Loss of Nrf1 rather than Nrf2 leads to inflammatory accumulation of lipids and reactive oxygen species in human hepatoma cells, which is alleviated by 2-bromopalmitate.

Since Nrf1 and Nrf2 are essential for regulating the lipid metabolism pathways, their dysregulation has thus been shown to be critically involved in the non-controllable inflammatory transformation into cancer. Herein, we have explored the molecular mechanisms underlying their distinct regulation of lipid metabolism, by comparatively analyzing the changes in those lipid metabolism-related genes in Nrf1α-/- and/or Nrf2-/- cell lines relative to wild-type controls. The results revealed that loss of Nrf1α leads to lipid metabolism disorders. That is, its lipid synthesis pathway was up-regulated by the JNK-Nrf2-AP1 signaling, while its lipid decomposition pathway was down-regulated by the nuclear receptor PPAR-PGC1 signaling, thereby resulting in severe accumulation of lipids as deposited in lipid droplets. By contrast, knockout of Nrf2 gave rise to decreases in lipid synthesis and uptake capacity. These demonstrate that Nrf1 and Nrf2 contribute to significant differences in the cellular lipid metabolism profiles and relevant pathological responses. Further experimental evidence unraveled that lipid deposition in Nrf1α-/- cells resulted from CD36 up-regulation by activating the PI3K-AKT-mTOR pathway, leading to abnormal activation of the inflammatory response. This was also accompanied by a series of adverse consequences, e.g., accumulation of reactive oxygen species (ROS) in Nrf1α-/- cells. Interestingly, treatment of Nrf1α-/- cells with 2-bromopalmitate (2BP) enabled the yield of lipid droplets to be strikingly alleviated, as accompanied by substantial abolishment of CD36 and critical inflammatory cytokines. Such Nrf1α-/- -led inflammatory accumulation of lipids, as well as ROS, was significantly ameliorated by 2BP. Overall, this study provides a potential strategy for cancer prevention and treatment by precision targeting of Nrf1, Nrf2 alone or both.

Single-cell RNA sequencing reveals a pro-metastatic subpopulation and the driver transcription factor NFE2L1 in ovarian cancer cells.

BACKGROUND: Although cytoreductive surgery followed by adjuvant chemotherapy is effective as a standard treatment for early-stage ovarian cancer, the majority of ovarian cancer cases are diagnosed at the advanced stages with dissemination to the peritoneal cavity, leading to a poor prognosis. Therefore, it is crucial to understand the cellular and molecular mechanisms underlying metastasis and identify novel therapeutic targets. OBJECTIVE: In this study, we aimed to elucidate the mechanisms underlying gene expression alterations during the acquisition of metastatic potential and characterize the metastatic subpopulations within ovarian cancer cells. METHODS: We conducted single-cell RNA sequencing of two human ovarian cancer cell lines: SKOV-3 and SKOV-3-13, a highly metastatic subclone of SKOV-3. Suppression of NFE2L1 expression was performed through siRNA-mediated knockdown and CRISPR-Cas9-mediated knockout. RESULTS: Clustering and pseudotime trajectory analysis revealed pro-metastatic subpopulation within these cells. Furthermore, gene set enrichment analysis and prognosis analysis indicated that NFE2L1 could be a key transcription factor in the acquisition of metastasis potential. Inhibition of NFE2L1 significantly reduced migration and viability of both cells. In addition, NFE2L1 knockout cells exhibited significantly reduced tumor growth in a mouse xenograft model, recapitulating in silico and in vitro results. CONCLUSION: The results presented in this study deepen our understanding of the molecular pathogenesis of ovarian cancer metastasis with the ultimate goal of developing treatments targeting pro-metastatic subclones prior to metastasis.

Unravelling the role of NFE2L1 in stress responses and related diseases.

The nuclear factor erythroid 2 (NF-E2)-related factor 1 (NFE2L1, also known as Nrf1) is a highly conserved transcription factor that belongs to the CNC-bZIP subfamily. Its significance lies in its control over redox balance, proteasome activity, and organ integrity. Stress responses encompass a series of compensatory adaptations utilized by cells and organisms to cope with extracellular or intracellular stress initiated by stressful stimuli. Recently, extensive evidence has demonstrated that NFE2L1 plays a crucial role in cellular stress adaptation by 1) responding to oxidative stress through the induction of antioxidative responses, and 2) addressing proteotoxic stress or endoplasmic reticulum (ER) stress by regulating the ubiquitin-proteasome system (UPS), unfolded protein response (UPR), and ER-associated degradation (ERAD). It is worth noting that NFE2L1 serves as a core factor in proteotoxic stress adaptation, which has been extensively studied in cancer and neurodegeneration associated with enhanced proteasomal stress. In these contexts, utilization of NFE2L1 inhibitors to attenuate proteasome "bounce-back" response holds tremendous potential for enhancing the efficacy of proteasome inhibitors. Additionally, abnormal stress adaptations of NFE2L1 and disturbances in redox and protein homeostasis contribute to the pathophysiological complications of cardiovascular diseases, inflammatory diseases, and autoimmune diseases. Therefore, a comprehensive exploration of the molecular basis of NFE2L1 and NFE2L1-mediated diseases related to stress responses would not only facilitate the identification of novel diagnostic and prognostic indicators but also enable the identification of specific therapeutic targets for NFE2L1-related diseases.

Ferroptosis regulation by the NGLY1/NFE2L1 pathway.

SignificanceFerroptosis is an oxidative form of cell death whose biochemical regulation remains incompletely understood. Cap'n'collar (CNC) transcription factors including nuclear factor erythroid-2-related factor 1 (NFE2L1/NRF1) and NFE2L2/NRF2 can both regulate oxidative stress pathways but are each regulated in a distinct manner, and whether these two transcription factors can regulate ferroptosis independent of one another is unclear. We find that NFE2L1 can promote ferroptosis resistance, independent of NFE2L2, by maintaining the expression of glutathione peroxidase 4 (GPX4), a key protein that prevents lethal lipid peroxidation. NFE2L2 can also promote ferroptosis resistance but does so through a distinct mechanism that appears independent of GPX4 protein expression. These results suggest that NFE2L1 and NFE2L2 independently regulate ferroptosis.

NFE2L1-mediated proteasome function protects from ferroptosis.

OBJECTIVE: Ferroptosis continues to emerge as a novel modality of cell death with important therapeutic implications for a variety of diseases, most notably cancer and degenerative diseases. While susceptibility, initiation, and execution of ferroptosis have been linked to reprogramming of cellular lipid metabolism, imbalances in iron-redox homeostasis, and aberrant mitochondrial respiration, the detailed mechanisms of ferroptosis are still insufficiently well understood. METHODS AND RESULTS: Here we show that diminished proteasome function is a new mechanistic feature of ferroptosis. The transcription factor nuclear factor erythroid-2, like-1 (NFE2L1) protects from ferroptosis by sustaining proteasomal activity. In cellular systems, loss of NFE2L1 reduced cellular viability after the induction of both chemically and genetically induced ferroptosis, which was linked to the regulation of proteasomal activity under these conditions. Importantly, this was reproduced in a Sedaghatian-type Spondylometaphyseal Dysplasia (SSMD) patient-derived cell line carrying mutated glutathione peroxidase-4 (GPX4), a critical regulator of ferroptosis. Also, reduced proteasomal activity was associated with ferroptosis in Gpx4-deficient mice. In a mouse model for genetic Nfe2l1 deficiency, we observed brown adipose tissue (BAT) involution, hyperubiquitination of ferroptosis regulators, including the GPX4 pathway, and other hallmarks of ferroptosis. CONCLUSION: Our data highlight the relevance of the NFE2L1-proteasome pathway in ferroptosis. Manipulation of NFE2L1 activity might enhance ferroptosis-inducing cancer therapies as well as protect from aberrant ferroptosis in neurodegeneration, general metabolism, and beyond.

Multiple myeloma cells depend on the DDI2/NRF1-mediated proteasome stress response for survival.

Multiple myeloma (MM) cells suffer from baseline proteotoxicity as the result of an imbalance between the load of misfolded proteins awaiting proteolysis and the capacity of the ubiquitin-proteasome system to degrade them. This intrinsic vulnerability is at the base of MM sensitivity to agents that perturb proteostasis, such as proteasome inhibitors (PIs), the mainstay of modern-day myeloma therapy. De novo and acquired PI resistance are important clinical limitations that adversely affect prognosis. The molecular mechanisms underpinning PI resistance are only partially understood, limiting the development of drugs that can overcome it. The transcription factor NRF1 is activated by the aspartic protease DNA damage inducible 1 homolog 2 (DDI2) upon proteasome insufficiency and governs proteasome biogenesis. In this article, we show that MM cells exhibit baseline NRF1 activation and are dependent upon DDI2 for survival. DDI2 knockout (KO) is cytotoxic for MM cells, both in vitro and in vivo. Protein structure-function studies show that DDI2 KO blocks NRF1 cleavage and nuclear translocation, causing impaired proteasome activity recovery upon irreversible proteasome inhibition and, thereby, increasing sensitivity to PIs. Add-back of wild-type, but not of catalytically dead DDI2, fully rescues these phenotypes. We propose that DDI2 is an unexplored promising molecular target in MM by disrupting the proteasome stress response and exacerbating proteotoxicity.

Nfe2l1 deficiency mitigates streptozotocin-induced pancreatic ß-cell destruction and development of diabetes in male mice.

Streptozotocin (STZ) is a pancreatic ß cell-specific toxicant that is widely used to generate models of diabetes in rodents as well as in the treatment of tumors derived from pancreatic ß cells. DNA alkylation, oxidative stress and mitochondrial toxicity have been recognized as the mechanisms for STZ-induced pancreatic ß cell damage. Here, we found that pancreatic ß cell-specific deficiency of nuclear factor erythroid-derived factor 2-related factor 1 (NFE2L1), a master regulator of the cellular adaptive response to a variety of stresses, in mice led to a dramatic resistance to STZ-induced hyperglycemia. Indeed, fifteen days subsequent to last dosage of STZ, the pancreatic ß cell specific Nfe2l1 knockout [Nfe2l1(ß)-KO] mice showed reduced hyperglycemia, improved glucose tolerance, higher plasma insulin and more intact islets surrounded by exocrine acini compared to the Nfe2l1-Flox control mice with the same treatment. Immunohistochemistry staining revealed a greater amount of insulin-positive cells in the pancreas of Nfe2l1(ß)-KO mice than those in Nfe2l1-Flox mice 15 days after the last STZ injection. In line with this observation, both isolated Nfe2l1(ß)-KO islets and Nfe2l1-deficient MIN6 (Nfe2l1-KD) cells were resistant to STZ-induced toxicity and apoptosis. Furthermore, pretreatment of the MIN6 cells with glycolysis inhibitor 2-Deoxyglucose sensitized Nfe2l1-KD cells to STZ-induced toxicity. These findings demonstrated that loss of Nfe2l1 attenuates pancreatic ß cells damage and dysfunction caused by STZ exposure, partially due to Nfe2l1 deficiency-induced metabolic switch to enhanced glycolysis.

Metformin leads to accumulation of reactive oxygen species by inhibiting the NFE2L1 expression in human hepatocellular carcinoma cells.

Metformin, as the first-line drug for the treatment of type 2 diabetes mellitus, has been shown to possess a capability to activate or inhibit the production of reactive oxygen species (ROS) in different ways. However, the detailed mechanisms of the opposite effect are poorly understood. Here we provide evidence that metformin induces accumulation of ROS by inhibiting the expression of a core antioxidant transcription factor nuclear factor erythroid 2 like 1 (NFE2L1/Nrf1) in human hepatocellular carcinoma HepG2 cells. In the present study, we originally found that the increased ROS induced by metformin was blunted in NFE2L1 knockdown cell line. Furtherly by examining the effects of metformin on endogenous and exogenous NFE2L1, we also found metformin could not only inhibit the transcription of NFE2L1 gene, but also promote the degradation of NFE2L1 protein at the post-transcriptional level, whereas this effect can be reversed by high glucose. The inhibitory effect of metformin on NFE2L1 was investigated to occur through the N-terminal domain (NTD) of NFE2L1 protein, and its downregulation by metformin was in an AMP-activated protein kinase (AMPK)-independent manner. But the activation of AMPK signaling pathway by metformin in NFE2L1 knockdown HepG2 cells is reversed, indicating that NFE2L1 may be an important regulator of AMPK signal. Altogether, this work provides a better understanding of the relationship between metformin and oxidative stress, and hence contributes to translational study of metformin through its hypoglycemic and tumor suppressive effects.

Long-isoform NRF1 protects against arsenic cytotoxicity in mouse bone marrow-derived mesenchymal stem cells by suppressing mitochondrial ROS and facilitating arsenic efflux.

Acute exposure to arsenic is known to cause bone marrow depression and result in anemia, in which the dusfunction of cells in the bone marrow niche such as mesenchymal stem cells (MSCs) is vital. However, the mechanism underlying response of MSCs to arsenic challange is not fully understood. In the present study, we investigated the role of nuclear factor erythroid 2-related factor (NRF) 1 (NRF1), a sister member of the well-known master regulator in antioxidative response NRF2, in arsenite-induced cytotoxicity in mouse bone marrow-derived MSCs (mBM-MSCs). We found that arsenite exposure induced significant increase in the protein level of long-isoform NRF1 (L-NRF1). Though short-isoform NRF1 (S-NRF1) was induced by arsenite at mRNA level, its protein level was not obviously altered. Silencing L-Nrf1 sensitized the cells to arsenite-induced cytotoxicity. L-Nrf1-silenced mBM-MSCs showed decreased arsenic efflux with reduced expression of arsenic transporter ATP-binding cassette subfamily C member 4 (ABCC4), as well as compromised NRF2-mediated antioxidative defense with elevated level of mitochondrial reactive oxygen species (mtROS) under arsenite-exposed conditions. A specific mtROS scavenger (Mito-quinone) alleviated cell apoptosis induced by arsenite in L-Nrf1-silenced mBM-MSCs. Taken together, these findings suggest that L-NRF1 protects mBM-MSCs from arsenite-induced cytotoxicity via suppressing mtROS in addition to facilitating cellular arsenic efflux.

Regulation of NRF1, a master transcription factor of proteasome genes: implications for cancer and neurodegeneration.

The ability to sense proteasome insufficiency and respond by directing the transcriptional synthesis of de novo proteasomes is a trait that is conserved in evolution and is found in organisms ranging from yeast to humans. This homeostatic mechanism in mammalian cells is driven by the transcription factor NRF1. Interestingly, NRF1 is synthesized as an endoplasmic reticulum (ER) membrane protein and when cellular proteasome activity is sufficient, it is retrotranslocated into the cytosol and targeted for destruction by the ER--associated degradation pathway (ERAD). However, when proteasome capacity is diminished, retrotranslocated NRF1 escapes ERAD and is activated into a mature transcription factor that traverses to the nucleus to induce proteasome genes. In this Perspective, we track the journey of NRF1 from the ER to the nucleus, with a special focus on the various molecular regulators it encounters along its way. Also, using human pathologies such as cancer and neurodegenerative diseases as examples, we explore the notion that modulating the NRF1-proteasome axis could provide the basis for a viable therapeutic strategy in these cases.

SLCO4A1-AS1 promotes cell growth and induces resistance in lung adenocarcinoma by modulating miR-4701-5p/NFE2L1 axis to activate WNT pathway.

Long noncoding RNAs (lncRNAs) possessed essential functions in the biological behaviors of various human cancers. SLCO4A1 antisense RNA 1 (SLCO4A1-AS1) is a lncRNA that has been reported as a oncogenic regulator in colorectal cancer and bladder cancer. However, whether it exerted functions in the gene expression and cellular processes in lung adenocarcinoma (LUAD) remains still obscure. In the present research, we unveiled the high level of SLCO4A1-AS1 in LUAD tissues and cells. Moreover, functional assays indicated that SLCO4A-AS1 facilitated LUAD cell proliferation, motility, and cisplatin-resistance. Besides, mechanism investigation revealed that miR-4701-5p could interact with SLCO4A1-AS1 and directly target to NFE2L1. The expression correlation between miR-4701-5p and SLCO4A1-AS1 or NFE2L1 was found to be negative. Moreover, NFE2L1 was expressed at a same tendency with SLCO4A1-AS1 in LUAD tissues and cells. In addition, it was confirmed that NFE2L1 was involved in SLCO4A1-AS1-mediated activation of WNT pathway. According to rescue assays, NFE2L1 could involve in SLCO4A1-AS1-mediated LUAD cell growth. Conclusively, our study demonstrated that SLCO4A1-AS1 facilitated cell growth and enhanced the resistance of LUAD cells to chemotherapy via activating WNT pathway through miR-4701-5p/NFE2L1 axis.

BET Inhibitors Synergize with Carfilzomib to Induce Cell Death in Cancer Cells via Impairing Nrf1 Transcriptional Activity and Exacerbating the Unfolded Protein Response.

Currently, proteasome inhibitors bortezomib, carfilzomib, and ixazomib are successfully used in clinics to treat multiple myeloma. However, these agents show limited efficacy against solid tumors. Identification of drugs that can potentiate the action of proteasome inhibitors could help expand the use of this therapeutic modality to solid tumors. Here, we found that bromodomain extra-terminal (BET) family protein inhibitors such as JQ1, I-BET762, and I-BET151 synergize with carfilzomib in multiple solid tumor cell lines. Mechanistically, BET inhibitors attenuated the ability of the transcription factor Nrf1 to induce proteasome genes in response to proteasome inhibition, thus, impeding the bounce-back response of proteasome activity, a critical pathway by which cells cope with proteotoxic stress. Moreover, we found that treatment with BET inhibitors or depletion of Nrf1 exacerbated the unfolded protein response (UPR), signaling that was initiated by proteasome inhibition. Taken together, our work provides a mechanistic explanation behind the synergy between proteasome and BET inhibitors in cancer cell lines and could prompt future preclinical and clinical studies aimed at further investigating this combination.

Identification of microRNA­363­3p as an essential regulator of chondrocyte apoptosis in osteoarthritis by targeting NRF1 through the p53­signaling pathway.

Osteoarthritis (OA) is a degenerative joint disease that affects the physical, and mental health of middle­aged and elderly people. The aims of the present study were to determine the biological function and molecular mechanisms of miR­363­3p in chondrocyte apoptosis. Exploration of the molecular mechanisms of OA may be helpful in the understand of the causes, and facilitating the prevention and treatment of OA. In the present study, the expression of nuclear respiratory factor1 (NRF1) was downregulated in the articular cartilage of OA rats in vivo and lipopolysaccharide (LPS)­treated chondrocytes in vitro. MicroRNAs (miRNA) are regulators of gene expression in the progression of OA. TargetScan software was used to predict that NRF1 was a potential target for miRNA (miR)­363, and this was confirmed in subsequent experiments. The expression of miR­363­3p was negatively correlated with the expression of NRF1, and its expression was significantly upregulated in OA model rats and in LPS­induced chondrocytes compared with the expression in the respective controls. In addition, the overexpression of miR­363­3p increased the levels of interleukin (IL)­1ß, IL­6 and tumor necrosis factor­α in vivo, and was demonstrated to promote chondrocyte injury and apoptosis by Safranin O staining and TUNEL. Moreover, the inhibition of miR­363­3p expression increased the expression of NRF1 and protected chondrocytes from apoptosis in vitro and in vivo, whereas the overexpression of miR­363­3p downregulated NRF1 expression and promoted LPS­induced chondrocyte apoptosis through the p53 pathway in vitro. The results of this study suggested that miR­363­3p­mediated inhibition of NRF1may be associated with chondrocyte apoptosis in OA.

Pro-renin receptor suppresses mitochondrial biogenesis and function via AMPK/SIRT-1/ PGC-1α pathway in diabetic kidney.

Abnormal mitochondrial biogenesis and function has been linked to multiple diseases including diabetes. Recently, we demonstrated the role of renal (Pro)renin receptor (PRR) in the dysregulation of mitochondria. We hypothesized that PRR contributes to the reduction of mitochondrial biogenesis and function in diabetic kidney via PGC-1α/AMPK/SIRT-1 signaling pathway. In vivo and in vitro studies were conducted in C57BL/6 mouse and mouse renal mesangial cells (mRMCs). Control and streptozotocin-induced diabetic mice were injected with scramble or PRR shRNA and followed for a period of eight weeks. PRR mRNA and protein expression increased by 44% and 39% respectively (P<0.05) in kidneys of diabetic mice, and in mRMCs exposed to high glucose by 43 and 61% respectively compared to their respective controls. These results were accompanied by reduced mRNA and protein expressions of PGC-1α (67% and 75%), nuclear respiratory factors (NRF-1, 48% and 53%), mitochondrial transcriptional factor A (mtTFA, 56% and 40%), mitochondrial DNA copy number by 75% (all, P<0.05), and ATP production by 54%, respectively in diabetic kidneys and in mRMCs exposed to high glucose. Compared to non-diabetic control mice, PRR knockdown in diabetic mice and in mRMCs, not only attenuated the PRR mRNA and protein expression but also normalized mRNA and protein expressions of PGC-1α, NRF-1, mtTFA, mitochondrial DNA copy number, and ATP production. Treatment with AMPK inhibitor, Compound C, or SIRT-1 inhibitor, EX-527, alone, or combined with PRR siRNA caused marked reduction of mRNA expression of PGC-1α, NRF-1 and mtTFA, and ATP production in mRMCs exposed to high glucose. In conclusion, our study demonstrated the contribution of the PRR to the reduction of mitochondrial biogenesis and function in diabetic kidney disease via decreasing AMPK/SIRT-1/ PGC-1α signaling pathway.

NRF1 and NRF2 mRNA and Protein Expression Decrease Early during Melanoma Carcinogenesis: An Insight into Survival and MicroRNAs.

The prognostic significance of the major redox regulator nuclear factor erythroid-2-related factor (NRF2) is recognized in many cancers, but the role of NRF1 is not generally well understood in cancer. Our aim was to investigate these redox transcription factors in conjunction with redox-related microRNAs in naevi and melanoma. We characterized the immunohistochemical expression of NRF1 and NRF2 in 99 naevi, 88 primary skin melanomas, and 67 lymph node metastases. In addition, NRF1 and NRF2 mRNA and miR-23B, miR-93, miR-144, miR-212, miR-340, miR-383, and miR-510 levels were analysed with real-time qPCR from 54 paraffin-embedded naevi and melanoma samples. The immunohistochemical expression of nuclear NRF1 decreased from benign to dysplastic naevi (p < 0.001) and to primary melanoma (p < 0.001) and from primary melanoma to metastatic lesions (p = 0.012). Also, NRF1 mRNA levels decreased from benign naevi to dysplastic naevi (p = 0.034). Similarly, immunopositivity of NRF2 decreased from benign to dysplastic naevi (p = 0.02) and to primary lesions (p = 0.018). NRF2 mRNA decreased from benign to dysplastic naevi and primary melanomas (p = 0.012). Analysis from the Gene Expression Omnibus datasets supported the mRNA findings. High nuclear immunohistochemical NRF1 expression in pigment cells associated with a worse survival (p = 0.048) in patients with N0 disease at the time of diagnosis, and high nuclear NRF2 expression in pigment cells associated with a worse survival (p = 0.033) in patients with M0 disease at the time of diagnosis. In multivariate analysis, neither of these variables exceeded the prognostic power of Breslow. The levels of miR-144 and miR-212 associated positively with ulceration (p = 0.012 and p = 0.027, respectively) while miR-510 levels associated positively with lymph node metastases at the time of diagnosis (p = 0.004). Furthermore, the miRNAs correlated negatively with the immunohistochemical expression of NRF1 and NRF2 but positively with their respective mRNA. Together, this data sheds new light about NFE2L family factors in pigment tumors and suggests that these factors are worth for further explorations.

T-2 toxin upregulates the expression of human cytochrome P450 1A1 (CYP1A1) by enhancing NRF1 and Sp1 interaction.

T-2 toxin is a major pollutant in crops and feedstuffs. Due to its high toxicity in a variety of organisms, T-2 toxin is of great concern as a threat to humans and to animal breeding. Overexpression of CYP1A1 may contribute to carcinogenesis, and CYP1A1 may be a promising target for the prevention and treatment of human malignancies. Therefore, it is essential to understand the regulatory mechanism by which T-2 toxin induces CYP1A1 expression in human cells. In this study, we confirmed that T-2 toxin (100 ng/mL) induced the expression of CYP1A1 in HepG2 cells through NRF1 and Sp1 bound to the promoter instead of through the well-recognized Aromatic hydrocarbon receptors (AhR). In cells treated with T-2 toxin, Sp1, but not NRF1, was significantly upregulated. However, T-2 toxin apparently promoted the interaction between NRF1 and Sp1 proteins, as revealed by IP analysis. Furthermore, in T-2 toxin-treated HepG2 cells, nuclear translocation of NRF1 was enhanced, while knockdown of Sp1 ablated NRF1 nuclear enrichment. Our results revealed that the upregulation of CYP1A1 by T-2 toxin in HepG2 cells depended on enhanced interaction between Sp1 and NRF1. This finding suggests the tumorigenic features of T-2 toxin might be related to the CYP1A1, which provides new insights to understand the toxicological effect of T-2 toxin.

Distinct isoforms of Nrf1 diversely regulate different subsets of its cognate target genes.

The single Nrf1 gene has capability to be differentially transcripted alongside with alternative mRNA-splicing and subsequent translation through different initiation signals so as to yield distinct lengths of polypeptide isoforms. Amongst them, three of the most representatives are Nrf1α, Nrf1ß and Nrf1γ, but the putative specific contribution of each isoform to regulating ARE-driven target genes remains unknown. To address this, we have herein established three cell lines on the base of the Flp-In T-REx system, which are allowed for the tetracycline-inducibly stable expression of Nrf1α, Nrf1ß and Nrf1γ. Consequently, the RNA-Sequencing results have demonstrated that a vast majority of differentially expressed genes (i.e. >90% DEGs detected) were dominantly up-regulated by Nrf1α and/or Nrf1ß following induction by tetracycline. By contrast, the other DEGs regulated by Nrf1γ were far less than those regulated by Nrf1α/ß (i.e. ~11% of Nrf1α and ~7% of Nrf1ß). However, further transcriptomic analysis revealed that the tetracycline-induced expression of Nrf1γ significantly increased the percentage of down-regulated genes in total DEGs. These statistical data were further validated by quantitative real-time PCR. The experimental results indicate that distinct Nrf1 isoforms make diverse and even opposing contributions to regulating different subsets of target genes, such as those encoding 26S proteasomal subunits and others involved in various biological processes and functions. Collectively, Nrf1γ acts as a major dominant-negative inhibitor competitively against Nrf1α/ß activity, such that a number of DEGs regulated by Nrf1α/ß are counteracted by Nrf1γ.

Autophagy controls neonatal myogenesis by regulating the GH-IGF1 system through a NFE2L2- and DDIT3-mediated mechanism.

Macroautophagy/autophagy is emerging as an important process in adult muscle stem cells functions: it regulates metabolic reprogramming during activation from a quiescent state, maintains stemness and prevents senescence. We now show that autophagy is specifically required for neonatal myogenesis and muscle development. Specific deletion of Atg7 in PAX7+ (paired box 7) precursors led in mice to a dwarf phenotype, with an effect restricted to the neonatal phase of muscle development. Atg7 knockdown suppressed neonatal satellite cell (nSC) proliferation and differentiation, downregulating the GH-IGF1 functions. When we disrupted autophagy, NFE2L2/NRF2 (nuclear factor, erythroid 2 like 2) accumulated in muscle and nSCs and negatively modulated DDIT3/CHOP (DNA-damage inducible transcript 3) expression. Lower levels of DDIT3 were responsible for reduced GHR expression leading to impaired local production of IGF1. Our results conclusively identify a novel autophagy-dependent pathway that regulates nSC behavior and indicate that autophagy is required for skeletal muscle development in the neonatal phase. Abbreviations: AKT/protein kinase B: Thymoma viral proto-oncogene; ASCs: adult stem cells; ATF4: activating transcription factor 4; ATG7: autophagy related 7; BAT: brown adipose tissue; BMP: bone morphogenetic protein; CEBPB: CCAAT/enhancer binding protein (C/EBP), beta; CSA: cross sectional area; CTNNB1: catenin (cadherin associated protein), beta 1; DDIT3: DNA-damage inducible transcript 3; DM: differentiation medium; E: embryonic stage; EIF2AK3/PERK; EIF4EBP1: eukaryotic translation initiation factor 2 alpha kinase 3; eukaryotic translation initiation factor 4E binding protein 1; ER: endoplasmic reticulum; FGF21: fibroblast growth factor 21; GH: growth hormone; GHR: growth hormone receptor; HSCs: hematopoietic stem cells; IGF1: insulin-like growth factor 1; ITGAM: integrin alpha M; KEAP1: kelch-like ECH-associated protein 1; LY6A/Sca-1; MAP1LC3: lymphocyte antigen 6 complex, locus A; microtubule-associated protein 1 light chain 3; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPK3/ERK1: mitogen-activated protein kinase 3; miRNAs: microRNAs; MSCs: mesenchymal stem cells; MTOR: mechanistic target of rapamycin kinase; mtUPR: mitochondrial unfolded protein response; MYF5: myogenic factor 5; MYH: myosin, heavy polypeptide; MYOD1: myogenic differentiation 1; MYOG: myogenin; NFE2L2: nuclear factor, erythroid derived 2, like 2; nSC: neonatal satellite cells; NSCs: neuronal stem cells; P: postnatal day; PAX7: paired box 7; PECAM1: platelet/endothelial cell adhesion molecule 1; PPARG: peroxisome proliferator activated receptor gamma; PTPRC: protein tyrosine phosphatase, receptor type, C; ROS: reactive oxygen species; RPS6: ribosomal protein S6; SCs: adult satellite cells; SQSTM1: sequestosome 1; STAT5: signal transducer and activator of transcription 5; TGFB1: transforming growth factor beta 1; WAT: white adipose tissue; WT: wild type.

Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex.

Inhibition of the proteasome leads to proteotoxic stress, which is characterized by the buildup of ubiquitinated proteins that cannot be degraded properly. The transcription factor Nrf1 (also called NFE2L1) counteracts proteotoxic stress by inducing transcription of proteasome subunit genes, resulting in the restoration of proteasome activity. Further understanding of the Nrf1 pathway is therefore of interest in both neurodegeneration, where proteasome activity could be enhanced, and cancer, where suppression of this pathway could potentiate the cell-killing effect mediated by proteasome inhibitor drugs. Here, to identify novel regulators of Nrf1, we performed an RNAi screen in an engineered cell line, reporting on Nrf1 transcriptional activity. In addition to validating known regulators, we discovered that the AAA+ ATPase RUVBL1 is necessary for Nrf1's transcriptional activity. Given that RUVBL1 is part of different multisubunit complexes that play key roles in transcription, we dissected this phenomenon further and found that the TIP60 chromatin-regulatory complex is essential for Nrf1-dependent transcription of proteasome genes. Consistent with these observations, Nrf1, RUVBL1, and TIP60 proteins were co-recruited to the promoter regions of proteasome genes after proteasome inhibitor treatments. More importantly, depletion of RUVBL1 or TIP60 in various cancer cells sensitized them to cell death induced by proteasome inhibition. Overall, our study provides a framework for manipulating the TIP60-Nrf1 axis to alter proteasome function in various human diseases, including cancer.

Mechanisms controlling the multistage post-translational processing of endogenous Nrf1α/TCF11 proteins to yield distinct isoforms within the coupled positive and negative feedback circuits.

To gain a better understanding of the multistep processing of Nrf1 to yield various isoforms with confused molecular masses, we herein establish a generally acceptable criterion required for identification of its endogenous full-length proteins and derivative isoforms expressed differentially in distinct experimental cell lines. Further work has been focused on the molecular mechanisms that dictate the successive post-translational modifications (i.e. glycosylation by OST, deglycosylation by NGLY, and ubiquitination by Hrd1) of this CNC-bZIP protein and its proteolytic processing to give rise to multiple proteoforms. Several lines of experimental evidence have demonstrated that the nascent Nrf1α/TCF11 polypeptide (non-glycosylated) is transiently translocated into the endoplasmic reticulum (ER), in which it becomes an inactive glycoprotein-A, and is folded in a proper topology within and around membranes. Thereafter, dynamic repositioning of the ER-resident domains in Nrf1 glycoprotein is driven by p97-fueled retrotranslocation into extra-ER compartments. Therein, Nrf1 glycoprotein is allowed for deglycosylation digestion by glycosidases into a deglycoprotein-B and its progressive proteolytic processing by cytosolic DDI-1/2 and proteasomes so as to generate N-terminally-truncated protein-C/D. This processing is accompanied by removal of a major N-terminal ~12.5-kDa polypeptide from Nrf1α. Interestingly, our present study has further unraveled that there exist coupled positive and negative feedback circuits between Nrf1 and cognate target genes, including those encoding its regulators p97, Hrd1, DDI-1 and proteasomes. These key players are differentially or even oppositely involved in diverse cellular signaling responses to distinct extents of ER-derived proteotoxic and oxidative stresses induced by different concentrations of proteasomal inhibitors.