Negatively-regulated necroptosis by autophagy required caspase-6 activation in TNFα-treated murine fibrosarcoma L929 cells.
Autophagy and necroptosis have been known to be interconnected, while the relationship between autophagy and necroptosis remains unclear. Here, we demonstrated that pan-caspase inhibitor z-VAD-fmk (zVAD) exacerbated TNFα-induced necroptosis and autophagy in murine fibrosarcoma L929 cells. And the RIP-1 inhibitor necrostatin-1 inhibited TNFα+zVAD-induced necroptosis and autophagy. Inhibition of autophagy by 3-methyladenine (3MA) or small interfering RNA (siRNA) against Beclin 1 augmented TNFα-induced necroptosis, while, autophagy inhibition did not influence TNFα+zVAD-induced necroptosis. These results suggested that autophagy was a downstream consequence of necroptosis, and had a negative-feedback function to necroptosis in TNFα-treated L929 cells, but not in the presence of zVAD. Subsequently, TNFα administration was accompanied with caspase-6 activation. Inhibition of caspase-6 activity by z-V-E(OMe)-I-D(OMe)-fmk (zVEID) or caspase-6 (p20) siRNA had no effect on necroptosis but promoted TNFα-induced autophagy. Meanwhile, autophagy inhibition further increased caspase-6 activation. Caspase-6 (p20) siRNA sequestered the increased necroptotic ratio by 3MA pretreatment in TNFα-treated L929 cells. In addition, caspase-6 activation induced by TNFα administration was inhibited by zVAD. Further, autophagy induced by higher concentration of zVAD did not negatively regulate necroptosis because caspase-6 was not activated. Collectively, our data indicated that autophagy was a downstream consequence of necroptosis, and negatively regulated necroptosis when caspase-6 was activated in TNFα-treated L929 cells.
China-Japan Research Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang 110016, China.
Yuan-Chao
YC
Ye
Hong-Ju
HJ
Wang
Ling
L
Chen
Wei-Wei
WW
Liu
Shin-Ichi
S
Tashiro
Satoshi
S
Onodera
Ming-Yu
MY
Xia
Takashi
T
Ikejima
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D053178:Caspase 6 / Q000378:metabolism*; D061945:Caspase Inhibitors / Q000494:pharmacology; D016923:Cell Death / Q000502:physiology; D007093:Imidazoles / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D007739:L Cells / Q000187:drug effects; D051379:Mice; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D007093:Imidazoles; D007211:Indoles; D014409:Tumor Necrosis Factor-alpha; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C507699:necrostatin-1; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; D053178:Caspase 6
Autophagy; Caspase-6; Necroptosis; TNFα; z-VAD-fmk