Restoration of estrogen-dependent progesterone receptor expression in a uterine myocyte cell line.
Although the in vivo effect of estrogen on myometrial differentiation is well documented, estrogen effects on primary myocytes in vitro have been difficult to demonstrate. To construct a stable uterine myocyte system, capable of direct estrogen responsiveness, we used a transformed hamster myometrial cell line. Since these cells expressed a low level of estrogen receptors (ERs), we have stably transfected them with a vector for the human ER. After transfection, ER concentration increased from less than 300 sites per cell to 17,000 +/- 2,000 sites per cell (mean +/- SEM). To test the functional integrity of the transfected receptors, a chloramphenicol acetyltransferase gene linked to an estrogen response element upstream of thymidine kinase promoter was transiently transfected, and the amount of chloramphenicol acetyltransferase activity, an indicator of estrogen responsiveness, was found to increase 20-fold in response to 17 beta-estradiol (1 nM for 48 h). Furthermore, we tested the ability of estrogen to activate endogenous genes by measuring progesterone receptor (PR) induction. PR concentration in the transfected cells was 3,700 +/- 800 and increased 9-fold to 33,000 +/- 6,000 with 17 beta-estradiol (2 nM). This receptor density increase was confirmed by immunoblotting. PR induction was maximal at 16 h, was concentration dependent, and was not elicited by tamoxifen or ICI 164,384. We conclude that transformed hamster myocytes transfected with an ER gene are capable of estrogen-dependent PR expression in vitro and may serve as a useful system to study estrogen effect on myocytes.
Sadovsky
Y
Y
Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco 94143.
Kushner
P J
PJ
Roberts
J M
JM
Riemer
R K
RK
D000818:Animals; D002461:Cell Line, Transformed; D004958:Estradiol / Q000502:physiology*; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism*; D011980:Receptors, Progesterone / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014599:Uterus / Q000166:cytology / Q000378:metabolism*
D011980:Receptors, Progesterone; D004958:Estradiol