A signal transduction pathway for activation of the mdr1 promoter involves the proto-oncogene c-raf kinase

To investigate the regulation of expression of the human mdr1 gene, the response of the mdr1 promoter to signals involved in cell proliferation was examined. The activity of the mdr1 promoter was measured in transiently transfected NIH 3T3 cells, which were stimulated to enter the cell cycle by addition of serum, platelet-derived growth factor, or transforming growth factor alpha. Addition of serum to quiescent NIH 3T3 cells resulted in a 6-8-fold activation of mdr1 promoter activity, which peaked at 10 h, just prior to S-phase. Treatment of serum-starved cells with the serum mitogens platelet-derived growth factor and transforming growth factor alpha also activated the mdr1 promoter. To define components of the signal cascade resulting in mdr1 promoter activation, the role of c-Raf kinase, a serine/threonine kinase important in mitogen-activated signal transduction, was examined. We measured the effects of a constitutively activated Raf kinase, v-raf, and a dominant negative Raf mutant, c-Raf-C4, on mdr1 promoter activity in NIH 3T3 and HepG2 cells. In serum-starved NIH 3T3 cells, v-raf increased mdr1 promoter activity approximately 10-fold compared to a v-raf frame-shift control. Raf responsiveness of the mdr1 promoter was localized to sequences between -69 and -41, relative to the initiation site. Serum stimulation of the mdr1 promoter was blocked by co-transfection of NIH 3T3 cells with the dominant negative Raf mutant c-Raf-C4. In the human hepatoma cell line HepG2, which has high endogenous Raf kinase activity (Bruder, J. T., Heidecker, G., and Rapp, U. R. (1992) Genes & Dev. 6, 545-556), co-transfection with c-Raf-C4 decreased mdr1 promoter activity 20-fold. Taken together, these data suggest that the mdr1 gene is transcriptionally regulated by normal cellular signaling events involving activation of c-Raf kinase.