Nobiletin protects against alcohol-induced mitochondrial dysfunction and liver injury by regulating the hepatic NRF1-TFAM signaling pathway.

OBJECTIVES: Alcohol and its metabolites, such as acetaldehyde, induced hepatic mitochondrial dysfunction play a pathological role in the development of alcohol-related liver disease (ALD). METHODS: In this study, we investigated the potential of nobiletin (NOB), a polymethoxylated flavone, to counter alcohol-induced mitochondrial dysfunction and liver injury. RESULTS: Our findings demonstrate that NOB administration markedly attenuated alcohol-induced hepatic steatosis, endoplasmic reticulum stress, inflammation, and tissue damage in mice. NOB reversed hepatic mitochondrial dysfunction and oxidative stress in both alcohol-fed mice and acetaldehyde-treated hepatocytes. Mechanistically, NOB restored the reduction of hepatic mitochondrial transcription factor A (TFAM) at both mRNA and protein levels. Notably, the protective effects of NOB against acetaldehyde-induced mitochondrial dysfunction and cell death were abolished in hepatocytes lacking Tfam. Furthermore, NOB administration reinstated the levels of hepatocellular NRF1, a key transcriptional regulator of TFAM, which were decreased by alcohol and acetaldehyde exposure. Consistent with these findings, hepatocyte-specific overexpression of Nrf1 protected against alcohol-induced hepatic Tfam reduction, mitochondrial dysfunction, oxidative stress, and liver injury. CONCLUSIONS: Our study elucidates the involvement of the NRF1-TFAM signaling pathway in the protective mechanism of NOB against chronic-plus-binge alcohol consumption-induced mitochondrial dysfunction and liver injury, suggesting NOB supplementation as a potential therapeutic strategy for ALD.

A transcriptional regulatory mechanism of genes in the tricarboxylic acid cycle in the heart.

The tricarboxylic acid (TCA) cycle plays a crucial role in mitochondrial ATP production in the healthy heart. However, in heart failure, the TCA cycle becomes dysregulated. Understanding the mechanism by which TCA cycle genes are transcribed in the healthy heart is an important prerequisite to understanding how these genes become dysregulated in the failing heart. PPARγ coactivator 1α (PGC-1α) is a transcriptional coactivator that broadly induces genes involved in mitochondrial ATP production. PGC-1α potentiates its effects through the coactivation of coupled transcription factors, such as estrogen-related receptor (ERR), nuclear respiratory factor 1 (Nrf1), GA-binding protein-a (Gabpa), and Yin Yang 1 (YY1). We hypothesized that PGC-1α plays an essential role in the transcription of TCA cycle genes. Thus, utilizing localization peaks of PGC-1α to TCA cycle gene promoters would allow the identification of coupled transcription factors. PGC-1α potentiated the transcription of 13 out of 14 TCA cycle genes, partly through ERR, Nrf1, Gabpa, and YY1. ChIP-sequencing showed PGC-1α localization peaks in TCA cycle gene promoters. Transcription factors with binding elements that were found proximal to PGC-1α peak localization were generally essential for the transcription of the gene. These transcription factor binding elements were well conserved between mice and humans. Among the four transcription factors, ERR and Gabpa played a major role in potentiating transcription when compared to Nrf1 and YY1. These transcription factor-dependent PGC-1α recruitment was verified with Idh3a, Idh3g, and Sdha promoters with DNA binding assay. Taken together, this study clarifies the mechanism by which TCA cycle genes are transcribed, which could be useful in understanding how those genes are dysregulated in pathological conditions.

Ubiquitin gets sweet: Sugar-mediated ubiquitination regulates Nrf1 function.

In this issue of Molecular Cell, Yoshida et al.1 report an unconventional sugar-dependent ubiquitination event on Nrf1 that disrupts Nrf1 transcriptional activation.

Sugar-mediated non-canonical ubiquitination impairs Nrf1/NFE2L1 activation.

Proteasome is essential for cell survival, and proteasome inhibition induces proteasomal gene transcription via the activated endoplasmic-reticulum-associated transcription factor nuclear factor erythroid 2-like 1 (Nrf1/NFE2L1). Nrf1 activation requires proteolytic cleavage by DDI2 and N-glycan removal by NGLY1. We previously showed that Nrf1 ubiquitination by SKP1-CUL1-F-box (SCF)FBS2/FBXO6, an N-glycan-recognizing E3 ubiquitin ligase, impairs its activation, although the molecular mechanism remained elusive. Here, we show that SCFFBS2 cooperates with the RING-between-RING (RBR)-type E3 ligase ARIH1 to ubiquitinate Nrf1 through oxyester bonds in human cells. Endo-ß-N-acetylglucosaminidase (ENGASE) generates asparagine-linked N-acetyl glucosamine (N-GlcNAc) residues from N-glycans, and N-GlcNAc residues on Nrf1 served as acceptor sites for SCFFBS2-ARIH1-mediated ubiquitination. We reconstituted the polyubiquitination of N-GlcNAc and serine/threonine residues on glycopeptides and found that the RBR-specific E2 enzyme UBE2L3 is required for the assembly of atypical ubiquitin chains on Nrf1. The atypical ubiquitin chains inhibited DDI2-mediated activation. The present results identify an unconventional ubiquitination pathway that inhibits Nrf1 activation.

Protective Effects of Hepatocyte Stress Defenders, Nrf1 and Nrf2, against MASLD Progression.

Progression of metabolic dysfunction-associated steatites liver disease (MASLD) to steatohepatitis (MASH) is driven by stress-inducing lipids that promote liver inflammation and fibrosis, and MASH can lead to cirrhosis and hepatocellular carcinoma. Previously, we showed coordinated defenses regulated by transcription factors, nuclear factor erythroid 2-related factor-1 (Nrf1) and -2 (Nrf2), protect against hepatic lipid stress. Here, we investigated protective effects of hepatocyte Nrf1 and Nrf2 against MASH-linked liver fibrosis and tumorigenesis. Male and female mice with flox alleles for genes encoding Nrf1 (Nfe2l1), Nrf2 (Nfe2l2), or both were fed a MASH-inducing diet enriched with high fat, fructose, and cholesterol (HFFC) or a control diet for 24-52 weeks. During this period, hepatocyte Nrf1, Nrf2, or combined deficiency for ~7 days, ~7 weeks, and ~35 weeks was induced by administering mice hepatocyte-targeting adeno-associated virus (AAV) expressing Cre recombinase. The effects on MASH, markers of liver fibrosis and proliferation, and liver tumorigenesis were compared to control mice receiving AAV-expressing green fluorescent protein. Also, to assess the impact of Nrf1 and Nrf2 induction on liver fibrosis, HFFC diet-fed C57bl/6J mice received weekly injections of carbon tetrachloride, and from week 16 to 24, mice were treated with the Nrf2-activating drug bardoxolone, hepatocyte overexpression of human NRF1 (hNRF1), or both, and these groups were compared to control. Compared to the control diet, 24-week feeding with the HFFC diet increased bodyweight as well as liver weight, steatosis, and inflammation. It also increased hepatocyte proliferation and a marker of liver damage, p62. Hepatocyte Nrf1 and combined deficiency increased liver steatosis in control diet-fed but not HFFC diet-fed mice, and increased liver inflammation under both diet conditions. Hepatocyte Nrf1 deficiency also increased hepatocyte proliferation, whereas combined deficiency did not, and this also occurred for p62 level in control diet-fed conditions. In 52-week HFFC diet-fed mice, 35 weeks of hepatocyte Nrf1 deficiency, but not combined deficiency, resulted in more liver tumors in male mice, but not in female mice. In contrast, hepatocyte Nrf2 deficiency had no effect on any of these parameters. However, in the 15-week CCL4-exposed and 24-week HFFC diet-fed mice, Nrf2 induction with bardoxolone reduced liver steatosis, inflammation, fibrosis, and proliferation. Induction of hepatic Nrf1 activity with hNRF1 enhanced the effect of bardoxolone on steatosis and may have stimulated liver progenitor cells. Physiologic Nrf1 delays MASLD progression, Nrf2 induction alleviates MASH, and combined enhancement synergistically protects against steatosis and may facilitate liver repair.

Isoquercitrin alleviates OGD/R-induced oxidative stress and impaired mitochondrial biogenesis in SH-SY5Y cells via the NRF1/TFAM pathway.

Isoquercitrin (ISO) is a traditional Chinese medicine extract, that has been found to possess potent neuroprotective properties. However, its precise role in the context of ischemic stroke (IS) remains to be fully elucidated. We constructed an in vitro model of IS induced by OGD/R in SH-SY5Y cells. Cell viability, the levels of oxidative stress-related indicators (8-OHDG, MDA, SOD, GSH, and GSH-Px), ROS, and mitochondrial membrane potential were measured by using detection kits. The protein levels of GPX1, SOD, Cytc were measured. The mRNA levels of mitochondrial biogenesis-related indicators (Cytb, CO1, ND2, ND5, and ND6), and mtDNA copy number were measured by RT-qPCR. ATP levels were measured. Molecular docking between ISO and NRF1, and Co-IP assay for NRF1 and TFAM interaction were performed. Expression of NRF1 and TFAM was evaluated. ISO treatment reversed the detrimental effects of OGD/R on cell viability, attenuated the elevation of oxidative stress markers, restored antioxidant levels, and alleviated the impairment of mitochondrial biogenesis in SH-SY5Y cells. ISO interacted with NRF1 and increased its expression along with TFAM. Silencing NRF1 reversed the protective effects of ISO, suggesting its involvement in mediating the neuroprotective effects of ISO. ISO alleviates oxidative stress and mitochondrial biogenesis damage induced by OGD/R in SH-SY5Y cells by upregulating the NRF1/TFAM pathway.

Proteasome gene expression is controlled by coordinated functions of multiple transcription factors.

Proteasome activity is crucial for cellular integrity, but how tissues adjust proteasome content in response to catabolic stimuli is uncertain. Here, we demonstrate that transcriptional coordination by multiple transcription factors is required to increase proteasome content and activate proteolysis in catabolic states. Using denervated mouse muscle as a model system for accelerated proteolysis in vivo, we reveal that a two-phase transcriptional program activates genes encoding proteasome subunits and assembly chaperones to boost an increase in proteasome content. Initially, gene induction is necessary to maintain basal proteasome levels, and in a more delayed phase (7-10 days after denervation), it stimulates proteasome assembly to meet cellular demand for excessive proteolysis. Intriguingly, the transcription factors PAX4 and α-PALNRF-1 control the expression of proteasome among other genes in a combinatorial manner, driving cellular adaptation to muscle denervation. Consequently, PAX4 and α-PALNRF-1 represent new therapeutic targets to inhibit proteolysis in catabolic diseases (e.g., type-2 diabetes, cancer).

DNA methylation promotes the expression of PPARγ transcript 1 at least in part by preventing NRF1 binding to the promoter P1 of chicken PPARγ gene.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a master regulator of adipogenesis. Our previous study revealed that chicken PPARγ has 3 alternative promoters named as P1, P2, and P3, and the DNA methylation of promoter P3 was negatively associated with PPARγ mRNA expression in abdominal adipose tissue (AAT). However, the methylation status of promoters P1 and P2 is unclear. Here we assessed promoter P1 methylation status in AAT of Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF). The results showed that promoter P1 methylation differed in AAT between the lean and fat lines of NEAUHLF at 7 wk of age (p < 0.05), and AAT expression of PPARγ transcript 1 (PPARγ1), which was derived from the promoter P1, was greatly higher in fat line than in lean line at 2 and 7 wk of age. The results of the correlation analysis showed that P1 methylation was positively correlated with PPARγ1 expression at 7 wk of age (Pearson's r = 0.356, p = 0.0242), suggesting P1 methylation promotes PPARγ1 expression. To explore the underlying molecular mechanism of P1 methylation on PPARγ1 expression, bioinformatics analysis, dual-luciferase reporter assay, pyrosequencing, and electrophoresis mobility shift assay (EMSA) were performed. The results showed that transcription factor NRF1 repressed the promoter activity of the unmethylated P1, but not the methylated P1. Of all the 4 CpGs (CpG48, CpG49, CpG50, and CpG51), which reside within or nearby the NRF1 binding sites of the P1, only CpG49 methylation in AAT was remarkably higher in the fat line than in lean line at 7 wk of age (3.18 to 0.57, p < 0.05), and CpG49 methylation was positively correlated with PPARγ1 expression (Pearson's r = 0.3716, p = 0.0432). Furthermore, EMSA showed that CpG49 methylation reduced the binding of NRF1 to the P1. Taken together, our findings illustrate that P1 methylation promotes PPARγ1 expression at least in part by preventing NRF1 from binding to the promoter P1.

Nuclear respiratory factor 1 regulates super enhancer-controlled SPIDR to protect hepatocellular carcinoma cells from oxidative stress.

BACKGROUND: Cellular response to oxidative stress plays significant roles in hepatocellular carcinoma (HCC) development, yet the exact mechanism by which HCC cells respond to oxidative stress remains poorly understood. This study aimed to investigate the role and mechanism of super enhancer (SE)-controlled genes in oxidative stress response of HCC cells. METHODS: The GSE112221 dataset was used to identify SEs by HOMER. Functional enrichment of SE-controlled genes was performed by Metascape. Transcription factors were predicted using HOMER. Prognosis analysis was conducted using the Kaplan-Meier Plotter website. Expression correlation analysis was performed using the Tumor Immune Estimation Resource web server. NRF1 and SPIDR expression in HCC and normal liver tissues was analyzed based on the TCGA-LIHC dataset. ChIP-qPCR was used to detect acetylation of lysine 27 on histone 3 (H3K27ac) levels of SE regions of genes, and the binding of NRF1 to the SE of SPIDR. To mimic oxidative stress, HepG2 and Hep3B cells were stimulated with H2O2. The effects of NRF1 and SPIDR on the oxidative stress response of HCC cells were determined by the functional assays. RESULTS: A total of 318 HCC-specific SE-controlled genes were identified. The functions of these genes was significant association with oxidative stress response. SPIDR and RHOB were enriched in the "response to oxidative stress" term and were chosen for validation. SE regions of SPIDR and RHOB exhibited strong H3K27ac modification, which was significantly inhibited by JQ1. JQ1 treatment suppressed the expression of SPIDR and RHOB, and increased reactive oxygen species (ROS) levels in HCC cells. TEAD2, TEAD3, NRF1, HINFP and TCFL5 were identified as potential transcription factors for HCC-specific SE-controlled genes related to oxidative stress response. The five transcription factors were positively correlated with SPIDR expression, with the highest correlation coefficient for NRF1. NRF1 and SPIDR expression was up-regulated in HCC tissues and cells. NRF1 activated SPIDR transcription by binding to its SE. Silencing SPIDR or NRF1 significantly promoted ROS accumulation in HCC cells. Under oxidative stress, silencing SPIDR or NRF1 increased ROS, malondialdehyde (MDA) and γH2AX levels, and decreased superoxide dismutase (SOD) levels and cell proliferation of HCC cells. Furthermore, overexpression of SPIDR partially offset the effects of NRF1 silencing on ROS, MDA, SOD, γH2AX levels and cell proliferation of HCC cells. CONCLUSION: NRF1 driven SPIDR transcription by occupying its SE, protecting HCC cells from oxidative stress-induced damage. NRF1 and SPIDR are promising biomarkers for targeting oxidative stress in the treatment of HCC.

Molecular mechanism of specific DNA sequence recognition by NRF1.

Nuclear respiratory factor 1 (NRF1) regulates the expression of genes that are vital for mitochondrial biogenesis, respiration, and various other cellular processes. While NRF1 has been reported to bind specifically to GC-rich promoters as a homodimer, the precise molecular mechanism governing its recognition of target gene promoters has remained elusive. To unravel the recognition mechanism, we have determined the crystal structure of the NRF1 homodimer bound to an ATGCGCATGCGCAT dsDNA. In this complex, NRF1 utilizes a flexible linker to connect its dimerization domain (DD) and DNA binding domain (DBD). This configuration allows one NRF1 monomer to adopt a U-turn conformation, facilitating the homodimer to specifically bind to the two TGCGC motifs in the GCGCATGCGC consensus sequence from opposite directions. Strikingly, while the NRF1 DBD alone could also bind to the half-site (TGCGC) DNA of the consensus sequence, the cooperativity between DD and DBD is essential for the binding of the intact GCGCATGCGC sequence and the transcriptional activity of NRF1. Taken together, our results elucidate the molecular mechanism by which NRF1 recognizes specific DNA sequences in the promoters to regulate gene expression.

Inhibitor of DNA Binding Protein 3 (ID3) and Nuclear Respiratory Factor 1 (NRF1) Mediated Transcriptional Gene Signatures are Associated with the Severity of Cerebral Amyloid Angiopathy.

Cerebral amyloid angiopathy (CAA) is a degenerative vasculopathy. We have previously shown that transcription regulating proteins- inhibitor of DNA binding protein 3 (ID3) and the nuclear respiratory factor 1 (NRF1) contribute to vascular dysregulation. In this study, we have identified sex specific ID3 and NRF1-mediated gene networks in CAA patients diagnosed with Alzheimer's Disease (AD). High expression of ID3 mRNA coupled with low NRF1 mRNA levels was observed in the temporal cortex of men and women CAA patients. Low NRF1 mRNA expression in the temporal cortex was found in men with severe CAA. High ID3 expression was found in women with the genetic risk factor APOE4. Low NRF1 expression was also associated with APOE4 in women with CAA. Genome wide transcriptional activity of both ID3 and NRF1 paralleled their mRNA expression levels. Sex specific differences in transcriptional gene signatures of both ID3 and NRF1 were observed. These findings were further corroborated by Bayesian machine learning and the GeNIe simulation models. Dynamic machine learning using a Monte Carlo Markov Chain (MCMC) gene ordering approach revealed that ID3 was associated with disease severity in women. NRF1 was associated with CAA and severity of this disease in men. These findings suggest that aberrant ID3 and NRF1 activity presumably plays a major role in the pathogenesis and severity of CAA. Further analyses of ID3- and NRF1-regulated molecular drivers of CAA may provide new targets for personalized medicine and/or prevention strategies against CAA.

NRF1 Alleviated Oxidative Stress of Glioblastoma Cells by Regulating NOR1.

Oxidored-nitro domain-containing protein 1 (NOR1) is a critical tumour suppressor gene, though its regulatory mechanism in oxidative stress of glioblastoma (GBM) remains unclear. Hence, further study is needed to unravel the function of NOR1 in the progression of oxidative stress in GBM. In this study, we evaluated the expression of NOR1 and nuclear respiratory factor 1 (NRF1) in GBM tissue and normal brain tissue (NBT) using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot (WB), and investigated their relationship. We then induced oxidative stress in U251 cells through H2O2 treatment and conducted Cell Count-ing Kit-8, Transwell and wound healing assays to analyse cell proliferation, invasion and migration. Cell apoptosis was assessed by flow cytometry and TUNEL staining. We also measured the activities of superoxide dismutase and catalase, as well as the level of reactive oxygen species (ROS) using biochemical techniques. Via qRT-PCR and WB, the mRNA and protein expression levels of NOR1 and NRF1 were determined. Chromatin immunoprecipitation (ChIP) assays were applied to validate NRF1's interaction with NOR1. Our results showed that the expression of NOR1 and NRF1 was low in GBM, and their expression levels were positively correlated. H2O2-induced oxidative stress reduced NRF1 and NOR1 expression levels and increased the ROS level. The ChIP assay confirmed the binding of NRF1 to NOR1. Over-expression of NRF1 attenuated the inhibitory effect of oxidative stress on the proliferation, migration and invasion of U251 cells, which was reversed by knockdown of NOR1.

Nuclear respiratory factor 1 drives hepatocellular carcinoma progression by activating LPCAT1-ERK1/2-CREB axis.

BACKGROUND: Nuclear respiratory factor 1 (NRF1) is a transcription factor that participates in several kinds of tumor, but its role in hepatocellular carcinoma (HCC) remains elusive. This study aims to explore the role of NRF1 in HCC progression and investigate the underlying mechanisms. RESULTS: NRF1 was overexpressed and hyperactive in HCC tissue and cell lines and high expression of NRF1 indicated unfavorable prognosis of HCC patients. NRF1 promoted proliferation, migration and invasion of HCC cells both in vitro and in vivo. Mechanistically, NRF1 activated ERK1/2-CREB signaling pathway by transactivating lysophosphatidylcholine acyltransferase 1 (LPCAT1), thus promoting cell cycle progression and epithelial mesenchymal transition (EMT) of HCC cells. Meanwhile, LPCAT1 upregulated the expression of NRF1 by activating ERK1/2-CREB signaling pathway, forming a positive feedback loop. CONCLUSIONS: NRF1 is overexpressed in HCC and promotes HCC progression by activating LPCAT1-ERK1/2-CREB axis. NRF1 is a promising therapeutic target for HCC patients.

NRF1-mediated mitochondrial biogenesis antagonizes innate antiviral immunity.

Mitochondrial biogenesis is the process of generating new mitochondria to maintain cellular homeostasis. Here, we report that viruses exploit mitochondrial biogenesis to antagonize innate antiviral immunity. We found that nuclear respiratory factor-1 (NRF1), a vital transcriptional factor involved in nuclear-mitochondrial interactions, is essential for RNA (VSV) or DNA (HSV-1) virus-induced mitochondrial biogenesis. NRF1 deficiency resulted in enhanced innate immunity, a diminished viral load, and morbidity in mice. Mechanistically, the inhibition of NRF1-mediated mitochondrial biogenesis aggravated virus-induced mitochondrial damage, promoted the release of mitochondrial DNA (mtDNA), increased the production of mitochondrial reactive oxygen species (mtROS), and activated the innate immune response. Notably, virus-activated kinase TBK1 phosphorylated NRF1 at Ser318 and thereby triggered the inactivation of the NRF1-TFAM axis during HSV-1 infection. A knock-in (KI) strategy that mimicked TBK1-NRF1 signaling revealed that interrupting the TBK1-NRF1 connection ablated mtDNA release and thereby attenuated the HSV-1-induced innate antiviral response. Our study reveals a previously unidentified antiviral mechanism that utilizes a NRF1-mediated negative feedback loop to modulate mitochondrial biogenesis and antagonize innate immune response.

NRF1 Regulates the Epithelial Mesenchymal Transition of Breast Cancer by Modulating ROS Homeostasis.

Introduction: Nuclear respiratory factor 1 (NRF1) is an important regulator involved in mitochondrial biogenesis and energy metabolism. However, the specific mechanism of NRF1 in anoikis and epithelial-mesenchymal transition (EMT) remains unclear. Methods: We examined the effect of NRF1 on mitochondria and identified the specific mechanism through transcriptome sequencing, and explored the relationships among NRF1, anoikis, and EMT. Results: We found that upregulated NRF1 expression led to increased mitochondrial oxidative phosphorylation (OXPHOS) and ATP generation. Simultaneously, a significant amount of ROS is generated during OXPHOS. Alternatively, NRF1 upregulates the expression of ROS-scavenging enzymes, allowing tumor cells to maintain low ROS levels and promoting anoikis resistance and EMT. We also found that exogenous ROS was maintained at a low level by NRF1 in breast cancer cells. Conclusion: our study provides mechanistic insight into the function of NRF1 in breast cancer, indicating that NRF1 may serve as a therapeutic target for breast cancer treatment.

The Regulatory Role of Nuclear Respiratory Factor 1 in Bladder Cancer Cells.

BACKGROUND/AIM: Nuclear respiratory factor 1 (NRF1) is a key mediator of genes involved in mitochondrial biogenesis and the respiratory chain; however, its role in bladder cancer remains unknown. Transitional cell carcinoma, also known as urothelial cell carcinoma, is the most common type of bladder cancer resistant to chemotherapy. An established high-grade and invasive transitional cell carcinoma line from patients with urinary bladder cancer, known as T24, has been extensively used in cancer research. In this study, we aimed to investigate the mechanisms through which NRF1 regulates proliferation and cell migration of bladder cancer cells using the T24 cell line. MATERIALS AND METHODS: Cells were transfected with plasmid cloning DNA for NRF1 to evaluate the effect of NRF1 overexpression on bladder cancer cells. Western blot was used to examine epithelial and mesenchymal markers (E-cadherin and α-smooth muscle actin), transcriptional regulators for epithelial-mesenchymal transition (snail family transcriptional repressors), components of transforming growth factor-ß1/SMADs signaling, high-mobility group box 1 (HMGB1), and receptor for advanced glycation end-products (RAGE). The in situ expression of E-cadherin, α-smooth muscle actin and SMAD7 was determined using immunofluorescence staining. Cell migration capacity was assessed by wound-healing assay. RESULTS: Transfection with NRF1 expression vector repressed the migration capacity of bladder cancer cells, diminishing HMGB1/RAGE expression and reducing transforming growth factor ß-associated epithelial-mesenchymal transition in T24 cells. CONCLUSION: Therapeutic avenues that increase NRF1 expression may serve as an adjunct to conventional treatments for bladder cancer.

Redox-metabolic reprogramming of skin in mice lacking functional Nrf2 under basal conditions and cold acclimation.

Adaptive responses to environmental and physiological challenges, including exposure to low environmental temperature, require extensive structural, redox, and metabolic reprogramming. Detailed molecular mechanisms of such processes in the skin are lacking, especially the role of nuclear factor erythroid 2-related factor 2 (Nrf2) and other closely related redox-sensitive transcription factors Nrf1, Nrf3, and nuclear respiratory factor (NRF1). To investigate the role of Nrf2, we examined redox and metabolic responses in the skin of wild-type (WT) mice and mice lacking functional Nrf2 (Nrf2 KO) at room (RT, 24 ± 1°C) and cold (4 ± 1°C) temperature. Our results demonstrate distinct expression profiles of major enzymes involved in antioxidant defense and key metabolic and mitochondrial pathways in the skin, depending on the functional Nrf2 and/or cold stimulus. Nrf2 KO mice at RT displayed profound alterations in redox, mitochondrial and metabolic responses, generally akin to cold-induced skin responses in WT mice. Immunohistochemical analyses of skin cell compartments (keratinocytes, fibroblasts, hair follicle, and sebaceous gland) and spatial locations (nucleus and cytoplasm) revealed synergistic interactions between members of the Nrf transcription factor family as part of redox-metabolic reprogramming in WT mice upon cold acclimation. In contrast, Nrf2 KO mice at RT showed loss of NRF1 expression and a compensatory activation of Nrf1/Nrf3, which was abolished upon cold, concomitant with blunted redox-metabolic responses. These data show for the first time a novel role for Nrf2 in skin physiology in response to low environmental temperature, with important implications in human connective tissue diseases with altered thermogenic responses.

Long noncoding RNA LINC01132 enhances immunosuppression and therapy resistance via NRF1/DPP4 axis in hepatocellular carcinoma.

BACKGROUND: Long noncoding RNAs (lncRNAs) are emerging as critical regulators of gene expression and play fundamental roles in various types of cancer. Current developments in transcriptome analyses unveiled the existence of lncRNAs; however, their functional characterization remains a challenge. METHODS: A bioinformatics screen was performed by integration of multiple omics data in hepatocellular carcinoma (HCC) prioritizing a novel oncogenic lncRNA, LINC01132. Expression of LINC01132 in HCC and control tissues was validated by qRT-PCR. Cell viability and migration activity was examined by MTT and transwell assays. Finally, our results were confirmed in vivo mouse model and ex vivo patient derived tumor xenograft experiments to determine the mechanism of action and explore LINC01132-targeted immunotherapy. RESULTS: Systematic investigation of lncRNAs genome-wide expression patterns revealed LINC01132 as an oncogene in HCC. LINC01132 is significantly overexpressed in tumor and associated with poor overall survival of HCC patients, which is mainly driven by copy number amplification. Functionally, LINC01132 overexpression promoted cell growth, proliferation, invasion and metastasis in vitro and in vivo. Mechanistically, LINC01132 acts as an oncogenic driver by physically interacting with NRF and enhancing the expression of DPP4. Notably, LINC01132 silencing triggers CD8+ T cells infiltration, and LINC01132 knockdown combined with anti-PDL1 treatment improves antitumor immunity, which may prove a new combination therapy in HCC. CONCLUSIONS: LINC01132 functions as an oncogenic driver that induces HCC development via the NRF1/DPP4 axis. Silencing LINC01132 may enhance the efficacy of anti-PDL1 immunotherapy in HCC patients.

Nuclear Respiratory Factor 1 Overexpression Inhibits Proliferation and Migration of PC3 Prostate Cancer Cells.

BACKGROUND/AIM: The role of nuclear respiratory factor 1 (NRF1) on the prostate cancer progression is controversial. We aimed to investigate the effect of NRF1 overexpression on the metastasis potential of PC3 prostate cancer cells and the associated molecular mechanisms. MATERIALS AND METHODS: Cell survival, migration capacity, mitochondrial biogenesis, the expression of TGF-ß signaling and EMT markers were examined after overexpression and silencing of NRF1 in PC3 cells. RESULTS: We found that NRF1-overexpressing cells exhibited a decreased cell viability and proliferation ability as well as a reduced migration capacity compared to control cells. Moreover, ectopic expression of NRF1 increased the mitochondrial biogenesis and inhibited the EMT characteristics, including a decrease in the mesenchymal marker, α-SMA and an increase in the epithelial cell marker, E-cadherin. We also demonstrated that overexpression of NRF1 suppressed the expression of TGF-ß signaling in PC3 cells. As expected, silencing of NRF1 reversed the abovementioned effects. CONCLUSION: This study demonstrated that upregulation of NRF1 holds the potential to inhibit the metastasis of prostate cancer, possibly through an elevation of mitochondrial biogenesis and the subsequent repression of TGF-ß-associated EMT. Therapeutic avenues that increase NRF1 expression may serve as an adjunct to conventional treatments of prostate cancer.

Differential Susceptibility of Retinal Neurons to the Loss of Mitochondrial Biogenesis Factor Nrf1.

The retina, the accessible part of the central nervous system, has served as a model system to study the relationship between energy utilization and metabolite supply. When the metabolite supply cannot match the energy demand, retinal neurons are at risk of death. As the powerhouse of eukaryotic cells, mitochondria play a pivotal role in generating ATP, produce precursors for macromolecules, maintain the redox homeostasis, and function as waste management centers for various types of metabolic intermediates. Mitochondrial dysfunction has been implicated in the pathologies of a number of degenerative retinal diseases. It is well known that photoreceptors are particularly vulnerable to mutations affecting mitochondrial function due to their high energy demand and susceptibility to oxidative stress. However, it is unclear how defective mitochondria affect other retinal neurons. Nuclear respiratory factor 1 (Nrf1) is the major transcriptional regulator of mitochondrial biogenesis, and loss of Nrf1 leads to defective mitochondria biogenesis and eventually cell death. Here, we investigated how different retinal neurons respond to the loss of Nrf1. We provide in vivo evidence that the disruption of Nrf1-mediated mitochondrial biogenesis results in a slow, progressive degeneration of all retinal cell types examined, although they present different sensitivity to the deletion of Nrf1, which implicates differential energy demand and utilization, as well as tolerance to mitochondria defects in different neuronal cells. Furthermore, transcriptome analysis on rod-specific Nrf1 deletion uncovered a previously unknown role of Nrf1 in maintaining genome stability.