A new system that analyzes erythropoietin-mediated early signal transduction: transfection of the c-fos enhancer.promoter-luciferase gene into a murine erythroid cell line.
Erythropoietin (Epo) exerts its effects by binding specific receptors on the surface of reactive cells. However, the signal transduction system after binding has not been well described. To develop a system to analyze the steps of signal transduction, we transfected the human c-fos-enhancer/promoter linked with the Photinus pyralis luciferase gene (pfosluc2) into a murine erythroleukemia cell line ELM-I-1, in which we previously showed that c-fos mRNA is rapidly induced upon Epo-stimulation. A stable transfectant was obtained. The cells transfected with pfosluc2 were stimulated with Epo and luciferase activity in the cells was measured as light intensity. The light intensity integrated for 2 min (LI2.0) was 3202 +/- 80 unit/1.5 x 10(5) cells before stimulation. This increased up to 5869 +/- 321 unit/1.5 x 10(5) cells by incubating the cells with 5 U/ml Epo for 2 h. After Epo stimulation, light intensity began to increase at 30 min, reached a peak (about 1.8 times the basal level) at 120 min, and then gradually dropped. The effect of Epo was dose-dependent; significant action occurred at as low as 0.5 U/ml, with a maximum at 5 U/ml. A similar response was observed when the cells were stimulated with interleukin-3 (IL-3) although the response was apparently lower than that with Epo. It was also found that IL-3 had an additive action with Epo on c-fos activity in this system. Thus, the above method was proven to be simple, rapid and sensitive enough to use to determine the early phase of signal transduction of Epo.
Tsuda
H
H
Division of Clinical Haematology/Immunology, Kumamoto City Hospital, Japan.
Huang
R W
RW
Konishi
M
M
Mizukami
T
T
Yoko-o
Y
Y
Hasegawa
M
M
Sakaguchi
M
M
Kawakita
M
M
Takatsuki
K
K
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004742:Enhancer Elements, Genetic; D004921:Erythropoietin / Q000502:physiology*; D005786:Gene Expression Regulation; D016762:Genes, fos; D005822:Genetic Vectors; D066298:In Vitro Techniques; D007377:Interleukin-3 / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D015398:Signal Transduction; D014158:Transcription, Genetic
D007377:Interleukin-3; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D004921:Erythropoietin; D008156:Luciferases