Molecular analysis of estrogen induction of preproenkephalin gene expression and its modulation by thyroid hormones

Estrogen receptors (ER) and thyroid hormone receptors (TR) are ligand-dependent nuclear transcription factors. Estrogen-induced preproenkephalin (PPE) gene expression in the hypothalamus is directly related to estrogen-induced lordosis behavior in the rat. In the present study, we showed that the PPE mRNA level in the ventromedial hypothalamus of female rats was significantly decreased by ovariectomy. This decrease was reversed by estrogen replacement in a dose- and time-dependent manner. Using transient transfection and electrophoretic mobility shift assays (EMSA), functional estrogen response elements (ERE) were identified between -437 and -145 base pairs (bp) of the rat PPE gene promoter region. Two ERE-like elements are present between -405 and -364 of the rat PPE gene promoter, which bind ERalpha as demonstrated by EMSA. Estrogen produced a dose-dependent increase in CAT activity in cotransfection assays with ERalpha expression vector and a 437PPE-CAT reporter construct containing 437 bp of the rat PPE gene promoter and the CAT reporter gene. This estrogen-induced PPE promoter activity was inhibited by liganded-TR in transient cotransfection assays. Analysis of DNA-protein interactions by EMSA revealed that both ERalpha and TR (alpha1 and beta1) could bind to the EREs in the rat PPE gene promoter. Furthermore, estrogen induction of PPE mRNA in the ventromedial hypothalamus of the ovariectomized female rat was significantly attenuated by concomitant administration of triiodothyronine. These results suggest that estrogen regulation of the hypothalamic PPE gene expression is mediated through an estrogen-receptor complex directly interacting with the functional EREs in its promoter region; and that this estrogen effect can be modified by thyroid hormones.