cell-fractionation-u937-cells-absence-high-speed

Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. ...

cell-fractionation-u937-cells-isopycnic-density-gradient

Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and ...

generation-rip1-knockout-u937-cell-line-using-crispr-cas9

Generation of a <em>RIP1</em> Knockout U937 Cell Line Using the CRISPR-Cas9 System

This protocol outlines a procedure for knocking out the RIP1 gene using CRISPR/Cas9 in the human monocyte U937 cell line. The method utilizes designated ...

Albany College of Pharmacy and Health Sciences

Apoptosis and necroptosis are the primary modes of eukaryotic cell death, with apoptosis being non-inflammatory while necroptosis is highly inflammatory. We previously demonstrated that, once activated, necroptosis is enhanced by hyperglycemia in several cell types. Here, we determine if hyperglycemia affects apoptosis similarly. We show that hyperglycemia does not enhance extrinsic apoptosis but potentiates a shift to RIP1-dependent necroptosis. This is due to increased levels and activity of RIP1, RIP3, and MLKL, as well as decreased levels and activity of executioner caspases under hyperglycemic conditions following stimulation of apoptosis. Cell death under hyperglycemic conditions was classified as necroptosis via measurement of markers and involvement of RIP1, RIP3, and MLKL. The shift to necroptosis was driven by RIP1, as mutation of this gene using CRISPR-Cas9 caused cell death to revert to apoptosis under hyperglycemic conditions. The shift of apoptosis to necroptosis depended on glycolysis and production of mitochondrial ROS. Importantly, the shift in PCD was observed in primary human T cells. Levels of RIP1 and MLKL increased, while executioner caspases and PARP1 cleavage decreased, in cerebral tissue from hyperglycemic neonatal mice that underwent hypoxia-ischemia (HI) brain injury, suggesting that this cell death shift occurs . This is significant as it demonstrates a shift from non-inflammatory to inflammatory cell death which may explain the exacerbation of neonatal HI-brain injury during hyperglycemia. These results are distinct from our previous findings where hyperglycemia enhanced necroptosis under conditions where apoptosis was inhibited artificially. Here we demonstrate a shift from apoptosis to necroptosis under hyperglycemic conditions while both pathways are fully active. Therefore, while our previous work documented that intensity of necroptosis is responsive to glucose, this work sheds light on the molecular balance between apoptosis and necroptosis and identifies hyperglycemia as a condition that pushes cells to undergo necroptosis despite the initial activation of apoptosis.

Hyperglycemia potentiates a shift from apoptosis to RIP1-dependent necroptosis.

Like nucleated cells, erythrocytes (red blood cells, RBCs) are capable of executing programmed cell death pathways. RBCs undergo necroptosis in response to CD59-specific pore-forming toxins (PFTs). The relationship between blood bank storage and RBC necroptosis was explored in this study.

Storage Primes Erythrocytes for Necroptosis and Clearance.

We have previously identified a shift from TNF-α-induced apoptosis to necroptosis that occurs under hyperglycemic conditions. This shift involves the downregulation or silencing of caspases and concurrent upregulation of necroptotic proteins leading to activation of the necrosome. In addition, under hyperglycemic conditions in vivo, this shift in cell death mechanisms exacerbates neonatal hypoxia-ischemia (HI) brain injury. Here, we identify two major factors that drive the hyperglycemic shift to necroptosis: (1) reactive oxygen species (ROS) and (2) receptor-interacting protein kinase 1 (RIP1). ROS, including mitochondrial superoxide, led to the oxidation of RIP1, as well as formation and activation of the necrosome. Concurrently, ROS mediate a decrease in the levels and activation of executioner caspases-3, -6, and -7. Importantly, hyperglycemia and mitochondrial ROS result in the oxidation of RIP1 and loss of executioner caspases prior to death receptor engagement by TNF-α. Moreover, RIP1 partially controlled levels of mitochondrial ROS in the context of hyperglycemia. As a result of its regulation of ROS, RIP1 also regulated necrosome activation and caspase loss. Mitochondrial ROS exacerbated neonatal HI-brain injury in hyperglycemic mice, as a result of the shift from apoptosis to necroptosis.

Mitochondrial ROS prime the hyperglycemic shift from apoptosis to necroptosis.

The ribosome-inactivating proteins include the biothreat agent, ricin toxin (RT). When inhaled, RT causes near complete destruction of the lung epithelium coincident with a proinflammatory response that includes TNF family cytokines, which are death-inducing ligands. We previously demonstrated that the combination of RT and TNF-related apoptosis inducing ligand (TRAIL) induces caspase-dependent apoptosis, while RT and TNF-α or RT and Fas ligand (FasL) induces cathepsin-dependent cell death in lung epithelial cells. We hypothesize that airway macrophages constitute a major source of cytokines that drive lung epithelial cell death.

Necroptosis of Lung Epithelial Cells Triggered by Ricin Toxin and Bystander Inflammation.

Apoptosis and necroptosis overlap in their initial signaling but diverge to produce non-inflammatory and pro-inflammatory outcomes, respectively. High glucose pushes signaling in favor of necroptosis producing a hyperglycemic shift from apoptosis to necroptosis. This shift depends on receptor-interacting protein 1 (RIP1) and mitochondrial reactive oxygen species (ROS). Here, we show that RIP1, mixed lineage kinase domain-like (MLKL) protein, Bcl-2 agonist/killer (Bak), Bcl-2 associated x (Bax) protein, and dynamin-related protein 1 (Drp1) traffic to the mitochondria in high glucose. RIP1 and MLKL appear in the mitochondria in their activated, phosphorylated states while Drp1 appears in its activated, dephosphorylated state in high glucose. Mitochondrial trafficking is prevented in  KO cells and upon treatment with N-acetylcysteine. Induction of ROS replicated the mitochondrial trafficking seen in high glucose. MLKL forms high MW oligomers in the outer and inner mitochondrial membranes while Bak and Bax form high MW oligomers in the outer mitochondrial membrane in high glucose, suggesting pore formation. MLKL, Bax, and Drp1 promoted cytochrome  release from the mitochondria as well as a decrease in mitochondrial membrane potential in high glucose. These results indicate that mitochondrial trafficking of RIP1, MLKL, Bak, Bax, and Drp1 are key events in the hyperglycemic shift from apoptosis to necroptosis. This is also the first report to show oligomerization of MLKL in the inner and outer mitochondrial membranes and dependence of mitochondrial permeability on MLKL.

Mitochondrial Trafficking of MLKL, Bak/Bax, and Drp1 Is Mediated by RIP1 and ROS which Leads to Decreased Mitochondrial Membrane Integrity during the Hyperglycemic Shift to Necroptosis.

ArticleTotal : 3	Year
Publication title Cited by 4	2019
Publication title Cited by 1	2021
Publication title	2025

Article	Year
Hyperglycemia potentiates a shift from apoptosis to RIP1-dependent necroptosis. Cell death discovery\| PubMed ID: 29760953	2018
Storage Primes Erythrocytes for Necroptosis and Clearance. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology\| PubMed ID: 31486324	2019
Mitochondrial ROS prime the hyperglycemic shift from apoptosis to necroptosis. Cell death discovery\| PubMed ID: 33298902	2020
Necroptosis of Lung Epithelial Cells Triggered by Ricin Toxin and Bystander Inflammation. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology\| PubMed ID: 36695077	2023
Mitochondrial Trafficking of MLKL, Bak/Bax, and Drp1 Is Mediated by RIP1 and ROS which Leads to Decreased Mitochondrial Membrane Integrity during the Hyperglycemic Shift to Necroptosis. International journal of molecular sciences\| PubMed ID: 37239951	2023

Matthew A. Deragon

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