Transcriptional regulation of cyclin D2 by the PKA pathway and inducible cAMP early repressor in granulosa cells

Luis C. Muñiz, Ghassan Yehia, Elisabeth Mémin, Pillarisetty V.A.L. Ratnakar, Carlos Molina

Research output: Contribution to journalArticleResearchpeer-review

18 Citations (Scopus)

Abstract

Cyclin D2 (Ccnd2) is an essential gene for folliculogenesis, as null mutation in mice impairs granulosa cell proliferation in response to FSH. Ccnd2 mRNA is induced during the estrus cycle by FSH and is rapidly inhibited by LH. Yet, the responsive elements and transcription factors accounting for the gene expression of cyclin D2 in the ovary have not been fully characterized. Using primary cultures of rat granulosa cells and immortalized mouse granulosa cells, we demonstrate a mechanism for the regulation of cyclin D2 at the level of transcription via a PKA-dependent signaling mechanism. The promoter activity of cyclin D2 was shown to be induced by FSH and the catalytic alpha subunit of PKA (PRKACA), and this activity was repressible by inducible cAMP early repressor (ICER), a cAMP response element (CRE) modulator isoform. In silico analysis of the mouse, rat, and human cyclin D2 promoters identified two CRE-binding protein sites, a conserved proximal element and a less conserved distal element relative to the translation start site. The mutation on the proximal element drastically decreases the effects of PRKACA and ICER on the promoter activity, whereas the mutation on the distal element did not contribute to the decrease in the promoter activity. Electrophoretic mobility shift assays and deoxyribonuclease footprint analysis confirmed ICER binding to the proximal element, and chromatin immunoprecipitation analysis demonstrated the occurrence of this binding in vivo. These results showed a CRE within the upstream region of Ccnd2 that is (at least partly) implicated in the stimulation and repression of cyclin D2 transcription. Finally, our data suggest that ICER involvement in the regulation of granulosa cell proliferation as overexpression of ICER results in the inhibition of PRKACA-induced DNA synthesis.

Original languageEnglish
Pages (from-to)279-288
Number of pages10
JournalBiology of Reproduction
Volume75
Issue number2
DOIs
StatePublished - 1 Aug 2006

Fingerprint

Cyclin D2
Granulosa Cells
Response Elements
Mutation
Cell Proliferation
Alpha Subunit Glycoprotein Hormones
Cyclic AMP Response Element-Binding Protein
Deoxyribonucleases
Chromatin Immunoprecipitation
Essential Genes
Estrus
Electrophoretic Mobility Shift Assay
Computer Simulation
Ovary
Catalytic Domain
Protein Isoforms
Transcription Factors
Binding Sites
Gene Expression
Messenger RNA

Keywords

  • Ccnd2
  • Cyclic adenosine monophosphate
  • Cyclin D2
  • Follicle-stimulating hormone
  • Folliculogenesis
  • Gene regulation
  • Granulosa
  • Granulosa cells
  • ICER
  • Mechanisms of hormone action
  • Ovarian
  • cAMP

Cite this

Muñiz, Luis C. ; Yehia, Ghassan ; Mémin, Elisabeth ; Ratnakar, Pillarisetty V.A.L. ; Molina, Carlos. / Transcriptional regulation of cyclin D2 by the PKA pathway and inducible cAMP early repressor in granulosa cells. In: Biology of Reproduction. 2006 ; Vol. 75, No. 2. pp. 279-288.
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title = "Transcriptional regulation of cyclin D2 by the PKA pathway and inducible cAMP early repressor in granulosa cells",
abstract = "Cyclin D2 (Ccnd2) is an essential gene for folliculogenesis, as null mutation in mice impairs granulosa cell proliferation in response to FSH. Ccnd2 mRNA is induced during the estrus cycle by FSH and is rapidly inhibited by LH. Yet, the responsive elements and transcription factors accounting for the gene expression of cyclin D2 in the ovary have not been fully characterized. Using primary cultures of rat granulosa cells and immortalized mouse granulosa cells, we demonstrate a mechanism for the regulation of cyclin D2 at the level of transcription via a PKA-dependent signaling mechanism. The promoter activity of cyclin D2 was shown to be induced by FSH and the catalytic alpha subunit of PKA (PRKACA), and this activity was repressible by inducible cAMP early repressor (ICER), a cAMP response element (CRE) modulator isoform. In silico analysis of the mouse, rat, and human cyclin D2 promoters identified two CRE-binding protein sites, a conserved proximal element and a less conserved distal element relative to the translation start site. The mutation on the proximal element drastically decreases the effects of PRKACA and ICER on the promoter activity, whereas the mutation on the distal element did not contribute to the decrease in the promoter activity. Electrophoretic mobility shift assays and deoxyribonuclease footprint analysis confirmed ICER binding to the proximal element, and chromatin immunoprecipitation analysis demonstrated the occurrence of this binding in vivo. These results showed a CRE within the upstream region of Ccnd2 that is (at least partly) implicated in the stimulation and repression of cyclin D2 transcription. Finally, our data suggest that ICER involvement in the regulation of granulosa cell proliferation as overexpression of ICER results in the inhibition of PRKACA-induced DNA synthesis.",
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Transcriptional regulation of cyclin D2 by the PKA pathway and inducible cAMP early repressor in granulosa cells. / Muñiz, Luis C.; Yehia, Ghassan; Mémin, Elisabeth; Ratnakar, Pillarisetty V.A.L.; Molina, Carlos.

In: Biology of Reproduction, Vol. 75, No. 2, 01.08.2006, p. 279-288.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Transcriptional regulation of cyclin D2 by the PKA pathway and inducible cAMP early repressor in granulosa cells

AU - Muñiz, Luis C.

AU - Yehia, Ghassan

AU - Mémin, Elisabeth

AU - Ratnakar, Pillarisetty V.A.L.

AU - Molina, Carlos

PY - 2006/8/1

Y1 - 2006/8/1

N2 - Cyclin D2 (Ccnd2) is an essential gene for folliculogenesis, as null mutation in mice impairs granulosa cell proliferation in response to FSH. Ccnd2 mRNA is induced during the estrus cycle by FSH and is rapidly inhibited by LH. Yet, the responsive elements and transcription factors accounting for the gene expression of cyclin D2 in the ovary have not been fully characterized. Using primary cultures of rat granulosa cells and immortalized mouse granulosa cells, we demonstrate a mechanism for the regulation of cyclin D2 at the level of transcription via a PKA-dependent signaling mechanism. The promoter activity of cyclin D2 was shown to be induced by FSH and the catalytic alpha subunit of PKA (PRKACA), and this activity was repressible by inducible cAMP early repressor (ICER), a cAMP response element (CRE) modulator isoform. In silico analysis of the mouse, rat, and human cyclin D2 promoters identified two CRE-binding protein sites, a conserved proximal element and a less conserved distal element relative to the translation start site. The mutation on the proximal element drastically decreases the effects of PRKACA and ICER on the promoter activity, whereas the mutation on the distal element did not contribute to the decrease in the promoter activity. Electrophoretic mobility shift assays and deoxyribonuclease footprint analysis confirmed ICER binding to the proximal element, and chromatin immunoprecipitation analysis demonstrated the occurrence of this binding in vivo. These results showed a CRE within the upstream region of Ccnd2 that is (at least partly) implicated in the stimulation and repression of cyclin D2 transcription. Finally, our data suggest that ICER involvement in the regulation of granulosa cell proliferation as overexpression of ICER results in the inhibition of PRKACA-induced DNA synthesis.

AB - Cyclin D2 (Ccnd2) is an essential gene for folliculogenesis, as null mutation in mice impairs granulosa cell proliferation in response to FSH. Ccnd2 mRNA is induced during the estrus cycle by FSH and is rapidly inhibited by LH. Yet, the responsive elements and transcription factors accounting for the gene expression of cyclin D2 in the ovary have not been fully characterized. Using primary cultures of rat granulosa cells and immortalized mouse granulosa cells, we demonstrate a mechanism for the regulation of cyclin D2 at the level of transcription via a PKA-dependent signaling mechanism. The promoter activity of cyclin D2 was shown to be induced by FSH and the catalytic alpha subunit of PKA (PRKACA), and this activity was repressible by inducible cAMP early repressor (ICER), a cAMP response element (CRE) modulator isoform. In silico analysis of the mouse, rat, and human cyclin D2 promoters identified two CRE-binding protein sites, a conserved proximal element and a less conserved distal element relative to the translation start site. The mutation on the proximal element drastically decreases the effects of PRKACA and ICER on the promoter activity, whereas the mutation on the distal element did not contribute to the decrease in the promoter activity. Electrophoretic mobility shift assays and deoxyribonuclease footprint analysis confirmed ICER binding to the proximal element, and chromatin immunoprecipitation analysis demonstrated the occurrence of this binding in vivo. These results showed a CRE within the upstream region of Ccnd2 that is (at least partly) implicated in the stimulation and repression of cyclin D2 transcription. Finally, our data suggest that ICER involvement in the regulation of granulosa cell proliferation as overexpression of ICER results in the inhibition of PRKACA-induced DNA synthesis.

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KW - Cyclic adenosine monophosphate

KW - Cyclin D2

KW - Follicle-stimulating hormone

KW - Folliculogenesis

KW - Gene regulation

KW - Granulosa

KW - Granulosa cells

KW - ICER

KW - Mechanisms of hormone action

KW - Ovarian

KW - cAMP

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U2 - 10.1095/biolreprod.105.049486

DO - 10.1095/biolreprod.105.049486

M3 - Article

VL - 75

SP - 279

EP - 288

JO - Biology of Reproduction

JF - Biology of Reproduction

SN - 0006-3363

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