Estrogen activates raf-1 kinase and induces expression of Egr-1 in MCF-7 breast cancer cells

We have investigated whether the raf-1 kinase, a downstream mediator of bot h receptor tyrosine kinase and protein kinase C signalling, is activated by estrogen (E2) in an estrogen receptor positive human breast cancer cell li ne. Autophosphorylation of raf-1 kinase was studied after treatment of MCF- 7 cells with E2. E2-deprived cells contained low levels of raf-1 kinase act ivity. Treatment of cells for 1 min with E2 resulted in raf-1 autophosphory lation which was maximal within 5 min. Western blot analysis showed that ra f-1 undergoes an electrophoretic mobility shift following E2 treatment. Egr -1 is a zinc finger-containing transcription factor which is expressed in a ssociation with raf-1 activation. Untreated MCF-7 cells expressed low level s of Egr-1 while E2 treatment resulted in an induction of egr-1 mRNA expres sion. These kinetics followed closely behind the E2 induction of c-myc mRNA . Egr-1 protein was similarly low in E2-deprived MCF-7 cells and was transi ently increased following E2 treatment. Several studies have suggested that kinase activity may play a role in estrogen receptor (ER) activation. Whil e activated v-raf failed to augment ER activation of transcription in trans ient transfection assays, a dominant negative mutant of raf-1 inhibited E2- induced transcription by 50% primarily as a result of increased baseline le vels of E2 independent transcription. The results show that E2 can induce r af-1 kinase activity in MCF-7 breast cancer cells associated with the expre ssion of an early growth response gene and modulation of ER signalling.