With the knowledge that estrogen replacement therapy can circumvent postmenopausal osteoporosis and with the discovery of estrogen receptors (ER) in cultures of normal osteoblast-like cells, extensive investigations have been directed toward understanding the role of the ER in normal bone homeostasis. ROS 17/2.8 and UMR-106-01, two established osteoblast-like cell lines derived from rat osteosarcomas, have been shown to have estrogen-regulated biologic responses. Only the ROS 17/2.8 cell 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, [125I]17β-estradiol. An initial immunoconcentrtion step before western blot analysis also allowed detection of the full-length ER protein. In addition, northern blot analysis indicated that the entire ER transcript was expressed and that the half-life of the ER message was increased following cycloheximide treatment. Message levels were also regulated by removal of serum and treatment with estradiol. An estrogen-regulated reporter vector, ERET81CAT, was transfected into the UMR-106-01 cells to determine whether the detected level of ER was transcriptionally functional. Using this assay, estrogen responsiveness was evident; however, 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 available to induce the CAT response. When the cells are responsive, the ICI 164,384 antagonist could block the estrogen-induced activation of CAT.

The ROS 17/2.8 cells were also analyzed in parallel with the UMR-106-01 cells to allow comparisons between these two osteoblast-like cell lines because they exhibit phenotypes for two unique stages of differentiation. ROS 17/2.8 cells were found to contain more receptor sites/cell by the 125I-E2 (estradiol) binding assays, as well as higher levels of ER-specific transcripts, than UMR-106-01 cells (two- to threefold). This level of ER was consistently able to modulate estrogen-induced 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 these osteoblast-like cells. These data also indicate that levels of ER that are low or undetectable by conventional methods are able to mediate biologic responses through direct interactions of the ER with the specific DNA response element.