title: Cyclic adenosine 3',5'-monophosphate activation of the rat prolactin promoter is restricted to the pituitary-specific cell type.
abstract: Pituitary lactotroph cell function and PRL gene expression are highly regulated by the cAMP-protein kinase-A (PKA) pathway. To further our understanding of the molecular mechanisms by which cAMP/PKA regulates rat (r) PRL promoter activity and to determine whether cAMP regulation is cell type specific, we 1) transected intact (-425), internal and 5'-deletion, and site-specific mutants of the rPRL promoter ligated to the firefly luciferase reporter gene into both pituitary and nonpituitary cell lines; and 2) assessed the role of the cAMP-cAMP response element-binding protein (CREB) pathway in GH4 rat pituitary cells. The data show that deleting the rPRL promoter from -425 to -116 did not abolish cAMP regulation, implying that proximal elements, such as the basal transcription element (-112/-80) or the pituitary-specific footprint (FP) I (-67/-45), mediate the cAMP response. However, nucleotide changes within FP I or FP II (-130/-120) did not alter the rPRL promoter response to 1 microM forskolin (FSK), despite the 77% and 26% reductions in basal rPRL promoter activity caused by these mutations, respectively. Furthermore, internal deletion of either the basal transcription element of FP I element also failed to affect cAMP regulation of the rPRL promoter, again despite the 90% and 93% reductions in basal promoter activity by these deletions, respectively. Since these internal deletion constructs otherwise contain rPRL promoter sequences from -425 to +73, including the up-stream pituitary-specific FPs III and IV, the data suggest that any one of these cell-specific elements is capable of imparting cAMP regulation to the proximal rPRL promoter. To directly test the implication that the cAMP response of the rPRL promoter is restricted to the pituitary-specific cell type, we took advantage of a 5'-deletion mutant truncated at position -116 and a FP II site-specific mutant, since constructs containing these rPRL promoters are active in nonpituitary cells. Despite the 6.6- and 18.5-fold stimulations over wild-type rPRL promoter activity in nonpituitary cells, respectively, these mutations remained completely unresponsive to FSK treatment. To document that the cAMP-CREB pathway was functional in GC/GH4 rat pituitary cells, CREB was affinity purified from GC rat pituitary cells, and DNase-I protection studies showed that it does not bind to the proximal rPRL promoter. Also, the human glycoprotein alpha-subunit promoter was induced 10-fold by FSK in GH4 rat pituitary cells.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Medicine and Biochemistry, University of Colorado Health Sciences Center, Denver 80262.
authors.forename: C A
authors.initials: CA
authors.lastname: Keech
authors.forename: S M
authors.initials: SM
authors.lastname: Jackson
authors.forename: S K
authors.initials: SK
authors.lastname: Siddiqui
authors.forename: K W
authors.initials: KW
authors.lastname: Ocran
authors.forename: A
authors.initials: A
authors.lastname: Gutierrez-Hartmann
mesh_terms: D000818:Animals; D001483:Base Sequence; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009928:Organ Specificity; D010903:Pituitary Gland, Anterior / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D010911:Pituitary Neoplasms; D011388:Prolactin / Q000096:biosynthesis* / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017384:Sequence Deletion; D014407:Tumor Cells, Cultured
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D011993:Recombinant Fusion Proteins; D005576:Colforsin; D011388:Prolactin; D003513:Cycloheximide; D000242:Cyclic AMP