GSK3β is a key regulator of the ROS-dependent necrotic death induced by the quinone DMNQ

Signaling pathways controlling necrosis are still mysterious and debated. We applied a shRNA-based viability screen to identify critical elements of the necrotic response. We took advantage from a small molecule (G5) that makes covalent adducts with free thiols by Michael addition and elicits multiple stresses. In cells resistant to apoptosis, G5 triggers necrosis through the induction of protein unfolding, glutathione depletion, ER stress, proteasomal impairments, and cytoskeletal stress. The kinase GSK3β was isolated among the top hits of the screening. Using the quinone DMNQ, a ROS generator, we demonstrate that GSK3β is involved in the regulation of ROS-dependent necrosis. Our results have been validated using siRNA and by knocking-out GSK3β with the CRISPR/Cas9 technology. In response to DMNQ GSK3β is activated by serine 9 dephosphorylation, concomitantly to Akt inactivation. During the quinone-induced pro-necrotic stress, GSK3β gradually accumulates into the nucleus, before the collapse of the mitochondrial membrane potential. Accumulation of ROS in response to DMNQ is impaired by the absence of GSK3β. We provide evidence that the activities of the obligatory two-electrons reducing flavoenzymes, NQO1 (NAD(P)H quinone dehydrogenase 1) and NQO2 are required to suppress DMNQ-induced necrosis. In the absence of GSK3β the expression of NQO1 and NQO2 is dramatically increased, possibly because of an increased transcriptional activity of NRF2. In summary, GSK3β by blunting the anti-oxidant response and particularly NQO1 and NQO2 expression, favors the appearance of necrosis in response to ROS, as generated by the quinone DMNQ.

Abstract 239: Parthanatos is Involved in Hydrogen Peroxide Induced Vascular Smooth Muscle Cell Death

Objectives: Oxidative stress underlies major vascular diseases including atherosclerosis and abdominal aortic aneurysm. Hydrogen peroxide (H2O2) is widely used to trigger oxidative stress in vitro for the study of apoptosis. However, we have previously shown that vascular smooth muscle cells (SMCs) respond to high concentrations (>1 mM) of H2O2 with necrosis. Traditionally regarded as incidental form of cell death, necrosis can occur through different mechanisms mediated by distinct intracellular signaling networks. The precise knowledge of death pathway is essential to the design of therapeutic strategy targeting cell death. The goal of the current study is to determine how H2O2 induces necrosis in SMCs. Methods: Mouse vascular aortic smooth muscle cell line, MOVAS, were treated with 3mM H2O2 for 2 hours, after which cell death was analyzed using flow cytometry and protein expression determined via western blot. Results: SMCs underwent apoptosis and necrosis in response to 0.3 and 3 mM H2O2, respectively. The 3mM H2O2 group died via a caspase-independent mechanism. Expression of common autophagy-associated proteins were unaffected. Additionally, different autophagy activators and inhibitors only moderately facilitated the pro-necrotic effect of H2O2. The H2O2-induced necrosis was not affected by necroptosis inhibitors including necrostatin-1s or by SiRNA silencing of necroptosis mediators RIP1, RIP3 and MLKL. Furthermore, ferroptosis and CypD inhibitors did not provide protection from necrosis induced by H2O2. In contrast, the necrotic response was attenuated by the PARP-1 inhibitor 3-aminobenzamide (37.10±13.72% vs 82.05±0.64%). Moreover, an PARP1 siRNA also reduced necrosis. PARP-1 is the central mediator of a necroptosis mechanism called parthanatos. Conclusions: Our data demonstrates that parthanatos constitutes a major mechanism underlying the necrotic response to high concentrations of H2O2. Current studies delineate the involvement of parthanatos in myocardial ischemia/reperfusion injury, cardiovascular ailments, and atherosclerosis. The present study may provide a new perspective on targeting PARP-1 for the protection of SMCs and likely cardiac myocytes against oxidative stress.

Regions of the Cf-9B Disease Resistance Protein Able to Cause Spontaneous Necrosis in Nicotiana benthamiana Lie Within the Region Controlling Pathogen Recognition in Tomato

The tomato Cf-9 and Cf-9B genes both confer resistance to the leaf mold fungus Cladosporium fulvum but only Cf-9 confers seedling resistance and recognizes the avirulence (Avr) protein Avr9 produced by C. fulvum. Using domain swaps, leucine-rich repeats (LRR) 5 to 15 of Cf-9 were shown to be required for Cf-9-specific resistance to C. fulvum in tomato, and the entire N-terminus up to LRR15 of Cf-9B was shown to be required for Cf-9B-specific resistance. Finer domain swaps showed that nine amino-acid differences in LRR 13 to 15 provided sufficient Cf-9-specific residues in a Cf-9B context for recognition of Avr9 in Nicotiana tabacum or sufficient Cf-9B residues in a Cf-9 context for a novel necrotic response caused by the expression of Cf-9B in N. benthamiana. The responses conferred by LRR 13 to 15 were enhanced by addition of LRR 10 to 12, and either region of Cf-9B was found to cause necrosis in N. benthamiana when the other was replaced by Cf-9 sequence in a Cf-9B context. As a consequence, the domain swap with LRR 13 to 15 of Cf-9 in a Cf-9B context gained the dual ability to recognize Avr9 and cause necrosis in N. benthamiana. Intriguingly, two Cf-9B-specific domain swaps gave differing results for necrosis assays in N. benthamiana compared with disease resistance assays in transgenic tomato. The different domain requirements in these two cases suggest that the two assays detect unrelated ligands or detect related ligands in slightly different ways. A heat-sensitive necrosis-inducing factor present in N. benthamiana intercellular washing fluids was found to cause a necrotic response in N. tabacum plants carrying Hcr9-9A, Cf-9B, and Cf-9 but not in plants carrying only Cf-9. We postulate that this necrosis-inducing factor is recognized by Cf-9B either directly as a ligand or indirectly as a regulator of Cf-9B autoactivity.