Characterization of estrogen receptor-beta (ERbeta) messenger ribonucleic acid and protein expression in rat granulosa cells.
We have examined estrogen-responsiveness of ovarian granulosa cells by focusing on estrogen receptor (ER) expression. Estrogen responsiveness was determined by examining the effect of 17beta-estradiol (1-10 nM) on luciferase reporter activity in rat granulosa cells transfected with an ERE-luciferase construct. The results demonstrate an estrogen-induced (approximately 3-fold) increase in luciferase reporter activity, indicating that granulosa cells contain functional estrogen response element (ERE)-binding transcriptional activators. Gel mobility shift assays in combination with ER antibodies show that ERbeta is the predominant ERE-binding protein in granulosa cells. Western blotting results show that granulosa cells contain ERbeta-immunoreactive protein(s) migrating at a size substantially larger than the recombinant protein generated from the originally proposed 485 amino acid open-reading frame. This size discrepancy is not due to granulosa cell expression of ERbeta isoforms with insertions within the coding region because RT-PCR assays revealed products with sizes expected for ERbeta, ERbetaB, and delta3 isoforms. This size discrepancy appears to be due to usage of a well-conserved, upstream in-frame translation initiation codon (ATG436) leading to a 530 amino acid open reading frame. ERbeta messenger RNA (mRNA) characterization using 5'-rapid amplification of complementary DNA ends (5'-RACE) show the presence of two different (P1- and P2-) 5'-ends of rat ERbeta mRNA encoding the full-length ERbeta protein. The generation of the P2-specific exon is likely due to initiation of transcription from an alternative promoter. Both P1- and P2-specific exon-containing ERbeta mRNAs are expressed in granulosa cells, and they are rapidly down-regulated by the cAMP-mediated intracellular signaling pathway in cultured granulosa cells. Taken together, our results show that rat granulosa cells produce two different 3',5'-cAMP-regulated ERbeta mRNA species and that these mRNA species are capable of encoding the full-length ERbeta protein.
O'Brien
M L
ML
Graduate Center for Toxicology, University of Kentucky, Lexington 40536-0084, USA.
Park
K
K
In
Y
Y
Park-Sarge
O K
OK
D000818:Animals; D001483:Base Sequence / Q000235:genetics; D002478:Cells, Cultured; D004958:Estradiol / Q000494:pharmacology; D047629:Estrogen Receptor beta; D005091:Exons / Q000502:physiology; D005260:Female; D006107:Granulosa Cells / Q000187:drug effects / Q000378:metabolism*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010053:Ovary / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011960:Receptors, Estrogen / Q000235:genetics* / Q000378:metabolism*; D014020:Tissue Extracts / Q000378:metabolism; D014158:Transcription, Genetic; D014599:Uterus / Q000737:chemistry
D047629:Estrogen Receptor beta; D012333:RNA, Messenger; D011960:Receptors, Estrogen; D014020:Tissue Extracts; D004958:Estradiol; D008156:Luciferases