GENERATION AND CHARACTERIZATION OF A HUMAN OSTEOSARCOMA CELL-LINE STABLY TRANSFECTED WITH THE HUMAN ESTROGEN-RECEPTOR GENE

Although 17 beta-estradiol (E(2)) replacement therapy has been shown t o be effective in treating postmenopausal osteoporosis, the underlying mechanism remains unclear. The presence of low levels of functional e ndogenous estrogen receptor (ER) in some osteoblastic cells has been d emonstrated, and the suggestion that the abundance of ER may be rate-l imiting in the action of E(2) on these cells has been made. To study t he mechanism of ER in regard to E(2)-mediated effects, we stably trans fected a human osteosarcoma cell line, SaOS-2, with an expression vect or, pMV-7-ER, containing the human ER gene. We characterized six of th e stably transfected clones. One of the stable clones, SaOS-2-ER, expr essed extra copies of ER genes integrated into the genome as detected by Southern blot analysis, showed a significantly increased level of E R mRNA by RT-PCR, and contained an increased level of ER cytosolic pro tein as detected by an ER-specific EIA. The overexpressed ER was funct ional and sensitive to E(2) in a dose-dependent fashion after transien t transfection with a vector containing an estrogen response element ( ERE) linked to a chloramphenicol acetyltransferase (CAT) reporter gene . Scatchard analysis revealed a single high-affinity binding site with a K-d similar to values obtained for the ER in MCF-7 breast cancer ce lls. These SaOS-2-ER cells had altered osteoblast phenotypic features including growth inhibition, decreased basal alkaline phosphatase acti vity, and decreased IL-6 expression and secretion. In response to E(2) , a greater than 2-fold increase in TGF-beta 1 mRNA was quantitatively measured in these ER-overexpressing osteoblasts. These cells may prov ide a sensitive and unique model for understanding the mechanism of E( 2) and ER in overall bone metabolism.