cGMP-mediated antioxidant signaling: a role for the c-Abl tyrosine kinase

Background

Oxidant injury to the pulmonary endothelium contributes to acute lung injury. We have shown that activation of PKG_I by cGMP increases protein levels of the antioxidant enzymes catalase and glutathione peroxidase-1 (GPx-1) and ameliorates oxidant injury in mouse lung endothelium [1]. Catalase and GPx-1 mRNA was not increased. The pathway downstream of PKG_I that leads to increases in catalase and GPx-1 is unknown. The c-Abl tyrosine kinase has been reported to regulate catalase and GPx-1; fibroblasts deficient in c-Abl have increased levels of catalase and resist oxidant injury [2–4]. We hypothesized that 1) activation of PKG_I would decrease c-Abl protein levels; and 2) inhibition of c-Abl with imatinib would increase antioxidant proteins and hydrogen peroxide (H₂O₂) degradation, attenuate H₂O₂-induced endothelial permeability, and decrease H₂O₂-induced cell death in mouse lung microvascular endothelial cells (MLVMEC).

Methods

MLMVEC were isolated from wild-type (wt) and PKG_I knock-out (PKG_I -/-) C57Bl/6 mice. MLMVEC were treated for 4 hours with 8pCPT-cGMP (50 μM) or imatinib. H₂O₂ scavenging was measured with a H₂O₂ electrode after addition of known concentrations of H₂O₂ to cells in suspension. Nuclear condensation was assessed using fluorescence microscopy. Transendothelial resistance (TER) was measured using electric cell-substrate impedance sensing (ECIS). Proteins were quantified by Western blotting.

Results

Treatment of wt MLMVEC with 8pCPT-cGMP significantly decreased protein levels of c-Abl by 29% (p=0.02). 8pCPT-cGMP had no effect on cAbl in PKG_I -/- MLMVEC. 7 days of sildenafil treatment (100mg/kg/day) [5] significantly decreased whole lung c-Abl protein by 37 % and increased whole lung catalase protein by 40% (p<0.05 for both). Treatment of wt MLMVEC with 10 and 20 µM imatinib increased catalase protein by 16% and 37% respectively, and GPx-1 protein levels by 24% and 36%, respectively (p<0.05 for all values). Imatinib (10 µM) decreased the measured peak H₂O₂ after addition of extracellular H₂O₂ (20, 50, and 100 µM) by 18%, 23%, and 9%, respectively (p ≤0.05 at all concentrations). Imatinib (10 µM) significantly attenuated 100 and 250 µM H₂O₂-induced nuclear condensation by 15% (p<0.05). Finally, imatinib (20 µM) attenuated the H₂O₂-induced TER decrease in monolayers of MLMVEC (p<0.05 by ANOVA).

Conclusion

These data suggest that cGMP, through PKG_I, increases MLMVEC antioxidant activity by down regulating the cAbl tyrosine kinase. Inhibition of c-Abl with imatinib attenuates oxidant-induced MLMVEC death and dysfunction, mimicking the antioxidant effects of cGMP-PKG_I signalling. The mechanism linking activated PKG_I and cAbl expression is unknown.