Upstream mediators of p53 in cigarette smoke induced endothelial apoptosis

BACKGROUND: Pulmonary endothelial cell (EC) apoptosis is an early determinant of lung destruction in emphysema. We set out to characterize the upstream molecular mediators required for p53 dependent cigarette smoke (CS) induced EC apoptosis and rationalize them as potential therapeutic targets. p38 mitogen activated protein kinase (MAPK) has been shown to be activated in alveolar walls in patients with COPD. We have observed that XOR activation is necessary for CS-induced EC apoptosis. XOR activation by mechanical stress or hypoxia is through p38 MAPK dependent pathways. We hypothesize that p38 MAPK activation and its upregulation of XOR activity are necessary upstream events in p53 dependent CS-induced EC apoptosis. METHODS: Pulmonary microvascular endothelial cells (PMVEC) were exposed to cigarette smoke extract (CSE) for 4 and 24 hours. Molecular and pharmacologic approaches were used to manipulate p38 MAPK, XOR, and p53 expression. Endpoints included Western blotting and quantification of EC apoptosis as well as XOR enzymatic activity using the pterin fluorimetric assay (Beckman et al., 1989). RESULTS: Exposure to CSE increases p38 MAPK activity, XOR activity, and p53 expression in our PMVEC. When EC were pretreated with inhibitors for p38 MAPK, XOR, or p53, they were protected from CSE-induced apoptosis. Inhibition of p38 MAPK blocked CSE-induced XOR activation. Suppression of the mitochondrial function of p53 with pifithrin-μ blocks CS-induced apoptosis. CONCLUSIONS: CS exposure induces increased apoptosis in PMVEC that is dependent on p53 expression and XOR activation. Inhibition of p38 MAPK prevents upregulation of XOR activation and p53 expression, protecting cells from CS-induced apoptosis.