PROMOTER AND SPECIES-SPECIFIC DIFFERENTIAL ESTROGEN-MEDIATED GENE-TRANSCRIPTION IN THE UTERUS AND CULTURED-CELLS USING STRUCTURALLY ALTEREDAGONISTS

Certain types of estrogenic compounds have been shown to have tissue-s pecific actions. In addition, some tissues may exhibit differential ge ne regulation by agonists and antagonists. Our previous studies using structurally modified estrogenic molecules had indicated differential effects on specific estrogen responses, indicating that the activity o f the estrogen receptor protein can be altered depending not only upon the structure of the bound ligand but also the regulated gene itself. The mechanism of differential induction, however, was not determined, and might involve altered binding to the estrogen response element (E RE), altered transcription, or post-transcriptional modification of ge ne products. Our previous studies indicated that differential inductio n by modified diethylstilbestrol (DES) agonists could not be accounted for by differences in ligand affinity for the estrogen receptor (ER) or differential binding of the ER to a consensus vitellogenin A2 (vit A2) ERE. To determine if this differential hormonal responsiveness was reflected at the level of transcription, we analyzed mouse uterine mR NA of several estrogen-responsive genes, including glucose-6-phosphate dehydrogenase (G6PD), ornithine decarboxylase (ODC) and lactoferrin, by Northern blot following injection with the modified agonists DES, i ndenestrol A (IA), indenestrol B (IB) and Z-pseudo DES (ZPD). All comp ounds induced the G6PD message, although IB and ZPD induced expression only transiently, while DES and IA maintained the message for 24 h. N o difference in induction was seen for ODC message, which was induced equally by all the compounds. In contrast, lactoferrin, a highly estro gen-responsive gene, was induced only by DES and IA and not by the oth er agonists IB or ZPD, showing that the lactoferrin gene was different ially regulated by these compounds. To determine whether this differen ce was due to altered transcriptional activity, the mouse lactoferrin estrogen-responsive module (mERM) linked to a chloramphenicol acetyl t ransferase (CAT) reporter gene was tested in transfected cells. Using the mouse estrogen receptor in RL95 cells, DES and IA induced expressi on of CAT, but IB did not, confirming the differential response seen i n vivo. To show whether this difference in transcription occurred beca use of altered binding to the lactoferrin ERE, which is not a perfect consensus ERE, a gel shift assay was used to examine DNA binding of ER bound to the agonists. All ligands produced equivalent binding to the lactoferrin ERE suggesting that differential regulation was not a res ult of altered DNA binding. Taken together, these observations indicat e that the differential induction of lactoferrin by these compounds oc curs via altered activation of the transcriptional components unique t o lactoferrin and is likely to involve altered interaction with co-act ivators. Surprisingly, unlike the mouse ER, the human estrogen recepto r activated and induced expression of lactoferrin estrogen-responsive module-CAT with all the compounds. Mouse ER is also known to vary from the human ER in its activity with the triphenylethylene estrogen tamo xifen, which has agonist activity with the mouse ER but mixed antagoni st/agonist activity with the human ER. The data show that human and mo use estrogen receptors are activated differently by this group of stil bestrol estrogen ligands when assayed on the lactoferrin response elem ent, which is the first description of this type of gene and species s pecific difference. Lactoferrin gene regulation by estrogen receptor c an be used as a model to study the mechanism of differential gene acti vation by different estrogen agonists and antagonists using a more phy siological situation than commonly used with in vitro gene reporter sy stems.