Cystic fibrosis transmembrane conductance regulator controls lung proteasomal degradation and nuclear factor-kappaB activity in conditions of oxidative stress

Abstract

Cystic fibrosis is a lethal inherited disorder caused by mutations in a single gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein, resulting in progressive oxidative lung damage. In this study, we evaluated the role of CFTR in the control of ubiquitin-proteasome activity and nuclear factor (NF)-kappaB/IkappaB-alpha signaling after lung oxidative stress. After a 64-hour exposure to hyperoxia-mediated oxidative stress, CFTR-deficient (cftr(-/-)) mice exhibited significantly elevated lung proteasomal activity compared with wild-type (cftr(+/+)) animals. This was accompanied by reduced lung caspase-3 activity and defective degradation of NF-kappaB inhibitor IkappaB-alpha. In vitro, human CFTR-deficient lung cells exposed to oxidative stress exhibited increased proteasomal activity and decreased NF-kappaB-dependent transcriptional activity compared with CFTR-sufficient lung cells. Inhibition of the CFTR Cl(-) channel by CFTR(inh-172) in the normal bronchial immortalized cell line 16HBE14o- increased proteasomal degradation after exposure to oxidative stress. Caspase-3 inhibition by Z-DQMD in CFTR-sufficient lung cells mimicked the response profile of increased proteasomal degradation and reduced NF-kappaB activity observed in CFTR-deficient lung cells exposed to oxidative stress. Taken together, these results suggest that functional CFTR Cl(-) channel activity is crucial for regulation of lung proteasomal degradation and NF-kappaB activity in conditions of oxidative stress.

Figure 1

Activities of caspase-3 and proteasome are modified in response to oxidative stress in CFTR-sufficient versus CFTR-deficient in vivo and in vitro systems. A and B: The effect of oxidative stress on the caspase-3 and proteasome activities in lungs of WT and cftr^−/− (CF) mice. Left: Images of immunohistochemical staining of active caspase-3 were representative of immunostainings for two WT and two CF mice placed either under basal conditions or in 95% O₂. Right: The proteasome activity assay was determined from the specific cleavage of the LLVY-AMC substrate by the proteasome. The enzymatic activity is plotted as the fold induction relative to the basal condition. Results are expressed as the mean ± SD of four animals [eight WT and eight CF mice split into two equal groups exposed to either room air (n = 4) or to 95% O₂ (n = 4)]. C and D: The effect of oxidative stress on the caspase-3 and proteasome activities in CFTR-sufficient versus CFTR-deficient lung epithelial cells. Subconfluent cells were placed either under basal conditions or in 95% O₂ for 24 hours. Left: The caspase-3 enzyme activity in cell lines was assayed by the cleavage of a luminescent substrate by active caspase-3 in total lysates and is presented as a fold induction compared to the basal condition. Values are means ± SD of four independent experiments in either cell line. Right: The proteasome activity was assayed as in A and B in at least four independent experiments in either cell type. *P < 0.05 compared to the basal condition.

Figure 2

Level of IκB-α protein is decreased in vivo as in vitro under oxidative stress in CFTR-sufficient systems but not in CFTR-deficient systems. A–D: The effect of oxidative stress on the level of IκB-α protein was analyzed in lungs from WT and CF mice (A and B, respectively) or in CFTR-sufficient and CFTR-deficient cells (C and D, respectively). A typical blot of IκB-α is shown for each system. As a control for equal loading, the same blot was stripped and reprobed with an antibody to β-actin. Densitometric analysis of four independent experiments is shown as a graph of fold induction of the level of IκB-α protein expression in the basal condition and in 95% O₂. *P < 0.05 compared to basal condition.

Figure 3

The NF-κB-dependent transcriptional activity is increased in CFTR-sufficient cells but decreased in CFTR-deficient cells under oxidative stress. The activation of NF-κB/IκB-α pathway was studied by analyzing the level of p-IκB-α, the nuclear translocation of p65-NF-κB fusion protein, and the NF-κB-dependent transcriptional activity. A and B: Level of IκB-α phosphorylation. CFTR-sufficient (A) and CFTR-deficient (B) cells were cultured under basal or oxidative stress conditions and the level of IκB-α phosphorylation was analyzed by the ELISA method. Results are expressed as the mean relative luminescence units (RLUs) ± SD of at least four experiments in two cultured cell types (*P < 0.05) compared to the control condition. C and D: Localization of the YFP-p65NF-κB fusion protein. CFTR-sufficient and CFTR-deficient cells were transiently transfected with a plasmid expressing the YFP-p65NF-κB fusion protein and exposed or not to 24 hours of 95% O₂. Images are representative of four independent experiments. E–G: NF-κB-dependent transcriptional activity. CFTR-sufficient and CFTR-deficient cells were transiently transfected with a NF-κB-firefly-luciferase reporter construct and a control R. reniformis-luciferase vector and exposed or not to 24 hours of 95% O₂. In G, transfected CFTR-deficient cells were preincubated at 27°C for 24 hours before their exposure to control or oxidative stress conditions at 27°C. The luciferase activity indicative of NF-κB-dependent transcriptional activity is expressed as a value relative to that observed under basal conditions. Results are shown as the mean ± SD of at least four independent experiments. *P < 0.05 compared to the control condition.

Figure 4

Implication of CFTR deficiency in the modulation of the proteasome activity in response to oxidative stress. A and B: Western blot analysis of the effect of oxidative stress alone or in combination with a 24-hour treatment of 500 nmol/L MG132 on the rate of ubiquitinated proteins and levels of expression of the ubiquitin-conjugating enzymes UbcH5 and UbcH9. To normalize the quantity of proteins, the same blots were stripped and reprobed with an anti-β-actin antibody. At least four experiments were conducted and a typical blot is shown in the figure. C and D: Inhibition of CFTR on normal bronchial epithelial cells 16HBE14o− and its consequence on the proteasome activity. 16HBE14o− cells were treated with the specific CFTR inhibitor (CFTR_inh-172) at 10 μmol/L for 0.5 hour or 48 hours before exposition to oxidative stress. Proteasome activity is plotted as fold induction relative to the basal condition. Results are expressed as the mean ± SD of at least four independent experiments. *P < 0.05 compared to the control condition. E: Effect of a rescue of CFTR on the proteasome activity. CFTR-deficient cells were preincubated at 27°C for 24 hours before exposure to control or oxidative stress conditions at 27°C. Proteasome activity of S9 cells, 16HBE14o−, and IB3-1 cells at 27°C is plotted as fold induction relative to the basal condition. Results are expressed as the mean ± SEM of at least four independent experiments. *P < 0.05 compared to the control condition.

Figure 5

Treatment with MG132 restores regulation of the IκB-α inhibitor and NF-κB-dependent transcription activity in CFTR-deficient cells to a similar level to that in CFTR-sufficient cells. A and B: Typical blots of the level of the IκB-α protein in cells incubated with 500 nmol/L MG132 are shown. As a control for equal loading, the same blots were stripped and reprobed with an antibody to β-actin. C and D: The luciferase activity indicative of NF-κB-dependent transcriptional activity in which MG132 is expressed as a value relative to that observed in the basal condition in CFTR-sufficient (C), and CFTR-deficient (D) cells. Results are shown as the mean ± SD of at least four independent experiments. *P < 0.05 compared to the control condition.

Figure 6

Inhibition of caspase-3 in CFTR-sufficient cells results in the same type of modulation of NF-κB-dependent transcription and proteasome activity as in oxidative-stressed CF systems. A and B: CFTR-sufficient cells were treated with 5 μmol/L of either the broad range caspase inhibitor Z-VAD FMK or the specific caspase-3 inhibitor Z-DQMD-FMK incubated either in basal or oxidative stress conditions (95% O₂) for a 24-hour period. A: Effect of caspase-3 inhibition on NF-κB-dependent transcriptional activity. The luciferase activity indicative of the NF-κB-dependent transcriptional activity of CFTR-sufficient cells under oxidative stress is shown as a value relative to that observed under the basal condition. Results are expressed as the mean ± SD of at least four independent experiments. *P < 0.05 compared to room air and *^#P < 0.05 compared to 95% O_2. B: Effect of caspase-3 inhibition on the proteasome-proteolytic activity. The proteasome activity is given as a value relative to that observed in the basal condition. Results are expressed as the mean ± SD of at least four independent experiments. *P < 0.05 compared to the control condition.