Stimulation of prodynorphin gene expression requires a functional protein kinase A.
To investigate the involvement of the cAMP/protein kinase A (PKA) second messenger system in mediating the hormonal regulation of prodynorphin expression, a normal, homologous cell transfection model was used. Ovarian granulosa cells, which express high levels of prodynorphin mRNA in response to hormonal stimulation, were obtained from the ovaries of gonadotropin-primed 26-day-old rats, pooled, and cultured under serum-free conditions. Cells were transfected with a plasmid construct (proDYN-CAT) containing approximately 2 kb of the rat prodynorphin gene (-1858 to +133 by relative to the transcription start site) fused to the chloramphenicol acetyltransferase (CAT) reporter gene. Cultures were cotransfected with either a control plasmid (pUC13) or the Mt-REV expression plasmid; Mt-REV overexpresses a mutated form of the RIalpha regulatory subunit of PKA which binds to and thereby inhibits the activity of the catalytic subunit. When granulosa cells were cotransfected with proDYN-CAT and a control plasmid, CAT activity was stimulated by human FSH (20 ng/ml), human chorionic gonadotropin (10 ng/ml), and 8-(4-chlorphenyl thio)-cAMP (0.5 mM) (10-, 11-, and 6-fold, respectively; P < 0.005). However, when cells were cotransfected with proDYN-CAT and Mt-REV there was a complete abolition of hormone and cAMP stimulation of CAT activity (P > 0.4 vs controls). The effect of Mt-REV cotransfection was specific since cotransfection with Mt-REVwt, a plasmid which overexpresses the normal, wild-type regulatory subunit, had no effect on hormonal responsiveness. Furthermore, cotransfection with Mt-CalphaEV or Mt-CbetaEV (which overexpress the alpha or beta catalytic subunits of PKA and thus mimic the effects of PKA activation) significantly stimulated prodynorphin promoter activity by 14-fold (P < 0.05). These results support the hypothesis that gonadotropin stimulation of granulosa cells increases prodynorphin promoter activity via a process which requires a functional PKA second messenger system. Thus, PKA may be a generalized mechanism by which stimulatory input increases cellular prodynorphin expression.
Kaynard
A H
AH
Divisions of Neuroscience and Reproductive Sciences, Oregon Regional Primate Research Center, Beaverton, Oregon 97006, USA.
Melner
M H
MH