Prevention of TNF-induced necrotic cell death by rottlerin through a Nox1 NADPH oxidase

Abstract

Previous studies have demonstrated that rottlerin, a specific PKCdelta inhibitor, potentiates death receptor- mediated apoptosis through a cytochrome c-dependent or -independent pathway. However, its ability to regulate necrotic cell death, as well as the underlying mechanism, remains unknown. We found that in murine fibrosarcoma L929 cells, treatment with rottlerin protected the cells against TNF-induced necrosis, whereas it sensitized the cells to apoptosis induced by co-treatment with Hsp90 inhibitor geldanamycin and TNF, in a manner independent of its ability to inhibit PKC-delta. TNF treatment induced rapid accumulation of mitochondrial superoxide (O2-) through the Nox1 NADPH oxidase when cells undergo necrosis. Moreover, pretreatment with rottlerin failed to induce the GTP-bound form of small GTPase Rac1 by TNF treatment, and subsequently suppressed mitochondrial O2- production and poly(ADP-ribose) polymerase activation, thus inhibiting necrotic cell death. Therefore, our study suggests that Nox1 NADPH oxidase is a new molecular target for anti-necrotic activity of rottlerin upon death-receptor ligation.

Figure 1

Rottlerin protects murine fibrosarcoma L929 cells against TNF-induced necrotic cell death in a PKC-δ-independent manner. (A) L929 cells were treated with 15 ng/ml TNF for 8 h in the presence or absence of 10 µM rottlerin, stained with FITC-labeled annexin V and PI, and analyzed by flow cytometry. (B) L929 cells were pretreated with rottlerin at the indicated concentrations (left) and times (right), and further treated with TNF for 8 h. Cell death was quantified by trypan blue exclusion assay. Each bar shows the mean ± SE of at least three independent experiments. ^#P < 0.05 compared with TNF-treated group. (C) After pretreatment with 10 µM rottlerin, 100 µM BHA and 10 mM NAC, L929 cells were treated with 500 µM H₂O₂ or 10 µM menadione (MD) for 12 h, and cell death was quantified as described in (B). ^*P < 0.05 compared with the H₂O₂-treated group. ^#P < 0.05 compared with the MD-treated group. (D) L929 cells were infected with wild type or dominant-negative type of PKC-δ adenovirus at an m.o.i. of 100. After 48 h incubation, cells were treated with TNF (15 ng/ml) for 8 h in the presence or absence of 10 µM rottlerin, and cell death was quantified as described in B. ^#P < 0.05 compared with the TNF-treated group. PKCδ activities in L929 cells infected with DN-PKCδ AdV or WT-PKCδ AdV were analyzed by immune complex kinase assay with [γ-³²P] ATP and MBP as a substrate. Phosphorylation of MBP was assessed by SDS-PAGE and autoradiography. PKCδ or actin protein content was analyzed by Western blotting with anti-PKCδ or actin antibody. (E) After pretreatment with 50 nM PMA, 10 µM Bis1, 10 µM Go6983 or 10 µM rottlerin, L929 cells were treated with TNF (15 ng/ml) for 8 h, and cell death was quantified as described in (B). ^#P < 0.05 compared with the TNF-treated group.

Figure 2

Rottlerin sensitizes murine fibrosarcoma L929 cells against GA plus TNF-induced apoptosis. (A) L929 cells were pretreated with GA (1 µM) for 12 h, followed by treatment with TNF (15 ng/ml) for 5 h. Cells were immediately photographed under electron microscopy. (B) L929 cells were pretreated with or without GA (1 µM) for 12 h, followed by treatment with TNF for various times. Cell extracts were analyzed by SDS-PAGE and Western blotting with antibodies against caspase-8, caspase-3, PARP and actin. (C) L929 cells were pretreated with rottlerin at the indicated concentrations (left) and times (right), and further treated with GA (12 h) plus TNF (5 h). Cell death was quantified as described in Figure 1B. ^*P < 0.05 compared with the GA-plus-TNF-treated group. (D) Mouse embryonic fibroblast (MEF) cells were pretreated with 50 µM z-VAD-FMK for 30 min and then treated with 15 ng/ml TNF or 15 ng/ml TNF plus 10 µg/ml CHX in the absence of presence of rottlerin for various concentrations as indicated. Cell death was quantified as described in Figure 1B. Each bar shows mean ± SE of at least three independent experiments. ^*P < 0.05, when compared with CHX/TNF-treated group. ^#P < 0.05, when compared with TNF/CHX/z-VAD-FMK-treated group.

Figure 3

Rottlerin suppresses mitochondrial O₂^- production induced by TNF, and subsequently block PARP activation. (A) L929 cells were treated with TNF (15 ng/ml) for various times as indicated in the presence or absence of rottlerin (10 µM) and BHA (100 µM). Mitochondrial O₂^- production was determined using superoxide-sensitive MitoSOX Red. (B) L929 cells were treated with TNF in the presence or absence of 100 µM BHA and 10 µM rottlerin. Cell extracts were analyzed by SDS-PAGE and Western blotting with antibodies against PAR, PARP and actin.

Figure 4

Mitochondrial Nox1 NADPH oxidase is expressed in murine fibrosarcoma L929 cells, and rottlerin inhibits Nox1 activity through down-regulation of GTP-bound Rac1. (A) L929 cells were treated with TNF for various times in the presence of 10 µM DPI, and mitochondrial O₂^- production was determined as described for Figure 3A. (B) Mitochondrial and plasma membrane fractions of L929 cells were obtained, and lysates were analyzed by SDS-PAGE, followed by Western blotting with antibodies against Nox1, COX-4 and TNFR1 (upper panel). After treatment with TNF in the presence of rottlerin (10 µM), BHA (100 µM) and DPI (10 µM), NADPH oxidase assays were conducted. (C) L929 cells were transfected with the Xp-tagged Nox1, NOXO1 and NOXA1 expression plasmids, and treated with PMA (50 nM) and/or TNF (15 ng/ml) for 30 min, in the presence or absence of rottlerin (10 µM). The PAK1 pull-down complex was analyzed by SDS-PAGE and Western blotting with antibodies against Rac1.