Activation Of Nuclear Respiratory Factor-1 Research Articles

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.

The clock gene Bmal1 inhibits kidney expression of SGLT2 and glucose reabsorption via the NR1d1/NRF1 pathway

Background & Aims: The nuclear receptor subfamily 1, group D, member 1 (Nr1d1, also called Rev-erbα) is highly expressed in the kidney, and its transcriptional activity is increased by the clock gene Bmal1 (ARNTL or MOP3). Here we studied the roles and functional interactions of Bmal1 and Nr1d1 in mouse kidney, particularly in regulating the expression of the glucose transporter SGLT2 (SLC5A2) and glucose reabsorption. Methods: We performed histologic and biochemical analyses of kidney tissues from male C57BL/6 and Bmal1-knockout mice and performed gene expression analyses to identify genes regulated by Bmal1. The effects of fasting and refeeding on renal expression of Sglt2 was examined in male C57BL/6, Bmal1-knockout, and Nr1d1-knockout mice. Some mice were given hemin to stimulate Nr1d1 gene expression (daily for 1 week) at 12:00 with 100 μg/kg/day; renal glucose reabsorption, and glucose and fatty acid levels were measured. Renal proximal tubule epithelial cells (RPTECs) were generated from C57BL/6 and Bmal1-knockout mice and glucose uptake determined. Luciferase reporter assays were performed using HK2 cell. Results: We found that kidney SGLT2 mRNA and protein expression and glucose absorption were increased in Bmal1-knockout mice compared with C57BL/6 mice. Expression of SGLT2 was affected by fasting and refeeding in C57BL/6 mice, but not in Bmal1-knockout or Nr1d1-knockout mice. Bmal1-knockout mice also presented with higher levels of renal fatty acids compared with C57BL/6 mice. Hemin injection reduced expression of SGLT2, decreased glucose re-absorption in RPTECs, and increased levels of fatty acid in kidney; these changes were not observed in Bmal1-knockout mice. Nuclear respiratory factor 1 (NRF1), which regulates glucose, activated transcription of Sglt2. Fasting caused MAPK signing pathway to phosphorylate Bmal1, which activated NR1d1 and inhibited NRF1 activity and thereby reduced Sglt2 expression. Conclusions: The data indicate that fasting activates Bmal1 and Nr1d1, which inhibits NRF1 and leads to repression of renal SGLT2 expression and glucose reabsorption. This is the first study that mechanistically links food intake to the regulation of kidney glucose transport through a circadian clock gene. NIH National Heart, Lung, and Blood Institute grant R56 HL137912-01, and American Heart Association grant-in-aid 16GRNT30960027 to PX; NIH R01DK112042 to VV This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Caveolin-1 accelerates hypoxia-induced endothelial dysfunction in high-altitude cerebral edema

BackgroundHigh-altitude cerebral edema (HACE) is a serious and potentially fatal brain injury that is caused by acute hypobaric hypoxia (HH) exposure. Vasogenic edema is the main pathological factor of this condition. Hypoxia-induced disruptions of tight junctions in the endothelium trigger blood‒brain barrier (BBB) damage and induce vasogenic edema. Nuclear respiratory factor 1 (NRF1) acts as a major regulator of hypoxia-induced endothelial cell injury, and caveolin-1 (CAV-1) is upregulated as its downstream gene in hypoxic endothelial cells. This study aimed to investigate whether CAV-1 is involved in HACE progression and the underlying mechanism.MethodsC57BL/6 mice were exposed to HH (7600 m above sea level) for 24 h, and BBB injury was assessed by brain water content, Evans blue staining and FITC-dextran leakage. Immunofluorescence, transmission electron microscope, transendothelial electrical resistance (TEER), transcytosis assays, and western blotting were performed to confirm the role and underlying mechanism of CAV-1 in the disruption of tight junctions and BBB permeability. Mice or bEnd.3 cells were pretreated with MβCD, a specific blocker of CAV-1, and the effect of CAV-1 on claudin-5 internalization under hypoxic conditions was detected by immunofluorescence, western blotting, and TEER. The expression of NRF1 was knocked down, and the regulation of CAV-1 by NRF1 under hypoxic conditions was examined by qPCR, western blotting, and immunofluorescence.ResultsThe BBB was severely damaged and was accompanied by a significant loss of vascular tight junction proteins in HACE mice. CAV-1 was significantly upregulated in endothelial cells, and claudin-5 explicitly colocalized with CAV-1. During the in vitro experiments, hypoxia increased cell permeability, CAV-1 expression, and claudin-5 internalization and downregulated tight junction proteins. Simultaneously, hypoxia induced the upregulation of CAV-1 by activating NRF1. Blocking CAV-1-mediated intracellular transport improved the integrity of TJs in hypoxic endothelial cells and effectively inhibited the increase in BBB permeability and brain water content in HH animals.ConclusionsHypoxia upregulated CAV-1 transcription via the activation of NRF1 in endothelial cells, thus inducing the internalization and autophagic degradation of claudin-5. These effects lead to the destruction of the BBB and trigger HACE. Therefore, CAV-1 may be a potential therapeutic target for HACE.E5r9nDKChFa7F_nrUkRwh3Video abstract

Quercetin-mediated SIRT1 activation attenuates collagen-induced mice arthritis

Ethnopharmacological relevanceHerba taxilli (HT, Sangjisheng in Chinese), which is composed of the dried stems and leaves of Taxillus chinensis (DC.) Danser, has been commonly used to treat inflammation and arthritis in traditional Chinese medicine (TCM). Quercetin (Que) is a major active flavonoid component isolated from HT and is one of the quality control indexes of HT. In the clinical practice of TCM, formulas containing HT are commonly used to treat rheumatoid arthritis (RA). Recent studies have shown that Que exerts antiarthritic effects. However, the mechanism by which Que treatment affects RA is not fully understood. Aim of the studyThis study aimed to explore the antiarthritic activity of Que in a collagen-induced arthritis (CIA) mouse model and investigate the underlying mechanisms. Materials and methodsThe antiarthritic activity of Que was evaluated in a CIA mouse model by determining the paw clinical arthritis scores and left ankle thicknesses and by conducting micro-PET imaging and histopathological analysis of ankle joint tissues. The proinflammatory cytokine (IL-6, TNF-α, IL-1β, IL-8, IL-13, IL-17) levels in the serum and ankle joint tissues were measured by ELISA. Mitochondrial oxidative stress was assessed by biochemical methods. Mitochondrial biogenesis was analysed by RT-qPCR. The protein levels of silent information regulator 1 (SIRT1), peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), nuclear respiratory factor 1 (NRF1), mitochondrial transcription factor A (TFAM), high-mobility group box 1 (HMGB1), Toll-like receptor 4 (TLR4), p38, phospho-p38, extracellular signal-regulated kinases (ERK)-1/2, phospho-ERK1/2, p65, and phospho-p65 in ankle joint tissues were detected by Western blot analysis. A total of 30 RA patients were recruited to investigate the relationship between the disease activity score (DAS28) and the SIRT1, PGC-1α, NRF1, and HMGB1 plasma levels. ResultsQue treatment decreased the clinical score and left ankle thickness of CIA mice, attenuated the synovial inflammation and hyperplasia and bone/cartilage destruction in ankle joints, and decreased the secretion of IL-6, TNF-α, IL-1β, IL-8, IL-13, and IL-17. Mechanistically, Que treatment improved impaired mitochondrial biogenesis and mitochondrial function by regulating the SIRT1/PGC-1α/NRF1/TFAM pathway and inhibited inflammation via the HMGB1/TLR4/p38/ERK1/2/NF-κB p65 pathway. Notably, epidemiological data revealed correlations between abnormal circulating levels of SIRT1, PGC-1α, NRF1, HMGB1 and RA disease activity in patients. ConclusionsOur data suggested a potential role of Que as a dietary therapeutic drug for RA treatment that may act through SIRT1 to target mitochondrial biogenesis. Additionally, the role of impaired mitochondrial biogenesis in RA was evaluated.

Abstract 4391: Aberrant transcription factor activity of NRF1, NFE2L2, E2F1, RFX1, and MEF2 associated with severity of astrocytoma

Abstract Astrocytoma is the most frequently occurring nervous system cancer in humans. Glioblastoma (GBM) is the most severe form of astrocytoma ([WHO] grade IV), and GBM patients rarely live beyond 5 years. Understanding how transcription factor regulatory networks contribute to the severity of astrocytoma will help identify novel therapeutic strategies for GBM. Nuclear Respiratory Factor 1 (NRF1) is a transcription factor (TF) that we have previously demonstrated as being active in cancer and having the ability to participate in crosstalk with other TFs such as the E2Fs and MYC. We have previously demonstrated that NRF1 gene networks are associated with increased NRF1 gene expression and GBM patient survival outcomes. Here, we examined the TF activity landscape that is differentially expressed in GBM compared to non-tumor tissue and in lower grade (I, II, and III) astrocytoma and their role in disease severity. Activities of 247 TFs including NRF1 were examined using patient mRNA expression data from four independent public datasets including the TCGA. The R package ‘limma' with ‘voom' was used to calculate the differential expression of each gene in the microarray and RNA-Seq datasets included in the present study, and then LRPath was employed to determine TF activity. NRF1 transcription factor (TF) activity is upregulated in glioma, correlates with disease aggressiveness, and is associated with cancer hallmarks and key functional pathways known to be dysregulated in cancer. Further analysis of data from all four patient cohorts including TCGA showed that NRF1 TF activity positively correlates with NFE2L2, E2F1, and RFX1, and negatively correlates with MEF2. Differential activity of these TFs is associated with astrocytoma disease severity. Protein-protein interaction networks (PPIN) were then generated for the TF combinations separately for male and female patients. Downstream gene ontology pathway analysis of the TF target genes revealed that DNA repair (GO:0006281), histone modification (GO:0016570), mitotic cell cycle (GO:0000278), and regulation of neurotransmitter level (GO: 0001505) genes regulated by NFE2L2, E2F1, and RFX1 are upregulated corresponding to increased NRF1 activity, while tissue development (GO: 0009888) genes regulated by MEF2 are down-regulated corresponding to increased NRF1 activity. These findings suggest that aberrant transcription factor activity of NRF1, NFE2L2, E2F1, MEF2, and RFX1 may be involved in the pathogenesis and severity of astrocytoma. Further analysis of these TF-regulated gene signatures combined with patient clinical outcomes will help pave the way for next generation targeted therapies and drug combination strategies for GBM patients. Citation Format: Kaumudi Bhawe, Quentin Felty, Changwon Yoo, Deodutta Roy. Aberrant transcription factor activity of NRF1, NFE2L2, E2F1, RFX1, and MEF2 associated with severity of astrocytoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4391.

Sensitivity to differential NRF1 gene signatures contributes to breast cancer disparities.

Nuclear respiratory factor 1 (NRF1) drives estrogen-dependent breast tumorigenesis. Herein we examined the impact of NRF1 activity on the aggressiveness and disparate molecular signature of breast cancer in Black, White, Asian, and Hispanic women. NRF1 activity by transcription factor target enrichment analysis and causal NRF1-target gene signatures by Bayesian Network Inference with Java Objects (BANJO) and Markov Chain Monte Carlo (MCMC)-based gene order were examined in The Cancer Genome Atlas (TCGA) breast cancer cohorts. We are the first to report increased NRF1 activity based on its differential effects on genome-wide transcription associated with luminal A and B, HER2+ and triple-negative (TN) molecular subtypes of breast cancer in women of different race/ethnicity. We observed disparate NRF1 motif-containing causal gene signatures unique to Black, White, Asian, and Hispanic women for luminal A breast cancer. Further gene order searches showed molecular heterogeneity of each subtype of breast cancer. Six different gene order sequences involving CDK1, HMMR, CCNB2, CCNB1, E2F1, CREB3L4, GTSE1, and LMNB1 with almost equal weight predicted the probability of luminal A breast cancer in whites. Three different gene order sequences consisting of CCNB1 and GTSE1, and CCNB1, LMNB1, CDK1 or CASP3 predicted almost 100% probability of luminal B breast cancer in whites; CCNB1 and LMNB1 or GTSE predicted 100% HER2+ breast cancer in whites. GTSE1 and TUBA1C combined together predicted 100% probability of developing TNBC in whites; NRF1, TUBA1B and BAX with EFNA4, and NRF1 and BTRC predicated 100% TNBC in blacks. High expressor NRF1 TN breast tumors showed unfavorable prognosis with a high risk of breast cancer death in white women. Our findings showed how sensitivity to high NRF1 transcriptional activity coupled with its target gene signatures contribute to racial differences in luminal A and TN breast cancer subtypes. This knowledge may be useful in personalized intervention to prevent and treat this clinically challenging problem.

Nuclear Respiratory Factor 1 (NRF1) Transcriptional Activity-Driven Gene Signature Association with Severity of Astrocytoma and Poor Prognosis of Glioblastoma.

Despite tremendous progress in understanding the pathobiology of astrocytoma, major gaps remain in our knowledge of the molecular basis underlying the aggressiveness of high-grade astrocytoma (glioblastoma - GBM). Recently, we and others have shown nuclear respiratory factor 1 (NRF1) transcription factor being highly active in human cancers, but its role in astrocytoma remains unknown. Therefore, the purpose of this study was to uncover the role of NRF1 in the progression of GBM. NRF1 has higher mRNA expression and transcription factor activity in astrocytoma compared to non-tumor brain tissue. NRF1 activity also correlated with the aggressiveness of cancer. Increased NRF1 TF activity coupled with overexpression of RHOG was associated with poor survival of GBM patients. NRF1 activity was associated with transcriptomic signatures of neurogenesis, cell stemness, epithelial-mesenchymal transition and cell cycle progression. Overexpression of CDK4, AKT1, APAF1, HDAC1, NBN, TGFB1, & TNFRSF1A and downregulation of CASP3, IL7, STXBP1 and OPA1 predicted GBM malignancy in high expressors of NRF1 activity. Increased expression of the NRF1 motif containing genes, H6PD, NAT10, NBEAL2, and RNF19B predicted poor survival of IDH1 wild-type GBM patients. Poor survival outcomes and resistance to Temozolomide therapy were associated with higher NRF1 expression including its targets - LDHA, ZMAT3, NSUN2, ARMC5, NDEL1, CLPTM1L, ALKBH5, YIPF5, PPP2CA, and TFG. These findings suggest that aberrant NRF1 activity may contribute to the pathogenesis of GBM and severity of astrocytoma. Further analyses of NRF1 gene signatures will pave the way for next generation targeted therapies and drug combination strategies for GBM patients.

Nuclear respiratory factor 1 (NRF-1) upregulates the expression and function of reduced folate carrier (RFC) at the blood-brain barrier.

Folates are important for neurodevelopment and cognitive function. Folate transport across biological membranes is mediated by three major pathways: folate receptor alpha (FRα), proton-coupled folate transporter (PCFT), and reduced folate carrier (RFC). Brain folate transport primarily occurs at the choroid plexus through FRα and PCFT; inactivation of these transport systems results in suboptimal folate levels in the cerebrospinal fluid (CSF) causing childhood neurological disorders. Our group has reported that upregulation of RFC at the blood-brain barrier (BBB) through interactions with specific transcription factors, that is, vitamin D receptor (VDR) could increase brain folate delivery. This study investigates the role of nuclear respiratory factor 1 (NRF-1) in the regulation of RFC at the BBB. Activation of NRF-1/PGC-1α signaling through treatment with its specific ligand, pyrroloquinoline quinone (PQQ), significantly induced RFC expression and transport activity in hCMEC/D3 cells. In contrast, transfection with NRF-1 or PGC-1α targeting siRNA downregulated RFC functional expression in the same cell system. Applying chromatin immunoprecipitation (ChIP) assay, we further demonstrated that PQQ treatment increased NRF-1 binding to putative NRF-1 binding sites within the SLC19A1 promoter, which encodes for RFC. Additionally, in vivo treatment of wild type mice with PQQ-induced RFC expression in isolated mouse brain capillaries. Together, these findings demonstrate that NRF-1/PGC-1α activation by PQQ upregulates RFC functional expression at the BBB and could potentially enhance brain folate uptake.

Abstract 2441: Association between NRF1 regulatory gene networks and cancer and aging

Abstract There is a growing convergence between molecular aspects of aging and cancer. Cellular senescence acts as a barrier through preventing the proliferation of cells under oncogenic stress. Therefore, we decided to examine the role of nuclear respiratory factor 1 (NRF1) transcription factor in human brain cancer and brain aging. In contrast to higher expression of NRF1 in human glioma tumors, lower NRF1 levels are observed in Alzheimer’s disease (AD) brain tissue. Using the glioma and AD transcriptomic data of NRF1 target genes, we conducted Transcription Factor Target Enrichment Analysis that detects transcription factor (TF) activity based on quantification of the collective transcriptional activation of their target genes. NRF1 TF activity was significantly lower in AD brain tissues from four independent cohorts compared to non-AD brain tissues. In contrast, we observed higher NRF1 transcription factor activity in glioma. NRF1 activity as well as expression of several cell cycle pathway genes including CDK1,2,6,9 and NRF1 target genes such as NR2E1, APOE and MAPT are significantly altered in these two population cohorts. We also discovered that expression of NRF1, RB1 and E2F influenced survival in glioma. Specifically, the mean overall survival for patients with a high NRF1, E2F1,2,6,7,8 and RB1 gene signature was 20.91 months (95% CI = 9.37-32.46), compared to 101.99 months (95% CI = 74.28-129.70), in high NRF1 expressing patients that lacked the specific gene signature. NRF1 based gene signatures combined with IDH1 mutation status are necessary and sufficient for explaining the observed differences in survival curves. We examined the association of NRF1, the E2Fs and RB1 through Bayesian graphical modeling and observed gender differences in gene networks between NRF1 and E2F family members in the glioma patient sub-populations as compared to normal aging human brain samples. Furthermore, the differentially expressed NRF1 regulated genes are part of the KEGG pathways associated with spliceosome, ribosome and lysosome function as well as a diverse array of other functions are enriched, indicating ways in which NRF1 may be involved in glioma biology. In summary these findings support our postulate that NRF1 is an important transcription factor in the human brain, where it plays an active role in both aging and cancer. Citation Format: Kaumudi Bhawe, Quentin Felty, Changwon Yoo, Deodutta Roy. Association between NRF1 regulatory gene networks and cancer and aging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2441.

Elucidation of the therapeutic role of mitochondrial biogenesis transducers NRF-1 in the regulation of renal fibrosis

BackgroundMitochondrial dysfunction is a newly established risk factor for the development of renal fibrosis. Cell survival and injury repair is facilitated by mitochondrial biogenesis. Nuclear respiratory factor 1 (NRF-1) is a transcriptional regulation factor that plays a central role in the regulation of mitochondrial biogenesis. However, the transcription factor of this process in renal fibrosis is unknown. Thus, we hereby discussed the correlations of NRF-1 and renal interstitial fibrosis. Materials and methodsIn vitro fibrosis model was established by treatment with transforming growth factor-β1 (TGF-β1) in NRK-49F (Normal Rat kidney fibroblast). We investigated the ROS production, mitochondrial biogenesis and fibrogenic marker (e.q. fibronectin) during the progression of renal fibrosis by kit and Western blotting assay. Here, we used that two distinct mechanisms regulate NRF-1 activation and degradation of NRF-1. NRF-1 was transfect by pcDNA-NRF-1 overexpression gene to evaluate the NRF-1 activity of the therapeutic effect in renal fibrosis. In addition, NRF-1 was silenced by shRNA-NRF-1 to evaluate the significance of NRF-1. ELISA was used to evaluate the secreted fibronectin. Immunofluorescence staining was used to assay the in situ expression of proteins (e.g. fibronectin, NRF-1). ResultsUnder renal fibrosis conditions, TGF-β1 (5ng/ml) increased ROS. Simultaneously, TGF-β1-induced extracellular fibronectin by ELISA assay. In addition, TGF-β1 decreased expression of mitochondrial biogenesis. This is the first time to demonstrate that expression of NRF-1 is significantly decreased in renal fibrosis. However, NRK49F was a transfection with pcDNA-NRF-1 (2μg/ml) expression vector dramatically reverse TGF-β1-induced cellular fibrosis concomitantly with the suppression of fibronectin (both intracellular and extracellular fibronectin). More importantly, transfection with shRNA-NRF-1 (2μg/ml) significantly increased the expression of fibronectin of both intercellular and extracellular origins in NRK-49F cells. DiscussionThese finding suggest that NRF-1 plays a pivotal role on renal cellular fibrosis. Moreover, NRF-1 might act as a novel renal fibrosis antagonist by down-regulating fibrosis signaling in renal fibroblast cells.

Abstract 4559: Phosphodeficient NRF1 mutant suppresses the susceptibility of the breast epithelial cells to develop tumors when exposed to estrogen - a major breast cancer risk factor

Abstract Although several nuclear regulatory proteins may be targeted by estrogen, our efforts have focused on the redox nuclear respiratory factor-1 (NRF-1), because our recent study showed that NRF-1 gene expression significantly increases with the progression of breast tumor grades. NRF-1 overexpression supported in vitro tumor formation. To determine if NRF-1 is required for 17 beta estradiol (E2)-induced neoplastic phenotype, we have generated NRF-1 mutants by site directed mutagenesis. We have generated stable clones that express NRF-1 mutant. The in vitro tumor formation was detected by anchorage independent growth and 3D spheroid assays. NRF-1 overexpression enhanced in vitro tumor spheroid formation, cell migration and cell invasion. Our flow cytometry analysis showed that overexpression of wild-type NRF-1 increased the percent of transformed MCF-10A cells in S phase compared to vector alone. Dominant negative NRF-1 protein showed reduction in the tumor formation. In addition, shRNA targeting NRF-1 resulted in the inhibition of anchorage independent growth of MCF-10A cells in both vehicle control and E2-treated cells. Since NRF-1 is a substrate of the kinase AKT, we determined whether NRF-1 phosphorylation was increased in MCF-10A cells after treatment with a carcinogenic regimen of E2. We observed more than a 2-fold increase in phospho-NRF-1 in E2 treated (100pg/ml for 30 min) MCF-10A cells and phosphorylation of NRF-1 was inhibited by co-treatment with either biological (CAT or MnSOD) or chemical (20μM ebselen) ROS modifiers. These results suggest that E2-induced phosphorylation of NRF-1 is influenced by ROS signaling messengers. We found that E2 treated MCF-10A cells showed increased NRF-1 binding to the promoters of Cdc2, PRC1, PCNA, Cyclin B1, and CDC25C genes. NRF-1 binding induced by E2 treatment was inhibited by the overexpression of CAT and MnSOD. NRF-1 phosphorylation site specific to kinase AKT, Thr-109, was mutated to Asp (D) or Ala (A), and the NRF-1 acetylation site specific to acetyl-transferase PCAF, Lys- 89, was mutated to Gln (glutamine)-Q or Ala (A). We examine the effect of one of the NRF-1 mutants on E2-induced in vitro tumor formation. We observed that the expression of NRF-1 (T109A) phosphodeficient mutant (NRF-1PMT) significantly inhibited E2-induced cell transformation, whereas empty vector did not influence E2-induced colony formation. Together these findings support the idea that NRF-1 may play an important role in E2-induced malignant transformation of breast epithelial cells. Thus, our data is consistent with the hypothesis that in addition to the estrogen receptor activity, NRF-1 activation contributes to the susceptibility to develop malignant phenotype in response to exposure to estrogen. This work was in part supported by a VA MERIT Review (VA BX001463) grant to DR Citation Format: Lazaro Mesa, Jayanta Das, Alok Deoraj, Victor Okoh, Deodutta Roy. Phosphodeficient NRF1 mutant suppresses the susceptibility of the breast epithelial cells to develop tumors when exposed to estrogen - a major breast cancer risk factor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4559. doi:10.1158/1538-7445.AM2015-4559

Redox signalling to nuclear regulatory proteins by reactive oxygen species contributes to oestrogen-induced growth of breast cancer cells.

Background:17β-Oestradiol (E2)-induced reactive oxygen species (ROS) have been implicated in regulating the growth of breast cancer cells. However, the underlying mechanism of this is not clear. Here we show how ROS through a novel redox signalling pathway involving nuclear respiratory factor-1 (NRF-1) and p27 contribute to E2-induced growth of MCF-7 breast cancer cells.Methods:Chromatin immunoprecipitation, qPCR, mass spectrometry, redox western blot, colony formation, cell proliferation, ROS assay, and immunofluorescence microscopy were used to study the role of NRF-1.Results:The major novel finding of this study is the demonstration of oxidative modification of phosphatases PTEN and CDC25A by E2-generated ROS along with the subsequent activation of AKT and ERK pathways that culminated in the activation of NRF-1 leading to the upregulation of cell cycle genes. 17β-Oestradiol-induced ROS by influencing nuclear proteins p27 and Jab1 also contributed to the growth of MCF-7 cells.Conclusions:Taken together, our results present evidence in the support of E2-induced ROS-mediated AKT signalling leading to the activation of NRF-1-regulated cell cycle genes as well as the impairment of p27 activity, which is presumably necessary for the growth of MCF-7 cells. These observations are important because they provide a new paradigm by which oestrogen may contribute to the growth of breast cancer.

Sphingosine kinase 2 and sphingosine-1-phosphate promotes mitochondrial function in dopaminergic neurons of mouse model of Parkinson’s disease and in MPP+-treated MN9D cells in vitro

Dysregulation of sphingolipid metabolism has been shown to trigger the pathophysiology of many neurodegenerative disorders. The present study focuses on the role of one of the two sphingosine kinases, Sphk2 and its metabolite sphingosine-1-phosphate (S1P) signaling in Parkinson’s disease (PD). Our study indicated a marked down regulation of Sphk2 expression in the substantia nigra region of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model and in the cellular PD model. Localization studies indicated that Sphk2 was predominantly present in mitochondria, proposing for its potential role in mitochondrial functions. Since mitochondrial dysfunction has been described to be the major pathological event in PD, the present study focused on the role of Sphk2/S1P signaling in promoting mitochondrial functions in the MPTP-induced mouse model of PD and in 1-methyl-4 phenylpyridinium (MPP+)-treated MN9D cells. Our study demonstrated that inhibition of Sphk2 decreased the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and its downstream targets nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM) which are the key genes regulating mitochondrial function. In addition, there was also a significant reduction in the total cellular adenosine triphosphate (ATP) and superoxide dismutase 2 (SOD 2) with an associated increase in levels of reactive oxygen species (ROS) in the absence of Sphk2. Interestingly, it was found that treating the cells with exogenous S1P along with MPP+ exerted a neuroprotective effect by activation of p-CREB, PGC-1α and NRF-1 in the MN9D cells. Moreover, the level of ATP was unaffected in the MPP+-treated cells in the presence of S1P. It was also observed that levels of ROS were significantly decreased in the MPP+-treated cells in the presence of exogenous S1P. Our study also demonstrated that S1P exerted its protective effect through the S1P1 receptor. Taken together, these results show that Sphk2/S1P has an important role to play in the survival of the dopaminergic neurons, in the pathogenesis of PD.

14,15-EET promotes mitochondrial biogenesis and protects cortical neurons against oxygen/glucose deprivation-induced apoptosis

14,15-Epoxyeicosatrienoic acid (14,15-EET), a metabolite of arachidonic acid, is enriched in the brain cortex and exerts protective effect against neuronal apoptosis induced by ischemia/reperfusion. Although apoptosis has been well recognized to be closely associated with mitochondrial biogenesis and function, it is still unclear whether the neuroprotective effect of 14,15-EET is mediated by promotion of mitochondrial biogenesis and function in cortical neurons under the condition of oxygen–glucose deprivation (OGD). In this study, we found that 14,15-EET improved cell viability and inhibited apoptosis of cortical neurons. 14,15-EET significantly increased the mitochondrial mass and the ratio of mitochondrial DNA to nuclear DNA. Key makers of mitochondrial biogenesis, peroxisome proliferator activator receptor gamma-coactivator 1 alpha (PGC-1α), nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM), were elevated at both mRNA and protein levels in the cortical neurons treated with 14,15-EET. Moreover, 14,15-EET markedly attenuated the decline of mitochondrial membrane potential, reduced ROS, while increased ATP synthesis. Knockdown of cAMP-response element binding protein (CREB) by siRNA blunted the up-regulation of PGC-1α and NRF-1 stimulated by 14,15-EET, and consequently abolished the neuroprotective effect of 14,15-EET. Our results indicate that 14,15-EET protects neurons from OGD-induced apoptosis by promoting mitochondrial biogenesis and function through CREB mediated activation of PGC-1α and NRF-1.

Diesel exhaust particulate extracts inhibit transcription of nuclear respiratory factor-1 and cell viability in human umbilical vein endothelial cells

Endothelial dysfunction precedes cardiovascular disease and is accompanied by mitochondrial dysfunction. Here we tested the hypothesis that diesel exhaust particulate extracts (DEPEs), prepared from a truck run at different speeds and engine loads, would inhibit genomic estrogen receptor activation of nuclear respiratory factor-1 (NRF-1) transcription in human umbilical vein endothelial cells (HUVECs). Additionally, we examined how DEPEs affect NRF-1-regulated TFAM expression and, in turn, Tfam-regulated mtDNA-encoded cytochrome c oxidase subunit I (COI, MTCO1) and NADH dehydrogenase subunit I (NDI) expression as well as cell proliferation and viability. We report that 17β-estradiol (E(2)), 4-hydroxytamoxifen (4-OHT), and raloxifene increased NRF-1 transcription in HUVECs in an ER-dependent manner. DEPEs inhibited NRF-1 transcription, and this suppression was not ablated by concomitant treatment with E(2), 4-OHT, or raloxifene, indicating that the effect was not due to inhibition of ER activity. While E(2) increased HUVEC proliferation and viability, DEPEs inhibited viability but not proliferation. Resveratrol increased NRF-1 transcription in an ER-dependent manner in HUVECs, and ablated DEPE inhibition of basal NRF-1 expression. Given that NRF-1 is a key nuclear transcription factor regulating genes involved in mitochondrial activity and biogenesis, these data suggest that DEPEs may adversely affect mitochondrial function leading to endothelial dysfunction and resveratrol may block these effects.

Abstract LB-12: Estrogen-induced redox signaling via NRF-1 is involved in the development of breast cancer

Abstract The purpose of this study was to determine the role of estrogen-induced reactive oxygen species (ROS) signaling pathways in the development of breast cancer. Here we tested the postulate that activation of a redox sensitive transcription factor, nuclear respiratory factor-1 (NRF-1) signaling is essential for 4-hydroxyestradiol (4-OH-E2)-induced tumorigenesis. We observed that (i) 4-OH-E2 treatment of MCF-10A or MCF-7 cells increased the formation of ROS, (ii) Estrogen-induced ROS activated redox sensitive transcription factor NRF-1, (iii) 4-OH-E2 through activating a redox sensitive serine-threonine kinase AKT phosphorylated NRF-1, (iv) Overexpression of catalase and MnSOD, as well as silencing of NRF-1 prevented 4-OH-E2-induced anchorage-independent growth of MCF-10A and MCF-7 cells and xenograft tumor formation, and (v) 4-OH-E2-induced ROS through stimulating AKT kinase and histone acetyltransferase of CREB binding protein-associated factor (P/CAF), respectively, controlled phosphorylation and acetylation of NRF-1. The results also showed that 4-OH-E2-induced post-translational changes lead to NRF-1 activation, and silencing of NRF-1 and overexpression of MnSOD or catalase prevented 4-OH-E2-induced tumor formation. Taken together, these findings suggest a new paradigm for estrogen-induced breast carcinogenesis that deregulation of phosphorylation and aceytylation of NRF-1 may represent a major mechanism of 4-OH-E2-induced neoplastic transformation and tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-12. doi:10.1158/1538-7445.AM2011-LB-12

Dynamic Mitochondrial Responses to Oxidative Stress in Primary AML Cells Involve Structural Damage and Repair Activity.

Mitochondrial DNA (mtDNA) is particularly susceptible to oxidative damage and mutations because of the high level of reactive oxygen species (ROS) generated and the inefficiency of the mtDNA repair system. The oxidative stress elicited by chronic inflammation increases the number of mtDNA mutations and might correlate with a cancerous status. We postulated that increased oxidative stress in primary AML cells might cause mtDNA damage, which can lead to mtDNA mutations, structural changes, perturbation of mtDNA repair and biogenesis. Many mutation and polymorphisms (a total of 606 mtDNA sequence variants) were identified from 48 matched AML bone marrow and buccal mucosa samples, and blood samples from 57 control subjects. There were profound alterations in the 303 poly C, 16184 poly C, and 514 CA repeats. The intracellular ROS generation of cells can be investigated using the 2′,7′-dichlorfluorescein-diacetate and flow cytometry. The results were expressed as mean fluorescence intensity (MFI). MFI in primary AML cells (4,435±709) was significantly higher than those in control blood cells (1,562±141) (P<0.05). After then, we checked mRNA expression of peroxisome proliferators activated receptor gamma coactivator-1α (PGC-1α), PGC-1-related coactivator (PRC) and nuclear respiratory factor 1 (NRF-1) because these have been identified as an important regulator of intracellular ROS level and crucial factors linking external stimuli to mitochondrial biogenesis. As compared to normal blood cells, about 2.0 fold increase in NRF-1 mRNA expression was noted in primary AML, whereas PGC-1α and PRC mRNA expression were not remarkably changed. The supercoiled and open relaxed forms of mtDNA reflect functional and damaged molecules. Thus, sensitive detection of the relaxed and total mtDNA from the same DNA templates should allow quantitative measurements on mtDNA damage, repair and copy number change in stressed cells (Nucleic Acids Res 2007; 35:1377–88). Primary AML cells and normal blood cells were exposed to sublethal concentration of hydrogen peroxide to study mtDNA damage and repair activity using real-time PCR quantification. When AML cells and normal blood cells were treated with 240uM hydrogen peroxide, an average of 2.1 and 2.2 fold decreases in mtDNA amplification of AML cells were detected in two mtDNA markers (HV2 and cytochrome b) after 1-h and 2-h exposure, respectively. The exposure time-dependent increase in mtDNA amplification due to increased proportion of open relaxed forms was observed in normal blood cells after 15 min to 2-h treatment of hydrogen peroxide. In conclusion, the high level of intracellular ROS in primary AML cells might cause mtDNA damage, which can lead to mtDNA mutations and increased mitochondrial biogenesis by NRF-1 activation. Although the limited repair capacity hypothesis has been validated experimentally in some experimental systems, this study showed elevated mtDNA repair activity compared to normal blood cells. Our data also support the possibility that NRF-1 targeting approach may aid in the treatment of AML.

Control of mitochondrial biogenesis during myogenesis

We used expression and reporter gene analysis to understand how changes in transcription factors impinge on mitochondrial gene expression during myogenesis of cultured murine myoblasts (C2C12 and Sol8). The mRNA levels for nuclear respiratory factor-1 (NRF-1) and NRF-2alpha increased 60% by the third day of myogenesis, whereas NRF-1 and NRF-2 reporter gene activity increased by fivefold over the same period. Although peroxisome proliferator activated receptor (PPARalpha) mRNA levels increased almost 10-fold, the activity of a PPAR reporter was unchanged during myogenesis. The PPAR coactivator PPAR-gamma coactivator-1alpha (PGC1alpha), a master controller of mitochondrial biogenesis, was not expressed at detectable levels. However, the mRNA for both PGC1alpha-related coactivator and PGC1beta was abundant, with the latter increasing by 50% over 3 days of differentiation. We also conducted promoter analysis of the gene for citrate synthase (CS), a common mitochondrial marker enzyme. The proximal promoter ( approximately 2,100 bp) of the human CS lacks binding sites for PPAR, NRF-1, or NRF-2. Deletion mutants, a targeted mutation, and an Sp1 site-containing reporter construct suggest that changes in Sp1 regulation also participate in mitochondrial biogenesis during myogenesis. Because most mitochondrial genes are regulated by PPARs, NRF-1, and/or NRF-2, we conducted inhibitor studies to further support the existence of a distinct pathway for CS gene regulation in myogenesis. Although both LY-294002 (a phosphatidylinositol 3-kinase inhibitor) and SB-203580 (a p38-MAPK inhibitor) blocked myogenesis (as indicated by creatine phosphokinase activity), only SB-203580 prevented the myogenic increase in cytochrome oxidase activity, whereas only LY-294002 blocked the increase in CS (enzyme and reporter gene activities). Collectively, these studies help delineate the roles of some transcriptional regulators involved in mitochondrial biogenesis associated with myogenesis and underscore an import role for posttranscriptional regulation of transcription factor activity.

Lipopolysaccharide Stimulates Mitochondrial Biogenesis via Activation of Nuclear Respiratory Factor-1

Exposure to bacterial lipopolysaccharide (LPS) in vivo damages mitochondrial DNA (mtDNA) and interferes with mitochondrial transcription and oxidative phosphorylation (OXPHOS). Because this damage accompanies oxidative stress and is reversible, we postulated that LPS stimulates mtDNA replication and mitochondrial biogenesis via expression of factors responsive to reactive oxygen species, i.e. nuclear respiratory factor-1 (NRF-1) and mitochondrial transcription factor-A. In testing this hypothesis in rat liver, we found that LPS induces NRF-1 protein expression and activity accompanied by mRNA expression for mitochondrial transcription factor-A, mtDNA polymerase gamma, NRF-2, and single-stranded DNA-binding protein. These events restored the loss in mtDNA copy number and OXPHOS gene expression caused by LPS and increased hepatocyte mitotic index, nuclear cyclin D1 translocation, and phosphorylation of pro-survival kinase, Akt. Thus, NRF-1 was implicated in oxidant-mediated mitochondrial biogenesis to provide OXPHOS for proliferation. This implication was tested in novel mtDNA-deficient cells generated from rat hepatoma cells that overexpress NRF-1. Depletion of mtDNA (rhoo clones) diminished oxidant production and caused loss of NRF-1 expression and growth delay. NRF-1 expression and growth were restored by exogenous oxidant exposure indicating that oxidative stress stimulates biogenesis in part via NRF-1 activation and corresponding to recovery events after LPS-induced liver damage.

Skeletal muscle overexpression of nuclear respiratory factor 1 increases glucose transport capacity.

Nuclear respiratory factor 1 (NRF-1) is a transcriptional activator of nuclear genes that encode a range of mitochondrial proteins including cytochrome c, various other respiratory chain subunits, and delta-aminolevulinate synthase. Activation of NRF-1 in fibroblasts has been shown to induce increases in cytochrome c expression and mitochondrial respiratory capacity. To further evaluate the role of NRF-1 in the regulation of mitochondrial biogenesis and respiratory capacity, we generated transgenic mice overexpressing NRF-1 in skeletal muscle. Cytochrome c expression was increased approximately twofold and delta-aminolevulinate synthase was increased approximately 50% in NRF-1 transgenic muscle. The levels of some mitochondrial proteins were increased 50-60%, while others were unchanged. Muscle respiratory capacity was not increased in the NRF-1 transgenic mice. A finding that provides new insight regarding the role of NRF-1 was that expression of MEF2A and GLUT4 was increased in NRF-1 transgenic muscle. The increase in GLUT4 was associated with a proportional increase in insulin-stimulated glucose transport. These results show that an isolated increase in NRF-1 is not sufficient to bring about a coordinated increase in expression of all of the proteins necessary for assembly of functional mitochondria. They also provide the new information that NRF-1 overexpression results in increased expression of GLUT4.