ERK activation protects against DNA damage and apoptosis in hyperoxic rat AEC2

The survival of type 2 alveolar epithelial cells (AEC2) in the lung after h yperoxic injury is regulated by signals from the cellular environment. Kera tinocyte growth factor and Matrigel can ameliorate the hallmarks of apoptos is seen in hyperoxic AEC2 after 24-h culture on plastic [S. Buckley, L. Bar sky; B. Driscoll, K. Weinberg, K. D. Anderson, and D. Warburton. Am. J. Phy siol. 274 (Lung Cell. Mel. Physiol. 18): L714-L720, 1998]. We used the same model of in vivo short-term hyperoxia to characterize the protective effec ts of substrate attachment. Culture of hyperoxic AEC2 on various biological adhesion substrates showed reduced DNA end labeling in cells grown on all biological substrates compared with growth on plastic. In contrast, the syn thetic substrate poly-D-lysine conferred no protection. Hyperoxic AEC2 cult ured on laminin showed an increased ratio of expression of Bcl-2 to interle ukin-1 beta-converting enzyme compared with culture on plastic. Laminin als o partially restored hyperoxia-depleted glutathione levels and conferred im proved optimal mitochondrial viability as measured by the 3-(4,5-dimethylth iazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Conversely, attach ment to the nonphysiological substrate poly-D-lysine afforded no such prote ction, suggesting that protection against hyperoxia-induced damage may be a ssociated with integrin signaling. Increased activation of extracellular si gnal-regulated kinase (ERK), as detected by increased ERK tyrosine phosphor ylation, was seen in hyperoxic AEC2 as soon as the cells started to attach to laminin and was sustained after 24 h of culture in contrast to that in c ontrol AEC2. To confirm that protection against DNA strand breakage and apo ptosis was being conferred by ERK activation, the cells were also plated in the presence of 50 mu M PD-98059, an inhibitor of the ERK-activating mitog en-activating kinase. Culture for 24 h with PD-98059 abolished the protecti ve effect of laminin. me speculate that after hyperoxic lung injury, signal s through the basement membrane confer specific protection against oxygen-i nduced DNA strand breakage and apoptosis through an ERK activation-dependen t pathway.