Calcium ions positively modulate follicle-stimulating hormone- and exogenous cyclic 3 ',5 '-adenosine monophosphate-driven transcription of the P450(scc) gene in porcine granulosa cells

Given the evident modulation of FSH-induced steroidogenesis by Ca2+ in gran ulosa cells, we here test the hypothesis that Ca2+ controls expression of t he enzymatically rate-limiting cytochrome P450(scc) (CYP11A) gene. To test this postulate, we quantitated the ability of Ca2+ to regulate: 1) transcri ptional activity of a transiently transfected luciferase reporter gene driv en by a 2.32-kb 5'-upstream fragment of the porcine P450(scc) gene promoter region; and 2) accumulation of endogenous P450(scc) transcripts in primary monolayer cultures of porcine granulosa cells. To this end, granulosa cell s were stimulated for 4 h with FSH (15 ng/ml, NIDDK-oFSH-20) or 8-Bromo-cAM P (8 Br-cAMP, 1 mM) in serum-free medium containing either 1.8 mM Ca2+ or n o added Ca2+ with 100 mu M EGTA or 100 mu M CoCl2. In the presence of extra cellular Ca2+, FSH and 8 Br-cAMP stimulated expression of the transfected P 450(scc) promoter-reporter fusion construct by 5.6 +/- 1.1 and 3.6 +/- 0.67 -fold, respectively over Ca2+-containing unstimulated control (P less than or equal to 0.04, n = 5-6 experiments). The foregoing two agonists augmente d 4-h progesterone production by cultured granulosa cells by 1.8 +/- 0.11 a nd 1.6 +/- 0.16-fold, respectively (P less than or equal to 0.001 for FSH a nd P less than or equal to 0.01 for 8 Br-cAMP). FSH and 8 Br-cAMP also sign ificantly elevated endogenous P450(scc) transcript levels as measured by ho mologous solution-hybridization RNase protection assay; i.e. by 3.1 +/- 0.4 9 and 2.9 +/- 0.45-fold, respectively (P less than or equal to 0.001). In C a2+-free/EGTA-supplemented medium, basal luciferase reporter-gene activity and endogenous P450(scc) messenger RNA accumulation in granulosa cells decl ined to 34 +/- 12% and 78 +/- 12%, respectively, of corresponding values in control (unstimulated Ca2+-containing) cultures-Extracellular Ca2+ depriva tion inhibited the stimulatory effect of FSH (and 8 Br-cAMP) on P450(scc) p romoter-luciferase reporter expression to 58 +/- 30% land 58 +/- 23%), and restrained endogenous P450(scc) message accumulation to 86 +/- 15% land 96 +/- 18%) of the value in Ca2+-containing control. Extracellular Ca2+ withdr awal suppressed FSH (and 8 Br-cAMP)-driven progesterone production over 4 h to basal levels but did not alter FSH-stimulated cAMP accumulation by gran ulosa cells. Ca2+-deprived cells exposed to serum-containing media regained P450(scc) responsiveness to both agonists. Antagonism of cellular uptake o f Ca2+ and other divalent cations via administration of cobalt chloride (10 0 mu M) inhibited FSH and 8 Br-cAMP's stimulation of endogenous (but not ex ogenous promoter-driven) P450(scc) gene expression. In contrast, granulosa- cell concentrations of messenger RNA's encoding sterol-carrier protein-2 (S CP-2) and the low density lipoprotein receptor were not altered by Ca2+ wit hdrawal. In summary, uptake of extracellular Ca2+ by porcine granulosa cells signifi cantly potentiates transactivation of the endogenously expressed and exogen ously transfected P450scc gene by FSH and 8 Br-cAMP. The agonistic impact o f Ca2+ on P450(scc) promoter activity is requisite downstream of FSH-induce d cAMP second-messenger signaling.