Characterization of estrogen receptor-β (ERβ) 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 17 beta-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 ER beta is the predominant ERE-binding protein in granulosa cells. Western blotting results show that granulosa cells contain ER beta-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 ER beta isoforms with insertions within the coding region because RT-PCR assays revealed products with sizes expected for ER beta, ER beta B, and delta 3 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. ER beta 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 ER beta mRNA encoding the full-length ER beta protein. The generation of the P2-specific exon is likely due to initiation of transcription from an alternative promoter. Both P1- and Pa-specific exon-containing ER beta mRNAs are expressed in granulosa cells, and they are rapidly downregulated 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 ER beta mRNA species and that these mRNA species are capable of encoding the full-length ER beta protein.