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, Randall Urban, G. Zhang, J. D. Veldhuis

Research output: Contribution to journalArticle

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Abstract

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
JournalEndocrinology
Volume141
Issue number7
DOIs
StatePublished - 2000

Fingerprint

Granulosa Cells
Follicle Stimulating Hormone
Adenosine Monophosphate
Swine
Ions
Calcium
Genes
Luciferases
Egtazic Acid
Reporter Genes
Progesterone
National Institute of Diabetes and Digestive and Kidney Diseases (U.S.)
Cholesterol Side-Chain Cleavage Enzyme
8-Bromo Cyclic Adenosine Monophosphate
Messenger RNA
LDL Receptors
Serum-Free Culture Media
Divalent Cations
Second Messenger Systems
Ribonucleases

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

Calcium ions positively modulate follicle-stimulating hormone- and exogenous cyclic 3′,5′-adenosine monophosphate-driven transcription of the P450scc gene in porcine granulosa cells. / Jayes, F. C L; Day, R. N.; Garmey, J. C.; Urban, Randall; Zhang, G.; Veldhuis, J. D.

In: Endocrinology, Vol. 141, No. 7, 2000, p. 2377-2384.

Research output: Contribution to journalArticle

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title = "Calcium ions positively modulate follicle-stimulating hormone- and exogenous cyclic 3′,5′-adenosine monophosphate-driven transcription of the P450scc gene in porcine granulosa cells",
abstract = "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.",
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TY - JOUR

T1 - Calcium ions positively modulate follicle-stimulating hormone- and exogenous cyclic 3′,5′-adenosine monophosphate-driven transcription of the P450scc gene in porcine granulosa cells

AU - Jayes, F. C L

AU - Day, R. N.

AU - Garmey, J. C.

AU - Urban, Randall

AU - Zhang, G.

AU - Veldhuis, J. D.

PY - 2000

Y1 - 2000

N2 - 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.

AB - 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.

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