MHY1485 ameliorates UV-induced skin cell damages via activating mTOR-Nrf2 signaling

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.


INTRODUCTION
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][2][3]. Our group [4][5][6][7][8][9] has been dedicated to understand the underlying mechanisms of UV-induced skin cell damages, and to develop possible anti-UV strategies.
Choi et al., recently developed a cell-permeable, small-molecule mTOR specific activator, named MHY1485 [14]. This compound has been shown to directly bind to mTOR, and to activate mTOR at μM concentrations [14]. 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.

MHY1485 inhibits UV-induced skin cell death
Here, we aim to understand the potential effect of MHY1485 on UV. Primary cultured human skin keratinocytes [7] were irradiated with UV (20 mJ/cm 2 ), MTT assay results in Figure 1A 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 1A). MHY1485 displayed a dosedependent response in protecting skin keratinocytes from UV ( Figure 1A). At a very low concentration (0.1 μM), MHY1485 failed to inhibit UV damages ( Figure 1A). MHY1485 alone, at tested concentrations (1-50 μM), failed to change cell survival ( Figure 1A). Since 10 μM MHY1485 displayed superior efficiency in protecting skin keratinocytes from UV ( Figure 1A), this concentration was selected for future mechanistic studies.
Skin keratinocytes were also irradiated with UV at other intensities (5-30 mJ/cm 2 ), MHY1485 (10 μM) pre-treatment was again cytoprotective under these UV doses ( Figure 1B). Results of the trypan blue staining assay showed that UV (20 mJ/cm 2 )-induced death of skin keratinocytes was ameliorated with pre-treatment of MHY1485 (10 μM) ( Figure 1C). The potential effect of MHY1485 on UV radiation in other skin cells was also examined. As displayed, in HaCaT keratinocytes ( Figure  1D) and primary human skin fibroblasts ( Figure 1E), MHY1485 (10 μM) remarkably inhibited UV (20 mJ/ cm 2 )-induced viability reduction. Together, these results demonstrate that MHY1485 pre-treatment inhibits UVinduced skin cell death.

MHY1485 attenuates UV-induced skin cell apoptosis
Next, using the methods described previously [7], we tested the potential effect of MHY1485 on UV-induced skin cell apoptosis. In line with our previous findings [7], UV radiation, at 10-20 mJ/cm 2 , increased caspase-3 activity (Figure 2A), TUNEL-positive nuclei ( Figure 2B) and Histone DNA ELISA optic density (OD) value ( Figure  2C) in skin keratinocytes, indicating profound apoptosis activation. Significantly, pre-treatment with MHY1485 (10 μM) largely attenuated UV-provoked apoptosis in skin keratinocytes ( fibroblasts E. pre-treated for 30 min with designated concentration of MHY1485 (1-50μM), were irradiated with UV at applied intensity (5-30 mJ/cm 2 ), cells were further cultured in the complete medium for 48 hours; Cell survival was tested by MTT assay (A, B, E and F); Cell death was tested by Trypan blue staining assay (C). The values were expressed as mean ± standard deviation (SD) (Same for the following figures). All experiments were repeated three times and similar results were obtained (Same for the following figures). "C" stands for medium-treated control group (Same for the following figures). * P < 0.05 vs. "C" group. # P < 0.05 vs. UV irradiation only group. cm 2 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 2D and 2E) and primary skin fibroblasts ( Figure 2F). MHY1485 (10 μM) alone didn't induce apoptosis in the skin cells (Figure 2A-2F).
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 3E). Consequently, MHY1485-induced mTOR activation was almost blocked by mTOR shRNAs ( Figure 3E). human skin fibroblasts F. pre-treated for 30 min with MHY1485 (10 μM), were irradiated with UV at applied intensity (5-20 mJ/cm 2 ), cells were further cultured in the complete medium for applied time; Cell apoptosis was tested by the listed assays. * P < 0.05 vs. "C" group. # P < 0.05 vs. UV irradiation only group. www.impactjournals.com/oncotarget Consequently, MHY1485-induced cytoprotection against UV was largely compromised in the mTOR-silenced keratinocytes ( Figure 3F). In another words, MHY1485 failed to protect skin keratinocytes when mTOR was silenced ( Figure 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 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).

MHY1485 attenuates UV-induced ROS production and DNA damages
Growth evidences have indicated that activation of Nrf2 signaling could inhibit UV-induced reactive oxygen species (ROS) production and DNA damages [24][25][26]. Our recent study demonstrated that gremlin activated Nrf2 and inhibited UV-induced ROS production and subsequent DNA single strand break (SSB) [7]. 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/cm 2 )-induced ROS production in skin keratinocytes ( Figure 5A). As a result, UV-induced DNA SSB was largely attenuated ( Figure 5B). The similar results were also observed in the skin fibroblasts, where MHY1485 (10 μM) decreased UV-induced oxidative stress ( Figure 5C) and DNA damages ( Figure 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.

DISCUSSION
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.
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][34][35][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.

Chemicals and reagents
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).

Cell culture and UV radiation
The culture of the primary human skin keratinocytes, HaCaT keratinocytes and human skin fibroblasts were described in detail in our previous studies [4][5][6][7]. UV radiation procedures were also described previously [4,8,9].

Cell survival and cell death assays
MTT cell viability assay and cell death trypan blue staining assay were described in our previous studies [4][5][6].

Cell apoptosis assay
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][5][6][7].

Real-time quantitative PCR ("RT-qPCR") analysis
RNA extraction and RT-qPCR were depicted in our previous studies [7]. All the primers of the Nrf2 genes were provided by Dr. Jiang [24,26].

Western blot assay
Western blot assay was depicted previously [4][5][6]. For detection of nuclear proteins, the cell nuclei were isolated by the nuclei isolation kit purchased from Sigma [23]. Indicated protein band (in total gray) was quantified via the ImageJ software [37].

Reactive oxygen species (ROS) detection
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].

Measure of DNA single strand breaks (SSB)
The detailed protocol for analyzing DNA SSB was described previously [4,7]. SSB intensity in UV-irradiated cells was always normalized to the control level.

Nrf2 knockdown and mutation
Nrf2 shRNA knockdown, S40T dominant negative mutation, and the stable cell selection were described in detail in our previous study [7].

Statistical analysis
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.