Abstract
Protein kinase C (PKC) triggers cellular signals that regulate proliferation or death in a cell- and stimulus-specific manner. Although previous studies have demonstrated that activation of PKC with phorbol 12-myristate 13-acetate (PMA) protects cells from apoptosis induced by a number of mechanisms, including death receptor ligation, little is known about the effect or mechanism of PMA in the necrotic cell death. Here, we demonstrate that PMA-mediated activation of PKC protects against tumor necrosis factor (TNF)-induced necrosis by disrupting formation of the TNF receptor (TNFR)1 signaling complex. Pretreatment with PMA protected L929 cells from TNF-induced necrotic cell death in a PKC-dependent manner, but it did not protect against DNA-damaging agents, including doxorubicin (Adriamycin) and camptothecin. Analysis of the upstream signaling events affected by PMA revealed that it markedly inhibited the TNF-induced recruitment of TNFR1-associated death domain protein (TRADD) and receptor-interacting protein (RIP) to TNFR1, subsequently inhibiting TNF-induced activation of nuclear factor-κB and c-Jun NH2-terminal kinase (JNK). However, JNK inhibitors do not significantly affect TNF-induced necrosis, suggesting that the inhibition of JNK activation by PMA is not part of the antinecrotic mechanism. In addition, PMA acted as an antagonist of TNF-induced reactive oxygen species (ROS) production, thereby suppressing activation of ROS-mediated poly(ADP-ribose)polymerase (PARP), and thus inhibiting necrotic cell death. Furthermore, during TNF-induced necrosis, PARP was significantly activated in wild-type mouse embryonic fibroblast (MEF) cells but not in RIP-/- or TNFR-associated factor 2-/-MEF cells. Taken together, these results suggest that PKC activation ensures effective shutdown of the death receptor-mediated necrotic cell death pathway by modulating formation of the death receptor signaling complex.
Footnotes
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This work was supported by Korea Research Foundation grant KRF-2004-041-E00078 and Chungnam National University Hospital Research Fund, 2005.
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ABBREVIATIONS: TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor; TRADD, tumor necrosis factor receptor 1-associated death domain; FADD, FAS-associated death domain; TRAF, tumor necrosis factor receptor-associated factor; RIP, receptor-interacting protein; NF-κB, nuclear factor-κB; NCD, necrotic cell death; FasL, Fas ligand; ROS, reactive oxygen species; MEF, mouse embryonic fibroblast; PKC, protein kinase C; DAG, diacylglycerol; PMA, phorbol 12-myristate 13-acetate; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; ERK, extracellular signal-regulated kinase; PARP, poly(ADP-ribose)polymerase; GFP, green fluorescent protein; PAR, poly(ADP-ribose); JNK, c-Jun NH2-terminal kinase; p-, phospho-; CHX, cycloheximide; BHA, butylated hydroxyanisole; BAY-11, BAY 11-7082; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole; MEK, mitogen-activated protein kinase kinase; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene; CM-H2 DCFDA, 2′,7′-dichlorodihydrofluorescein diacetate; HEK, human embryonic kidney; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; NP-40, Nonidet P-40; FITC, fluorescein isothiocyanate; PI, propidium iodide; DCF-DA, 27-dichlorofluorescein diacetate; Ro 31-8220, 3-[1-[3-(amidinothio)propyl-1H-indol-3-yl]-3-(1-methyl-1H-indol-3-yl) maleimide (bisindolylmaleimide IX); 3-AB, 3-aminobenzamide; DPQ, 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1-(2H)-isoquinolinone; Gö6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole; SP600125, anthra(1,9-cd)pyrazol-6(2H)-one; z-VAD-FMK, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone.
- Received April 6, 2006.
- Accepted June 23, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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