PEA3 sites within the progression elevated gene-3 (PEG-3) promoter and mitogen-activated protein kinase contribute to differential PEG-3 expression in Ha-ras and v-raf oncogene transformed rat embryo cells

Transformation of normal cloned rat embryo fibroblast (CREF) cells with cel lular oncogenes results in acquisition of anchorage-independent growth and oncogenic potential in nude mice. These cellular changes correlate with an induction in the expression of a cancer progression-promoting gene, progres sion elevated gene-3 (PEG-3). To define the mechanism of activation of PEG- 3 as a function of transformation by the Ha-ras and v-raf oncogenes, evalua tions of the signaling and transcriptional regulation of the similar to2.0 kb promoter region of the PEGS gene, PEG-From, was undertaken. The full-len gth and various mutated regions of the PEG-From were linked to a luciferase reporter construct and tested for promoter activity in CREF and oncogene-t ransformed CREF cells. An analysis was also performed using CREF cells doub ly transformed with Ha-ras and the Ha-ras specific suppressor gene Krev-1, which inhibits the transformed phenotype in vitro. These assays document an association between expression of the transcription regulator PEA3 and PEG S. The levels of PEA3 and PEG-3 RNA and proteins are elevated in the oncoge nically transformed CREF cells, and reduced in transformation and tumorigen ic suppressed Ha-ras/Krev-1 doubly transformed CREF cells. Enhanced tumorig enic behavior, PEG-3 promoter function and PEG-3 expression in Ha-ras trans formed cells were all dependent upon increased activity within the mitogen- activated protein kinase (MAPK) pathway. Electrophoretic mobility shift ass ays and DNase I footprinting experiments indicate that PEA3 binds to sites within the PEG-From in transformed rodent cells in an area adjacent to the TATA box in a MAPK-dependent fashion. These findings demonstrate an associa tion between Ha-ras and v-raf transformation of CREF cells with elevated PE A3 and PEG-3 expression, and they implicate MAPK signaling via PEA3 as a si gnaling cascade involved in activation of the PEG-From.