Gene expression profile of human airway epithelium induced by hyperoxia in vivo

Abstract

Hyperoxia leads to oxidative modification and damage of macromolecules in the respiratory tract with loss of biological functions. Given the lack of antioxidant gene induction with acute exposure to 100% oxygen, we hypothesized that clearance pathways for oxidatively modified proteins may be induced and serve in the immediate cellular response to preserve the epithelial layer. To test this, airway epithelial cells were obtained from individuals under ambient oxygen conditions and after breathing 100% oxygen for 12 h. Gene expression profiling identified induction of genes in the chaperone and proteasome-ubiquitin-conjugation pathways that together comprise an integrated cellular response to manage and degrade damaged proteins. Analyses also revealed gene expression changes associated with oxidoreductase function, cell cycle regulation, and ATP synthesis. Increased HSP70, protein ubiquitination, and intracellular ATP were validated in cells exposed to hyperoxia in vitro. Inhibition of proteasomal degradation revealed the importance of accelerated protein catabolism for energy production of cells exposed to hyperoxia. Thus, the human airway early response to hyperoxia relies predominantly upon induction of cytoprotective chaperones and the ubiquitin-proteasome-dependent protein degradation system to maintain airway homeostatic integrity.

Figure 1.

Genes significantly different under conditions of hyperoxia versus normoxia in five patients for expression of 9,787 genes evaluated in bronchial epithelial cells. The P values were calculated for each gene called “Present” and found in at least three of the four microarrays in each condition using the Welch t test. Ordinate, P value for each gene; abscissa, geometric mean of values in normoxia and hyperoxia, that is, 0.5 × (log₂Nor + log₂Hyp) (68). Genes significantly overexpressed in hyperoxia are in red, and significantly underexpressed in hyperoxia in green.

Figure 2.

Hierarchical clustering of all 9,787 genes (A), or the subset of 135 genes differentially expressed according to a P value below 0.05 between hyperoxia and normoxia (B). Gene and condition trees were generated by hierarchical clustering of genes called “Present” in at least three of the four microarrays using the Pearson correlation. N refers to normoxia, H to hyperoxia, with numbers identifying volunteers. Using all genes (A), clustering occurs by pairs; for example, N3 and H3 are paired normoxia and hyperoxia of Volunteer no. 3. Using the subset of genes (B), clustering is by normoxia and hyperoxia groups. Individuals N1 and H5 are unpaired conditions of normoxia and hyperoxia, respectively.

Figure 3.

Selected cellular pathways triggered by hyperoxia according to the significant biological processes (A) and the molecular functions (B) involved using the GOMINER approach. Upregulated genes are represented in red and downregulated genes in green. P value of functional categories, using Fisher's exact test, are presented in parentheses. See tables for gene descriptions.

Figure 3.

Selected cellular pathways triggered by hyperoxia according to the significant biological processes (A) and the molecular functions (B) involved using the GOMINER approach. Upregulated genes are represented in red and downregulated genes in green. P value of functional categories, using Fisher's exact test, are presented in parentheses. See tables for gene descriptions.

Figure 4.

Ubiquitination of proteins in BET1A cells exposed to 100% O₂ increases over time. Western blot shows increase in ubiquitination of proteins over time, with β-actin as control for protein loading. Graph shows average ± SD of three independent experiments.

Figure 5.

Time course of HSP70 mRNA expression in transformed bronchial epithelial cells (BET1A) and primary HAEC exposed to 100% O₂. The representative Northern blot analysis of total 10μg RNA/lane demonstrates an increase of HSP70 at 24 h. Ribosome 18S is shown as control. Results are representative of four independent experiments.

Figure 6.

(A) ATP levels in BET1A cells exposed to 100% O₂ over time with (closed circles) or without (open circles) ALLN pretreatment. Hyperoxia induces a significant and early increase of ATP in BET1A cells without ALLN pretreatment (*P = 0.01). The use of ALLN prior to hyperoxia significantly reduces ATP levels within BET-1A cells. (B) Increased activation of cell death pathway in BET1A cells exposed to hyperoxia over time with ALLN pretreatment. Western blot shows an increase in cleaved Caspase 8 protein, with β-actin as control for protein loading. Results are representative of three independent experiments.