Abstract
BackgroundThe extrinsic apoptotic pathway initiates when a death ligand, such as the Fas ligand, interacts with its cell surface receptor (ie., Fas/CD95), forming a death-inducing signaling complex (DISC). The Fas-dependent apoptotic pathway has been implicated in several models of lung or vascular injury. Carbon monoxide, an enzymatic product of heme oxygenase-1, exerts antiapoptotic effects at low concentration in vitro and in vivo.MethodsUsing mouse lung endothelial cells (MLEC), we examined the antiapoptotic potential of carbon monoxide against apoptosis induced by the Fas/CD95-activating antibody (Jo2). Carbon monoxide was applied to cell cultures in vitro. The expression and/or activation of apoptosis-related proteins and signaling intermediates were determined using Western Immunoblot and co-immunoprecipitation assays. Cell death was monitored by lactate dehydrogenase (LDH) release assays. Statistical significance was determined by student T-test and a value of P < 0.05 was considered significant.ResultsTreatment of MLEC with Fas-activating antibody (Jo2) induced cell death associated with the formation of the DISC, and activation of caspases (-8, -9, and -3), as well as the pro-apoptotic Bcl-2 family protein Bax. Exposure of MLEC to carbon monoxide inhibited Jo2-induced cell death, which correlated with the inhibition of DISC formation, cleavage of caspases-8, -9, and -3, and Bax activation. Carbon monoxide inhibited the phosphorylation of the Fas-associated death domain-containing protein, as well as its association with the DISC. Furthermore, carbon monoxide induced the expression of the antiapoptotic protein FLIP and increased its association with the DISC.CO-dependent cytoprotection against Fas mediated apoptosis in MLEC depended in part on activation of ERK1/2-dependent signaling.ConclusionsCarbon monoxide has been proposed as a potential therapy for lung and other diseases based in part on its antiapoptotic effects in endothelial cells. In vitro, carbon monoxide may inhibit both Fas/caspase-8 and Bax-dependent apoptotic signaling pathways induced by Fas-activating antibody in endothelial cells. Strategies to block Fas-dependent apoptotic pathways may be useful in development of therapies for lung or vascular disorders.
Highlights
The extrinsic apoptotic pathway initiates when a death ligand, such as the Fas ligand, interacts with its cell surface receptor, forming a death-inducing signaling complex (DISC)
We previously reported that the expression of FLIP protected against cell death in pulmonary epithelial and endothelial cells subjected to hyperoxia [17,18], or in endothelial cells subjected to hypoxia/reoxygenation [19]
We investigated the effect of Carbon monoxide (CO) on cell death induced by Fas-activating antibody (Jo2) in mouse lung endothelial cells (MLEC), using lactate dehydrogenase (LDH) release as a marker
Summary
The extrinsic apoptotic pathway initiates when a death ligand, such as the Fas ligand, interacts with its cell surface receptor (ie., Fas/CD95), forming a death-inducing signaling complex (DISC). Two apoptotic pathways have been identified by which cells can initiate and execute the cell death process: an intrinsic (mitochondria-dependent) pathway and an extrinsic (death receptor-dependent) pathway [6,7,8]. Extrinsic apoptosis initiates with the plasma membrane assimilation of the death-inducing signaling complex (DISC), consisting of Fas, FADD, and caspase, by ligand-dependent (ie., Fas ligand, FasL) or independent mechanisms. Fas (Apo-1/CD95), is a transmembrane cell surface receptor containing three cysteine-rich extracellular domains at the amino-terminus, which are responsible for ligand binding, and an intracytoplasmic death domain (DD) of ~80 amino acids essential for transducing the apoptotic signal [9]. Autoproteolytic activation of caspase-8 results in the processing of Bid to tBid, which assimilates into the mitochondria to trigger cytochrome c release, and may facilitate Bax activation [11]
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