Shohei Murakami

Roles of Nrf2 in cell proliferation and differentiation.

The Keap1-Nrf2 system plays pivotal roles in defense mechanisms by regulating cellular redox homeostasis. Nrf2 is an inducible transcription factor that activates a battery of genes encoding antioxidant proteins and phase II enzymes in response to oxidative stress and electrophilic xenobiotics. The activity of Nrf2 is regulated by Keap1, which promotes the ubiquitination and subsequent degradation of Nrf2 under normal conditions and releases the inhibited Nrf2 activity upon exposure to the stresses. Though an impressive contribution of the Keap1-Nrf2 system to the protection from exogenous and endogenous electrophilic insults has been well established, a line of evidence has suggested that the Keap1-Nrf2 system has various novel functions, particularly in cell proliferation and differentiation. Because the proliferation and differentiation of diverse cell types are often influenced and modulated by the cellular redox balance, Nrf2 has been considered to control these cellular processes by regulating the cellular levels of reactive oxygen species (ROS). In addition, analyses of the genome-wide distribution of Nrf2 have identified new sets of Nrf2 target genes whose products are involved in cell proliferation and differentiation but not necessarily in the regulation of oxidative stress. Considering the most characteristic features of Nrf2 as an inducible transcription factor, a newly emerged concept proposes that the Keap1-Nrf2 system translates environmental stresses into regulatory network signals in cell fate determination. In this review, we introduce the contribution of Nrf2 to lineage-specific differentiation, maintenance and differentiation of stem cells, and proliferation of normal and cancer cells, and we discuss how the response to fluctuating environments modulates cell behavior through the Keap1-Nrf2 system.

Keap1-Nrf2 system regulates cell fate determination of hematopoietic stem cells

Nrf2 is a major transcriptional activator of cytoprotective genes against oxidative/electrophilic stress, and Keap1 negatively regulates Nrf2. Emerging works have also suggested a role for Nrf2 as a regulator of differentiation in various cells, but the contribution of Nrf2 to the differentiation of hematopoietic stem cells (HSCs) remains elusive. Clarifying this point is important to understand Nrf2 functions in the development and/or resolution of inflammation. Here, we established two transgenic reporter mouse lines that allowed us to examine Nrf2 expression precisely in HSCs. Nrf2 was abundantly transcribed in HSCs, but its activity was maintained at low levels due to the Keap1‐mediated degradation of Nrf2 protein. When we characterized Keap1‐deficient mice, their bone marrow cells showed enhanced granulocyte‐monocyte differentiation at the expense of erythroid and lymphoid differentiation. Importantly, Keap1‐null HSCs showed lower expression of erythroid and lymphoid genes than did control HSCs, suggesting granulocyte‐monocyte lineage priming in Keap1‐null HSCs. This abnormal lineage commitment was restored by a concomitant deletion of Nrf2, demonstrating the Nrf2‐dependency of the skewing. Analysis of Nrf2‐deficient mice revealed that the physiological level of Nrf2 is sufficient to contribute to the lineage commitment. This study unequivocally shows that the Keap1‐Nrf2 system regulates the cell fate determination of HSCs.

Activation of the NRF2 pathway and its impact on the prognosis of anaplastic glioma patients

BACKGROUND Nuclear factor erythroid 2-related factor 2 (NRF2) plays pivotal roles in cytoprotection. We aimed at clarifying the contribution of the NRF2 pathway to malignant glioma pathology. METHODS NRF2 target gene expression and its association with prognosis were examined in 95 anaplastic gliomas with or without isocitrate dehydrogenase (IDH) 1/2 gene mutations and 52 glioblastomas. To explore mechanisms for the altered activity of the NRF2 pathway, we examined somatic mutations and expressions of the NRF2 gene and those encoding NRF2 regulators, Kelch-like ECH-associated protein 1 (KEAP1) and p62/SQSTSM. To clarify the functional interaction between IDH1 mutations and the NRF2 pathway, we introduced a mutant IDH1 to T98 glioblastoma-derived cells and examined the NRF2 activity in these cells. RESULTS NRF2 target genes were elevated in 13.7% and 32.7% of anaplastic gliomas and glioblastomas, respectively. Upregulation of NRF2 target genes correlated with poor prognosis in anaplastic gliomas but not in glioblastomas. Neither somatic mutations of NRF2/KEAP1 nor dysregulated expression of KEAP1/p62 explained the increased expression of NRF2 target genes. In most cases of anaplastic glioma with mutated IDH1/2, NRF2 and its target genes were downregulated. This was reproducible in IDH1 R132H-expressing T98 cells. In minor cases of IDH1/2-mutant anaplastic gliomas with increased expression of NRF2 target genes, the clinical outcomes were significantly poor. CONCLUSIONS The NRF2 activity is increased in a significant proportion of malignant gliomas in general but decreased in the majority of IDH1/2-mutant anaplastic gliomas. It is plausible that the NRF2 pathway plays an important role in tumor progression of anaplastic gliomas with IDH1/2 mutations.

Glucocorticoid receptor signaling represses the antioxidant response by inhibiting histone acetylation mediated by the transcriptional activator NRF2

NRF2 (nuclear factor erythroid 2-related factor 2) is a key transcriptional activator that mediates the inducible expression of antioxidant genes. NRF2 is normally ubiquitinated by KEAP1 (Kelch-like ECH-associated protein 1) and subsequently degraded by proteasomes. Inactivation of KEAP1 by oxidative stress or electrophilic chemicals allows NRF2 to activate transcription through binding to antioxidant response elements (AREs) and recruiting histone acetyltransferase CBP (CREB-binding protein). Whereas KEAP1-dependent regulation is a major determinant of NRF2 activity, NRF2-mediated transcriptional activation varies from context to context, suggesting that other intracellular signaling cascades may impact NRF2 function. To identify a signaling pathway that modifies NRF2 activity, we immunoprecipitated endogenous NRF2 and its interacting proteins from mouse liver and identified glucocorticoid receptor (GR) as a novel NRF2-binding partner. We found that glucocorticoids, dexamethasone and betamethasone, antagonize diethyl maleate-induced activation of NRF2 target genes in a GR-dependent manner. Dexamethasone treatment enhanced GR recruitment to AREs without affecting chromatin binding of NRF2, resulting in the inhibition of CBP recruitment and histone acetylation at AREs. This repressive effect was canceled by the addition of histone deacetylase inhibitors. Thus, GR signaling decreases NRF2 transcriptional activation through reducing the NRF2-dependent histone acetylation. Consistent with these observations, GR signaling blocked NRF2-mediated cytoprotection from oxidative stress. This study suggests that an impaired antioxidant response by NRF2 and a resulting decrease in cellular antioxidant capacity account for the side effects of glucocorticoids, providing a novel viewpoint for the pathogenesis of hypercorticosteroidism.

NRF2 Is a Key Target for Prevention of Noise-Induced Hearing Loss by Reducing Oxidative Damage of Cochlea

Noise-induced hearing loss (NIHL) is one of the most common sensorineural hearing deficits. Recent studies have demonstrated that the pathogenesis of NIHL is closely related to ischemia-reperfusion injury of cochlea, which is caused by blood flow decrease and free radical production due to excessive noise. This suggests that protecting the cochlea from oxidative stress is an effective therapeutic approach for NIHL. NRF2 is a transcriptional activator playing an essential role in the defense mechanism against oxidative stress. To clarify the contribution of NRF2 to cochlear protection, we examined Nrf2–/– mice for susceptibility to NIHL. Threshold shifts of the auditory brainstem response at 7 days post-exposure were significantly larger in Nrf2–/– mice than wild-type mice. Treatment with CDDO-Im, a potent NRF2-activating drug, before but not after the noise exposure preserved the integrity of hair cells and improved post-exposure hearing levels in wild-type mice, but not in Nrf2–/– mice. Therefore, NRF2 activation is effective for NIHL prevention. Consistently, a human NRF2 SNP was significantly associated with impaired sensorineural hearing levels in a cohort subjected to occupational noise exposure. Thus, high NRF2 activity is advantageous for cochlear protection from noise-induced injury, and NRF2 is a promising target for NIHL prevention.

Systemic Activation of NRF2 Alleviates Lethal Autoimmune Inflammation in Scurfy Mice

ABSTRACT The transcription factor NRF2 (nuclear factor [erythroid-derived 2]-like 2) plays crucial roles in the defense mechanisms against oxidative stress and mediates anti-inflammatory actions under various pathological conditions. Recent studies showed that the dysfunction of regulatory T cells (Tregs) is directly linked to the initiation and progression of various autoimmune diseases. To determine the Treg-independent impact of NRF2 activation on autoimmune inflammation, we examined scurfy (Sf) mice, which are deficient in Tregs and succumb to severe multiorgan inflammation by 4 weeks of age. We found that systemic activation of NRF2 by Keap1 (Kelch-like ECH-associated protein 1) knockdown ameliorated tissue inflammation and lethality in Sf mice. Activated T cells and their cytokine production were accordingly decreased by Keap1 knockdown. In contrast, NRF2 activation through cell lineage-specific Keap1 disruption (i.e., in T cells, myeloid cells, and dendritic cells) achieved only partial or no improvement in the inflammatory status of Sf mice. Our results indicate that systemic activation of NRF2 suppresses effector T cell activities independently of Tregs and that NRF2 activation in multiple cell lineages appears to be required for sufficient anti-inflammatory effects. This study emphasizes the possible therapeutic application of NRF2 inducers in autoimmune diseases that are accompanied by Treg dysfunction.

IL-11 contribution to tumorigenesis in an NRF2 addiction cancer model

The interaction between cancer cells and their microenvironment is an important determinant of the pathological nature of cancers, particularly their tumorigenic abilities. The KEAP1-NRF2 system, originally identified as a critical defense mechanism against oxidative stress, is often dysregulated in various human cancers forming solid tumors, resulting in the aberrant activation of NRF2. Increased accumulation of NRF2 in cancers is strongly associated with the poor prognoses of cancer patients, including those with lung and breast cancers. Multiple lines of evidence suggest that aberrantly activated NRF2 in cancer cells drives their malignant progression and that the cancer cells consequently develop ‘NRF2 addiction.’ Although the downstream effectors of NRF2 that are responsible for cancer malignancy have been extensively studied, mechanisms of how NRF2 activation contributes to the aggressive tumorigenesis remains to be elucidated. In this study, we found a significant correlation between NRF2 and IL-11 status in breast cancer patients. Based on a recent report demonstrating that IL-11 is induced downstream of NRF2, we examined the significance of IL-11 in NRF2-driven tumorigenesis with a newly established NRF2 addiction cancer model. Expression of Il11 was elevated during the tumorigenesis of the NRF2 addiction cancer model, but intriguingly, it was hardly detected when the cancer model cells were cultured in vitro. These results imply that a signal originating from the microenvironment cooperates with NRF2 to activate Il11. To the best of our knowledge, this is the first report showing the influence of the microenvironment on the NRF2 pathway in cancer cells and the contribution of NRF2 to the secretory phenotypes of cancers. Disruption of Il11 in the NRF2 addiction cancer model remarkably inhibited the tumorigenesis, suggesting an essential role of IL-11 in NRF2-driven tumorigenesis. Thus, this study suggests that IL-11 is a potential therapeutic target for NRF2-addicted breast cancers.

Hematopoietic Stem and Progenitor Cell Activation During Chronic Dermatitis Provoked by Constitutively Active Aryl-Hydrocarbon Receptor Driven by Keratin 14 Promoter

Polycyclic aromatic hydrocarbons (PAHs) activate aryl-hydrocarbon receptor (AhR). Because PAHs are known as a risk factor for allergic diseases, PAH-induced AhR activation is expected to be involved in the development of the pathology. We previously generated transgenic mice expressing a constitutively active AhR (AhR-CA) under the control of Keratin 14 (K14) promoter (AhR-CA mouse). The mice develop chronic dermatitis with immune imbalance toward Th2 predominance, indicating that the AhR activation driven by K14 promoter provokes allergic response. Because hematopoietic cells actively participate in the development of allergic inflammation, it is important to understand the hematopoietic status under allergic conditions. To clarify how the K14 promoter-driven AhR activation influences hematopoiesis, we analyzed bone marrow and spleen of AhR-CA mice. We verified that AhR-CA was expressed in keratinocytes and thymic epithelial cells but not in hematopoietic cells. The AhR-CA mice with full-blown dermatitis exhibited leukocytosis and skewed differentiation of hematopoietic progenitor cells toward granulocyte-monocyte lineages. They also showed a significant expansion of short-term hematopoietic stem cells and multipotent progenitors and a subtle reduction in long-term hematopoietic stem cells (LT-HSCs). Their spleens were enlarged and abundantly accumulated hematopoietic stem and progenitor cells. AhR-CA mice at the early stage of dermatitis did not show leukocytosis or splenomegaly but exhibited the granulocyte-monocyte skewing and the reduction in LT-HSCs. Thus, AhR activation driven by K14 promoter already alters the hematopoietic differentiation and reduces LT-HSCs at the initial stage of dermatitis development. These results suggest that nonhematopoietic exposure to PAHs triggers allergic response and concomitantly affects hematopoiesis.

Nucleomethylin deficiency impairs embryonic erythropoiesis

Nucleomethylin (NML) has been shown to contribute to ribosome formation through regulating transcription and post-transcriptional modification of rRNA. Based on the observation that NML-/- mice are frequently embryonic lethal, we analyzed NML-/- embryos to clarify the role of NML in embryogenesis. We found that NML deficiency leads to lethality at the time point between E10.5 and E12.5. Most of E10.5 NML-/- embryos exhibited growth retardation and/or malformation with marked impairment of erythropoiesis. Consistent with a previous study, the m1A in 28S rRNA was dramatically reduced in NML-/- foetal liver (FL) cells. Because the previous study demonstrated p53-dependent apoptosis of NML-knockdown cells, and because we observed upregulation of p21, one of the p53 target genes, in NML-/- FL cells, we tested whether p53 disruption cancelled the NML-deficient phenotypes. Contrary to our expectation, suppression of p53 did not rescue the lethality or impaired erythropoiesis of NML-/- embryos, suggesting that p53-independent mechanisms underlie the NML-deficient phenotypes. These results clarify an essential role of NML during embryogenesis, particularly in erythropoiesis. We surmise that embryonic erythropoiesis is particularly sensitive to impaired protein synthesis, which is caused by the defective methylation of rRNA and consequent failure of ribosome formation.