AMPKα pathway to promote cell survival under oxidative stress

Journal Pre-proof NAMPT maintains mitochondria content via NRF2-PPARα/ AMPKα pathway to promote cell survival under oxidative stress

An Yu, Ronghua Zhou, Benzeng Xia, Weiwei Dang, Zaiqing Yang, Xiaodong Chen PII:

S0898-6568(19)30292-X

DOI:

https://doi.org/10.1016/j.cellsig.2019.109496

Reference:

CLS 109496

To appear in:

Cellular Signalling

Received date:

12 September 2019

Revised date:

5 December 2019

Accepted date:

5 December 2019

Please cite this article as: A. Yu, R. Zhou, B. Xia, et al., NAMPT maintains mitochondria content via NRF2-PPARα/AMPKα pathway to promote cell survival under oxidative stress, Cellular Signalling(2019), https://doi.org/10.1016/j.cellsig.2019.109496

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© 2019 Published by Elsevier.

Journal Pre-proof NAMPT maintains mitochondria content via NRF2-PPARα/AMPKα pathway to promote cell survival under oxidative stress An Yu2,3 , Ronghua Zhou2 , Benzeng Xia1 Weiwei Dang3 , Zaiqing Yang2 , Xiaodong Chen1,* [email protected] 1

Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of

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Education, College of Animal Science and Technology & College of Veterinary Medicine,

College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070,

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Huazhong Agricultural University, Wuhan, 430070, P.R. China

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P.R. China

Huffington Center on Aging, Baylor College of Medicine, Houston, TX 77030, USA

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Corresponding author at: College of Animal Science and Technology & College of

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3

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Abstract

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Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, P.R. China.

Mitochondria plays a key role in regulating cell death process under stress conditions and it has been indicated that NAMPT overexpression promotes cell survival under genotoxic stress by maintaining mitochondrial NAD+ level. NAMPT is a rate-limiting enzyme for NAD+ production in mammalian cells and it was suggested that NAMPT and NMNAT3 are responsible for mitochondrial NAD+ production to maintain mitochondrial NAD+ pool. However, subsequent studies suggested mitochondrial may lack the NAMPT-NMANT3 pathway to maintain NAD+ level. Therefore, how NAMPT overexpression rescues mitochondrial NAD+ content to promote

Journal Pre-proof cell survival in response to genotoxic stress remains elusive. Here, we show that NAMPT promotes cell survival under oxidative stress via both SIRT1 dependent p53-CD38 pathway and SIRT1 independent NRF2-PPARα/AMPKα pathway, and the NRF2-PPARα/AMPKα pathway plays a more profound role in facilitating cell survival than the SIRT1-p53-CD38 pathway does. Mitochondrial content and membrane potential were significantly reduced in response to H2O2 treatment, whereas activated NRF2-PPARα/AMPKα pathway by NAMPT overexpression

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rescued the mitochondrial membrane potential and content, suggesting that maintained

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mitochondrial content and integrity by NAMPT overexpression might be one of the key

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mechanisms to maintain mitochondrial NAD+ level and subsequently dictate cell survival under

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oxidative stress. Our results indicated that NRF2 is a novel down-stream target of NAMPT, which mediates anti-apoptosis function of NAMPT via maintaining mitochondrial content and

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membrane potential.

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1. Introduction

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Keywords: NAMPT; Mitochondria content; Oxidative stress; Cell apoptosis

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Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme which catalyzes the conversion of nicotinamide (NAM) and phosphoribosyl-pyrophosphates to nicotinamide mononucleotide (NMN) in the mammalian nicotinamide adenine dinucleotide (NAD+) synthetic salvage pathway. NMN produced by NAMPT is further converted into NAD+ by nicotinamide mononucleotide adenylyltransferases (NMNAT1-3). Nmnat1 is a nuclear protein, whereas NMNAT2 and NMNAT3 are localized to the Golgi complex and the mitochondria, respectively [1]. It was suggested that NAMPT and NMNAT3 are responsible for mitochondrial NAD+ production [2]. A key mechanism for intensive genotoxic stress to induce cell death is cellular NAD+ pool depletion by hyperactivation of NAD+ consuming enzymes such as

Journal Pre-proof poly(ADP-ribose) polymerase-1 (PARP-1) and CD38 , while NAMPT overexpression is able to promote cell survival by preventing mitochondrial NAD+ pool from depletion under genotoxic stress [3]. Since NAMPT is a rate-limiting enzyme for NAD+ production, its overexpression may help to maintain cellular NAD+ pool by producing more NAD+ directly. However, under intensive genotoxic stress, NAMPT overexpression is not able to prevent nuclear and cytoplasm NAD+ pool from depletion [3], although the mitochondrial NAD+ pool was maintained by

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NAMPT overexpression, subsequent studies suggested mitochondria is lack of the

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canonical NAD+ synthetic salvage pathway [4] and NMNAT3 is dispensable in the maintenance

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of mitochondrial NAD+ levels [5]. Therefore, in which way NAMPT overexpression rescued

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mitochondrial NAD+ pool from depletion to dictate cell survival under genotoxic stress remains

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to be investigated.

Genotoxic stress could be induced by several DNA damaging reagents such as methylmethane

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sulfonate (MMS), etoposide and hydrogen peroxide (H2O2). MMS and etoposide are exogenous

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chemicals, while H2O2 is one of the endogenous reactive oxygen species (ROS), therefore, in the present study, H2O2 was used as the inducer of genotoxic stress, which may generate results

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with more physiological relevance than MMS and etoposide do. CD38 is a major NADase in mammalian tissues [6], it is capable of cleaving NAD+ into cADPR and hydrolyzing cADPR to ADPR [7]. It was reported that tissue levels of NAD+ in CD38 deficient mice are 10- to 20-fold higher than in wild-type animals [6] and the mouse embryonic fibroblasts (MEFs) from CD38(-/-) mice were significantly resistant to oxidative stress such as H2O2 injury [8]. SIRT1 and CD38 are both NAD+ consuming enzymes. It has been suggested that SIRT1 activity is significantly increased by CD38 knockout due to the higher NAD+ level [9], therefore CD38 negatively

Journal Pre-proof regulates SIRT1 activity by limiting NAD+ level, whether CD38 is in turn regulated by SIRT1 remains unknown. Mammalian sirtuins (SIRT1-7) are considered as the major down-stream effectors of NAMPT or its product NMN [10], especially, SIRT1 mediates many functions of NAMPT/NMN such as insulin secretion and metabolism regulations [11]. Very recently, it has been reported that the product of NAMPT, NMN, is able to rescue liver fibrosis caused by dysfunctional telomeres in a

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partially SIRT1-dependent manner [12]. However, SIRT1 may not be the major mediator of

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NAMPT overexpression to dictate cell survival under genotoxic stress, because a potent SIRT1

Therefore, identifying the major mediator of NAMPT to promote cell survival under

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[3].

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inhibitor, EX-527, did not abolish the effect of NAMPT overexpression to promote cell survival

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genotoxic stress is of interest. Nuclear Factor Erythroid-derived 2-like 2 (NRF2) is a master regulator of cellular anti-oxidative stress response, which is relatively independent on SIRT1 [13].

Therefore, whether NRF2 is another down-stream effector of NAMPT becomes an

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interesting question.

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[15-17].

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Similar to SIRT1 [14], NRF2 was also reported to be a key regulator for mitochondrial biogenesis

In the present study, we hypothesized that maintaining mitochondrial content and integrity under oxidative stress is a key step to preserve mitochondrial NAD+ pool and consequently to promote cell survival by NAMPT overexpression. We demonstrated that H2O2 treatment significantly reduced mitochondrial number and mitochondrial membrane potential, while NAMPT overexpression restored mitochondrial number and membrane potential in response to H2O2 to dictate cell survival. We identified that NRF2 is a key mediator of NAMPT to maintain mitochondrial content and membrane potential under oxidative stress, and NRF2 overexpression mimics the effect of NAMPT overexpression to promote cell survival under oxidative stress.

Journal Pre-proof Among the three important factors for mitochondrial biogenesis, AMPKα and PPARα, but not PGC-1α, are required for NRF2 mediated mitochondrial content and membrane potential maintenance in response to oxidative stress. 2. Materials and methods 2.1 Materials

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DMEM media and fetal bovine serum were purchased from Gibco (Beijing, China). NAMPT

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antibody, PPARα antibody, AMPKα antibody and β-actin antibody were purchased from Abcam

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(HKSP, N.T. Hong Kong), PGC-1α antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), NRF2 antibody was purchased from Cell Signaling Technology (Beverly,

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MA). p53 phosphorylation and acetylation antibodies were purchased from Proteintech Group,

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Inc (Wuhan, China). Mito Tracker red and JC-1 mitochondrial staining kit were purchased from

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Thermo Fisher. NAD+/NADH kit was purchased from Abbkine. Nicotinamide mononucleotide (NMN), FK866, H2O2, G418, EX-527 and all other chemicals were purchased from Sigma

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2.2 Cell culture

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(Shanghai, China).

HEK293 and IMR-90 cells were maintained in DMEM with 10% feta bovine serum and were incubated at 37℃ in 5% CO2. 2.3 Transfection and knockdown Both NAMPT and NRF2 overexpression are stable overexpression. The coding sequence (CDS) of NAMPT and NRF2 were cloned and constructed into pcDNA3.1+ overexpression vector, then the overexpression vectors were sequenced to verify mutations free constructs. The primers and

Journal Pre-proof restriction enzymes for human NAMPT and NRF2 overexpression vectors were showed in supplemental Figure 2B. HEK293 cells were transfected with the constructs by lipo-transfection reagents, one day after transfections, cells were split into 96 wells plates using media containing G418 (1mM) to select single cell derived positive overexpression cell strains in next weeks. The control cells are transfected with empty pcDNA3.1+ vector and also treated by G418 selection. All siRNAs used for knockdown were constructed into psilencer-CMV-4.1 vector, the control

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knockdown sequence, NAMPT, NRF2, AMPKα and PPARα knockdown sequence are shown in

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Supplemental Table 1. The knockdown efficiency of these genes is indicated in Supplemental

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Figure 1A, B (SFig. 1A, B).

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2.4 Quantitative real time PCR (qPCR) and western blots

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2 μg total RNA of each sample were used to perform reverse transcription. qPCR was carried out in a total volume of 20 μL with SYBR-Green mix (Takara, Dalian, China) on an IQ5 thermal

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cycler (Bio-Rad, Hercules, CA). Human β-actin was used as the internal references and

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amplified in parallel. The PCR conditions were 95℃ for 3 min, followed by 40 cycles of 95℃ for 15 s, 60℃ for 15 s, and 72℃ for 15 s. Cycle threshold values were normalized to that of the

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internal references, and the relative gene expression levels were calculated by the 2 -ΔΔCt method. The sequences of all qPCR primers are showed in the Supplemental table 2. For the mitochondrial DNA qPCR, human nuclear 28s DNA was used as the internal references and human mitochondrial DNA was used as the target to be quantified. The primers sequence of mitochondrial DNA and 28s DNA are showed in the Supplemental table 3. Proteins were extracted by RIPA buffer. Aliquots containing 60 μg of protein from each sample were separated by 10% SDS–PAGE then all proteins were transferred onto polyvinylidene difluoride membranes. The membranes were blocked with TBST buffer (20 mM Tris–HCl, 137

Journal Pre-proof mM NaCl, and 0.05% Tween-20) containing nonfat dried milk at room temperature for 60 min then incubated overnight with a 1:1000 dilution of appropriate primary antibodies. After extensive washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz; 1:4000) for 1 h at room temperature and visualized using an ECL Western blotting detection system (Tiangen, Beijing, China). 2.5 Annexin V/PI apoptosis staining, NAD+ level analysis, Mito Tracker and JC-1 staining

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carried out strictly according the instructions of the kits.

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Annexin V/PI apoptosis staining, NAD+ level analysis, Mito Tracker and JC-1 staining were

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2.6 Statistical analysis

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All experiments were repeated at least three times with similar results. Data are presented as means ± SD. Student’s t test was used for statistical comparison. p < 0.05 was considered as

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statistically significant.

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3. Results

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3.1 NAMPT overexpression promotes cell survival under oxidative stress

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To verify if H2O2 is able to induce cell death in HEK293 cells, the cells were treated with 1mM H2O2 for 3 h then were checked by Annexin V/PI apoptosis staining. 3 h H2O2 treatment generated stronger green fluorescence on cell membrane and red fluorescence in cell nucleus (Fig. 1A), which are signs of apoptosis. Therefore, like other genotoxic reagents such as methylmethane sulfonate (MMS) and etoposide [3], H2O2 is a potent cell death inducer in HEK293 cells. To test the effect of NAMPT overexpression on cell death regulation under oxidative stress, a NAMPT stable overexpression HEK293 cell strain was generated by pcDNA3.1-NAMPT transfection and followed by G418 selection. The mRNA and protein expression levels were determined by qPCR (Fig. 1 B) and western blotting experiments (Fig. 1

Journal Pre-proof C) respectively. Both mRNA and protein of NAMPT were significantly increased in NAMPT stable overexpression cells and the increase of NAMPT mRNA level is quite dramatical (Fig. 1B). Both NAMPT overexpressed cells and control cells were treated with 1 mM H2O2 for 3 h then tested by Annexin V/PI apoptosis staining, the results showed that in control cells 3 h H2O2 treatment induced strong apoptosis fluorescent signals and in NAMPT overexpression cells the fluorescent signals were largely eliminated upon H2O2 treatment (Fig. 1D). Because NAMPT is

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also a cytokine (known as VISFATIN or PBEF), to determine whether the anti-apoptosis

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function of NAMPT is dependent on its enzymatic activity, NAMPT overexpressed cells were

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treated by its enzymatic inhibitor (FK866 100nM) with H2O2 together. The result showed that

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FK866 greatly compromised the anti-apoptosis function of NAMPT in response to H2O2 (Fig. 1D), suggesting that the anti-apoptosis function of NAMPT largely depends on its enzymatic

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activity. FK866 (100nM) alone did not induce obvious cell death (supplemental Figure 2A).

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3 h H2O2 treatment is a relative short time treatment, to gain a longer time effect of NAMPT on

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cell death regulation, both control HEK293 cells and NAMPT overexpressed HEK293 cells were treated with 1 mM H2O2 for 24 h then the cell alive ratios were calculated. 24 h H2O2 treatment

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caused dramatical cell death, whereas NAMPT overexpression significantly rescued cell death (Fig. 1E). NAMPT enzymatic inhibitor, FK866, significantly compromised the of NAMPT overexpression (Fig. 1E). Since HEK293 is a cell line, human primary fibroblasts IMR-90 cells were also used in the test. The transfection efficiency is much lower in IMR-90 cells compared to the efficiency in HEK293 cells, and nicotinamide mononucleotide (NMN) is the direct enzymatic product of NAMPT, therefore, to mimic NAMPT overexpression, NMN (200μM) was added into the media together with H2O2 (1mM). The results showed that 24 h H2O2 treatment induced significantly cell death in IMR-90 cells, and NMN significantly reduced the cell death

Journal Pre-proof caused by H2O2 (Fig. 1F). Collectively, these results indicate 1mM H2O2 significantly induce cell death while NAMPT overexpression significantly prevents cell death from oxidative stress and this anti-apoptosis function of NAMPT depends on its enzymatic activity. 3.2 Suppressed CD38 expression by NAMPT moderately contributes to cell survival under oxidative stress

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In addition to sirtuins (SIRT1-7), CD38 and poly (ADP-ribose) polymerases (PARPs) are major

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NAD+ consuming enzymes. Under genotoxic stress condition, hyperactivation of PARPs may cause cell death by depletion of NAD+ pool [18,19]. It has been reported that CD38 expression

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was upregulated in response to H2O2 treatment [20]. Whether CD38 is regulated by NAMPT

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overexpression and whether it is involved in NAMPT overexpression mediated cell survival

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under oxidative stress are poorly investigated. To address these questions, both CD38 and PARP-1 mRNA level were determined in control HEK293 cells and NAMPT overexpression

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HEK293 cells in response to 3 h H2O2 treatment. The results showed that both CD38 and

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PARP-1 were upregulated after 3 h H2O2 treatment and NAMPT overexpression significantly

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suppressed the upregulation of these two genes (Fig. 2A and B). Importantly, SIRT1 inhibitor EX-527 significantly counteracted the effect of NAMPT overexpression on CD38 mRNA level (Fig. 2A) suggesting that CD38 expression level may be regulated by SIRT1 under oxidative stress. To determine whether suppressed CD38 expression by NAMPT under oxidative stress contributes to NAMPT overexpression mediated cell survival, a potent CD38 enzymatic inhibitor 78c [21] was added into the media together with H2O2 for 24 h and the cell death ratios were calculated. The result showed that CD38 inhibition significantly but moderately increased the cell survival ratio in response to H2O2 treatment (Fig. 2 C).

Journal Pre-proof p53 is a key cancer repressor and a key cellular stress responsive factor, in response to H2O2 treatment, p53 is one of the most significantly upregulated factors, which mediates apoptosis [22]. NAMPT/NMN-SIRT1 pathway not only suppresses p53 transcriptional activity [23-25] but also lowers its protein level [12]. Considering that there is a putative p53 binding site on CD38 promoter (SFig. 1C), we speculate that CD38 upregulation by H2O2 and its downregulation by NAMPT may be mediated by p53. CD38 mRNA level was determined in the condition of p53

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overexpression with or without NAMPT overexpression. The results showed that p53

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overexpression significantly upregulated CD38 level while NAMPT overexpression significantly

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counteracts p53 overexpression caused CD38 upregulation (Fig. 2D). The overexpression of

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NAMPT and p53 is showed in Fig. 2G. To further confirm the involvement of p53, p53 mRNA and protein level was determined by qPCR and western blotting experiments. The results

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indicate that H2O2 significantly increased p53 mRNA and this increase was suppressed by

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NAMPT overexpression (Fig.2 E). Importantly, SIRT1 inhibitor EX-527 significantly counteracted the effect of NAMPT on p53 expression (Fig.2 E). Consistent with the mRNA

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results, p53 protein level was increased in response to H2O2 treatment and NAMPT

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overexpression restored its protein level (Fig. 2F). H2O2 treatment also increases the phosphorylation and acetylation levels of p53 and NAMPT overexpression compromised the effects of H2O2 treatment (supplemental Fig. 1D). Taken together, these results indicate that SIRT1-p53-CD38 pathway significantly but moderately contributes to increased cell survival under oxidative stress by NAMPT overexpression. Both NAMPT overexpression and CD38 inhibition may cause increased NAD+ level, which might alleviate NAD+ pool depletion caused by genotoxic stress and rescue the cell death. And also, the promoting cell survival effect of NAMPT depends on its enzymatic activity (Fig. 1E)

Journal Pre-proof suggesting that CD38 inhibition may have similar effect to NAMPT overexpression. However, unlike NAMPT overexpression (Fig. 1E), CD38 activity inhibition only moderately promotes cell survival under H2O2 treatment (Fig. 2C) suggesting that, in addition to more NAD+ producing, other factors activated by NAMPT overexpression may be required for efficiently keeping cells alive under oxidative stress. The cellular NAD+ levels under different experiment conditions were tested by a NAD+ / NADH

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kit (abbkine), as expected, the results showed that H2O2 treatment significantly reduced the

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NAD+ level while NAMPT overexpression did not rescue the NAD+ level under H2O2 treatment

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(Fig. 2H).

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3.3 Mitochondrial content and NRF2 expression are regulated by NAMPT under oxidative

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stress

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Considering that mitochondrial NAD+ pool was preserved from genotoxic stress induced depletion [3] in NAMPT overexpression cells and NAMPT-NMNAT3 may not be the key

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pathway for mitochondrial NAD+ pool maintenance [4,5]; we speculated that mitochondrial

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content and integrity may be affected by intensive genotoxic stress and NAMPT overexpression may preserve mitochondrial content and integrity under stress conditions thus preserve mitochondrial NAD+ pool via a SIRT1 independent pathway. To test this hypothesis, both control and NAMPT overexpression HEK293 cells were treated with 1 mM H2O2 for 3 h then the mitochondrial DNA was measured by qPCR, the mitochondria was measured by Mito Tracker staining and the mitochondrial membrane potential was measured by JC-1 staining. The results showed that mitochondrial DNA level was significantly decreased in response to H2O2 treatment and NAMPT overexpression maintained mitochondrial DNA level (Fig. 3A). Consistent with the results of mitochondrial DNA, Mito Tracker staining revealed that the

Journal Pre-proof mitochondrial content was decreased by H2O2 treatment and NAMPT overexpression maintained mitochondrial content under oxidative stress (Fig. 3B). The mitochondrial membrane potential was also impaired by H2O2 treatment (Fig. 3C). These results suggest oxidative stress impairs mitochondria while its content maintenance by NAMPT overexpression may be a key step to preserve its NAD+ pool and subsequently to dictate cell survival. As a key down-stream effector of NAMPT, SIRT1 has been reported to promote mitochondrial

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biogenesis and improve mitochondrial function [26]; however previous study demonstrated that

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SIRT1 is not the key mediator of NAMPT to maintain mitochondrial NAD+ pool and

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subsequently to dictate cell survival under genotoxic stress condition [3]. Therefore, another

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factor with similar function to SIRT1 might be a significant candidate to mediate NAMPT

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effects on mitochondria and cell survival under stress conditions. Both SIRT1 and NRF2 are master regulators of cellular anti-oxidative response; and similar to SIRT1, NRF2 plays a key

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role in promoting mitochondrial biogenesis and regulating mitochondrial function [15-17]; more

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importantly, NRF2 function is relative independent on SIRT1 [13]. Based on these facts, we speculate that NRF2 might be a key factor that mediates NAMPT functions. To test this

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speculation, whether NRF2 expression is regulated by NAMPT was investigated. Firstly, both control and NAMPT overexpression cells were treated by H2O2 with different concentrations (01 mM), then NRF2 mRNA was determined by qPCR. The results showed that NRF2 was downregulated by NAMPT overexpression without H2O2 treatment, whereas, under the high concentration H2 O2 treatment, NRF2 expression was significantly increased in NAMPT overexpression cells (Fig. 3D), indicating NRF2 was upregulated by NAMPT under oxidative stress condition. Secondly, NAMPT overexpression cells were treated by 1 mM H2O2 with either NAMPT enzymatic inhibitor FK866 or SIRT1 enzymatic inhibitor EX-527. The results

Journal Pre-proof showed that, upon H2O2 treatment, NAMPT overexpression upregulated NRF2 expression and this upregulation was blocked by NAMPT inhibitor FK866, whereas SIRT1 inhibitor has no effect on NRF2 upregulation induced by NAMPT overexpression (Fig. 3E), indicating that under oxidative stress NRF2 is a target of NAMPT and NAMPT enzymatic activity is necessary for NRF2 upregulation. While NRF2 upregulation caused by NAMPT overexpression under oxidative stress is independent on SIRT1 activity. NRF2 protein levels in NAMPT

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overexpression cells are consistent with its mRNA level results. Without oxidative stress,

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NAMPT overexpression decreased NRF2 expression, whereas with 1 mM H2O2 treatment

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NRF2 expression was increased by NAMPT overexpression (Fig. 3F). SIRT1 inhibition did not

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affect NRF2 upregulation by NAMPT overexpression (Fig. 3F). These results indicate that NRF2

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is a down-stream target of NAMPT in a SIRT1 independent manner. 3.4 NRF2 mediated the effects of NAMPT on mitochondrial content and membrane potential

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To determine whether NRF2 is the key mediator of NAMPT overexpression to dictate cell

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survival under oxidative stress, NRF2 stable overexpression and NAMPT stable overexpression

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HEK293 cells were treated by 1mM H2O2 with or without CD38 inhibitor 78c for 24 h, then cell alive ratios were calculated. The results showed that NRF2 overexpression significantly increased the cell alive ratio and the effect of NRF2 overexpression to promote cell survival is more profound than that of CD38 inhibition (Fig. 4A). Notably, the cell survival ratio of combining NRF2 overexpression and CD38 inhibition is comparable to the cell survival ratio of NAMPT overexpression (Fig. 4A). These results indicate that NRF2 is the major mediator of NAMPT overexpression to dictate cell survival under oxidative stress while CD38 suppression moderately contributes to cell survival mediated by NAMPT overexpression.

Journal Pre-proof Similar to SIRT1, NRF2 plays important roles in promoting mitochondrial biogenesis and regulating mitochondrial function [15-17]. To test whether NRF2 is the down-stream target of NAMPT to maintain mitochondrial content and membrane potential, which is indicative of mitochondrial integrity, NAMPT and NRF2 overexpression cells were treated by H2O2 with or without NRF2 knockdown. Then mitochondrial content was measured by Mito Tracker staining, mitochondrial DNA was measured by qPCR and mitochondrial membrane potential was

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indicated by JC-1 staining. As expected, under oxidative stress, NRF2 overexpression maintain

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mitochondrial content as NAMPT overexpression did (Fig. 4B). While NRF2 knockdown in

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NAMPT overexpression cells blocked the mitochondrial maintenance effect (Fig. 4B) suggesting

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that, under oxidative stress, mitochondrial content maintenance by NAMPT overexpression depends on NRF2. The results of mitochondrial DNA measurement are consistent with the

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results of Mito Tracker staining. Under oxidative stress, both NAMPT overexpression and NRF2

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overexpression are able to maintain mitochondrial DNA level while NRF2 knockdown in Nampt overexpression cells blocked the ability of NAMPT to maintain mitochondria level (Fig. 4C).

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Notably, it has been suggested that NAMPT H427A is an enzymatic inactivation mutant of

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NAMPT [27], overexpression of NAMPT enzymatic inactivation mutant failed to maintain mitochondrial DNA level under oxidative stress (Fig. 4C). Previous experiment showed that H2O2 treatment decreased mitochondrial membrane potential (Fig. 3C), while both NAMPT and NRF2 overexpression increased mitochondrial membrane potential in response to H2O2 treatment (Fig. 4D) and NRF2 knockdown in NAMPT overexpression cells blocked the ability of NAMPT to increase mitochondrial membrane potential (Fig. 4D). Taken together, these results indicate that NRF2 is a functional down-stream target of NAMPT, which mediates cell survival by promoting mitochondrial maintenance under oxidative stress.

Journal Pre-proof 3.5 Both AMPKα and PPARα are required for NRF2 mediated mitochondrial maintenance and cell survival under oxidative stress AMP-activated protein kinase (AMPK) [28], peroxisome proliferator-activated receptor α (PPARα) [29,30] and peroxisome proliferator-activated receptor-γ coactivator (PGC-1α) [31-33] are three key factors that regulate mitochondrial biogenesis, homoeostasis and function. To explore whether they are required for NRF2 mediated mitochondrial maintenance under

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oxidative stress, their expression level in response to NAMPT and NRF2 overexpression was

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tested and their function upon NRF2 overexpression was also investigated. The western blotting

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experiments results showed that the protein levels of AMPKα, PGC-1α and PPARα are all

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decreased upon 6 h 1mM H2O2 treatment (Fig. 4E), while NAMPT overexpression maintained

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their protein levels under oxidative stress (Fig. 4E), suggesting the three genes are all possibly involved in NAMPT overexpression mediated mitochondrial maintenance under H2O2 treatment.

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The NAMPT knockdown experiments confirmed that the mRNA levels of all these three genes were downregulated in NAMPT knockdown cells under H2O2 treatment (Fig. 5A, C, E).

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However, only the mRNA levels of PPARα and AMPKα were downregulated in NRF2

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knockdown cells (Fig. 5A, E), unlike in NAMPT knockdown cells, PGC-1α mRNA level was not changed in response to NRF2 knockdown (Fig. 5C). Under H2O2 treatment, the mRNA levels of all three genes were upregulated by NAMPT overexpression (Fig. 5B, D, F), this result is consistent with the western blotting results (Fig. 4E). However, NRF2 knockdown in NAMPT overexpression cells only blocked the upregulation of PPARα and AMPKα (Fig. 5B, F) and the upregulation of PGC-1α by NAMPT overexpression was not blocked by NRF2 knockdown (Fig. 5D). NAMPT H427A is a enzymatic inactivation mutant of NAMPT [27]. The NAMPT enzymatic inactivation mutant overexpression failed to upregulate these three genes (Fig. 5B, D,

Journal Pre-proof F). These results suggest all of these three genes are regulated by NAMPT, while only PPARα and AMPKα are down-stream targets of NRF2. Under 3 h 1 mM H2O2 treatment, the mitochondrial DNA level was upregulated by NRF2 overexpression, while either PPARα knockdown or AMPKα knockdown significantly compromised the ability of NRF2 overexpression to upregulated mitochondrial DNA level (Fig. 6A). The Mito tracker staining and JC-1 staining experiments also showed that the mitochondrial content and membrane potential

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maintenance by NRF2 overexpression was greatly weakened upon PPARα knockdown or

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AMPKα knockdown (Fig. 6B).

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Taken together, these results demonstrate that PPARα and AMPKα are down-stream effectors of

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NRF2 and they are required for NRF2 mediated mitochondrial maintenance under oxidative

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stress.

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4. Discussion

Under intensive cellular stress including H2O2 induced oxidative stress [19], hyperactivation of

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NAD+ consuming enzymes is thought to be a key mechanism leading to cell death due to cellular

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NAD+ pool depletion [18]. While overexpression of the rate-limiting enzyme in NAD+ synthetic pathway, NAMPT, significantly prevented cell death from genotoxic stress [3], leading to an intuitive impression that cell death might be prevented by replenishment of the depleting NAD+ pool. However, under intensive genotoxic stress, NAMPT overexpression is not able to prevent nuclear and cytoplasm NAD+ pool from depletion [3] implying that there is an alternative mechanism for NAMPT to mediate cell survival. Although mitochondrial NAD+ pool was prevented from depletion by NAMPT overexpression [3], other studies suggested NAMPTNMNAT3 NAD+ synthetic pathway may be dispensable for mitochondrial NAD+ producing [4,5], which excludes the possibility that NAMPT overexpression directly replenish the mitochondrial

Journal Pre-proof NAD+ pool under stress conditions. In the present study we found that H2O2 induced oxidative stress impaired mitochondrial content (Fig. 3A, B) and mitochondrial membrane potential (Fig. 3C), while NAMPT overexpression under oxidative stress is able to counteract the effects of oxidative stress on mitochondria (Fig. 3A, B and Fig. 4D). These results provide a new alternative insight that under oxidative stress NAMPT overexpression preserved mitochondrial NAD+ pool and subsequently dictated cell survival by protecting mitochondrial content and

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integrity instead of replenishing mitochondrial NAD+ directly.

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SIRT1 mediates many functions of NAMPT/NMN such as insulin secretion and metabolism

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regulations [11]. However, under genotoxic stress, SIRT1 inhibition by EX-527 did not greatly

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compromise the increased cell survival by NAMPT overexpression [3], implying another SIRT1

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independent NAMPT down-stream effector mediated the mitochondria protection. In this study, we identified NRF2 as a previously unknown target of NAMPT, which mediates the major anti-

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apoptosis function (Fig. 4A) and mitochondria protection (Fig. 4B, C, D) of NAMPT in a SIRT1

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independent manner (Fig. 3E, F). Nevertheless, SIRT1-p53-CD38 pathway still slightly but significantly contributes to the anti-apoptosis function of NAMPT (Fig. 2A, C); SIRT1-p53-

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CD38 pathway working together with NRF2-PPARα/AMPKα pathway (Fig. 6A, B) recapitulates full function of NAMPT overexpression to promote cell survival under oxidative stress (Fig. 4A and Fig. 6C). 5. Conclusion NRF2 is a novel down-stream target of NAMPT, which mediates the major anti-apoptosis function of NAMPT by protecting mitochondria. Acknowledgements

Journal Pre-proof This work was supported by National Key Research and Development Program of China (Project No. 2018YFD0500401-03), Natural Science Foundation of China (31572382), and the Fundamental Research Funds for the Central Universities (No. 2662018PY020 and No. 2662017JC034). Author contribution

Conflicts of Interest

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We declare that there is no conflict of interest.

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An Yu, Ronghua Zhou and Xiaodong Chen designed the project. An Yu, Ronghua Zhou and Benzeng Xia performed all the experiments. Weiwei Dang and Zaiqing Yang gave suggestions. Weiwei Dang, Zaiqing Yang and Xiaodong Chen provided money and conditions for all experiments.

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Supplementary data

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Supplementary tables

Supplemental Figure 1: Knockdown efficiencies. A. The knockdown efficiencies of NAMPT and

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human CD38 promoter.

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NRF2. B. The knockdown efficiencies of AMPKα and PPARα. C. Putative p53 binding site on

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Figure 1. NAMPT overexpression promotes cell survival under oxidative stress. (Ctrl: Control; OE: overexpression; Data represent mean ± SD, n = 3, **p < 0.01, *p<0.05). A. The apoptosis of HEK293 cells were determined by Annexin V/PI apoptosis staining kit after

Journal Pre-proof 3 h 1 mM H2O2 treatment. Enhanced fluorescent signal is indicative of apoptosis. B. The mRNA level of NAMPT stable overexpression (Data represent mean ± SD, n = 3, **p < 0.01). C. The protein level of NAMPT stable overexpression. D. NAMPT overexpression suppressed H2O2 induced apoptosis in HEK293 cells. FK866: NAMPT inhibitor 100 nM. Fluorescent signals are obtained by Annexin V/PI apoptosis staining after 3 h 1 mM H2O2 treatment. E. Cell alive ratios of HEK293 cells after 24 h 1 mM H2O2 treatment. FK866 100 nM. (Data represent mean ± SD,

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n = 3, **p < 0.01). F. Cell alive ratios of IMR-90 cells after 24 h 1 mM H2 O 2 treatment. NMN

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200 μM.

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Figure 2. SIRT1-p53-CD38 pathway moderately but significantly contributes to NAMPT

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mediated cell survival under oxidative stress. (Ctrl: Control; OE: overexpression; EX-527: SIRT1 inhibitor 15 μM, 78c: CD38 inhibitor 100 nM; Data represent mean ± SD, n = 3, **p <

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0.01, *p<0.05). A. NAMPT overexpression suppressed H2O2 induced CD38 upregulation in a

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SIRT1 dependent manner. B. NAMPT overexpression suppressed H2O2 induced PARP-1 upregulation. C. Cell alive ratios under oxidative stress and CD38 enzymatic inhibition. D. CD38

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mRNA level in response to NAMPT and p53 overexpression. E. p53 mRNA level was downregulated by NAMPT overexpression in a SIRT1 dependent manner. F. p53 protein level was downregulated by NAMPT under oxidative stress.

Figure 3. NRF2 expression is regulated by NAMPT under oxidative stress. (Ctrl: Control; OE: overexpression; EX-527: SIRT1 inhibitor 15 μM; FK866: NAMPT inhibitor 100 nM; Data represent mean ± SD, n = 3, **p < 0.01, *p<0.05). A. NAMPT overexpression abolishes H2O2 induced mitochondrial DNA decrease. B. Mitochondrial content was maintained by NAMPT

Journal Pre-proof overexpression under oxidative stress. C. Mitochondrial membrane potential was decreased in response to H2O2 treatment. D. NRF2 expression was regulated by NAMPT in response to different H2O2 concentration. E. Under oxidative stress, NRF2 mRNA was upregulated by NAMPT in a SIRT1 independent manner. F. Under oxidative stress, NRF2 protein was upregulated by NAMPT in a SIRT1 independent manner.

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Figure 4. Mitochondrial content and membrane potential were maintained by Nrf2 under

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oxidative stress. (Ctrl: Control; OE: overexpression; KD: knockdown; Data represent mean ±

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SD, n = 3, **p < 0.01, *p<0.05). A. Nrf2 overexpression promotes HEK293 cell survival in

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response to 24 h 1 mM H2O2 treatment. B. NAMPT maintains mitochondrial content under oxidative stress via NRF2. C. Under oxidative stress, mitochondrial DNA content was restored

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by NAMPT overexpression through NRF2. D. Under oxidative stress, NAMPT overexpression

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restored mitochondrial membrane potential by NRF2. E. NAMPT overexpression restored

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AMPKα, PPARα and PGC-1α protein level in response to 6h 1 mM H2O2 treatment.

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Figure 5. AMPKα and PPARα are down-stream targets of NRF2. (KD: knockdown; Data represent mean ± SD, n = 3, **p < 0.01, *p<0.05). A. Under oxidative stress, PPARα mRNA was down regulated by NAMPT and NRF2 knockdown. B. Under oxidative stress, NRF2 is required for PPARα mRNA upregulation induced by NAMPT overexpression. C. Under oxidative stress, PGC-1α mRNA was downregulated by NAMPT knockdown while NRF2 knockdown does not affect its expression. D. NRF2 overexpression or knockdown does not affect PGC-1α expression under oxidative stress. E. Under oxidative stress, AMPKα mRNA was down regulated by NAMPT and NRF2 knockdown. F. Under oxidative stress, NRF2 is required

Journal Pre-proof for NAMPT mediated AMPKα mRNA upregulation.

Figure 6. Under oxidative stress, mitochondrial maintenance by NAMPT overexpression requires both AMPKα and PPARα. (OE: overexpression; KD: knockdown; Data represent mean ± SD, n = 3, **p < 0.01, *p<0.05). A. Under oxidative stress, both AMPKα and PPARα are required for NAMPT mediated mitochondrial DNA level maintenance. B. Both AMPKα and PPARα are

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necessary for NAMPT to maintain mitochondrial content and membrane potential. C. Working

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model for NRF2 and CD38 in NAMPT mediated cell survival under oxidative stress.

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Highlights

NAMPT suppresses CD38 expression via SIRT1 and p53

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NRF2 is a novel down-stream target of NAMPT

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NRF2 protects mitochondria under oxidative stress

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Figure 1

Figure 2

Figure 3

Figure 4

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Figure 6