Effect of estrogen agonists and antagonists on induction of progesterone receptor in a rat hypothalamic cell line

Estrogen is essential in the hypothalamus for the central regulation of rep roduction. To understand the molecular mechanism(s) of estrogen action in t he hypothalamus, immortalized rat embryonic hypothalamic cell lines were ch aracterized for steroid receptors and subcloned. Scatchard analysis of the D12 subclone demonstrated one high affinity estrogen receptor-binding site (K-d = 31.3 +/- 1.9 pM) with a B-max of 30.8 +/- 0.8 fmol/mg. Estrogen rece ptor-alpha protein was identified by Western blot and gel shift analyses. T reatment with estradiol (48 h) stimulated progesterone receptor (PR) messen ger RNA expression and binding to [H-3]R5020, a synthetic progestin. Becaus e the agonist or antagonist activity of estrogen mimetics can be cell type dependent, the activities of various estrogen mimetics were determined in D 12 cells. ICI 182,780 (IC50 = 0.63 nM), raloxifene (IC50 = 1 nM), enclomiph ene (IC50 = 77 nM), and tamoxifen (IC50 = 174 nM) inhibited the induction o f PR by estradiol, and none of these compounds significantly stimulated PR when given alone. In contrast, 17 alpha-ethynyl estradiol (EC50 = 0.014 nM) , zuclomiphene (EC50 = 100 nM), and genistein (EC50 = 17.5 nM) functioned a s estrogen agonists in these cells. In addition, the estrogen-induced proge sterone receptor activated a progesterone response element reporter constru ct in response to progestins. Thus, the D12 rat hypothalamic cell line prov ides a useful model for characterizing tissue-selective estrogenic compound s, identifying estrogen- and progesterone-regulated hypothalamic genes, and understanding the molecular mechanisms of steroid action in various physio logical processes mediated by the hypothalamus.