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  • Title: Reconstitution of protein kinase A regulation of the rat prolactin promoter in HeLa nonpituitary cells: identification of both GHF-1/Pit-1-dependent and -independent mechanisms.
    Author: Rajnarayan S, Chiono M, Alexander LM, Gutierrez-Hartmann A.
    Journal: Mol Endocrinol; 1995 Apr; 9(4):502-12. PubMed ID: 7659093.
    Abstract:
    Expression of the rat PRL (rPRL) gene is highly restricted to pituitary lactotroph cells and is induced by the cAMP-dependent protein kinase A (PKA) pathway. Current data indicate that this PKA effect requires at least one of the redundant pituitary-specific elements of the proximal rPRL promoter, suggesting the involvement of the pituitary-specific transcription factor, GHF-1/Pit-1. To directly determine whether GHF-1 is necessary and sufficient to mediate the PKA activation of the rPRL promoter, we established a cotransfection reconstitution assay whereby the activity of an intact and site-specific mutants of the (-425 to +73) rPRL promoter-luciferase reporter gene was reconstituted by cotransfecting expression vectors encoding for either the PKA beta catalytic subunit, GHF-1, or both, into HeLa nonpituitary cells. Cotransfection of PKA beta alone significantly stimulated rPRL promoter activity in HeLa cells in a GHF-1-independent manner, and this PKA beta effect was mapped to the most proximal GHF-1 site [footprint (FP) I; -67/-36]. Site-specific alterations of either FP II (-130/-120), or of the basal transcription element (BTE; -112/-80), did not significantly affect the PKA beta response. As expected, the transactivation effect of cotransfected GHF-1 mapped to the GHF-1/Pit-1 binding sites, FP I and/or FP III, of the rPRL promoter. Finally, cotransfection of PKA beta and GHF-1 resulted in a marked synergistic response of the rPRL promoter, and this response also localized to the FP I site. These data confirm not only that GHF-1/Pit-1 and the FP I site are involved in mediating the PKA response, but also imply that a distinct and possibly ubiquitous factor is involved by binding to FP I and functionally interacting with GHF-1 to modulate PKA beta regulation of the rPRL promoter.
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