105 in the herpes viral thymidine kinase promoter. Promoter sequences containing both cAMP response elements (CREs) are required for synergism. As synergistic activation was abolished by coexpression of an inhibitor of protein kinase-A (PKA), the combined effects of DEX and TPA also appear to require PKA-mediated phosphorylation. The binding activity of nuclear proteins to a CRE was not affected by treatment of GH(4)C(1) cells with DEX and TPA. We propose that a DEX-induced gene product along with phosphorylation events involving protein kinase-C and PKA participate in the activation of the rat Pit-1 gene promoter through sequences containing two CRE motifs.