Transcriptional regulation of cyclin D2 by the PKA pathway and inducible cAMP early repressor in granulosa cells.
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.
Muñiz
Luis C
LC
Department of Biochemistry and Molecular Biology and Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, New Jersey 07103, USA.
Yehia
Ghassan
G
Mémin
Elisabeth
E
Ratnakar
Pillarisetty V A L
PV
Molina
Carlos A
CA
D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D051786:Cyclic AMP Response Element Modulator / Q000235:genetics / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D056742:Cyclin D2; D016213:Cyclins / Q000235:genetics* / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis; D005260:Female; D005786:Gene Expression Regulation; D006107:Granulosa Cells / Q000502:physiology*; D006801:Humans; D051379:Mice; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D015398:Signal Transduction; D014158:Transcription, Genetic
C486197:Ccnd2 protein, rat; C497412:Crem protein, rat; D056742:Cyclin D2; D016213:Cyclins; D012097:Repressor Proteins; D051786:Cyclic AMP Response Element Modulator; D004247:DNA; D017868:Cyclic AMP-Dependent Protein Kinases