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

Jimmy Fourtounis 0 I-Ming Wang 2 Marie-Claude Mathieu 0 David Claveau 0 Tenneille Loo 0 Aimee L Jackson 2 Mette A Peters 2 Alex G Therien 1 Yves Boie 0 Michael A Crackower 0 0 Department of Respiratory and Immunology , Merck Research Laboratories, BMB10-128, 33 Avenue Louis Pasteur, Boston, Massachusetts 02115 , USA 1 Infectious Diseases , Merck Research Laboratories, Kenilworth, New Jersey , USA 2 Exploratory and Translational Sciences, Merck Research Laboratories , West Point, Pennsylvania , USA 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. - Background Oxidative stress in tissues leads to the generation of reactive oxygen species which can interfere with normal cellular function and homeostasis and can contribute to the pathophysiology of many diseases including cancer, atherosclerosis, ischemia reperfusion injury, neurodegenerative disorders and aging [1]. The lung is highly susceptible to oxidant stress since it is exposed to high amounts of oxygen [2] and exogenous oxidants found in environmental pollution such as ozone or diesel exhaust particles [3]. As such, markers of oxidative stress are present in the lungs of people with many pathological conditions including asthma [4,5], COPD [6] and acute lung injury [7,8]. There is a large body of evidence from clinical and preclinical studies that this oxidative stress is a key contributor to the disease pathophysiology [9-17] and can modulate responses to pharmacological respiratory therapeutics [18]. Since oxidative stress can have such detrimental effects to the health of the organism, there has evolved an extensive endogenous intracellular and extracellular antioxidant system to maintain redox homeostasis [1]. One of the key regulators of this endogenous antioxidant system is the transcription factor nuclear factor (erythroid-derived 2)-like 2 (NFE2L2, NRF2). NRF2 is basic leucine zipper (bZIP) transcription factor that regulates the expression of numerous genes that encode anti-oxidant and detoxifying phase II enzymes through the binding to cis-acting anti-oxidant response elements (AREs) found in the promoters of these genes. Thus, NRF2 acts as the master regulator of the cellular response to oxidant injury [19]. In order to ensure that the antioxidant response is appropriately regulated, under conditions of redox homeostasis NRF2 is sequestered in the cytoplasm by binding through its N-terminal Neh2 domain to Kelch-like ECH-associated protein 1 (KEAP1) [20,21]. KEAP1 also functions as a substrate adaptor for the cullin-dependent E3 ligase and targets NRF2 for ubiquitination and degradation by the 26S proteasome [22,23]. Several stimuli including oxidants, toxic agents and electrophilic agents can lead to an oxidation of key sulphydryl groups on KEAP1 leading to the release of NRF2 where it can enter the nucleus and activate the antioxidant machinery [24,25]. In support of this, it has been shown that KEAP1 deficiency results in constitutive activation of NRF2 responsive gene expression [26]. There is significant data suggesting a critical role for NRF2 in preventing lung disease. Studies in COPD patients have shown that NRF2 dependent genes are activated in disease [27], but that as disease progresses there is a defect in this antioxidant response [28]. In preclinical species, there is increased expression of NRF2 regulated genes in cigarette smoke induced models of COPD [29] and in allergic lung models [17] implicating NRF2 as an endogenous regulator of oxidative stress in these models. This critical role has been confirmed in studies using Nrf2 deficient mice. In an allergen-induced model of airway inflammation, loss of Nrf2 has been shown to result in an increase in cellular recruitment to the lung, mucus hypersecretion and airway hyperresponsiveness [30]. Similarly, in cigarette smoke-induced models of COPD, Nrf2 deficiency leads to an increase in inflammation and emphysema [31,32]. Additionally, Nrf2 deficient mice have also been shown to have increased susceptibility to acute lung injury [33] and Respiratory Syncytial virus infection [34]. Importantly, treatment of mice with pharmacological agents that can activate NRF2 can lead to the inhibition of cigarette smoke [35] and allergen induced pathology in the lung [17]. Thus, there is a clear demonstration of the critical role of the endogenous anti-oxidant response and NRF2 in regulating airway disease. In order to understand the precise mechanisms of the NRF2 induced anti-oxidant response, researchers have largely turned to expression profiling experiments to determine those genes that mediate NRF2 activity in the tissue or model of interest. Most of these studies have utilized Nrf2 deficient mice or pharmacological treatment of various NRF2 activating compounds to define the NRF2 responsive genes [36-41]. These studies have lead to a well established group of NRF2 regulated genes, however, many novel or differentially regulated genes have been identified suggesting that there are species, tissue and model dependent differences in NRF2 regulated gene expression [42-47]. In t (...truncated)