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Ultra Violet (UV)-caused skin cell damage is a main cause of skin cancer. Here, we studied the activity of MHY1485, a mTOR activator, in UV-treated skin cells. In primary human skin keratinocytes, HaCaT keratinocytes and human skin fibroblasts, MHY1485 ameliorated UV-induced cell death and apoptosis. mTOR activation is required for MHY1485-induced above cytoprotective actions. mTOR kinase inhibitors (OSI-027, AZD-8055 and AZD-2014) or mTOR shRNA knockdown almost abolished MHY1485-induced cytoprotection. Further, MHY1485 treatment in skin cells activated mTOR downstream NF-E2-related factor 2 (Nrf2) signaling, causing Nrf2 Ser-40 phosphorylation, stabilization/upregulation and nuclear translocation, as well as mRNA expression of Nrf2-dictated genes. Contrarily, Nrf2 knockdown or S40T mutation almost nullified MHY1485-induced cytoprotection. MHY1485 suppressed UV-induced reactive oxygen species production and DNA single strand breaks in skin keratinocytes and fibroblasts. Together, we conclude that MHY1485 inhibits UV-induced skin cell damages via activating mTOR-Nrf2 signaling.
Ultra Violet (UV) radiation in skin keratinocytes and fibroblasts would lead to oxidative stress and DNA damages, along with activation of several signal transduction pathways that are important for cancer initiation and progression [1–3]. Our group [4–9] has been dedicated to understand the underlying mechanisms of UV-induced skin cell damages, and to develop possible anti-UV strategies.
mammalian target of rapamycin (mTOR) is a vital pro-survival signaling . There are two functionally distinct multi-protein mTOR complexes, namely the mTOR complex 1 (mTORC1) and the mTOR complex 2 (mTORC2) . mTORC1 shall be inhibited by rapamycin or its analogs, and is formed with mTOR, Raptor, mLST8 and several others [10–12]. mTORC1 phosphorylates p70S6K1 (S6K1) and eukaryotic-translation initiation factor 4E-binding protein 1 (4E-BP1) to promote protein translation, energy metabolism and cell survival [11, 13]. On the other hand, the rapamycin-insensitive mTORC2 is compose of mTOR, Rictor and Sin1 [10–12]. The complex serves as the upstream kinase of Akt (Ser-473), a major pro-survival signaling [11, 13]. It has been increasingly clear that both complexes are important for cell survival [10, 12].
Choi et al., recently developed a cell-permeable, small-molecule mTOR specific activator, named MHY1485 . This compound has been shown to directly bind to mTOR, and to activate mTOR at μM concentrations . MHY1485 could induce phosphorylation of mTOR (at Ser-2448) to significantly increase its activity [14, 15]. In the current study, we show that MHY1485 inhibits UV-induced skin cell damages via activating mTOR signaling.
Here, we aim to understand the potential effect of MHY1485 on UV. Primary cultured human skin keratinocytes  were irradiated with UV (20 mJ/cm2), MTT assay results in Figure Figure1A1A showed that cell survival (MTT OD) was decreased sharply (over 50%) following UV radiation. Remarkably, pre-treatment with MHY1485 (1-50 μM) significantly attenuated UV-induced viability reduction (Figure (Figure1A).1A). MHY1485 displayed a dose-dependent response in protecting skin keratinocytes from UV (Figure (Figure1A).1A). At a very low concentration (0.1 μM), MHY1485 failed to inhibit UV damages (Figure (Figure1A).1A). MHY1485 alone, at tested concentrations (1-50 μM), failed to change cell survival (Figure (Figure1A).1A). Since 10 μM MHY1485 displayed superior efficiency in protecting skin keratinocytes from UV (Figure (Figure1A),1A), this concentration was selected for future mechanistic studies.
Skin keratinocytes were also irradiated with UV at other intensities (5-30 mJ/cm2), MHY1485 (10 μM) pre-treatment was again cytoprotective under these UV doses (Figure (Figure1B).1B). Results of the trypan blue staining assay showed that UV (20 mJ/cm2)-induced death of skin keratinocytes was ameliorated with pre-treatment of MHY1485 (10 μM) (Figure (Figure1C).1C). The potential effect of MHY1485 on UV radiation in other skin cells was also examined. As displayed, in HaCaT keratinocytes (Figure (Figure1D)1D) and primary human skin fibroblasts (Figure (Figure1E),1E), MHY1485 (10 μM) remarkably inhibited UV (20 mJ/cm2)-induced viability reduction. Together, these results demonstrate that MHY1485 pre-treatment inhibits UV-induced skin cell death.
Next, using the methods described previously , we tested the potential effect of MHY1485 on UV-induced skin cell apoptosis. In line with our previous findings , UV radiation, at 10-20 mJ/cm2, increased caspase-3 activity (Figure (Figure2A),2A), TUNEL-positive nuclei (Figure (Figure2B)2B) and Histone DNA ELISA optic density (OD) value (Figure (Figure2C)2C) in skin keratinocytes, indicating profound apoptosis activation. Significantly, pre-treatment with MHY1485 (10 μM) largely attenuated UV-provoked apoptosis in skin keratinocytes (Figure 2A–2C). UV radiation at 5 mJ/cm2 was unable to induce significant apoptosis activation (Figure 2A–2C). The similar anti-apoptosis activity by MHY1485 was also observed in UV-irradiated HaCaT keratinocytes (Figure (Figure2D2D and and2E)2E) and primary skin fibroblasts (Figure (Figure2F).2F). MHY1485 (10 μM) alone didn't induce apoptosis in the skin cells (Figure 2A–2F).
MHY1485 is a novel small-molecular mTOR activator [14, 16]. Western blot assay results in Figure Figure3A3A demonstrated that MHY1485 treatment in skin keratinocytes indeed activated mTOR, which was evidenced by phosphorylation (“p-”) of mTOR (Ser-2448), S6K1 (Thr-389), and Akt (Ser-473) (Figure (Figure3A).3A). As discussed, p-S6K1 was the indicator of mTORC1 activation, and p-Akt at Ser-473 reflected mTORC2 activation [10–12]. MHY1485 displayed dose-dependent response in activating mTOR (Figure (Figure3A).3A). Notably, to exclude the influence of medium serum on mTOR activation, cells were starved with warm PBS before MHY1485 treatment .
To study the link between mTOR activation and MHY1485-induced skin cell protection, various mTOR kinase inhibitors were applied, including OSI-027, AZD-8055 and AZD-2014 [18, 19]. Expectably, these mTOR inhibitors almost completely blocked MHY1485-induced mTOR activation (p-mTOR/S6K1/Akt Ser473) in skin keratinocytes (Figure (Figure3B).3B). Remarkably, MHY1485-induced cytoprotection against UV was almost nullified in the presence of above mTOR inhibitors (Figure (Figure3C3C and and3D).3D). Intriguingly, these mTOR blockers also aggravated UV-induce skin keratinocyte cell death (Figure (Figure3C)3C) and apoptosis (Figure (Figure3D),3D), indicating the function of basal mTOR activation in promoting cell survival against UV.
The above pharmacological evidences suggest that mTOR activation is required for MHY1485-induced cell protection against UV. To further support this hypothesis, shRNA strategy was applied. As described, a total of three different mTOR shRNAs (“shmTOR1/2/3”) targeting non-overlapping sequence of mTOR were utilized. Each of the applied mTOR shRNA led to dramatic mTOR downregulation in skin keratinocytes (Figure (Figure3E).3E). Consequently, MHY1485-induced mTOR activation was almost blocked by mTOR shRNAs (Figure (Figure3E).3E). Consequently, MHY1485-induced cytoprotection against UV was largely compromised in the mTOR-silenced keratinocytes (Figure (Figure3F).3F). In another words, MHY1485 failed to protect skin keratinocytes when mTOR was silenced (Figure (Figure3F).3F). These results provided genetic evidence to show that mTOR activation is required for MHY1485-induced cytoprotection against UV. Again, skin keratinocytes with mTOR shRNA were more sensitive to UV damages (Figure (Figure3F),3F), further support the cytoprotective effect of mTOR against UV radiation. The above pharmacological and genetic experiments were repeated in human skin fibroblasts, and similar results were obtained (Data not shown).
Recent studies have suggested that mTOR could activate its potential downstream NF-E2-related factor 2 (Nrf2), a key anti-oxidant signaling [20–22], to inhibit oxidative stress and promote cell survival [23, 24]. We therefore analyzed Nrf2 signaling in MHY1485-treated skin keratinocytes. Western blot assay results demonstrated that MHY1485 dose-dependently induced Nrf2 phosphorylation (at Ser-40) and cytosol accumulation in skin keratinocytes (Figure (Figure4A).4A). Further, Nrf2 nuclear translocation was also observed following MHY1485 treatment (Figure (Figure4A).4A). Consequently, mRNA expressions of Nrf2-dictated genes, including heme oxygenase-1 (HO1), NAD(P)H quinone oxidoreductase 1 (NQO1) and γ-glutamyl cystine ligase catalytic subunit (GCLC) , were significantly increased with MHY1485 (1-50 μM) treatment (Figure (Figure4B).4B). Importantly, MHY1485 (10 μM)-induced transcription of above genes was largely inhibited by the mTOR kinase inhibitor OSI-027 or mTOR shRNA (Figure (Figure4C).4C). These results suggest that MHY1485 activated mTOR downstream Nrf2 signaling in skin keratinocytes.
To study the function of Nrf2 activation in MHY1485-induced cytoprotection, we again utilized genetic strategies from our previous study  to interfere Nrf2 activation. As demonstrated, Nrf2 shRNA knockdown (Figure (Figure4D)4D) or S40T mutation (Figure (Figure4D)4D) almost abolished MHY1485 (10 μM)-induced mRNA expression of HO1 (Figure (Figure4E4E), NQO1 (Figure (Figure4F)4F) and GCLC (Data not shown). More intriguingly, Nrf2 silence or mutation in skin keratinocytes almost abolished MHY1485-induced cytoprotection against UV (Figure (Figure4G4G and and4H).4H). MHY1485 was largely in-effective against UV when Nrf2 was silenced or mutated (Figure (Figure4G4G and and4H).4H). These results imply that Nrf2 Ser40 phosphorylation and activation, as the downstream of mTOR, is required for MHY1485-induced cytoprotection. In line with our previous results , skin keratinocytes with Nrf2 silence or mutation were more vulnerable to UV (Figure (Figure4G4G and and4H),4H), once again confirming the cytoprotective effect of mTOR in skin cells. The above experiments were also repeated in human skin fibroblasts, and similar results were obtained (Data not shown).
Growth evidences have indicated that activation of Nrf2 signaling could inhibit UV-induced reactive oxygen species (ROS) production and DNA damages [24–26]. Our recent study demonstrated that gremlin activated Nrf2 and inhibited UV-induced ROS production and subsequent DNA single strand break (SSB) . Since MHY1485 activated Nrf2 signaling, its potential anti-oxidant activity was analyzed next. As demonstrated, pre-treatment with MHY1485 (10 μM, 30 min) indeed dramatically attenuated UV (20 mJ/cm2)-induced ROS production in skin keratinocytes (Figure (Figure5A).5A). As a result, UV-induced DNA SSB was largely attenuated (Figure (Figure5B).5B). The similar results were also observed in the skin fibroblasts, where MHY1485 (10 μM) decreased UV-induced oxidative stress (Figure (Figure5C)5C) and DNA damages (Figure (Figure5D).5D). MHY1485 (10 μM) alone, as expected, didn't change ROS content and SSB level (Figure 5A–5D). Collectively, these results demonstrate that MHY1485 attenuates UV-induced ROS production and DNA damages in skin cells.
Here, we found that MHY1485 activated mTOR and significantly attenuated UV-induced death and apoptosis of skin keratinocytes, HaCaT keratinocytes and skin fibroblasts. Activation of mTOR is required for MHY1485-induced above actions. mTOR inhibitors (OSI-027, AZD-8055 and AZD-2014) or mTOR shRNAs almost completely abolished MHY1485-exerted cytoprotection against UV.
As one of the uppermost anti-oxidant signalings in mammalian cells, Nrf2 dictates transcription of multiple key anti-oxidant genes to protect cells from oxidative stress [22, 27–29]. Intriguingly, recent studies have suggested that mTOR could also function as a potential upstream signaling for Nrf2 [23, 24, 26]. For instance, Zhang et al., demonstrated that Nrf2 activation by Salvianolic acid requires mTOR activation . Salvianolic acid A induced mTOR-dependent Nrf2 phosphorylation (at Ser-40) and accumulation . Li et al., demonstrated that 3H-1,2-dithiole-3-thione (D3T) activated Nrf2 via phosphorylation at Ser-40 in a mTOR-dependent manner . Our previous study also showed that gremlin activated Akt-mTOR and Nrf2 signaling, then protected skin cells from UV .
In the current study, we provided compelling evidences to support that MHY1485 activated Nrf2 signaling in skin cells. MHY1485 induced Nrf2 phosphorylation at Ser-40, which might cause it departure from its suppressor KEAP1 and subsequent stabilization [23, 24, 30, 31]. Indeed, Nrf2 expression was increased in MHY1485-treated cells. Further, Nrf2 nuclear localization was noticed following MHY1485 treatment in skin keratinocytes, which presumably led to transcription of several Nrf2 genes (HO1, NQO1, GCLC). Nrf2 S40T mutation or shRNA knockdown almost abolished above gene expression by MHY1485. Importantly, activation of Nrf2 is important for MHY1485-induced actions in skin keratinocytes. Nrf2 knockdown or mutation almost abolished MHY1485-induced cytoprotection against UV. Thus, we propose that mTOR downstream Nrf2 activation mediates MHY1485-induced skin cell protection against UV.
Groups including ours [4, 7, 32] have been focusing on the development of the agents that may inhibit or even reverse UV-induced DNA damages, which might discontinue the transformation process [33–36]. Here, we found that MHY1485 significantly inhibited UV-induced ROS production and following DNA damages in skin keratinocytes and fibroblasts. Thus, this novel mTOR activator might be further tested as a promising strategy for skin cancer prevention.
MHY1485 andmTOR kinase inhibitors OSI-027, AZD-8055 and AZD-2014 were obtained from MCE China (Shanghai, China). All antibodies of this study were obtained from Cell Signaling Technology (Nanjing, China). The cell culture regents were purchased from Gibco (Suzhou, China).
The culture of the primary human skin keratinocytes, HaCaT keratinocytes and human skin fibroblasts were described in detail in our previous studies [4–7]. UV radiation procedures were also described previously [4, 8, 9].
Following treatment of cells, apoptosis was tested by Histone DNA apoptosis ELISA assay,TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) staining assay, or the caspase-3 activity assay. The detailed protocols of these assays were described previously [4–7].
Western blot assay was depicted previously [4–6]. For detection of nuclear proteins, the cell nuclei were isolated by the nuclei isolation kit purchased from Sigma . Indicated protein band (in total gray) was quantified via the ImageJ software .
Following treatment of cells, the fluorescent dye dihydrorhodamine (DHR) was applied to test cellular ROS content via the FACS machine (Beckton Dickinson FACScan, Suzhou, China). The ROS fluorescent intensity of treatment group was normalized to that of untreated control group [4, 7].
The three different mTOR shRNAs (“shmTOR1/2/3”) with non-overlapping sequence were produced by constructing the GV248 vector (Genepharm, Shanghai, China) with a puromycin resistance gene and targeted shRNA. Stable knockdown by shRNA was described in detail previously [4–6]. Briefly, skin cells were cultured with 50-60% confluence. mTOR shRNA or the scramble control shRNA (“shSCR”, Santa Cruz Biotech) was added to the cells for 24 hours. Cells were then selected by puromycin (5.0 μg/mL) for 4-5 days. Afterwards, knockdown of mTOR was confirmed by Western blot assay.
Nrf2 shRNA knockdown, S40T dominant negative mutation, and the stable cell selection were described in detail in our previous study .
All data were normalized to control values of each assay and were presented as mean ± standard deviation (SD). Data were analyzed by one-way ANOVA with SPSS 16.0 software (SPSS Inc., Chicago, IL). Significance was chosen as P< 0.05.
This research was supported by grants from the National Natural Science Foundation of China (No.81673066 and No.81473684), the Natural Science Foundation of Fujian Province (No.2013J01297 and No.2016J01534), Joint Funds for the innovation of science and Technology of Fujian province (No.2016Y91020018), and the Outstanding Young Scientist Project of Fujian Province (No. 2015B028 and No. 2016-ZQN-47).
CONFLICTs OF INTEREST
The authors declare that they have no competing interests.
Author ContributionsAll authors carried out the experiments, participated in the design of the study and performed the statistical analysis, conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.