Nicotinamide Riboside

Journal of Photochemistry & Photobiology, B: Biology

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Journal of Photochemistry & Photobiology, B: Biology 221 (2021) 112238
Restoring NAD+ by NAMPT is essential for the SIRT1/p53-mediated Image survival of UVA- and UVB-irradiated epidermal keratinocytes
Takeshi Katayoshi *, Takahisa Nakajo , Kentaro Tsuji-Naito
DHC Corporation Laboratories, Division 2, 2-42 Hamada, Mihama-ku, Chiba 261-0025, Japan


Nicotinamide phosphoribosyltransferase Nicotinamide adenine dinucleotide Ultraviolet irradiation
Poly(ADP-ribose) polymerase
Normal human epidermal keratinocytes


Nicotinamide adenine dinucleotide (NAD+) is a crucial coenzyme in energy production. The imbalance of NAD+
synthesis has been found to trigger age-related diseases, such as metabolic disorders, cancer, and neurodegen- erative diseases. Also, UV irradiation induces NAD+ depletion in the skin. In mammals, nicotinamide phos- phoribosyltransferase (NAMPT) is the rate-limiting enzyme in the NAD+ salvage pathway and essential for NAD+
homeostasis. However, but few studies have focused on the role of NAMPT in response to UV irradiation. Here, we show that NAMPT prevents NAD+ depletion in epidermal keratinocytes to protect against the mild-dose UVA
and UVB (UVA/B)-induced proliferation defects. We showed that poly(ADP-ribose) polymerase (PARP) inhibitor rescued the NAD+ depletion in UVA/B-irradiated human keratinocytes, confirming that PAPR transiently ex-hausts cellular NAD+ to repair DNA damage. Notably, the treatment with a NAMPT inhibitor exacerbated the UVA/B-induced loss of energy production and cell viability. Moreover, the NAMPT inhibitor abrogated the sirtuin-1 (SIRT1)-mediated deacetylation of p53 and significantly inhibited the proliferation of UVA/B-irradiatedcells, suggesting that the NAMPT-NAD+-SIRT1 axis regulates p53 functions upon UVA/B stress.
The supplementation with NAD+ intermediates, nicotinamide mononucleotide and nicotinamide riboside, rescued the UVA/B-induced phenotypes in the absence of NAMPT activity. Therefore, NAD+ homeostasis is likely essential
for the protection of keratinocytes from UV stress in mild doses. Since the skin is continuously exposed to UVA/B irradiation, understanding the protective role of NAMPT in UV stress will help prevent and treat skin photoaging.

1. Introduction
Nicotinamide adenine dinucleotide (NAD+) is an essential coenzyme
in energy production. Many studies have observed that NAD+ levels decline with age in tissues and organs, such as heart, lung, liver, kidney,
spleen, skeletal muscle, brain, blood, and skin [1–5], likely due to the imbalance between NAD+ synthesis and degradation during aging, thus
trigger age-related diseases, including metabolic disorders, cancer, and neurodegenerative diseases. In addition to chronological aging, UV
irradiation triggers NAD+ depletion in the epidermis [6].

In mammals, NAD+ is generated via three routes, the de novopathway from tryptophan, the Preiss–Handler pathway from nicotinic acid (NA), and the salvage pathway from nicotinamide (NAM). Thesalvage pathway is considered the primary pathway for maintainingcellular NAD+ levels [7]. The NAD+ precursor, NAM, restores epidermal cells from UV-induced cellular energy loss, DNA damage, and immu-
nosuppression [8–10], which is presumably attributed to NAD+ regain. In the salvage pathway, NAM phosphoribosyltransferase (NAMPT) is the
rate-limiting enzyme that catalyzes the critical first step of NAD+ syn- thesis from NAM. The downregulation of NAMPT expression during
aging reduces intracellular NAD+ levels, leading to cellular senescence [11]. The specific inhibition of NAMPT by FK866 results in similar
cellular senescence phenotypes in rat bone marrow mesenchymal stem cells and human fibroblasts [12,13]. Therefore, considerable attention
has been focused on molecules that help boost NAD+ levels; for example,
nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR)

Abbreviations: 3-AB, 3-aminobenzamide; ADPr, ADP-ribose; DAPI, 4′-6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HRP, horseradish peroxidase; LDH, lactate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NA, nicotinic acid; NAM, nicotinamide;
NAD+, nicotinamide adenine dinucleotide; NAMPT, nicotinamide phosphoribosyltransferase; NHEKs, normal human epidermal keratinocytes; NMN, nicotinamide
mononucleotide; NR, nicotinamide riboside; PARP, poly ADP-ribose polymerase; PBS, phosphate-buffered saline; qPCR, quantitative PCR; SEM, standard error of the mean; SDS, sodium dodecyl sulfate; SIRT, sirtuin; TBS-T, Tris-buffered saline with 0.1% Tween-20; XPA, xeroderma pigmentosum group A.

* Corresponding author.
E-mail address: [email protected] (T. Katayoshi).

Received 4 January 2021; Received in revised form 6 May 2021; Accepted 1 June 2021
Available online 12 June 2021
1011-1344/© 2021 Elsevier B.V. All rights reservedare converted to NAD+ through a NAMPT independent pathway. Animal studies have shown that NMN and NR have beneficial effects in multiple age-related degenerative diseases [14]. In addition, several human studies have demonstrated the safety and efficacy of the orally admin- istered NMN and NR [15–17]. However, the effects of these agents on skin tissue have not yet been examined.
NAD+ acts as a substrate for a wide range of NAD+-consuming en-
zymes, such as poly ADP-ribose (ADPr) polymerase (PARP), sirtuin (SIRT), and cyclic (ADPr) synthases (e.g., CD38 and CD157). In response to genotoxic stresses such as UV irradiation, activated-PARP cleaves
NAD+ into NAM and ADPr to catalyze the poly(ADP-ribosyl)ation of
specific acceptor proteins and PARP itself, recruiting repair factors to DNA damage sites. PARP activation presumably triggers NAD+exhaustion to lower activities of other NAD+-consuming enzymes. Fang et al.

showed that the mitochondrial abnormalities in xeroderma pig-
mentosum group A (XPA), a DNA repair disorder, is caused by SIRT1 inactivation due to excessive activation of PARP1 [18]. PARP1 inhibi-
tion and interventions using NAD+-boosting molecules restored SIRT1
function and mitochondrial activities in XPA deficient cells, indicating
that the aberrant activation of PARP1 caused by defective DNA repair triggers NAD+ consumption to lower SIRT1 activity [18]. SIRT1 belongs to the NAD+-dependent deacetylase family and regulates critical cellularprocesses, such as aging, metabolism, stress response, and tumorigen- esis. SIRT1 has also been implicated in DNA repair through deacetyla- tion of various DNA repair-related proteins, including Ku70, Werner syndrome protein, Nijmegen breakage syndrome 1, and XPA [19–22].

SIRT1 also catalyzes the deacetylation of p53 [23,24]. The tumor suppressor p53 is activated and stabilized through its post-translational modification, such as phosphorylation and acetylation, leading to the transcriptional activation of target genes [25]. In the skin, p53 is acti- vated in response to UV irradiation to suppress cell cycle progression, allowing time for the cell to repair DNA damage [26]. While p53 plays an essential role in maintaining a cell’s proliferative capacity, it induces apoptosis if the level of cellular damage reaches a critical threshold. Ming et al. reported that keratinocyte-specific conditional Sirt1 knockout mice exhibit increased sensitivity to UV damage, concomitant with the accumulation of acetylated p53 [27], implicating that SIRT1 negatively regulates p53 activity to prevent epidermis from UV stress.
NAMPT is an essential enzyme in maintaining NAD+ homeostasis foNAD+-consuming enzymes, such as PARP and SIRT. It remains unclear, however, how NAMPT affects NAD+ levels in skin cells upon UV irra- diation. Thisreport showed that NAMPT restores cellular NAD+ levels in
normal human epidermal keratinocytes (NHEKs) after mild-dose UVA and UVB (UVA/B) irradiation to promote cell survival. In addition, we uncovered the mechanism underlying the protective effects of NAMPT on cell survival upon UVA/B irradiation, i.e., the suppression of p53- mediated proliferation defects through SIRT1 activation. Our findingsindicate that NAMPT is a master regulator of intracellular NAD+ under
UV stress.

2. Materials and Methods
2.1. Reagents and Antibodies
FK866 (AdooQ Bioscience, Irvine, CA, USA), 3-aminobenzamide (3- AB) (Tokyo Chemical Industry, Tokyo, Japan), actinomycin D (Adip- oGen Life Sciences, Liestal, Switzerland), EX527 and anti-β-actin (ab8226) and anti-Ki67 (ab16667) antibodies (Abcam, Cambridge, MA, USA), DAPI solution (Dojindo Laboratories, Kumamoto, Japan), and NMN (Oriental Yeast, Tokyo, Japan) were purchased. In addition, NR (Carbosynth, Compton, UK), anti-poly/mono-ADPr (#83732), anti- NAMPT (#86634), anti-cleaved caspase-3 (#9664S), anti-caspase-3 (#9662), anti-PARP (#9532S), anti-acetyl p53 (#2525), and horse- radish peroxidase (HRP)-conjugated anti-rabbit IgG antibodies (Cell Signaling Technology, Danvers, MA, USA) were procured. Also, the HRP-conjugated anti-mouse antibody (GE Healthcare, Chicago, IL, USA)
and Alexa Fluor 488- and 594-labeled anti-rabbit IgG antibodies (Thermo Fisher Scientific, Waltham, MA, USA) were obtained.

2.2. Cell Cultures
Neonatal foreskin-derived NHEKs were purchased from Thermo Fisher Scientific (#C0015C). The cells were maintained in the EpiLife™ medium containing 60 μM calcium chloride (Thermo Fisher Scientific) and Human Keratinocyte Growth Supplement (Thermo Fisher Scientific)
in a humidified incubator at 37 ◦C and 5% CO2. The medium was
changed every day, and the cells were passaged when they reached 80% confluency. The cells were used at 70–80% confluence and not used beyond passage
2.3. UV Irradiation
The semi-confluent NHEKs in calcium and phenol red-free EpiLife™ medium (Thermo Fisher Scientific) were irradiated once with UVA/B (280–400 nm; emission peak 361 nm; UVA: 0, 1, 2, and 4 J/cm2 and
UVB: 0, 13, 27, and 54 mJ/cm2) at room temperature using a Solar
Simulator (Wacom, Saitama, Japan) equipped with xenon lamp. Doses of UVA/B irradiation were calibrated with the MS-211-I UV monitor
(EKO, Tokyo, Japan) and the average UVA/B intensities for all mea- surements were 16.5 mW/cm2 and 0.22 mW/cm2, respectively. After irradiation, the cells were incubated in a maintenance medium for the
indicated time.

2.4. NAD+ Quantification
Cellular NAD+ levels were quantified using a NAD+/NADH Assay Kit-WST (Dojindo Laboratories). Briefly, 1.0 105 NHEKs were seeded in 12-well plates. After 24 h, the cells were irradiated with UVA (0, 1, 2,
and 4 J/cm2) and UVB (0, 13, 27, and 54 mJ/cm2) and cultured for 1–8
h. The cells were lysed with the NAD+/NADH Extraction Buffer and ultra-filtrated using a Nanosep 3 K centrifugal filtration device (Pall
Corporation, Washington, NY, USA). The filtrates were subjected to the enzymatic reaction at 37 ◦C for 1 h. Then, the samples were measured with the absorbance at 450 nm using an ARVO Series Multilabel Counter
(Perkin-Elmer, Waltham, MA, USA). The total NAD+/NADH levels were
normalized to their respective protein concentrations determined by the
Pierce BCA protein assay (Thermo Fisher Scientific). For the PARP in- hibition, the cells were pretreated with a PARP inhibitor 3-AB at 2.5 mM for 30 min, then irradiated with combined UVA/B (4 J/cm2 and 54 mJ/
cm2, respectively). After incubation for 1 h, the cellular NAD+ levels
were quantified.

2.5. Immunoblotting
The NHEKs were lysed with a lysis buffer containing 150 mM NaCl, 50 mM Tris pH 8.0, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), a phosphatase inhibitor cocktail (Thermo Fisher Scientific), and a protease inhibitor cocktail (Hoffmann-La Roche, Basel, Switzerland). The proteins in each lysate were separated by SDS- polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes using a semi-dry blotting apparatus (Bio-Rad Lab- oratories, Hercules, CA, USA). After the transfer, membranes were blocked in 5% dried skimmed milk in Tris-buffered saline with 0.1% Tween-20 (TBS-T) for 1 h to reduce nonspecific binding. Then, the
membranes were incubated with primary antibodies overnight at 4 ◦C,
washed, and incubated with HRP-conjugated secondary antibodies for 1
h. The proteins were detected using the ECL Prime Western Blotting Detection Reagent (GE Healthcare).

2.6. Immunochemistry
The NHEKs were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min, washed thrice with PBS, and per- meabilized using 0.1% Triton X-100 in PBS for 20 min. After three PBS washes, cells were blocked with 5% skim milk in TBS-T, washed with TBS-T, and stained for ADPr and Ki67 with 1: 200 diluted anti-poly/ mono-ADPr antibody and 1:800 diluted anti-Ki67 antibody, respec-
tively, overnight at 4 ◦C. Next, the cells were washed thrice with TBS-T
and incubated with 1:400 diluted Alexa Fluor 488- or 594-labeled anti- rabbit IgG antibodies for 1 h. For nuclear counterstaining, cells were treated with a 1:400 diluted 4′-6-diamidino-2-phenylindole (DAPI) so-
lution for 20 min. After washing with TBS-T, they were mounted with Fluoromount/Plus™ (Diagnostic BioSystems, Pleasanton, CA, USA). The fluorescence signal of the cells was imaged using a Leica confocal fluo- rescence microscope (Leica Microsystems Japan, Tokyo, Japan). The percentage of Ki67-positive cells was determined by evaluation of more than 200 cells in randomly selected images from each experimental group.

2.7. NAMPT Activity

The intracellular NAMPT activity of the NHEKs was evaluated with a modified NMN production assay [28]. Briefly, 1.0 106 cells were seeded in a 100-mm dish. After 24 h, cells were irradiated with 4 J/cm2 UVA and 54 mJ/cm2 UVB and cultured for 2–8 h and lysed with the
immunoprecipitation lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 5 mM EDTA, and 1% Triton X-100) containing phosphatase and protease inhibitor cocktails. The cell extracts were incubated with anti- NAMPT monoclonal antibody-coated Dynabeads Protein G (Thermo Fisher Scientific). After the supernatant was removed, and the precipi- tated proteins were washed with PBS and resuspended in lysis buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, and 2 mM DTT). The protein samples were incubated with 150 μM NAM, 300 μM phosphoribosyl
pyrophosphate, and 2 mM ATP at 30 ◦C for 60 min to produce NMN. A
volume of 100 μl of the resultant mixtures was then mixed with 50 μl of 20% acetophenone dissolved in ethanol and 135 μl of 1 M KOH. After incubating on ice for 15 min, 135 μl of 88% formic acid was added to each sample to incubate at 100 ◦C for 5 min. Next, the samples were transferred to a 96-well plate to measure fluorescence (Ex/Em 368/
438 nm) using an Infinite 200 Pro plate reader (Tecan, Ma¨nnedorf, Switzerland).
2.8. Quantitative PCR (qPCR)
Total RNA was isolated from cultured cells using an RNeasy Mini Kit (Qiagen, Mississauga, Canada) and stored in RNase-free water at 80 ◦C. The first-strand cDNA was reverse transcribed with 1 μg of total
RNA using a PrimeScript II 1st strand cDNA Synthesis Kit (Takara Bio, Shiga, Japan). The mRNA expression level of the target genes was measured using an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and the NAMPT (Hs00237184_m1), SIRT1 (Hs01009006_m1), and GAPDH
(Hs02786624_g1) TaqMan gene expression assays. All the reactions were performed in triplicates. GAPDH was used as the housekeeping gene for normalization. The comparative CT method was used to calculate the relative amount of mRNA.

2.9. ATP Quantification
The concentration of ATP in cell lysates were determined using the ATPLite assay (Perkin-Elmer, Japan). The luminescence of the samples was measured using an ARVO Series Multilabel Counter. The cellular ATP levels were normalized to the protein concentrations.2.10. Cell Viability Assay
The NHEKs were seeded in a 96 well plate at 1.0 104 cells per well and incubated for 24 h. The cells were then irradiated with 4 J/cm2 of
UVA and 54 mJ/cm2 of UVB and incubated with or without 5 nM FK866. After 24 h, the cells were incubated with 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) at 1 mg/ml at 37 ◦C for 3 h.
After three washes with PBS, the cells were incubated with 0.1 ml dimethyl sulfoxide at room temperature for 2 h. The absorbance of the cell extracts was measured at 560 nm using an ARVO Series Multilabel Counter.2.11. Cytotoxicity Assay
Cell death was assessed by measuring the release of lactate dehy- drogenase (LDH) by the NHEKs with a Cytotoxicity LDH Assay Kit-WST (Dojindo Laboratories). The absorbance of the samples was measured at 490 nm using an ARVO Series Multilabel Counter. The 100% cell death was determined by rupturing all the cells using the cell lysis buffer provided in the kit.

2.12. Statistical Analyses
The data were presented as mean SEM (standard error of the mean) from at least three independent experiments. The data were analyzed by
one-way ANOVA; these differences among means were analyzed using a Tukey–Kramer test. Statistical significance was set at p < 0.05 and p <
0.01. Asterisks and daggers indicate statistical significance when
compared to the control group (*p < 0.05 and **p < 0.01) and the indicated group (†p < 0.05 and ††p < 0.01).

3. Results

3.1. Intracellular NAD+ Level is Decreased by UVA/B Irradiation
We first performed kinetic analyses of the NAD+ content in UVA/B- irradiated NHEKs to investigate the variance of NAD+ levels in photo- damaged skin cells. The intracellular amounts of NAD+ were markedly
reduced within 1 h after UVA/B irradiation in a dose-dependent manner
(Fig. 1A). The cell viability significantly decreased at 24 h after irradi- ation with 4 J/cm2 UVA and 54 mJ/cm2 UVB, but not with 1 J/cm2 UVA and 13 mJ/cm2 UVB or 2 J/cm2 UVA and 27 mJ/cm2 UVB (Fig. 1B). Since significant NAD+ level and viability reductions were observed after the irradiation, we applied irradiation of 4 J/cm2 UVA and 54 mJ/ cm2 UVB for further experiments. The level of NAD+ was maximally
decreased at 1 h after UVA/B irradiation and gradually restored after- ward, reaching 70–80% of the control levels at 8 h (Fig. 1C). UV irra- diation immediately activates PARP in the epidermis [29]. Therefore, we examined the PARP activation within a short time frame by moni- toring the amounts of ADP-ribosylated proteins. The ADPr- modified proteins were elevated in the NHEKs at 10 min after UVA/B stimulation and continued to decline afterward (Fig. 1D). The immunofluorescent analysis showed that UVA/B irradiation rapidly caused a subnuclear accumulation of ADPr in the NHEKs (Fig. 1E). We employed 3-AB, aPARP inhibitor, to evaluate whether PARP activation caused NAD+
exhaustion. We observed that blocking PARP activity with 3-AB signif-
icantly prevented the decrease of relative NAD+ amount after UVA/B irradiation (Fig. 1F). These results suggest that a large amount of cellular
NAD+ is rapidly consumed by PARP after UVA/B irradiation to repair DNA damage.

3.2. NAMPT Prevents UVA/B-Induced Severe Energy Loss and Proliferation Defects
NAMPT is considered the primary regulator of intracellular NAD+ level in mammals through the NAD+ salvage pathway. Thus, we hy-
pothesized that NAMPT restored the NAD+ depleted by UVA/B irradi-
ation. We applied NAMPT specific inhibitor FK866 to the cells after 4 J/ cm2 UVA and 54 mJ/cm2 UVB irradiation and found that FK866 completely blocked NAD+ recovery at 2–8 h, resulting in severe NAD+ Cellular NAD+ is rapidly consumed by PARP after UVA/B irradiation in NHEKs. A, B. The NHEKs were exposed to UVA/B irradiation at the indicated dosages. The cellular NAD+ levels and cell viability were determined at 1 and 24 h post-irradiation using NAD+/NADH and MTT assays, respectively. C-E. The NHEK cells were irradiated with 4 J/cm2 UVA and 54 mJ/cm2 UVB. C. The cellular NAD+ levels were quantified at the indicated times after irradiation. D. NHEKs were harvested at the indicated times after irradiation. The level of ADP-ribosylated protein was analyzed using immunoblotting with an antibody specific for ADPr.
β-actin was used as the loading control. E. The subcellular distribution of ADP-ribosylated proteins were visualized by staining with an antibody to ADPr (green) and the nuclear counterstain DAPI (blue). Scale bar: 10 μm. F. The cells were pretreated with a PARP inhibitor 3-AB at 2.5 mM for 30 min and then irradiated with 4 J/ cm2 UVA and 54 mJ/cm2 UVB. After incubation for 1 h, the cellular NAD+ levels were quantified with the NAD+ assay. The data were indicated as mean ± SEM (n =3); the means were expressed as the number of fold-change compared to that of the control cells. *p < 0.05 and ** p < 0.01, the comparison to the control. †† p < 0.01,the comparison between the indicated groups. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)deficiency (Fig. 2A). We further tested whether intracellular NAMPT was activated by UVA/B exposure. UVA/B irradiation elevated NAMPT expression in a dose- and time-dependent manner (Fig. 2B, C). In addition, we used an anti-NAMPT monoclonal antibody to immune- precipitate endogenous NAMPT in the NHEKs and measured its
enzyme activity for NMN production. NAMPT activity was increased after 4 J/cm2 UVA and 54 mJ/cm2 UVB irradiation (Fig. 2D). To confirm NAMPT’s effect on ATP production and cell viability upon UVA/Birradiation, we evaluated the energy production capacity and cell viability of UVA/B-irradiated NHEKs in the presence or absence of

FK866. UVA/B irradiation decreased the cellular level of ATP regardless of FK866, but FK866-treated cells had more severe defects in ATP pro- duction (Fig. 2E). Using the MTT assay, we showed that the irradiated cells treated with FK866 exhibited a more profound decline in cell viability than the untreated cells (Fig. 2F). Thus, the intracellular ATP
level and cell viability are likely correlated. These results suggest that NAMPT improves subcellular NAD+-deficient conditions induced by
UVA/B irradiation via the salvage pathway, lowering the effect of en- ergy loss on cell viability.
UV irradiation causes apoptosis in epidermal keratinocytes [30]. We

NAMPT inhibition after UVA/B irradiation causes continuous NAD+ depletion, leading to severe ATP loss and proliferation defects. A. The NHEKs were exposed to 4 J/cm2 UVA and 54 mJ/cm2 UVB irradiation and incubated with or without 5 nM of the NAMPT inhibitor FK866 for the indicated times. The cellular NAD+ levels were quantified with the NAD+ assay. B. The cells were exposed to different UVA/B dosages and harvested 8 h later to assay the NAMPT mRNA levels. C. The level of NAMPT mRNA was quantified by qPCR at the indicated times after 4 J/cm2 UVA and 54 mJ/cm2 UVB irradiation. D. Whole-cell lysates collected at the indicated times after 4 J/cm2 UVA and 54 mJ/cm2 UVB irradiation were immunoprecipitated using an antibody to NAMPT. The activity of intracellular NAMPT was measured by quantifying NMN production. E, F. After 4 J/cm2 UVA and 54 mJ/cm2 UVB irradiation, cells were incubated for 24 h in the presence or absence of FK866. The cells’ ATP levels and viability were determined using an ATP detection kit and MTT assay, respectively. G, H. The cells were treated with 4 J/cm2 UVA and 54 mJ/cm2 UVB irradiation or 10 nM of the potent apoptosis inducer actinomycin D and incubated in the presence or absence of FK866 for 24 h. The levels of cell
toxicity were assessed by the LDH assay. The level of apoptosis was assayed by immunoblotting with antibodies against apoptosis markers, cleaved caspase-3 and PARP. An antibody specific for β-actin was used as the loading control. In the LDH assay, the means were expressed as the percentages of the control, which had 100%cell death. The data were indicated as mean ± SEM (n = 3); the means were expressed as the number of fold-change compared to that of the control cells. *p < 0.05 and ** p < 0.01, the comparison to the control. †† p < 0.01, the comparison between the indicated groups.
measured the amount of extracellular LDH released from dead cells to investigate whether the lowered cell viability (Fig. 2F) was attributed to cell death. There was no difference in LDH release between the control and UVA/B-irradiated cells treated with FK866, while actinomycin D, used as a positive control, significantly elicited LDH release (Fig. 2G). Moreover, apoptosis markers, such as cleaved caspase-3 and PARP, were detected only in actinomycin D-treated cells and not in the UVA/B- irradiated cells (Fig. 2H). In the absence of FK866, although both en- ergy production and cell viability decreased at 24 h after UVA/B irra- diation, they were restored in almost each control level by 48 h after

In contrast, FK866-treated cells exhibited continuous decline until 72 h after UVA/B irradiation (Supplementary Fig. S1A, B). Nevertheless, the significant cell death was not detected until 72 h after UVA/B-irradiation in the presence of FK866 (Supplementary Fig. S1C). These data indicate that the intensity of UVA/B irradiation in our experiment is insufficient to induce apoptosis and that the absence of NAMPT activity in NHEKs enhances UVA/B-induced proliferation ar- rest. These results collectively suggest that NAMPT alleviates mild dose
UVA/B-induced NAD+ depletion and prevents ATP deficiency and pro-
liferation defects.

The NAMPT and SIRT1 inhibitors induce the continuous activation of p53 and proliferation defects after UVA/B irradiation. A-D. The NHEKs were exposed to 4 J/cm2 UVA and 54 mJ/cm2 UVB irradiation. A. The SIRT1 mRNA levels at different time points after UV irradiation were quantified by qPCR. B. After incubation with or without 5 nM of FK866 or 10 μM of SIRT1 inhibitor EX527 for the indicated times, the level of acetylated p53 was analyzed using immunoblotting with an antibody specific for acetyl-p53. β-actin was used as the loading control. C, D. After incubation with or without FK866 or EX527 for 24 h, the rates of cell proliferation were measured by co-immunostaining with an antibody against Ki67 (red) and the nuclear counterstain DAPI (blue). Scale bar: 20 μm. The data were presented as the
mean ± SEM (n = 3). ** p < 0.01, compared to the control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version
of this article.)

3.3. NAMPT Regulates SIRT1-Mediated p53 Deacetylation during UVA/ B Stress
UV and singlet oxygen elevate SIRT1 expression in epidermal kera- tinocytes [31]. Our experiment confirmed the UVA/B irradiation- induced upregulation of SIRT1 expression (Fig. 3A). SIRT1 inhibits cell proliferation arrest and apoptosis by catalyzing the deacetylation of p53
[32]. Hence, we presumed that NAD+ deficiency induced by 4 J/cm2
UVA and 54 mJ/cm2 UVB irradiation could reduce SIRT1 activity in NHEKs, leading to reduced cell viability associated with the continuous activation of p53. We tried to confirm this assumption by evaluating the influences of NAMPT and SIRT1 inhibitors on the deacetylation of p53. The level of acetyl-p53 protein was rapidly increased, peaking at 2–4 h after UVA/B irradiation (Fig. 3B, upper panels). While the level of acetyl-p53 protein decreased in the absence of any inhibitor at 8 h after UVA/B irradiation, the acetylation of p53 was still observed in FK866- or EX527-treated NHEKs (Fig. 3B). These results indicate that the absence of SIRT1 activity affects the deactivation of UVA/B-activated p53, resulting in the accumulation of acetyl-p53. Furthermore, we stained the cells using an intracellular proliferative marker, Ki67, to assess whether SIRT1 activity affected cell proliferation arrest. Both NAMPT and SIRT1 inhibitors significantly reduced the rate of cell proliferation upon UVA/ B irradiation (Fig. 3C, D). These data suggest that NAMPT negatively regulates p53 activity through SIRT1 activation to prevent proliferation defects in keratinocytes after mild-dose UVA/B irradiation.

3.4. NAD+ is Required for Recovery from UVA/B-Induced Proliferation Arrest
The exogenous supplementation of NMN and NR, which bypass the
rate-limiting reaction of NAMPT in the salvage pathway, could boost the intracellular NAD+ levels (Fig. 4A). Since keratinocytes rapidly exhaust
to induce apoptosis in epidermal keratinocytes [30]. Under our experi- mental conditions, apoptotic or cytotoxic markers were not detected in the UVA/B-irradiated NHEKs, indicating that the intensity of UVA/B
irradiation was relatively mild. Nevertheless, UVA/B irradiation decreased the intracellular NAD+ levels by more than 50%, suggesting
that the transient reduction in NAD+ levels in the skin occurs continu- ously in our daily life. Hence, NAMPT plays a vital role in maintaining
NAD+ levels in the epidermis under UV stress.
The deacetylation of activated p53 is crucial for cell survival upon mild genotoxic stress [34]. We found that SIRT1 mediated p53 deace- tylation upon UVA/B stress. We also observed that SIRT1 inhibition resulted in severe proliferation defects after mild-dose UVA/B irradia- tion, suggesting that the persistent activation of p53 shifts the cells to a continuous state of proliferation arrest. The deacetylation of acetyl-p53 was also inhibited by a NAMPT inhibitor, indicating that the restoration
of NAD+ by NAMPT plays an essential role in maintaining SIRT1 activity
and preventing the irreversible proliferation arrest under mild UVA/B stress (Fig. 5).

In contrast to SIRT1, PARP positively regulates p53 [35]. Inhibiting PARP activity decreases the transcription of p53-responsive genes [36,37]. Additionally, Won et al. showed that the poly(ADP-ribosyl) ation of p53 by PARP increased the stability of p53 protein in low- dose UV-irradiated cells [38]. Therefore, SIRT1 and PARP likely
modulate p53 activity antagonistically in the epidermis, highlighting the importance of maintaining adequate NAD+ levels during the response to
UV damage. Massudi et al. showed that PARP activity in human pelvic skin was significantly increased in elderly subjects compared to young adults [4]. Hence, environmental UV exposure may deplete cellular
NAD+ in the elderly more than in younger adults.
To counteract UV-induced oxidative stress, skin cells possess multi- ple antioxidant systems comprising glutathione (GSH), thioredoxin (TRX), and antioxidant enzymes (e.g., catalase (CAT), superoxide dis-much of NAD+ after 4 J/cm2 UVA and 54 mJ/cm2 UVB irradiation and
mutase (SOD), and GSH peroxidase). Recent reports showed thatexhibit proliferation defects after adding a NAMPT inhibitor, we verified
whether supplementing NAD+ intermediates could rescue keratinocytes from the proliferation defects. We treated UVA/B-irradiated NHEKs with
NMN or NR in the presence of FK866. The decrease in cellular NAD+ and ATP levels by FK866 treatment after UVA/B irradiation was suppressed
by the addition of NMN or NR (Fig. 4B, C); the supplementation of these intermediates also inhibited the accumulation of acetyl-p53 protein in the same cells (Fig. 4D). Furthermore, NMM or NR supplementation significantly preserved the number of Ki67-positive cells compared to the cell without the NMM or NR supplement. (Fig. 4E, F). These results
suggest that intracellular NAD+ is required to protect keratinocytes from
UV stress by preventing excessive p53-mediated proliferation arrest.

4. Discussion
Many studies have shown that NAD+ levels decline in various tissues
and organs during chronological aging, including skin [1–5]. Here, we investigated how the epidermal NAD+ level was maintained in response to UV damage. We showed that the intracellular NAD+ level in the
epidermal keratinocytes was rapidly reduced by UVA/B irradiation but
gradually restored afterward. Also, we found that the inhibition of NAMPT abolished the restoration of NAD+ levels. In addition, the
decline in cellular ATP levels and cell viability by UVA/B exposure was exacerbated by the NAMPT inhibitor FK866. Thus, these data suggest
that NAMPT prevents ATP loss via the NAD+ salvage pathway to protect
cells from UV stress.

NAD+ is an essential substrate for enzymes, such as SIRT and PARP. A previous study has shown that hydrogen peroxide activates PARP to
repair DNA damage in normal human lung fibroblasts, leading to the transient depletion of cellular NAD+ [33]. Here, we also showed that UVA/B irradiation reduced NAD+ levels rapidly through PAPR activa-
tion. These data suggest that regardless of cell types, genotoxic stimuli could decrease cellular NAD+ levels transiently. UV irradiation is known
NAMPT inhibition downregulated antioxidant proteins, resulting in in- crease in the susceptibility to oxidative stress [39,40]. Furthermore, Hong et al. showed that colorectal cancer cells protect themselvesagainst detrimental oxidative stress through upregulation of NAMPT expression that increases in the NAD+ pool [41]. Thus, NAMPT isconsidered a key cellular capacity driver that tolerates oxidative stress. Until now, two possible mechanisms have been postulated to explain therole of NAMPT in cellular antioxidant systems: NAMPT upregulates the cellular NAD+ level, (i) providing NADPH essential for antioxidantsystems, such as GSH [13], and (ii) enhancing the gene expression of antioxidant enzymes, including SOD2 and CAT, through the NAD+-dependent activation of SIRT1 [42]. We showed that NAMPT inhibition exacerbated cellular damage by UVA/B irradiation, which may be at
least partially due to reduced cellular antioxidant capacity by NAD+

The medical importance of NAM, a NAD+ precursor, has been established in the late 1990s by the discovery of pellagra being caused
by the deficiency of niacin, including NAM and NA. Since pellagra’s typical symptom is photosensitive dermatitis, many studies have focused on NAM’s protective effects against skin photodamage [8–10].
Sincemost NAD+ synthesis in mammals depends on the NAM salvage
pathway, dietary NAM is commonly supplemented to replete NAD+ levels. However, NAM may not be available for NAD+ maintenance in
adults because the level of NAMPT decreases with age [11]. Moreover, NAM inhibits enzymes that require NAD+, such as SIRT, PARP, and
CD38, and is suspected to influence long-term cell survival [43]. NMN and NR are also NAD+ precursors, but their conversion to NAD+ is in-
dependent of catalysis by NAMPT. Few studies have evaluated the ef- ficacy of NMN and NR in the skin. In the present study, we showed that, in the absence of NAMPT activity, NMN and NR could maintain cellular
NAD+ levels as NAD+-boosting agents and prevent the keratinocytes
from UVA/B-induced proliferation defects. Our results suggest that these intermediates can effectively prevent photodamage and age-related

NAD+ intermediates, NMN and NR, rescue the UV-damaged cells in the absence of NAMPT activity. A. The salvage pathway of NAD+ biosynthesis. B-F. The NHEKs were exposed to 4 J/cm2 UVA and 54 mJ/cm2 UVB irradiation and incubated for 8 (B, D) or 24 h (C, E, and F) in the presence or absence of 5 nM of FK866, 100 μM of NMN, or 50 μM of NR. B, C. The cellular NAD+ and ATP levels were quantified using the NAD+/NADH assay and ATP detection kit, respectively. The means were expressed as the number of fold-change compared to the control cells. D. The level of acetylated p53 was analyzed using immunoblotting with anantibody specific for acetyl-p53. β-actin was used as the loading control. E, F. The rate of cell proliferation was measured by co-immunostaining with an antibody against Ki67 (red) and the nuclear counterstain DAPI (blue). Scale bar: 20 μm. The data were represented as the mean ± SEM (n = 3). ** p < 0.01, compared to the control. † p < 0.05 and †† p < 0.01, compared to the indicated group. (For interpretation of the references to color in this figure legend, the reader is referred to theweb version of this article.)

The putative mechanisms underlying the protective effects of NAMPT against proliferation defects upon mild-dose UVA/B irradiation. (i) In response to DNA damage triggered by mild-dose UVA/B irradiation, a stress sensor PARP is rapidly activated within 10 min. Activated PARP attaches poly-ADPr chains
to the damage site concomitant with large consumption of NAD+, recruiting
other DNA repair proteins. (ii) P53 is then acetylated for enzymatic activation within 1 h after irradiation and causes cell cycle arrest to allow DNA repair. (iii) UVA/B-activated NAMPT resolves the transient NAD+ depletion caused by
PARP 2–8 h after irradiation, maintaining intracellular NAD+ level enough to
activate NAD+-dependent deacetylase SIRT1. (iv) Upon completing the repair, SIRT1 represses p53 activity via its deacetylation to return the cell to the proliferative state. (v) NAMPT inhibition after UVA/B irradiation induces the breakdown of the NAD+-SIRT1-p53 axis, resulting in irreversible prolifera-tion arrest.pathophysiological changes in the skin. Thereby, we plan to evaluate the availability of oral and topical administration of NMN and NR by focusing on their permeability to the epidermis.

In general, NAD+ depletion is considered to cause necrotic cell death
[44–46]. Indeed, NAMPT inhibition efficiently induces cell death in several cancer models; therefore, it is considered a potential cancer therapeutic option [39,47–49]. On the other hand, the therapeutic po- tential of targeted NAMPT inhibition may not apply to epidermal ker-
atinocytes. Tan et al. have shown that FK866-induced NAD+ depletion
triggers the premature differentiation and senescence of keratinocytes
without significant cell death [50]. The present study showed that mild- dose UVA/B exposure markedly affected the intracellular NAD+ levels in
the NHEKs and that the addition of FK866 inhibited the proliferation. Keratinocyte proliferation and differentiation are reciprocally regulated.
Thus, NAD+ depletion in keratinocytes likely shifts the cells from pro-
liferation to anomalous differentiation instead of inducing cell death.
Previous studies have reported that niacin supplementation and SIRT1 promote keratinocyte differentiation [51–53]; NAD+ seems to be
required for normal differentiation. Thus, we will investigate the asso- ciation of the NAMPT activity with normal keratinocyte differentiation in future studies.

To our knowledge, we are the first to show that NAMPT is activated
both transcriptionally and enzymatically by UVA/B irradiation. Naka- hata et al. have shown that NAMPT expression and cellular NAD+ levels
are regulated by the circadian rhythms driven by the CLOCK–BMAL1 complex [54]. In addition, previous studies have demonstrated that low- dose UVB induces a rhythmic expression of the circadian rhythms- related genes in primary and immortalized human keratinocytes [55,56]. These research papers did not mention but provided the pos- sibility that UV irradiation triggers the NAMPT expression in keratino- cytes. Indeed, we showed that UVA/B irradiation increased NAMPT gene expression. Besides, we presume that the enzymatic activity of
NAMPT can be augmented by UVA/B-induced NAD+ loss.
A previousreport showed that the NAM-NMN conversion by NAMPT is suppressed
by abundant NAD+, indicating a robust feedback mechanism to balance
intracellular NAD+ levels [57]. Further investigations are needed to determine whether the UVA/B-induced NAMPT enzymatic activation
results from the release from the feedback inhibition of NAMPT activity by NAD+ as mentioned above or another activation mechanism by UVA/
B irradiation.

5. Conclusion
Our results indicate that NAMPT plays a pivotal role in the skin cells’
recovery from UV damage in mild doses. NAMPT protects skin cells from UVA/B-mediated stress by restoring the transient depletion of NAD+,
thus maintaining SIRT1 activity. Our findings not only provide a conclusive explanation for the involvement of NAMPT in skin protection against daily UVA/B exposure but also identifies novel candidate mol- ecules, NMN and NR, as potential therapeutic and preventive agents for age-associated skin disorders and functional decline.
Supplementary data to this article can be found online at https://doi. org/10.1016/j.jphotobiol.2021.112238.

Funding Sources
This work was supported by a research grant from the DHC Corporation.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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