INDUCTION OF CHINOOK SALMON GROWTH-HORMONE PROMOTER ACTIVITY BY THE ADENOSINE-3',5'-MONOPHOSPHATE (CAMP)-DEPENDENT PATHWAY INVOLVES 2 CAMP-RESPONSE ELEMENTS WITH THE CGTCA MOTIF AND THE PITUITARY-SPECIFIC TRANSCRIPTION FACTOR PIT-1

In this study, the functional role of two cAMP-response elements (CRE) in the promoter of the chinook salmon GH gene and their interactions with the transcription factor Pit-1 in regulating GH gene expression w ere examined. A chimeric construct of the chloramphenicol acetyltransf erase (CAT) reporter gene with the CRE-containing GH promoter (pGH.CAT ) was transiently transfected into primary cultures of rainbow trout p ituitary cells. The expression of CAT activity was stimulated by an ad enylate cyclase activator forskolin as well as a membrane-permeant cAM P analog 8-bromo-cAMP. Furthermore, these stimulatory responses were i nhibited by a protein kinase A inhibitor H89, suggesting that these CR Es are functionally coupled to the adenylate cyclase-cAMP-protein kina se A cascade. This hypothesis is supported by parallel studies using G H(4)ZR(7) cells, a rat pituitary cell line stably transfected with dop amine D2 receptors. In this cell line, D2 receptor activation is known to inhibit adenylate cyclase activity and cAMP synthesis. Stimulation with a nonselective dopamine agonist, apomorphine, or a D2-specific a gonist, Ly171555, suppressed the expression of pGH.CAT in GH(4)ZR(7) c ells, and this inhibition was blocked by simultaneous treatment with f orskolin. These results indicate that inhibition of the cAMP-dependent pathway reduces the basal promoter activity of the CRE-containing pGH -.CAT. The functionality of these CREs was further confirmed by deleti on analysis and site-specific mutagenesis. In trout pituitary cells, t he cAMP inducibility of pGH.CAT was inhibited after deleting the CRE-c ontaining sequence from the GH promoter. When the CRE-containing seque nce was cloned into a CAT construct with a viral thymidine kinase prom oter, a significant elevation of cAMP inducibility was observed. This stimulatory response, however, was abolished by mutating the core sequ ence, CGTCA, in these CREs, suggesting that these cis-acting elements confer cAMP inducibility to the salmon GH gene. The interactions betwe en CREs and the transcription factor Pit-1 in mediating GH gene expres sion were also examined. In HeLa cells, a human cervical cancer cell l ine deficient in Pit-1, both basal and cAMP-induced expression of pGH. CAT were apparent only with the cotransfection of a Pit-1 expression v ector. These results taken together indicate that the two CREs in the chinook salmon GH gene are functionally associated with the cAMP-depen dent pathway and that their promoter activity is dependent on the pres ence of Pit-1.