Niacin and its related derivatives have been shown to have effects on cellular activities. However, the molecular mechanism of its reduced immunosuppressive effects and photoprotective effects remains unclear. In this study, we investigated the molecular mechanism of the photoprotective effect of niacin in ultraviolet (UV)-irradiated human skin keratinocytes (HaCaT cells). We found that niacin effectively suppressed the UV-induced cell death and cell apoptosis of HaCaT cells. Existing data have shown that AKT activation is involved in the cell survival process. Yet, the potential mechanism of niacin in protection against UV-induced skin damage has thus far not fully been eluvidated. We observed that niacin pretreatment enhances UV induced activation of AKT (Ser473 phosphorylation) as well as that of the downstream signal mTOR (S6 and 4E-BP1 phosphorylation). The PI3K/AKT inhibitor, LY294002, and the mTOR inhibitor, rapamycin, largely neutralized the protective effects of niacin, suggesting that AKT and downstream signaling mTOR/S6 activation are necessary for the niacin-induced protective effects against UV-induced cell death and cell apoptosis. Collectively, our data suggest that niacin may be utilized to prevent UV-induced skin damage and provide a novel mechanism of its photoprotective effects against the UV radiation of sunlight by modulating both AKT and downstream mTOR signaling pathways.
Niacin has long been used in the treatment of high cholesterol, coronary heart disease, the skin disease Pellagra and age-related macular degeneration (AMD) (
Niacin has the potential to influence cellular processes including DNA repair, genomic stability, the immune system, stress responses, signaling, transcription, apoptosis, metabolism, differentiation, chromatin structure and life span. In addition to its well-known redox functions in energy metabolism, niacin and its derivatives in the form of NAD and NADP, are required for the synthesis of cyclic ADP-ribose and NAADP, which are two major mediators of intracellular calcium signaling pathways (
A previous study (
Furthermore, the identification of the nicotinic acid receptor in human skin keratinocytes provides a further link to niacin’s role as a potential skin cancer prevention agent and supports the role of the nicotinic acid receptor as a potential target for skin cancer prevention agents (
UV is divided into UVC (200–280 nm), UVB (280–320 nm) and UVA (320–400 nm). UVB is of environmental significance, penetrating into the papillary area of the dermis and inducing DNA damages to the residing dendritic cells (DC)(
Apoptosis is the process of programmed cell death, which involves a series of morphological changes, including cell detachment, cell shrinkage, mitochondria leakage, chromatin condensation, and DNA fragmentation. This process is controlled by the balance between pro-apoptotic and anti-apoptotic signaling pathways (
Previous studies in dendritic cells as well as keratinocytes have demonstrated that the cellular response to UV is composed of transactivation of cell surface growth factors, such as EGFR, and their downstream signal transduction machinery such as MAPK and PI3K/AKT (
It is possible that niacin affects the cell status by protecting against UV-induced apoptosis and death. To determine the mechanism of niacin in HaCaT cells under UV, we measured the level of AKT and MAPK after UV irradiation. In this study, we demonstrate that UV radiation induces both AKT and MAPK activation. AKT and TSC2 are required for UV-induced mTOR/S6 activation (S6K and 4E-BP1 phosphorylation). Niacin pretreatment protects against UV-induced cell death and apoptosis in keratinocytes (HaCaT cells) by enhancing AKT/mTOR and S6 activation. Niacin may have a functional role in the pro-survival mechanism through activating the AKT/mTOR/S6 signaling pathway.
Niacin (nicotinic acid) and DMSO were obtained from Sigma (St. Louis, MO, USA). Monoclonal mouse anti-β-actin, goat anti-rabbit IgG-HRP and goat anti-mouse IgG-HRP antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The Annexin V-FITC kit was purchased from BD Biosciences (San Jose, CA, USA). The JC-1 probe cell apoptosis detection kit was provided by the Beyotime Institute of Biotechnology (Haimen, China). The p-EGFR (Tyr1068) antibody, and the p-AKT (Ser473), p-AKT (Thr308), p-S6K (Thr389), p-4E-BP1 (Ser65), p-S6 (S235/236), p-mTOR (Ser2448), p-p38 (Thr180/Tyr182), p-JNK(Thr183/Tyr185), p-ERK1/2 (Thr202/Tyr 204), and p-TSC2 (Ser 1462) antibodies, the AKT inhibitor LY294002, the mTOR inhibitor rapamycin, the Tuberin/TSC2 siRNA, and the p38 MAPK, JNK and the AKT1/2 antibodies were all from Cell Signaling Technology (Beverly, MA, USA). The EGFR inhibitor, PD153035 was purchased from Invitrogen (Carlsbad, CA, USA).
The spontaneously immortalized human keratinocytes (HaCaT cell line) were cultured at 37°C in RPMI-1640 supplemented with 10% fetal bovine serum and 100 U/ml of penicillin/streptomycin as previously reported (
Cell viability was measured by the 3-[4,5-dimethylthylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) method (
As described previously (
As previously described (
A cell apoptosis detection kit was used for Annexin V and PI staining. HaCaT cells were cultured in 35-mm dishes and were exposed to different treatments. Cells were harvested after up to 24 h incubation and washed in PBS twice. Cells were re-suspended in the binding buffer, and then the fluorescein-conjugated Annexin V and PI reagent were added to cell suspensions. After incubation in the dark for 15 min the percentage of apoptotic cells and necrotic cells were assessed by a flow cytometer (FACSCalibur, BD Biosciences, USA).
The JC-1 probe was used to measure mitochondrial depolarization. Briefly, cells were cultured in 6-well plates up to ~50% confluency, and after the indicated treatments cells were incubated with 1 ml of JC-1 staining solution (5 μg/ml) at 37°C for 20 min and rinsed twice with PBS. Mitochondrial membrane potentials were monitored by determining the relative amounts of dual emissions from mitochondrial JC-1 monomers or aggregates using an Olympus fluorescent microscope under Argon-ion 488 nm laser excitation. Mitochondrial depolarization is indicated by an increase in the green/red fluorescence intensity.
The values in the figures are expressed as the means ± standard deviation (SD). All experiments were repeated at least three times. Experimental groups were compared by one-way ANOVA analysis of SPSS 16.0. Mean differences were considered significant (P<0.05) and highly significant (P<0.01).
By using the cell viability MTT assay, first we investigated the possible protective effects of niacin against UV-induced cell death. As indicated in
We next examined whether the AKT cascade is activated upon UV radiation. The results showed that UV radiation activates the EGFR/AKT/mTOR pathway in a time- and dose-dependent manner. UV radiation (30 mJ/cm2) activates AKT at the highest level. The peaks of EGFR/AKT/mTOR activation take place between 30 and 60 min at the UV dose of 30 mJ/cm2 (
As demonstrated, niacin protects against UV-induced cell death and cell apoptosis. The AKT cascade activation is induced by UV radiation. Taken together, we conclude that the AKT cascade signal activation serves as a critical upstream signal against UV, serving as a pro-survival signal against UV-induced cell apoptosis. To determine whether the AKT signaling pathway is involved in the pro-survival and anti-apoptotic effects of niacin, cells were treated with AKT cascade kinase inhibitors independently, such as the EGFR inhibitor, PD153035, the AKT inhibitor, LY294002 and the mTOR inhibitor, rapamycin. As previously shown, treatment with PD153035, LY294002 and rapamycin resulted in the increase of UV-induced apoptosis. Living cells were decreased successively from 93.21±2.74% to 80.08±3.20% while the percentages of apoptotic cells were successively increased from 6.35±0.32% in the control to 18.39±0.55% (P<0.01) in the rapamycin group. Independent treatment with LY294002 and rapamycin seemed to affect cells efficiently. The contribution of the AKT cascade signal activation after UV was further detected by pretreating cells with inhibitors before UV exposure. Treatment with LY294002 and rapamycin were found to have less influence on the UV-induced loss of cell viability in the cells in comparison with PD98059. However, LY294002 has a more critical role in the UV effect. The UV-induced apoptosis ratio was 36.71±3.67% compared to others. Last, we treated niacin-treated cells with inhibitors prior to UV. This led to an insignificant decrease of UV-induced cell apoptosis (P>0.05) except for PD98059 which increaed apoptotic cells (P<0.05).
We have shown that niacin pretreatment protects against UV-induced cell death and apoptosis in keratinocytes (HaCaT cells) (
Despite previous research showing that niacin reduces the immunosuppressive effects of UV both
To understand the molecular mechanism of the anti-apoptotic effect of niacin in UV-treated keratinocytes, first we analyzed several signaling pathways related to UV-induced apoptosis in HaCaT cells. We found that the short time-dependent regulation of the AKT cascade and MAPK feedback activation is involved in HaCaT cells after UV exposure. AKT is well known to be differentially activated depending on the type of extracellular stimuli. As suggested in previous studies, MAPK/JNK/p38 activation may be essential for UV-induced apoptosis, but the activation of AKT, mTOR, S6 can be the pro-surviving signals against UV-induced apoptosis. Some plant flavonoids with anti-apoptotic properties activate the MAPK/AKT/NF-κB pathway (
In addition to stimulating cell growth, mTOR also promotes cell survival. eIF4E serves as an important downstream effectors of mTOR in the control of cell survival through the 4E-BP1 (
Although the PI3K/AKT and its downstream substrate mTOR (S6K and 4E-BP1 phosphorylation, rapamycin sensitive) pathway are well-established, the identity of the kinase responsible for phosphorylating AKT at Ser473 remains elusive until in recent years, when it was revealed to be mTORC2 (
In conclusion, we found for the first time that niacin pretreatment protects against UV-induced cell death and apoptosis by enhancing the pro-survival pathways including AKT, mTOR and S6 in skin keratinocytes (HaCaT cells). Oral and external niacin and nicotinamide are both safe and inexpensive and appear to be promising chemopreventive supplements for reducing the mutagenic, immunosuppressive and cell damage effects of sunlight (
This research was supported by a grant from the National Natural Science Foundation of China (30872280 to Dr Aie Xu).
Niacin protects against UVB induced cell death in HaCaT cells. HaCaT cells were pretreated with niacin (0, 5, 10, 20 and 40 μM) for 1 h, followed by 30 mJ/cm2 UV radiation. After incubation at 37°C for 24 h, cell viability was detected by the MTT assay. The values in the figure are expressed as the means ± standard deviation (SD). *P<0.05, **P<0.01 vs. the UV-treated niacin-free group. All experiments were repeated at least three times and similar results were obtained.
AKT, TSC2, mTOR and S6 are required for UV-induced AKT cascade activation. (A and B) HaCaT cells were exposed to different dosages of UV radiation (0, 5, 15, 30, 60, 120 mJ/cm2) for 30 min and for different durations of 30 mJ/cm2 UV respectively. p-EGFR (Tyr1068), p-AKT (Ser473), p-4E-BP1 (Ser65) were detected by western blotting. (C) HaCaT cells were pretreated with 20 μM niacin, followed by UV radiation (0, 15, 30, 60 mJ/cm2), then stained with the JC-1 probe and imaged by fluorescent microscope. The individual red and green average fluorescence intensities are expressed as the ratio of green to red fluorescence. An increase on the fluorescence green/red ratio indicates a shift increase in mitochondrial depolarization as an early apoptosis label. HaCaT cells were pre-treated with (D) the EGFR inhibitor, PD153035 (PD, 1 μM); (E) with the PI3K/AKT inhibitor LY294002 (LY, 10 μM) and mTOR inhibitor rapamycin (Rapa, 100 nM), for 1 h, with UV (30 mJ/cm2) radiation and cultured for 15, 30 and 60 min. (F) Cells were transfected with Tuberin/TSC2 siRNAII (100 nM) for 48 h prior to UV radiation (30 mJ/cm2), for 30 or 60 min. p-EGFR (Tyr1068), p-AKT (Ser473), p-AKT (Thr308), p-TSC2 (Thr1462), p-mTOR (Ser2448), p-S6K (Thr389), p-S6 (Ser235/236), p-4E-BP1 (Ser65) and total AKT antibody were used by western blotting. All experiments were repeated at least three times and similar results were obtained.
Niacin protects HaCaT cells from UV-induced apoptosis cooperated with different AKT cascade inhibitors. HaCaTs were pre-treated with 20 μM niacin (NA) for 30 min, and then incubated with different inhibitors for 30 min until 30 mJ/cm2 of UV radiation. PD, LY, RaPa are abbreviations for PD153035, LY294002, and rapamycin respectively. Twenty-four hours post-treatment the number and percentage of apoptotic and necrotic cells were quantifled by FACS after cells were washed and stained with Annexin V and PI. Apoptotic cells were determined by counting the percentage of early apoptotic cells Annexin V(+), PI(−) cells plus the percentage of late apoptotic Annexin V(+), PI(+) cells. Normal living cells are presented as Annexin V(−), PI(−), respectively. The values in the figure are shown as mean ± SD of at least three separate experiments. *P<0.05, **P<0.01 vs. the control group; #P<0.05, ##P<0.01 vs. the UV only group; and &P<0.05 when comparing the PD+UV and PD+NA+UV group.
Niacin protects against UV-induced apoptosis via enhancement of AKT/mTOR/S6 activation. (A) HaCaT cells were treated with niacin (NA, 20 μM) and cultured for 5, 15, 30, 60 and 120 min. p-EGFR (Tyr1068), p-AKT (Ser473), p-mTOR (Ser2448), p-S6K (Thr389), p-S6 (Ser235/236), p-4E-BP1 (Ser65), total AKT and β-actin antibody were used for western blotting. (B and C) HaCaT cells were pretreated with or without niacin (20 μM) for 1 h, followed by UV (30 mJ/cm2) radiation and cultured for the indicated time, p-EGFR (Tyr1068), p-AKT (Ser473), p-mTOR (Ser2448), p-S6K (Thr389), p-S6 (Ser235/236), p-4E-BP1 (Ser65), p-p38 (Thr180/Tyr182), p-JNK (Thr183/Tyr185), p-ERK1/2 (Thr202/Tyr204), p38 MAPK, JNK total AKT and β-actin were detected by western blotting. (D) HaCaT cells were pretreated with niacin (20 μM) for 1 h, followed by UV radiation (30 mJ/cm2) and cultured for 1 h, p-AKT (Ser473) and p-S6 (Ser235/236) were detected by confocal microscopy. (E) HaCaT cells were pre-treated with niacin and LY294002 separately or in combination, after UV (30 mJ/cm2) radiation; mitochondrial depolarization was observed the same way in