Gene expression profiling following NRF2 and KEAP1 siRNA knockdown in human lung fibroblasts identifies CCL11/Eotaxin-1 as a novel NRF2 regulated gene

Abstract

Background: Oxidative Stress contributes to the pathogenesis of many diseases. The NRF2/KEAP1 axis is a key transcriptional regulator of the anti-oxidant response in cells. Nrf2 knockout mice have implicated this pathway in regulating inflammatory airway diseases such as asthma and COPD. To better understand the role the NRF2 pathway has on respiratory disease we have taken a novel approach to define NRF2 dependent gene expression in a relevant lung system.

Methods: Normal human lung fibroblasts were transfected with siRNA specific for NRF2 or KEAP1. Gene expression changes were measured at 30 and 48 hours using a custom Affymetrix Gene array. Changes in Eotaxin-1 gene expression and protein secretion were further measured under various inflammatory conditions with siRNAs and pharmacological tools.

Results: An anti-correlated gene set (inversely regulated by NRF2 and KEAP1 RNAi) that reflects specific NRF2 regulated genes was identified. Gene annotations show that NRF2-mediated oxidative stress response is the most significantly regulated pathway, followed by heme metabolism, metabolism of xenobiotics by Cytochrome P450 and O-glycan biosynthesis. Unexpectedly the key eosinophil chemokine Eotaxin-1/CCL11 was found to be up-regulated when NRF2 was inhibited and down-regulated when KEAP1 was inhibited. This transcriptional regulation leads to modulation of Eotaxin-1 secretion from human lung fibroblasts under basal and inflammatory conditions, and is specific to Eotaxin-1 as NRF2 or KEAP1 knockdown had no effect on the secretion of a set of other chemokines and cytokines. Furthermore, the known NRF2 small molecule activators CDDO and Sulphoraphane can also dose dependently inhibit Eotaxin-1 release from human lung fibroblasts.

Conclusions: These data uncover a previously unknown role for NRF2 in regulating Eotaxin-1 expression and further the mechanistic understanding of this pathway in modulating inflammatory lung disease.

Figure 1

Changes in mRNA expression following NRF2 and KEAP1 gene-specific siRNA transfection in NHLFs. (A,C-E) Cells were transfected with negative control siRNA targeting firefly luciferase (FFL), and 2 pools of 10 siRNAs (KEAP1 pool 1 and pools 2) or an esiRNA preparation targeting KEAP1 (esiRNA KEAP1) and evaluated for KEAP1, MRP2/ABCC-2, HMOX1 and NQO1 mRNA expression by QPCR. (B-E) Cells were transfected with negative control siRNA targeting firefly luciferase (FFL), and 3 pools of 10 siRNAs targeting NRF2 (NRF2 pool 1, pool 2, pool 3) and evaluated for NRF2, MRP2/ABCC-2, HMOX1 and NQO1 mRNA expression by QPCR. Data is expressed as expression levels relative to the FFL transfection. n=4 for each data point. A student’s t-test was performed on each data set relative to the FFL data (*p < 0.5, **p < 0.01). Data are represented as mean ± standard deviation.

Figure 2

K-means clustering of gene signature modulated by either KEAP1 or NRF2 siRNAs. (A) K-means clustering of gene signature modulated by either KEAP1 or NRF2 siRNAs at 48 hours post-transfection. Three replicates were profiled for each siRNA pool. One way ANOVA (NRF2 or KEAP1 siRNA-transfected vs. mock-transfected samples) was performed to identify genes up- or down-regulated by NRF2 or KEAP1 siRNA at p ≤ 0.01. Data from all three replicates of each siRNA pool were combined in silico and further filtered by absolute fold change ≥ 1.15. (B) K-means clustering of the anti-correlated gene signature modulated by KEAP1 and NRF2 siRNAs. The gene signature was obtained by combining anti-correlated genes modulated by KEAP1 and NRF2 siRNAs at 30 hours (308 genes at replicates combined p<=0.05) and 48 hours (893 genes at replicates combined p<=0.05). We further removed a 43 interferon-inducible gene set resulting from a KEAP1-3 siRNA pool to obtain a final signature gene set of 1,045 sequences with 361 sequences down-regulated by NRF2 siRNAs and 684 sequences down-regulated by KEAP1 siRNAs.

Figure 3

Modulation of Eotaxin-1 expression following KEAP1 and NRF2 siRNA knockdown. Cells were transfected with negative control siRNA targeting firefly luciferase (FFL), 1 siRNA pool targeting KEAP1 (KEAP1), and 1 siRNA pool targeting NRF2 (NRF2) and evaluated for (A) mRNA expression and (B) secreted potein levels of Eotaxin-1. Data is expressed levels relative to the FFL transfection. n=4 for each data point. A student’s t-test was performed on each data set relative to the FFL data (*p<0.5, **p<0.01). Data are represented as mean ± standard deviation.

Figure 4

Modulation of IL-1β induced cytokine/chemokine secretion following KEAP1 and NRF2 siRNA knockdown. Cells were transfected with negative control siRNA targeting firefly luciferase (FFL), 1 siRNA pool targeting KEAP1 , and 1 siRNA pool targeting NRF2. (A,B) 24 hours following transfection cells were stimulated with IL-1β and select cytokine and chemokine release was evaluated. Data is expressed as protein levels found in tissue culture supernatants. n=2 for each data point. (C) Eotaxin-1 mRNA expression was also evaluated following IL-1β challenge. Data is expressed as mRNA levels relative to baseline FFL transfection ( see Figure 5). N=4 for each data point. A student’s t-test was performed on each data set relative to the FFL data (*p<0.5, **p<0.01). Data are represented as mean ± standard deviation.

Figure 5

Modulation of IL-1β induced cytokine/chemokine by the IKK-β inhibitor Compound A [61]. Cells were treated with Compound A for 24 hours prior to IL-1β stimulation. Following IL-1β challenge select cytokine and chemokine release was evaluated. Data is expressed as protein levels found in tissue culture supernatants. n=3 for each data point. A student’s t-test was performed on each data set relative to the untreated data set (*p<0.5, **p<0.01). Data are represented as mean ± standard deviation.

Figure 6

Modulation of IL-1β, TNFα and IL-13 induced Eotaxin-1 release by CDDO and Sulforaphane. Cells were treated with Compound 1 hour prior to stimulation. Secreted Eotaxin-1 was measured 24 hours following challenge. (A) Dose dependent inhibition of IL-1β induced Eotaxin-1 release. (B) Dose dependent inhibition of TNFα induced Eotaxin-1 release. (C) Dose dependent inhibition of IL-13 induced Eotaxin-1 release. Data is expressed as protein levels found in tissue culture supernatants. n=3 for each data point. A student’s t-test was performed on each data set relative to the untreated data set (*p<0.5, **p<0.01). Data are represented as mean ± standard deviation.