Role of NRF2 in immune modulator expression in developing lung

Abstract

After birth, the alveolar epithelium is exposed to environmental pathogens and high O₂ tensions. The alveolar type II cells may protect this epithelium through surfactant production. Surfactant protein, SP-A, an immune modulator, is developmentally upregulated in fetal lung with surfactant phospholipid synthesis. Herein, we observed that the redox-regulated transcription factor, NRF2, and co-regulated C/EBPβ and PPARγ, were markedly induced during cAMP-mediated differentiation of cultured human fetal lung (HFL) epithelial cells. This occurred with enhanced expression of immune modulators, SP-A, TDO2, AhR, and NQO1. Like SP-A, cAMP induction of NRF2 was prevented when cells were exposed to hypoxia. NRF2 knockdown inhibited induction of C/EBPβ, PPARγ, and immune modulators. Binding of endogenous NRF2 to promoters of SP-A and other immune modulator genes increased during HFL cell differentiation. In mouse fetal lung (MFL), a developmental increase in Nrf2, SP-A, Tdo2, Ahr, and Nqo1 and decrease in Keap1 occurred from 14.5 to 18.5 dpc. Developmental induction of Nrf2 in MFL was associated with increased nuclear localization of NF-κB p65, a decline in p38 MAPK phosphorylation, increase in the MAPK phosphatase, DUSP1, induction of the histone acetylase, CBP, and decline in the histone deacetylase, HDAC4. Thus, together with surfactant production, type II cells protect the alveolar epithelium through increased expression of NRF2 and immune modulators to prevent inflammation and oxidative stress. Our findings further suggest that lung cancer cells have usurped this developmental pathway to promote immune tolerance and enhance survival.

Keywords: NRF2; SP-A; development; fetal lung; immune modulators; oxygen tension.

Figure 1.. Expression of SP-A and NRF2 are temporally induced by cAMP in HFL epithelial cells in an O₂-dependent manner.

Epithelial cells isolated from mid-gestation HFL were cultured for 24 - 72 h with (+) or without (−) Bt₂cAMP in an atmosphere of 20% or 2% O₂. Expression of NRF2 and SP-A mRNA was temporally increased in a 20% O₂ environment, but failed to increase in hypoxia (2% O₂). Data are the mean ± standard error of the mean (SEM) (n = 3). ***p<0.001; ****p<0.0001 (ANOVA) compared to 24 h vehicle control.

Figure 2.. C/EBPβ and PPARγ are temporally upregulated with NRF2 in HFL epithelial cells during culture and are inhibited by NRF2 knockdown.

(A) HFL epithelial cells were cultured for 24 - 72 h in the absence or presence of Bt₂cAMP. Using RT-qPCR, we observed that expression of NRF2, C/EBPβ and PPARγ mRNA was increased with time in culture and was further stimulated by cAMP treatment. Data are the mean ± SEM (n = 3). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (ANOVA), compared to 24 h vehicle control. (B) HFL epithelial cells were transfected with NRF2 siRNA or a non-targeting (NT) siRNA and cultured for 48 h in the presence of Bt₂cAMP. Using RT-qPCR, we observed that siRNA-mediated NRF2 knockdown decreased C/EBPβ and PPARγ mRNA expression compared to cells transfected with a non-targeting siRNA control (NT-siRNA). Data are mean ± SEM (n = 3). *p<0.05; **p<0.01; (ANOVA), compared to NT-siRNA cells.

Figure 3.. Expression of immune modulators is temporally increased by cAMP during HFL cell differentiation in culture and is inhibited by NRF2 knockdown.

(A) HFL epithelial cells were cultured for 24 – 72 h ± Bt₂cAMP. Expression of SP-A, TDO2, AhR and NQO1 mRNA levels were increased by cAMP as a function of time in culture. Data are mean ± SEM (n = 3). *p<0.05; **p<0.01; ***p<0.001 (ANOVA), compared to 24 h vehicle control. (B) HFL epithelial cells were transfected with NRF2 siRNA or a non-targeting (NT) siRNA and cultured for 48 h in the presence of Bt₂cAMP. NRF2 knockdown decreased SP-A, TDO2, AhR and NQO1 mRNA expression as compared to cells transfected with a non-targeting siRNA control (NT-siRNA). Data are mean ± SEM (n = 3). *p<0.05; **p<0.01 (ANOVA), compared to NT-siRNA cells.

Figure 4.. Endogenous NRF2 binds to SFTPA, C/EBPB, PPARG, AhR, and NQO1 promoters in HFL cells during differentiation in culture in the presence of Bt₂cAMP.

HFL epithelial cells were cultured for up to 72 h in the presence of Bt₂cAMP and subjected to ChIP-qPCR using antibodies to NRF2. Binding of endogenous NRF2 to the SFTPA, C/EBPB, PPARG, AhR, and NQO1 promoters was increased as a function of time in culture with cAMP. Data are mean ± SEM (n = 3). *p<0.05; **p<0.01; ***p<0.001 (ANOVA) compared to NRF2 binding in cells before culture (BC).

Figure 5.. Expression of Nrf2, Pparγ, C/ebpβ and immune modulators increase in mouse fetal lung tissues with advancing gestation.

Mouse fetal lungs at 14.5 –18.5 days post-coitum (dpc) were analyzed for SP-A, Nrf2, Pparγ, C/ebpβ, Ahr and Tdo2 mRNA by RT-qPCR; values were normalized to m36B4 or m18s and expressed as the fold increase over values at 14.5 dpc. Data are mean ± SEM (n = 10 mouse fetal lungs at each gestation time point). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (ANOVA) compared to values at 14.5 dpc.

Figure 6.. Factors regulating NRF2 expression and activity during MFL development.

(A) Mouse fetal lungs at 15.5 –18.5 dpc were analyzed by immunoblotting for the levels of Nrf2, NF-κB-p65, Keap1, p-p38 MAPK, p38 MAPK, p-DUSP1, DUSP1, HDAC4 and CBP. Shown are representative immunoblots. Histone H3 and β-actin were used as loading controls for nuclear and cytoplasmic proteins, respectively. (B) Densitometric quantification of scans of immunoblots (relative to β-actin and H3 as loading controls) from three biological replicates corresponding to the representative immunoblots shown in Panel A. Data are the mean ± SEM (n=3). *p<0.05, **p<0.01, ***p<0.001, compared to 15.5 dpc.

Figure 7.. Expression of NRF2 and immune modulators is increased in lung adenocarcinoma cell lines, which have hijacked a developmental pathway to enhance their survival.

(A) Expression of NRF2, NQO1, AhR and TDO2 is higher in lung adenocarcinoma cell lines, H1838, H2228, H2347 and HCC4150, compared to normal human bronchial human epithelial cells, HBEC3-KT. (B) cAMP increases NRF2, NQO1, AhR and TDO2 mRNA expression in cultured HCC4150 human lung adenoCa cells after 72 h. Data are mean ± SEM. **p<0.01; ***p<0.001; ****p<0.0001 (ANOVA), compared to Control (Con) (n = 3). (C) NRF2 knockdown in cultured HCC4150 human adenoCa cells inhibits NQO1, AhR and TDO2 mRNA at 72 h, compared to untransfected cells or cells transfected with nontargeting (NT) siRNA. Data are mean ± SEM (n = 3) *p<0.05; **p<0.01 (ANOVA), compared to NT-siRNA cells. (D) A conserved developmentally-regulated pathway in fetal lung involving transcription factors NF-κB, NRF2, C/EBPβ, PPARγ and immune modulators that protect the alveolar epithelium from oxidative stress and inflammation after birth is co-opted by lung adenocarcinoma cells to possibly protect lung cancer metastases from attack by the host immune system. Shown in the top panels are fetal lung tissues from mid-gestation (90 dpc, Left) and term (175 dpc, Right) fetal baboons immunostained for SP-A (Texas Red).