Calcium ions positively modulate follicle-stimulating hormone- and exogenous cyclic 3′,5′-adenosine monophosphate-driven transcription of the P450scc gene in porcine granulosa cells

F. C.L. Jayes, R. N. Day, J. C. Garmey, R. J. Urban, G. Zhang, J. D. Veldhuis

Research output: Contribution to journalArticlepeer-review

31 Scopus citations


Given the evident modulation of FSH-induced steroidogenesis by Ca2+ in granulosa cells, we here test the hypothesis that Ca2+ controls expression of the enzymatically rate-limiting cytochrome P450scc (CYP11A) gene. To test this postulate, we quantitated the ability of Ca2+ to regulate: 1) transcriptional activity of a transiently transfected luciferase reporter gene driven by a 2.32-kb 5′-upstream fragment of the porcine P450scc gene promoter region; and 2) accumulation of endogenous P450scc transcripts in primary monolayer cultures of porcine granulosa cells. To this end, granulosa cells were stimulated for 4h with FSH (15 ng/ml, NIDDK-oFSH-20) or 8-Bromo-cAMP (8 Br-cAMP, 1 mM) in serum-free medium containing either 1.8 mM Ca2+ or no added Ca2+ with 100 μM EGTA or 100 μ CoCl2. In the presence of extracellular Ca2+, FSH and 8 Br-cAMP stimulated expression of the transfected P450scc promoter-reporter fusion construct by 5.6 ± 1.1 and 3.6 ± 0.67-fold, respectively over Ca2+-containing unstimulated control (P ≤ 0.04, n = 5-6 experiments). The foregoing two agonists augmented 4-h progesterone production by cultured granulosa cells by 1.8 ± 0.11 and 1.6 ± 0.16-fold, respectively (P ≤ 0.001 for FSH and P ≤ 0.01 for 8 Br-cAMP). FSH and 8 Br-cAMP also significantly elevated endogenous P450scc transcript levels as measured by homologous solution-hybridization RNase protection assay; i.e. by 3.1 ± 0.49 and 2.9 ± 0.45-fold, respectively (P ≤ 0.001). In Ca2+-free/EGTA-supplemented medium, basal luciferase reporter-gene activity and endogenous P450scc messenger RNA accumulation in granulosa cells declined to 34 ± 12% and 78 ± 12%, respectively, of corresponding values in control (unstimulated Ca2+-containing) cultures. Extracellular Ca2+ deprivation inhibited the stimulatory effect of FSH (and 8 Br-cAMP) on P450scc promoter-luciferase reporter expression to 58 ± 30% (and 58 ± 23%), and restrained endogenous P450scc message accumulation to 86 ± 15% (and 96 ±18%) of the value in Ca2+-containing control. Extracellular Ca2+ withdrawal suppressed FSH (and 8 Br-cAMP)-driven progesterone production over 4 h to basal levels but did not alter FSH-stimulated cAMP accumulation by granulosa cells. Ca2+-deprived cells exposed to serum-containing media regained P450scc responsiveness to both agonists. Antagonism of cellular uptake of Ca2+ and other divalent cations via administration of cobalt chloride (100 μM) inhibited FSH and 8 Br-cAMP's stimulation of endogenous (but not exogenous promoter-driven) P450scc gene expression. In contrast, granulosa-cell concentrations of messenger RNA's encoding sterol-carrier protein-2 (SCP-2) and the low density lipoprotein receptor were not altered by Ca2+ withdrawal. In summary, uptake of extracellular Ca2+ by porcine granulosa cells significantly potentiates transactivation of the endogenously expressed and exogenously transfected P450scc gene by FSH and 8 Br-cAMP. The agonistic impact of Ca2+ on P450scc promoter activity is requisite downstream of FSH-induced cAMP second-messenger signaling.

Original languageEnglish (US)
Pages (from-to)2377-2384
Number of pages8
Issue number7
StatePublished - 2000

ASJC Scopus subject areas

  • Endocrinology


Dive into the research topics of 'Calcium ions positively modulate follicle-stimulating hormone- and exogenous cyclic 3′,5′-adenosine monophosphate-driven transcription of the P450scc gene in porcine granulosa cells'. Together they form a unique fingerprint.

Cite this