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 cellular 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, progression 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, evaluations of the signaling and transcriptional regulation of the approximately 2.0 kb promoter region of the PEG-3 gene, PEG-Prom, was undertaken. The full-length and various mutated regions of the PEG-Prom were linked to a luciferase reporter construct and tested for promoter activity in CREF and oncogene-transformed CREF cells. An analysis was also performed using CREF cells doubly 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-3. The levels of PEA3 and PEG-3 RNA and proteins are elevated in the oncogenically transformed CREF cells, and reduced in transformation and tumorigenic suppressed Ha-ras/Krev-1 doubly transformed CREF cells. Enhanced tumorigenic behavior, PEG-3 promoter function and PEG-3 expression in Ha-ras transformed cells were all dependent upon increased activity within the mitogen-activated protein kinase (MAPK) pathway. Electrophoretic mobility shift assays and DNase I footprinting experiments indicate that PEA3 binds to sites within the PEG-Prom in transformed rodent cells in an area adjacent to the TATA box in a MAPK-dependent fashion. These findings demonstrate an association between Ha-ras and v-raf transformation of CREF cells with elevated PEA3 and PEG-3 expression, and they implicate MAPK signaling via PEA3 as a signaling cascade involved in activation of the PEG-Prom.
Su
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Department of Urology, Department of Pathology and Department of Neurosurgery, Herbert Irving Comprehensive Cancer Center, Columbia University, College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032, USA.
Shi
Y
Y
Friedman
R
R
Qiao
L
L
McKinstry
R
R
Hinman
D
D
Dent
P
P
Fisher
P B
PB
D000818:Animals; D000943:Antigens, Differentiation; D001483:Base Sequence; D002461:Cell Line, Transformed; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D003260:Contact Inhibition / Q000187:drug effects; D004247:DNA / Q000235:genetics / Q000378:metabolism; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005419:Flavonoids / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D011905:Genes, ras / Q000235:genetics*; D020935:MAP Kinase Signaling System / Q000187:drug effects; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D027384:Oncogene Proteins v-raf; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D020218:Response Elements / Q000235:genetics*; D015682:Retroviridae Proteins, Oncogenic / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism*
D000943:Antigens, Differentiation; D004268:DNA-Binding Proteins; D005419:Flavonoids; D009363:Neoplasm Proteins; C489287:Ppp1r15a protein, rat; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D015682:Retroviridae Proteins, Oncogenic; D014157:Transcription Factors; C065788:transcription factor PEA3; D004247:DNA; D027384:Oncogene Proteins v-raf; D020928:Mitogen-Activated Protein Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one