Receptor-Interacting Protein Kinases 1 and 3, and Mixed Lineage Kinase Domain-Like Protein Are Activated by Sublytic Complement and Participate in Complement-Dependent Cytotoxicity

Abstract

The complement system participates in the pathogenesis of many diseases. Complement activation produces several active protein complexes and peptides, including the terminal C5b-9 complexes. It was reported that C5b-9 complexes insert into the plasma membrane and cause membrane perturbation, intracellular calcium surge, metabolic depletion, and osmotic lysis. Previously, we showed that complement-dependent cytotoxicity (CDC) is regulated by JNK and Bid. Here, we demonstrate that three mediators in TNFα-induced necroptosis (regulated necrosis), the receptor-interacting protein kinases, receptor-interacting protein kinase 1 (RIPK1) and receptor-interacting protein kinase 3 (RIPK3), and mixed-lineage kinase domain-like protein (MLKL), are activated by complement and contribute to CDC. Cell treatment with necrostatin-1 (Nec-1), a RIPK1 inhibitor, GSK’872, a RIPK3 inhibitor, or necrosulfonamide and GW806742X, MLKL inhibitors, restrain CDC. These findings were confirmed by using specific siRNAs targeting the synthesis of these proteins. Mouse fibroblasts lacking RIPK3 or MLKL were found to be less sensitive to C5b-9 than were wild-type (WT) fibroblasts. Enhanced CDC was achieved by RIPK1 or RIPK3 overexpression but not by the overexpression of a RHIM-RIPK1 mutant nor by a kinase-dead RIPK3 mutant. Nec-1 reduces the CDC of WT but not of RIPK3-knockout fibroblasts. Cells treated with a sublytic dose of complement exhibit co-localization of RIPK3 with RIPK1 in the cytoplasm and co-localization of RIPK3 and MLKL with C5b-9 at the plasma membrane. Data supporting cooperation among the RIP kinases, MLKL, JNK, and Bid in CDC are presented. These results provide a deeper insight into the cell death process activated by complement and identify potential points of cross talk between complement and other inducers of inflammation and regulated necrosis.

Astrocyte- and neuron-derived extracellular vesicles from Alzheimer’s disease patients effect complement-mediated neurotoxicity

“…Treatment of neurons with NEVs and AEVs from 4 AD compared to 4 control participants resulted in a significantly increased EthD-1 fluorescence fold change over vehicle [For NEVs, F (1, 24.020) = 4.525, p = 0.044; AD vs. Normal, p = 0.044; for AEVs, F (1, 24.872) = 5.804, p = 0.024; AD vs. controls, p = 0.024], suggesting that AD NEVs and AEVs mediate neuronal membrane disruption; there were no differences in EthD-1 fluorescence over vehicle in neurons treated with CD81+ EVs (F (1, 31.429) = 0.969, p = 0.332) from AD compared to control participants. MAC-dependent cytotoxicity is accompanied by activation of the regulated necrosis pathway known as necroptosis, which involves phosphorylation of the mixed-lineage kinase domain-like protein (MLKL) (32,33). To further explore the mechanism of neurotoxicity induced by AEVs and NEVs, we evaluated whether EV-treated neurons exhibit necroptosis and apoptosis cell death markers.…”

Pathomechanisms of Posttraumatic Osteoarthritis: Chondrocyte Behavior and Fate in a Precarious Environment

“…While mechanical stress has been shown to induce both primary necrosis and apoptosis [38,82], injury-related necroptosis was found in vivo [122] but seemed to play a minor role in our ex vivo cartilage trauma models under serum-free conditions [113]. We concluded that the activation of the necroptotic pathway might require further co-factors, such as TNFa or certain serum-components [109,123], and concurrent inhibition of the caspase cascade [113]. However, we could provide evidence that necroptosis occurs in highly degenerated human cartilage, implying a potential role of necroptosis in OA disease [113].…”

Angiotensin II triggers RIPK3-MLKL-mediated necroptosis by activating the Fas/FasL signaling pathway in renal tubular cells

“…The cells were cultured in DMEM/F12 medium (Gibco Life Technologies, Carlsbad, CA, USA) containing 10% FBS (HyClone, U.S.) supplemented with 1% penicillin and streptomycin (Beyotime, Shanghai, China) in a humidified incubator with 5% CO2 at 37˚C. Cells were grown to 70-80% confluency and stimulated with AngII (Sigma, USA) at a concentration range of 10 −10 -10 −5 M for 24 h and exposed to 10 −9 M AngII for 12 h, 24 h, 36 h, 48 h, and 72 h. To further assess the necroptosis of HK-2 cells, cells were pretreated with RIP1 inhibitor (100 μM Nec-1 [5]) or the pan-caspase inhibitor (10 mM zVAD [5]) for 30 min, RIPK3 blocker (0.25 μM GSK'872 [19]) or MLKL inhibitor (1 μM NSA, Selleck, USA [8]) for 1 h. To elucidate the relevant mechanisms, cells were treated with an AT1R antagonist (10 μM losartan [20,21]) and an AT2R antagonist (PD123319 [20,21]) for 30 min or with FasL inhibitor (3 μg/ml [22,23] the neutralizing Human Fas Ligand/TNFSF6 Antibody (RD Systems, USA)) for 2 h. After pretreatment for the assigned duration, HK-2 cells were exposed to 10 −9 M AngII for 24 h. The treated cells were collected at the assigned time for western blot analysis, TEM, and immunofluorescence staining.…”