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

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- κ 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. little is known about the effects or regulatory mechanisms of PKC in NCD. In this study, our data demonstrates that PMA-induced activation of PKC suppresses TNF-induced NCD by disrupting the recruitment of TRADD and RIP to the TNFR1 complex. Moreover, our results indicate that PMA inhibits TNF-induced ROS production, thereby suppressing activation of ROS-mediated poly(ADP-ribose)polymerase (PARP) leading to the inhibition of NCD. to a lesser extent than did BHA. These results suggest that, although ROS-dependent PARP activation is not essential for TNF-induced NCD, it may contribute to this process. Other factors, in addition to PARP activation, may participate in the TNF-induced NCD pathway. phospho-JNK, I κ B- , and actin. MEF cells were pretreated with z-VAD-fmk (50 µM) for 30 and then treated with TNF (15 ng/ml), TNF (15 ng/ml) plus CHX (10 or H 2 O 2 (200 µM) for and cell death was quantified by trypan blue exclusion Each shows mean SE of at least of three independent experiments. ∗ when compared with TNF/CHX/z-VAD-fmk-treated group. when compared with H 2 O 2 -treated wild-type MEF cells. with and cell extracts were analyzed by SDS-PAGE and Western with and