The classical and a non-classical pathways associated with NF-kappaB are involved in estrogen-mediated regulation of calbindin-D9k gene in rat pituitary cells.
Calbindin-D9k (CaBP-9k) is a high affinity calcium binding protein that is highly expressed in the duodenum, kidney, uterus, and pituitary glands. Previous studies have shown that CaBP-9k expression is regulated by several steroid hormones, such as 1,25-dihydroxyvitamin D3, glucocorticoids, 17beta-estradiol (E2), and progesterone (P4), in a tissue-specific manner. However, the promoter elements that mediate transcriptional regulation by these steroid hormones are not clearly understood, mainly due to the lack of an appropriate cell line expressing CaBP-9k. Recently it was shown that CaBP-9k was constitutively expressed in the rat pituitary gland, and is expressed in an E2-dependent manner in a pituitary gland tumor-derived cell line, GH3. In the current study, we examined the activity of the estrogen responsive element (ERE) in rat CaBP-9k gene in GH3 cells, using a luciferase gene reporter assay, electrophoretic mobility shift assay, and mutagenesis. A nuclear factor-kappaB (NF-kappaB) binding site in the CaBP-9k promoter region was identified (nucleotides -848 to -834 from the transcriptional start site), and we demonstrated that addition of an NF-kappaB blocker to GH3 cells reduced E2-induced CaBP-9k transcription. In the present study, we further showed a previously reported imperfect ERE (nucleotides +51 to +65) between exon I and intron A of CaBP-9k, indicating that the interaction of estrogen receptor (ER) alpha with this region is involved in the regulation of CaBP-9k promoter activity and its expression. Taken together, these results suggest that in GH3 cells, both the classical ERalpha-ERE pathway and a non-classical pathway involving NF-kappaB are involved in E2-mediatd regulation of CaBP-9k expression in the pituitary gland.
Lee
Geun-Shik
GS
Laboratory of Veterinary Biochemistry and Molecular Biology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk 361-763, Republic of Korea.
Choi
Kyung-Chul
KC
Han
Ho-Jae
HJ
Jeung
Eui-Bae
EB
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D064026:Calbindins; D047628:Estrogen Receptor alpha / Q000378:metabolism; D004967:Estrogens / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D010902:Pituitary Gland / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D020218:Response Elements; D064030:S100 Calcium Binding Protein G / Q000235:genetics* / Q000378:metabolism*; D051996:Transcription Factor RelA / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects