FLIP Protects against Hypoxia/Reoxygenation-Induced Endothelial Cell Apoptosis by Inhibiting Bax Activation

Abstract

Hypoxia/reoxygenation causes cell death, yet the underlying regulatory mechanisms remain partially understood. Recent studies demonstrate that hypoxia/reoxygenation can activate death receptor and mitochondria-dependent apoptotic pathways, involving Bid and Bax mitochondrial translocation and cytochrome c release. Using mouse lung endothelial cells (MLEC), we examined the role of FLIP, an inhibitor of caspase 8, in hypoxia/reoxygenation-induced cell death. FLIP protected MLEC against hypoxia/reoxygenation by blocking both caspase 8/Bid and Bax/mitochondrial apoptotic pathways. FLIP inhibited Bax activation in wild-type and Bid−/− MLEC, indicating independence from the caspase 8/Bid pathway. FLIP also inhibited the expression and activation of protein kinase C (PKC) (α, ζ) during hypoxia/reoxygenation and promoted an association of inactive forms of PKC with Bax. Surprisingly, FLIP expression also inhibited death-inducing signal complex (DISC) formation in the plasma membrane and promoted the accumulation of the DISC in the Golgi apparatus. FLIP expression also upregulated Bcl-XL, an antiapoptotic protein. In conclusion, FLIP decreased DISC formation in the plasma membrane by blocking its translocation from the Golgi apparatus and inhibited Bax activation through a novel PKC-dependent mechanism. The inhibitory effects of FLIP on Bax activation and plasma membrane DISC formation may play significant roles in protecting endothelial cells from the lethal effects of hypoxia/reoxygenation

Carbon Monoxide Protects against Hyperoxia-induced Endothelial Cell Apoptosis by Inhibiting Reactive Oxygen Species Formation

“…However, the precise mechanisms underlying DISC formation and the translocations of Fas, FADD, and caspase-8 are not entirely known. In the hypoxia/reoxygenation model, we previously reported that the DISC forms initially in the Golgi complex and then translocates to the plasma membrane (31,45). DISC formation in the Golgi complex is important for initiating apoptotic signaling (31), whereas its formation in the plasma membrane is critical for caspase-8 activation.…”

Interferon regulatory factor-1-induced apoptosis mediated by a ligand-independent fas-associated death domain pathway in breast cancer cells

“…Crosslinked 'killer' TRAIL was purchased from Axxora. The construction and characterization of E1, E3-deleted non-replicating recombinant adenoviruses Ad-C5 (empty vector control, Ad-Null), Ad-IRF-1 and Ad-EGFP (enhanced green fluorescent protein) were described previously , as were recombinant adenovirus expressing c-FLIP and LacZ (Wang et al, 2005), and were provided by Dr Andrea Gambotto from the Vector Core at the University of Pittsburgh (www.vectorcore.pitt.edu). Dimethylsulfoxide (DMSO) was obtained from ATCC (Manassas, VA, USA), and ZVAD (irreversible pan-caspase inhibitor) from Promega (Madison, WI, USA).…”

Cellular FLICE-Like Inhibitory Protein (C-FLIP): A Novel Target for Cancer Therapy

“…As discussed above, c-FLIP has been identified as a protease-dead, procaspase-8-like regulator of death ligand-induced apoptosis [27,45,52,53]. It has been shown that c-FLIP interferes with TNF-α, Fas-L, and TRAIL-induced signaling pathways by binding to FADD and/or caspase-8/10 in a ligand-dependent fashion, which in turn prevents DISC formation and subsequent activation of the caspase cascade [27,53,54].…”

“…It has been shown that c-FLIP interferes with TNF-α, Fas-L, and TRAIL-induced signaling pathways by binding to FADD and/or caspase-8/10 in a ligand-dependent fashion, which in turn prevents DISC formation and subsequent activation of the caspase cascade [27,53,54]. However, studies using c-FLIP-deficient mice support a dual function for c-FLIP by confirming a role for c-FLIP in Fas L-and TNF-α-induced apoptosis, and revealing that c-FLIP has a similar function to caspase-8 in heart development [55].…”

“…In fact, c-FLIP S inhibits TRAILinduced DISC formation and apoptosis [56], while c-FLIP L is responsible for the above described dual functions whereby it inhibits Fas-induced caspase-8 activation when expressed at high levels, but enhances caspase-8 activation when its expression level is low [53][54][55][56][57][58][59]. These opposing c-FLIP L functions may reflect observations that c-FLIP L activates caspases-8 and -10 in vitro by forming heterodimeric enzyme molecules with a substrate specificity and catalytic activity indistinguishable from caspase-8 homodimers, despite the fact that c-FLIP L is protease dead [60].…”