C/EBP{alpha} is required for pulmonary cytoprotection during hyperoxia

Abstract

A number of transcriptional pathways regulating fetal lung development are active during repair of the injured lung. We hypothesized that C/EBPalpha, a transcription factor critical for lung maturation, plays a role in protection of the alveolar epithelium following hyperoxic injury of the mature lung. Transgenic Cebpalpha(Delta/Delta) mice, in which Cebpalpha was conditionally deleted from Clara cells and type II cells after birth, were developed. While no pulmonary abnormalities were observed in the Cebpalpha(Delta/Delta) mice (7-8 wk old) under normal conditions, the mice were highly susceptible to hyperoxia. Cebpalpha(Delta/Delta) mice died within 4 days of exposure to 95% oxygen in association with severe lung inflammation, altered maturation of surfactant protein B and C, decreased surfactant lipid secretion, and abnormal lung mechanics at a time when all control mice survived. mRNA microarray analysis of isolated type II cells at 0, 2, and 24 h of hyperoxia demonstrated the reduced expression of number of genes regulating surfactant lipid and protein homeostasis, including Srebf, Scap, Lpcat1, Abca3, Sftpb, and Napsa. Genes influencing cell signaling or immune responses were induced in the lungs of Cebpalpha(Delta/Delta) mice. C/EBPalpha was required for the regulation of genes associated with surfactant lipid homeostasis, surfactant protein biosynthesis, processing and transport, defense response to stress, and cell redox homeostasis during exposure to hyperoxia. While C/EBPalpha did not play a critical role in postnatal pulmonary function under normal conditions, C/EBPalpha mediated protection of the lung during acute lung injury induced by hyperoxia.

Fig. 1.

Decreased C/EBPα expression in adult Cebpα^Δ/Δ mice in type II cells (A) and conducting airway epithelial cells (B). Dams of Cebpα^Δ/Δ mice were maintained on doxycycline (in chow) from embryonic day 0. Cebpα^Δ/Δ mice survived postnatally and were treated with doxycycline until 14 days of age. In littermate control mice, C/EBPα was detected in alveolar type II cells (arrowheads) and conducting airways, whereas nuclear staining of respiratory epithelial cells was absent or markedly decreased in Cebpα^Δ/Δ mice assessed at 7 wk of age. Immunohistochemical staining for C/EBPα was detected in alveolar macrophages (arrows) in both control and Cebpα^Δ/Δ mice, indicating the specificity of gene deletion in the respiratory epithelium. There were no changes in lung morphology in Cebpα^Δ/Δ mice under normal conditions. C: decreased expression of Cebpα mRNA in 7-wk-old Cebpα^Δ/Δ mice lungs. Cebpα mRNA in lung was decreased in Cebpα^Δ/Δ mice compared with that in control mice analyzed by RT-PCR (n = 3/group). D: decreased C/EBPα protein (by Western blot) in isolated type II cells. The majority of microscopically identified contaminating cells in isolated type II cells was alveolar macrophages in which C/EBPα was detected in Cebpα^Δ/Δ mice by immunohistochemistry (A). In this system, nearly 70% of Cebpα gene deletion occurs in respiratory epithelial cells from Cebpα^Δ/Δ mice. Type II cells isolated from 1 male and 1 female mouse were pooled. N = 3 pool/group, *P < 0.01 vs. control. E: electron microscopy was performed on lungs from control and Cebpα^Δ/Δ mice in room air at 7 wk of age. The ultrastructure of the lung of Cebpα^Δ/Δ mice, including type II cells, is similar to that of control mice. Shown are representative electron microphotographs of n = 3 mice/group.

Fig. 2.

A: Kaplan-Meier plot of survival of Cebpα^Δ/Δ and control mice in hyperoxia. Seven-week-old Cebpα^Δ/Δ mice and littermate control mice were exposed to 95% O₂. Survival of Cebpα^Δ/Δ mice in hyperoxia (n = 12) was significantly decreased compared with controls (n = 11). P < 0.01 by log-rank test. B: lung morphology (hematoxylin and eosin staining) in room air and after 69-h exposure to 95% O₂. Deletion of Cebpα did not influence lung morphology and was similar between control and Cebpα^Δ/Δ mice in room air (AIR). After exposure to hyperoxia (O₂), control mice showed only minor histological changes (perivascular edema). More severe lung inflammation was observed in Cebpα^Δ/Δ mice in hyperoxia, including thickened alveoli, epithelial necrosis, and air space enlargement. Photomicrographs are representative of n = 4/group. C: increased protein from bronchoalveolar lavage fluid (BALF) in Cebpα^Δ/Δ mice exposed to hyperoxia. Total protein contents were similarly low in control and Cebpα^Δ/Δ mice in room air. After oxygen exposure, protein was significantly increased in the BALF from Cebpα^Δ/Δ mice, suggesting increased alveolar capillary leak. N = 4/group.

Fig. 3.

A: increased IL-1β, IL-6, and MIP-2 in BALF and lung homogenate of Cebpα^Δ/Δ mice after exposure to 95% O₂ for 69 h. Levels of each cytokine were significantly increased in the Cebpα^Δ/Δ mice after hyperoxia. *P < 0.01 vs. others by ANOVA. ND, not detectable; n = 4/group. B: abnormal pulmonary mechanics in Cebpα^Δ/Δ mice following hyperoxia. Lung mechanics were assessed using FlexiVent System in control and Cebpα^Δ/Δ mice exposed to room air or 95% O₂ for 69 h. Lung mechanics were similar in control and Cebpα^Δ/Δ mice in room air. After exposure to 95% O₂, airway resistance, airway elastance, tissue damping, and tissue elastance were increased and compliance decreased significantly in Cebpα^Δ/Δ mice. *P < 0.01 vs. others, ^tP < 0.05 vs. air groups by ANOVA. N = 4/group.

Fig. 4.

A: while surfactant metabolism in Cebpα^Δ/Δ mice was normal in air, saturated phosphatidylcholine (Sat PC) was decreased in Cebpα^Δ/Δ mice BALF after 24-h exposure to hyperoxia. Sat PC in lung tissue after BAL was similar between control and Cebpα^Δ/Δ mice. *P < 0.05 vs. others, n = 5/group. B: intraperitoneally injected [³H]palmitic acid after 24-h exposure to hyperoxia, incorporated into Sat PC 8 h after injection was significantly decreased in BALF. Percentage of [³H]Sat PC in BALF relative to total lung was decreased in Cebpα^Δ/Δ mice suggesting decreased surfactant Sat PC secretion in hyperoxia. *P < 0.05 vs. control, n = 5/group. C: under 24-h hyperoxia stress, deletion of Cebpα influenced lamellar body formation in type II epithelial cells. C, a: size and number of lamellar bodies were unchanged in type II cells from control mice after 24-h exposure to hyperoxia. C, b and c: representative electron micrographs of heterogeneity of lamellar body formation in type II cells from Cebpα^Δ/Δ mice after 24-h hyperoxia are shown. Cebpα^Δ/Δ mice exhibited decreased number of lamellar bodies (b) or smaller-size lamellar bodies (c). Shown are representative electron micrographs of n = 3/groupscale; bars = 2 μm.

Fig. 5.

A: decreased mature SP-B and SP-C in Cebpα^Δ/Δ mice following hyperoxia. Mature surfactant proteins in the same volume BALF were quantitated by Western blot analyses. After exposure to 95% O₂ for 69 h, SP-B and SP-C were markedly decreased in Cebpα^Δ/Δ mice. Values are presented as relative to control mice in air (value given as 1). *P < 0.05 vs. others; n = 3/group. B: pro-SP-B immunostaining on control and Cebpα^Δ/Δ mice lungs in room air and after 69-h exposure to 95% O₂. Pro-SP-B immunostaining in alveolar type II epithelial cells (arrowheads) was similar for all the groups, suggesting altered maturation of SP-B from pro-SP-B in Cebpα^Δ/Δ mice in hyperoxia. Shown are representative microphotographs of n = 4/group. C: representative Western blot for SP-A, SP-B, SP-C, and SP-D in air and after 69-h exposure to hyperoxia; n = 3/group. D: representative Western blot for pro-SP-B after 0- (baseline), 24-, 48-, and 69-h exposure to 95% O₂. E: relative expression of Napsa by RT-PCR in Cebpα^Δ/Δ and control mice lungs during exposure to hyperoxia. Decrease in Napsa mRNA was significantly faster in Cebpα^Δ/Δ mice than control mice in hyperoxia.*P < 0.05 vs. control; n = 5 or 6/group.

Fig. 6.

Comparison analysis of genomic responses induced by exposure to 95% O₂ for 0 h (baseline), 2 h, and 24 h. Differences in mRNAs in the lung of Cebpα^Δ/Δ and control mice were identified through microarray analysis and subject to functional enrichment analysis using Ingenuity Pathway Analysis (IPA). Highly significant genes were selected by Fisher's Exact Test; P < 1.0E-4. The statistical significances in each biological function were presented using negative log transform of P value; n indicates the total gene numbers in each biological function category.

Fig. 7.

Dynamic mRNA profiling between Cebpα^Δ/Δ and control mice after 0-, 2-, and 24-h hyperoxia exposure. A: the heat map of hierarchical clustering results. B: corresponding line graph of each cluster calculated by group mean and standard deviation after 0-, 2-, and 24-h hyperoxia.

Fig. 8.

Cluster 2 genes significantly overlapped with those in the eicosanoid signaling pathway (P = 4.6E-05). The significance is calculated using IPA software based on the likelihood measurement for a given pathway associated with the dataset by random chance. After 24-h hyperoxia challenge, genes increased in Cebpα^Δ/Δ mice are indicated by up arrows; genes decreased in Cebpα^Δ/Δ mice are indicated by short down arrows.

Fig. 9.

Biological association networks of cluster 3 genes revealed by IPA. Genes/proteins are represented by nodes, and the biological relationship between 2 nodes is represented by an edge (line). A solid line indicates direct interaction; a dashed line is indirect interaction; A B indicates binding; A → B indicates A is the cause of B; A B indicates A inhibits B, and indicates self-regulation. The mRNA expression of cluster 3 genes was negatively correlated with the length of hyperoxia exposure time and color coded in green. Positively correlated genes are color coded in pink. Many genes in the network were known to be involved in synthesis, processing, and regulation of surfactant proteins and lipids.

Fig. 10.

Summary of microarray network. Nkx2-1, Srefb1/2, and IL6 are likely the key coregulators of the C/EBPα network. C/EBPα and Nkx2-1 regulate genes involved in surfactant biosynthesis, processing, transport, and assembly. In the network, C/EBPα interacts with Srefb1/2 to control phospholipid and cholesterol biosynthesis and with IL6 to regulate genes in acute/defense response and influences cell redox states.