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Calcium Ions Positively Modulate Follicle-Stimulating Hormone- and Exogenous Cyclic 3',5'-Adenosine Monophosphate-Driven Transcription of the P450_scc Gene in Porcine Granulosa Cells¹

Division of Endocrinology (F.C.L.J., R.N.D., J.C.G., G.Z., J.D.V.), Department of Internal Medicine or Cell Biology (F.C.L.J.), NIH Specialized Cooperative Center in Reproduction Research, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908; and Department of Medicine (R.J.U.), University of Texas Medical Branch, Galveston, Texas 77555

Address all correspondence and requests for reprints to: Johannes D. Veldhuis, M.D., Division of Endocrinology, Department of Internal Medicine, University of Virginia Health System, P.O. Box 800202, Charlottesville, Virginia 22908-0202. E-mail: jdv{at}virginia.edu

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 P450_scc (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 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 cells were stimulated for 4 h 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 µM CoCl₂. In the presence of extracellular Ca²⁺, FSH and 8 Br-cAMP stimulated expression of the transfected P450_scc 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 P450_scc 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 P450_scc 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 P450_scc promoter-luciferase reporter expression to 58 ± 30% (and 58 ± 23%), and restrained endogenous P450_scc 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 P450_scc 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) P450_scc 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 P450_scc promoter activity is requisite downstream of FSH-induced cAMP second-messenger signaling.

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