Common target for 12-O-tetradecanoylphorbol-13-acetate and ras in the transcriptional enhancer of the growth factor-inducible JE gene.

JE gene expression in the mouse osteoblast cell line MC3T3-E1 is activated transiently by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). In ras-transformed MC3T3-E1 cells the JE gene is constitutively expressed at a high level, whereas in their raf-transformed counterparts it is not constitutively expressed or inducible by TPA. Using these cells, we investigated a specific sequence recognized by nuclear factors in the 5'-upstream region of the rat JE gene. By gel-mobility shift assays, we determined that the amount of nuclear factors that bind to the region -130 to -96 bp upstream from the cap site of the rat JE gene (JE-1 probe) increased after TPA treatment of MC3T3-E1 cells. However, in the ras transformants it was elevated constitutively, and in the raf transformants it was not detectable. Both unlabeled JE-1 probe and a probe containing a TPA-responsive element (TGACTCA) competed with the binding of these nuclear factors. Preincubation of the nuclear extracts with fos- or jun-specific antibodies interfered with the binding of the factors to the JE-1 probe. The essential sequence in the JE-1 element for the binding of nuclear factors was found to be TTACTCA. c-fos and c-jun proteins synthesized in vitro could bind to the DNA fragment containing this sequence, but the binding was weaker than to the TPA-responsive element (TGACTCA). These results suggest that the sequence TTACTCA in the JE-1 element is a common target of TPA and ras and that protein complexes containing fos- and jun-related proteins recognize the sequence.