Okadaic acid, a protein phosphatase inhibitor, enhances transcription of a receptor gene containing sequence A of the human prolactin promoter.
Human PRL (hPRL) gene expression is controlled by cAMP and Ca2+. This control is mediated by two cis-elements: a Pit-1 binding site (-62 to -35) and sequence A (-110 to -85), present in the hPRL promoter. We have investigated whether protein phosphatases could be involved in this regulation. GC-type rat pituitary tumor cells were transfected with sequence -138 to -35 of the hPRL gene promoter, upstream from a thymidine kinase promoter and a chloramphenicol acetyltransferase (CAT) reporter gene. Addition of okadaic acid (OA), a specific inhibitor of protein phosphatases 1 and 2A, stimulates transient expression of the CAT gene. The dose-response curve shows a maximal effect at 25 nM OA (2.2-fold stimulation above controls). The OA effect is also observed with a natural 4500-base pair hPRL promoter. A single copy of the hPRL promoter sequence -115 to -85 (sequence A) confers to a thymidine kinase-CAT construct an identical response to OA, whereas a single copy of the proximal Pit-1 binding site does not. Synergism is observed between cAMP and OA in activating PRL gene transcription. This synergism is also observed with a single copy of sequence A. The effect of cAMP is not mediated by an L-type Ca2+ channel, since addition of the Ca2+ channel antagonist verapamil does not decrease it, nor does complexing extracellular Ca2+ significantly reduce it. Furthermore, OA and the Ca2+ channel opener BAY K8644 exert additive effects.
Wera
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S
Laboratoire de Biologie Moléculaire et de Génie Génétique, Institut de Chimie B6, Université de Liège, Sart-Tilman, Belgium.
Belayew
A
A
Martial
J A
JA
D002118:Calcium / Q000494:pharmacology / Q000502:physiology; D015220:Calcium Channels / Q000502:physiology; D000242:Cyclic AMP / Q000494:pharmacology / Q000502:physiology; D004357:Drug Synergism; D004988:Ethers, Cyclic / Q000494:pharmacology*; D006801:Humans; D019319:Okadaic Acid; D011388:Prolactin / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics*
D015220:Calcium Channels; D004988:Ethers, Cyclic; D011993:Recombinant Fusion Proteins; D019319:Okadaic Acid; D011388:Prolactin; D000242:Cyclic AMP; D002118:Calcium