The circadian clock regulates rhythmic activation of the NRF2/glutathione-mediated antioxidant defense pathway to modulate pulmonary fibrosis

Abstract

The disruption of the NRF2 (nuclear factor erythroid-derived 2-like 2)/glutathione-mediated antioxidant defense pathway is a critical step in the pathogenesis of several chronic pulmonary diseases and cancer. While the mechanism of NRF2 activation upon oxidative stress has been widely investigated, little is known about the endogenous signals that regulate the NRF2 pathway in lung physiology and pathology. Here we show that an E-box-mediated circadian rhythm of NRF2 protein is essential in regulating the rhythmic expression of antioxidant genes involved in glutathione redox homeostasis in the mouse lung. Using an in vivo bleomycin-induced lung fibrosis model, we reveal a clock "gated" pulmonary response to oxidative injury, with a more severe fibrotic effect when bleomycin was applied at a circadian nadir in NRF2 levels. Timed administration of sulforaphane, an NRF2 activator, significantly blocked this phenotype. Moreover, in the lungs of the arrhythmic Clock(Δ19) mice, the levels of NRF2 and the reduced glutathione are constitutively low, associated with increased protein oxidative damage and a spontaneous fibrotic-like pulmonary phenotype. Our findings reveal a pivotal role for the circadian control of the NRF2/glutathione pathway in combating oxidative/fibrotic lung damage, which might prompt new chronotherapeutic strategies for the treatment of human lung diseases, including idiopathic pulmonary fibrosis.

Keywords: NRF2; bleomycin; circadian clock; glutathione; pulmonary fibrosis.

Figure 1.

Circadian expression of NRF2 protein in mouse lungs and fibroblast cells. (A,B) Representative Western blots of rhythmic NRF2 levels in total lung lysates and nuclear extracts from wild-type (WT) mice kept in DD. NRF2 densitometry (mean ± SEM) was normalized to β-actin or histone H1, and the highest expression set as 100%. (Gray bar) Subjective day; (black bar) subjective night. One-way ANOVA for the effect of time, (*) P < 0.05. (C) Representative micrographs of histological lung sections showing temporal NRF2 immunoreactivity at ZT0 versus ZT12. Brown NRF2 staining was localized mainly to bronchial epithelium (BE) and alveolar epithelium (AE). Mean ± SEM; (***) P < 0.001, t-test. Bar, 100 μM. (D) Representative Western blots of temporal NRF2 levels in total lysates of Rat1 fibroblasts following serum shock. NRF2 densitometry (mean ± SEM) was normalized to tubulin. Harvest of cells started at 12 h after serum shock to exclude the initial gene induction. (**) P < 0.01, one-way ANOVA for the effect of time. (E, F) Representative immunofluorescent micrographs and quantification of endogenous NRF2 in Rat1 cells at 24 h or 36 h after serum shock. Nuclei were counterstained with DAPI. Bar, 50 μM. (***) P < 0.001, t-test.

Figure 2.

Circadian clock components control Nrf2 transcription via a putative E-box element in vitro and in vivo. (A) A conserved putative E-box element (shown in red) in the proximal promoters of the mouse, rat, and human Nrf2 gene. Sequences were aligned using the Clustal W algorithm and shaded with the Boxshade algorithm to highlight the conserved regions (shown in black). (B, C) Effects of CLOCK/BMAL1 overexpression on wild-type (WT) or ΔE-box Nrf2∷luc promoter activity in Rat1 cells. Data (mean ± SEM) were normalized to the β-galactosidase reporter gene and expressed relative to pCDNA3 control. The Per1∷luc reporter served as a positive control. (*) P < 0.05; (**) P < 0.01; (ns) not significant, t-test. (D) Effects of PER2 or CRY1 overexpression in suppressing the CLOCK/BMAL1-activated Nrf2∷luc promoter activity in Rat1 cells. (*) P < 0.05; (**) P < 0.01, t-test. (E) Temporal mRNA expression of Nrf2 in wild-type and Clock^Δ19 lungs. Data (mean ± SEM) were normalized to Gapdh. (*) P < 0.05 for wild-type lungs;( ns) not significant for Clock^Δ19 lung; one-way ANOVA for the effect of time. (F) Temporal CLOCK binding to E-boxes in Nrf2 and Dbp gene promoters in wild-type and Clock^Δ19 lungs using CLOCK-specific ChIP. IgG served as a negative control. Data (mean ± SEM) were expressed as percent input. (*) P < 0.05; (ns) not significant, t-test. (White bar) Light phase; (black bar) dark phase.

Figure 3.

Circadian rhythm of NRF2 binding to ARE drives rhythmic oscillations of genes involved in glutathione synthesis and utilization. (A) Temporal mRNA levels of NRF2 targets in wild-type (WT) mouse lungs (DD). Data (mean ± SEM) were normalized to Gapdh, and the lowest expression was set as 1. (*) P < 0.05, one-way ANOVA. (B) Rhythmic NRF2 occupancy on AREs of antioxidant gene promoters in wild-type mouse lungs by NRF2-specific ChIP. The position of each ARE in relation to the TSS is shown. Mean ± SEM. (*) P < 0.05; (**) P < 0.01, t-test. (White bars) Light phase; (black bars) dark phase. (C) Temporal mRNA profiles of NRF2 target genes and clock gene Dbp in MEFs from wild-type and Nrf2 knockout (KO) mice following serum shock. Harvest of cells started at 28 h after serum shock. Data (mean ± SEM) were normalized to Gapdh. (*) P < 0.05 for all six genes in wild type; not significant for Nrf2 knockout MEFs (except Hmox1 and Dbp; [*] P < 0.05), one-way ANOVA for the effect of time. (D) Temporal levels of reduced glutathione (GSH) in MEFs from wild-type and Nrf2 knockout mice following serum shock. Data (mean ± SEM) were normalized to cellular counts and quantified using standard curve for reduced GSH. (**) P < 0.01; (ns) not significant, t-test.

Figure 4.

Time-of-day-dependent lung response to bleomycin challenge is coupled to temporal NRF2 activity. (A) Representative mouse lung sections were stained with Masson's Trichrome solution to reveal collagen fiber deposition (blue staining) and overall fibrosis. Wild-type (WT) mice were treated with bleomycin (n = 8) or saline (n = 5) at either ZT0 or ZT12 and, 7 d later, were sacrificed for tissue processing. Bar, 200 μM. (B) Lung sections in A were scored for fibrosis according to the Ashcroft scoring system. Mean ± SEM; (***) P < 0.001, saline versus bleomycin, t-test; (*) P < 0.05, ZT0 versus ZT12, two-way ANOVA. (C) Fold induction in wild-type lung mRNA levels of NRF2 targets (Gclc, Gsta3, and Hmox1) following bleomycin delivered at ZT0 or ZT12. Data (mean ± SEM) were expressed relative to saline controls at the respective time points. (**) P < 0.01, two-way ANOVA. (D,E) SFN treatment partially rescues the lung fibrosis phenotype, as revealed by Masson's Trichrome staining (with lower Ashcroft score) and reduced gene induction for fibrotic markers (Timp1, Col1a2, Mmp3, and Ctgf). Wild-type mice were treated with either saline (n = 3), bleomycin (n = 4), or SFN plus bleomycin (n = 5) and, 7 d later, were sacrificed for histological and gene expression analysis. Mean ± SEM; (*) P < 0.05; (**) P < 0.01, t-test.

Figure 5.

Loss of rhythmic NRF2 pathway activity in Clock^Δ19 lungs. (A) Representative Western blots of diurnal NRF2 levels in total lung lysates from wild-type (WT) and Clock^Δ19 mice. NRF2 densitometry (mean ± SEM) was normalized to tubulin and expressed relative to wild-type ZT0. (*) P < 0.05 for wild type; not significant for Clock^Δ19, one-way ANOVA for the effect of time. (White bar) Light phase; (black bar) dark phase. (B) Temporal (ZT0 vs. ZT12) ChIP assays for the ARE regions of NRF2 targets (Gclm and Gsta3) in wild-type and Clock^Δ19 lungs using NRF2-specific antibody. Data (mean ± SEM) were expressed as percent input. (*) P < 0.05; (ns) not significant, t-test. (C) Temporal mRNA levels of Gclm and Gsta3 in wild-type and Clock^Δ19 lungs at ZT0 versus ZT12. Data (mean ± SEM) were normalized to Gapdh. (*) P < 0.05; (ns) not significant, t-test.

Figure 6.

A fibrotic-like injury in the lungs of Clock^Δ19 mice is associated with increased oxidative damage. (A) Representative wild-type (WT) and Clock^Δ19 mouse lung sections processed for Masson's Trichrome staining. Bar, 200 μM. Quantification of lung fibrosis in wild-type (n = 4) and Clock^Δ19 (n = 5) mice was performed by the Ashcroft scoring system. (***) P < 0.001, t-test. (B) mRNA levels of fibrotic markers (Ctgf and Mmp3) in wild-type versus Clock^Δ19 lungs. Data (mean ± SEM) were normalized to Gapdh. (**) P < 0.01, t-test. (C) Temporal levels of reduced glutathione (GSH) in wild-type and Clock^Δ19 lungs at ZT0 versus ZT12. Data (mean ± SEM) were normalized to total protein concentration and quantified using a standard curve for reduced GSH. (**) P < 0.01; (ns) not significant, t-test. (D) Protein carbonyl levels in wild-type and Clock^Δ19 lungs at ZT0 versus ZT12 using oxy blotting with anti-dinitrophenyl (DNP) antibody. (+DNPH ) Proteins derivatized with 2,4-dinitrophenylhydrazine (DNPH); (−DNPH) proteins prepared in the absence of DNPH. Data (mean ± SEM) were normalized to tubulin and expressed relative to wild-type at ZT12. (**) P < 0.01; (ns) not significant, t-test.

Figure 7.

A hypothetical model depicting how the circadian clock exerts regulation of the NRF2 pathway under physiological conditions.