Received for publication April 11, 2006.
Revised June 22, 2006.
Accepted for publication June 23, 2006.

Phorbol 12-myristate 13-acetate protects against TNF-induced necrotic cell death by modulating the recruitment of TRADD and RIP into the TNF receptor 1 signaling complex: Implication for the regulatory role of PKC

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 TNF-induced necrosis by disrupting formation of the TNF receptor 1 (TNFR1) signaling complex. Pretreatment with PMA protected L929 cells from TNF-induced necrotic cell death in a PKC dependent manner, but did not protect against DNA damaging agents, including 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 NF-B and 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 anti-necrotic 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 MEF cells but not in RIP-/- or TRAF2-/- 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.

Key words: Tumor necrosis factor, Protein Kinase C, MAP Kinase, Jun Kinase, NFkappaB, Apoptosis, Oxidative stress, Mechanisms of cell killing/apoptosis

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