ESTROGEN RESPONSE IN THE HFOB-1.19 HUMAN FETAL OSTEOBLASTIC CELL-LINESTABLY TRANSFECTED WITH THE HUMAN ESTROGEN-RECEPTOR GENE

The gene coding for the human wild-type estrogen receptor (ER) was sta bly transfected into the human fetal osteoblastic cell line hFOB 1.19, a clonal cell line which is conditionally immortilized with a tempera ture sensitive mutant of SV40 large T antigen (tsA58). Five subclones were obtained which express various levels of ER mRNA and protein. The subclone with the highest level of functional (nuclear bound) ER, hFO B/ER9, contained 3,931 (+/-1,341) 17 beta-estradiol molecules bound/nu cleus as determined by the nuclear binding (NB) assay. Using the dextr an coated charcoal (DCC) method, the level of total cytosolic ER measu red was 204 (+/-2) fmol/mg protein. This subclone was examined further for estradiol (E(2)) responsiveness. The ER expressed in hFOB/ER9 cel ls was shown to be functional using a transiently transfected ERE-TK-l uciferase construct. Expression of luciferase from this construct incr eased similar to 25-fold in hFOB/ER9 cells following 10(-9)M E(2) trea tment. This effect on ERE-TK-luciferase expression was both dose and s teroid dependant. Further, treatment of hFOB/ER9 cells with 10(-9)M E( 2) resulted in a 2.5-4.0-fold increase in endogenous progesterone rece ptor (PR) levels detected by steroid binding assays, and a noticeable increase in both the A and B forms of PR by western blot assay. The es tablishment of this estrogen responsive human osteoblastic cell line s hould provide an excellent model system for the study of estrogen acti on on osteoblast function. (C) 1995 Wiley-Liss, Inc.