CORRELATION BETWEEN LOW-LEVELS OF ESTROGEN-RECEPTORS AND ESTROGEN RESPONSIVENESS IN 2 RAT OSTEOBLAST-LIKE CELL-LINES

With the knowledge that estrogen replacement therapy can circumvent po stmenopausal osteoporosis and with the discovery of estrogen receptors (ER) in cultures of normal osteoblast-like cells, extensive investiga tions have been directed toward understanding the role of the ER in no rmal bone homeostasis. ROS 17/2.8 and UMR-106-01, two established oste oblast-like cell lines derived from rat osteosarcomas, have been shown to have estrogen-regulated biologic responses. Only the ROS 17/2.8 ce ll line has been reported to contain ER. In this study, high-affinity, saturable binding sites characteristic of the ER were detected in UMR -106-01 cells by binding assays with the high-affinity ligand, [I-125] 17 beta-estradiol, An initial immunoconcentrtion step before western b lot analysis also allowed detection of the full-length ER protein. In addition, northern blot analysis indicated that the entire ER transcri pt was expressed and that the half-life of the ER message was increase d following cycloheximide treatment. Message levels were also regulate d by removal of serum and treatment with estradiol. An estrogen-regula ted reporter vector, ERET81CAT, was transfected into the UMR-106-01 ce lls to determine whether the detected level of ER was transcriptionall y functional. Using this assay, estrogen responsiveness was evident; h owever, the response was inconsistent. Multiple factors, such as serum , estradiol, and cell density, influence the ER levels in these cells and probably cause fluctuations in the abundance of receptors availabl e to induce the CAT response. When the cells are responsive, the ICI 1 64,384 antagonist could block the estrogen-induced activation of CAT. The ROS 17/2.8 cells were also analyzed in parallel with the UMR-106-0 1 cells to allow comparisons between these two osteoblast-like cell li nes because they exhibit phenotypes for two unique stages of different iation. ROS 17/2.8 cells were found to contain more receptor sites/cel l by the I-125-E(2) (estradiol) binding assays, as well as higher leve ls of ER-specific transcripts, than UMR-106-01 cells (two- to threefol d). This level of ER was consistently able to modulate estrogen-induce d stimulation of the reporter CAT vector. Therefore, functional ER is expressed in both cell types, but the higher level of receptors found in the ROS 17/2.8 cell line improves the estrogen responsiveness of th ese osteoblast-like cells. These data also indicate that levels of ER that are low or undetectable by conventional methods are able to media te biologic responses through direct interactions of the ER with the s pecific DNA response element.