Regulation of phorbol ester-mediated TRAF1 induction in human colon cancer cells through a PKC/RAF/ERK/NF-kappaB-dependent pathway.
Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are cytoplasmic adapter proteins that link a wide variety of cell surface receptors to the apoptotic signaling cascade. The purpose of this study was to delineate the signaling pathways and TRAF1 promoter elements responsible for phorbol ester-mediated TRAF1 induction in human colon cancers. Here, we found that the PKC activators, phorbol 12-myristate 13-acetate (PMA) and bryostatin I, induced TRAF1 mRNA expression; pretreatment with actinomycin D blocked PMA-mediated TRAF1 expression suggesting induction at the transcriptional level. In contrast, expression of other TRAFs (TRAF2, 3 and 4) was minimally altered by PMA. Various PKC isoform-selective inhibitors blocked PMA-mediated TRAF1 mRNA and promoter stimulation; rottlerin, a selective PKCdelta inhibitor, had no effect suggesting that Ca(2+)-dependent PKC isoforms (e.g., PKCalpha and betaI) play a role in TRAF1 regulation. In addition, the MEK/ERK inhibitors, PD98059 and UO126, suppressed PMA-stimulated TRAF1 promoter activity indicating a role for ERK in TRAF1 induction. Moreover, cotransfection of a dominant-negative Raf-1 (Raf-C4) significantly reduced PMA-stimulated TRAF1 promoter activity whereas transfection of dominant-negative Ras or treatment with Ras inhibitors had minimal to no effect on TRAF1 induction suggesting dependence on Raf, but not Ras, activation. Finally, site-specific mutagenesis of functional NF-kappaB sites (particularly the most proximal site) in the TRAF1 promoter significantly decreased PMA-mediated promoter activity. In conclusion, our results demonstrate selective induction of TRAF1 in human colon cancer cells through a Ca(2+)-dependent PKC/Raf-1/ERK/NF-kappaB-dependent pathway.
Department of Surgery and Sealy Center for Cancer Cell Biology, The University of Texas Medical Branch, Galveston, TX 77555, USA.
Xiaofu
X
Wang
Qingding
Q
Wang
Wanqin
W
Hu
B Mark
BM
Evers
D045744:Cell Line, Tumor; D003110:Colonic Neoplasms; D003609:Dactinomycin / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000378:metabolism; D011506:Proteins / Q000187:drug effects / Q000235:genetics*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D047990:TNF Receptor-Associated Factor 1; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects
D004791:Enzyme Inhibitors; D005419:Flavonoids; D016328:NF-kappa B; D011506:Proteins; D047990:TNF Receptor-Associated Factor 1; D003609:Dactinomycin; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D013755:Tetradecanoylphorbol Acetate; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one