Correlation between low levels of estrogen receptors and estrogen responsiveness in two rat osteoblast-like cell lines.
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]17 beta-estradiol. An initial immunoconcentration 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Davis
V L
VL
Receptor Biology Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina.
Couse
J F
JF
Gray
T K
TK
Korach
K S
KS
D000818:Animals; D001665:Binding Sites; D015152:Blotting, Northern; D015153:Blotting, Western; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004958:Estradiol / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D004965:Estrogen Antagonists / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D005822:Genetic Vectors; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D012516:Osteosarcoma / Q000473:pathology; D053284:Polyunsaturated Alkamides; D051381:Rats; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured
D004965:Estrogen Antagonists; D053284:Polyunsaturated Alkamides; D011960:Receptors, Estrogen; D004958:Estradiol; C051781:ICI 164384; D015500:Chloramphenicol O-Acetyltransferase