The classical and a non-classical pathways associated with NF-κB are involved in estrogen-medicated 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, 17β-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-κB (NF-κB) 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-κB 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) α 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 ERα-ERE pathway and a non-classical pathway involving NF-κB are involved in E2-mediatd regulation of CaBP-9k expression in the pituitary gland.