Carbon monoxide inhibits Fas activating antibody-induced apoptosis in endothelial cells

Abstract

Background: 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). 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.

Methods: Using 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.

Results: Treatment 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.

Conclusions: Carbon 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.

Figure 1

CO inhibits cell death and caspase activation induced by Fas activating antibody Jo2. MLEC cultures were pretreated in the absence or presence of CO (250 ppm) for 2 h prior to the addition of antibody Jo2 (200 ng/ml) for the indicated times. 200 μl of supernatant medium was removed for LDH assays as described in Methods (A). The data represent an average of two independent experiments with each sample in triplicate (n = 3). Data from Jo2 treated cells in the presence of CO were compared with control (Jo2 treatment alone) cells at each time point using Student's T-test (*P < 0.05). The total lysates were subjected to Western blot to detect caspase-9 and caspase-3. β-actin served as the standard (B). Westerns are representative of three independent experiments.

Figure 2

CO inhibited Jo2-dependent caspase-8 and Bax activation. MLEC cultures were pretreated in the absence or presence of CO (250 ppm) for 2 h prior to the addition of antibody Jo2 (200 ng/ml) for the indicated times. The total lysates were subjected to immunopreciptiation (IP) with anti-Fas, followed by immunoblotting (IB) to detect caspase-8. Total Fas served as the standard. The control lane (c) represents cell lysate taken immediately after Jo2 addtion (A). Lysates were subjected to Western blot analysis to detect caspase-8 (B). MLEC cultures were pretreated in the absence or presence of CO (250 ppm) for 2 h prior to the addition of antibody Jo2 (200 ng/ml) for the indicated times. Lysates were subjected to immunoprecipitation with antibody 6A7 that specifically recognizes the activated form of Bax, followed by immunoblotting with anti-Bax. Total Bax served as the standard (C). Westerns are representative of three independent experiments.

Figure 3

CO upregulated FLIP and increased DISC associated FLIP. MLEC cultures were pretreated in the absence or presence of CO (250 ppm) for 2 h prior to the addition of antibody Jo2 (200 ng/ml) for the indicated times. The total lysates were subjected to Western blot analysis to detect FLIP (A), or to immunoprecipitation (IP) with anti-Fas followed by immunoblotting (IB) to detect FLIP. β-Actin (A) or Total Fas (B) served as the standard. Westerns are representative of three independent experiments.

Figure 4

CO inhibited FADD phosphorylation via blockage of JNK signaling. MLEC cultures were pretreated in the absence or presence of CO (250 ppm) for 2 h prior to the addition of antibody Jo2 (200 ng/ml) for the indicated times. The total lysates were subjected to Western blot analysis to detect phosphorylated and total FADD (A), or to immunoprecipitation (IP) with anti-Fas followed by immunoblotting to detect phospho-FADD (B). Lysates were subjected to immunoblotting to detect phospho- and total JNK. (C). MLEC cultures were treated with antibody Jo2 (200 ng/ml) for the indicated times, in the absence or presence of JNK inhibitor (20 μM). The total lysates were subjected to immunoprecipitation with anti-Fas followed by immunoblotting to detect phospho-FADD (D). Total FADD (A, B, D) or total JNK (C) served as the standards. Westerns are representative of three independent experiments.

Figure 5

CO activated ERK and NF-κB signaling. MLEC cultures were pretreated in the absence or presence of CO (250 ppm) for 2 h prior to the addition of antibody Jo2 (200 ng/ml) for the indicated times. The total lysates were subjected to Western blot to detect ERK or NF-κB p65 phosphorylation. Total p65 served as the standard. Westerns are representative of three independent experiments.

Figure 6

CO-dependent cytoprotection requires ERK1/2 pathway. MLEC cultures were pretreated with room air (RA) or CO (250 ppm) for 2 h prior to the addition of antibody Jo2 (200 ng/ml). Additionally, PD 98059 (25 μM) or vehicle (DMSO) was added to the cultures for 30 min prior to Jo2 addition. Cells were then incubated an additional 24 hours in the absence or presence of CO. 200 μl of supernatant medium was removed for LDH assays as described in Methods (A). The data represent triplicate (n = 3) determinations. Comparisons between the treatment groups were performed using Student's T-test (*P < 0.05). The total lysates were subjected to Western blot to detect caspase-3. β-actin served as the standard (B).

Figure 7

Proposed pathways of the antiapoptotic effects of CO in Jo2-dependent apoptosis.