An TGACG motif mediates GHF1/Pit-1-dependent cAMP regulation of the rainbow trout growth hormone promoter

The mechanisms involved in the regulation of the rainbow trout growth hormone (tGH) gene promoter by the pituitary-specific transcription factor GHF1 (growth hormone factor 1), also called Pit1 (pituitary transcriptional activator 1), and cAMP have been investigated in mammalian and fish cells. The -340 to +24 5'-flanking Fegion of the tGH gene focused to the luciferase gene was activated in rat pituitary GC cells and in HeLa cells cotransfected with an effector plasmid encoding rat GHFI. GC cell nuclear extracts produced four GHFI-specific footprints (sites Fl to F4) on the tGH promoter, each containing multiple W4NCAT (W, A or T) or closely related motifs. Mutational analysis performed in GC cells indicated that the proximal Fl site alone can direct transcription, but that the region encompassing the F2 and F3 sites is necessary for optimal activation and contains a TGACG motif (cAMP-response element, CRE) conferring cAMP responsiveness. The role of the TGACG motif in mediating cAMP regulation of the tGH promoter was confirmed in primary cultures of trout pituitary cells. Cotransfection studies in carp EPC cells using an effector plasmid encoding trout GHF1 demonstrated the GHF1 dependence of cAMP stimulation. Gel shift and southwestern experiments revealed nuclear proteins of 43 kDa and 30 kDa in GC and fish cells, respectively, that bind specifically to the tGH CRE, suggesting the involvement of CRE-binding-protein/activating-transcription-factor-l-related peptides in cAMP response. Incidentally, and in contrast with previous reports, we found the rat GH promoter, that lacks TGACG motifs, unresponsive to cAMP. Thus, the CAMP stimulation of the tGH gene is more similar to its human counterpart. that is also GHF1 dependent and mediated by TGACG motifs in the promoter. It is suggested that control of GH gene expression has evolved modularly, through various assortments of the same regulatory units, rather than molecularly, through innovative units.