Hypoxia modulates pulmonary epithelial cell turnover and surfactant gene expression. † 1576

Intermittent and sustained hypoxia are important clinical problems that occur frequently in the newborn period. Effects of hypoxia on pulmonary epithelial cell turnover and function are not well described. We tested the hypothesis that hypoxia decreases pulmonary epithelial cell growth and surfactant protein expression. We examined the cellular effects of various O₂ concentrations (21%, 7%, 3%, and 1%) and durations(2-24h) using the human pulmonary epithelial cell line, NCI-H441. This line retains certain phenotypic features of pulmonary type II and clara cells. Cell death was assessed by trypan blue uptake and a LDH release assay. Cell proliferation was assessed by a colorimetric tetrazolium dye assay. Expression of surfactant proteins A & B (SP-A, SP-B) and the housekeeping gene GAPDH was assessed by northern blot hybridization with [^(32)P]-labeled antisense cRNA probes. We found that exposure to 7%, 3%, and 1% O₂ for 8h increased cell death by, respectively, 9%, 14%, and 18% after 8h and by 14%, 20%, and 34%; after 24h (each point p<0.05 compared to normoxic controls, n=4). Exposure to graded hypoxia decreased cell proliferation by 22%, 35%, and 38% after 8h and by 12%, 17%, and 29% after 24h (each point p<0.05, n=3). Exposure to graded hypoxia was also associated with decreases in steady-state SP-B mRNA levels (normalized to the corresponding GAPDH signal intensities and to 28s rRNA ethidium bromide fluorescence). SP-B expression was decreased 4.5-fold (3% O₂) and 8.6 -fold (1% O₂) after 8h and was decreased 6.9-fold after 24h exposure to both 3% and 1% O₂. In contrast, under these experimental conditions, hypoxia had no significant effect on SP-A expression. These findings indicate that hypoxia can modulate pulmonary epithelial cell growth and lung-specific gene expression. The molecular mechanism(s) and signal transduction pathways involved in hypoxia-mediated pulmonary epithelial cell turnover and differential gene expression remain to be determined. We speculate that some of these changes may be mediated through hypoxia-induced apoptosis and alterations in oxygen-regulated intracellular effector mollecules.