The Antiaging Activity of Ergothioneine in UVA-Irradiated Human Dermal Fibroblasts via the Inhibition of the AP-1 Pathway and the Activation of Nrf2-Mediated Antioxidant Genes

Abstract

UVA irradiation induced ROS-mediated photo damage to the human skin leading to coarseness, wrinkling, pigmentation, and cutaneous malignancies. We investigated the dermatoprotective efficacies of submicromolar concentrations of ergothioneine (EGT, 0.125-0.5 μM), which occurs naturally as a sulfur-containing amino acid, in the mechanisms in human skin fibroblast (HSF) cells. UVA-induced AP-1 (c-Fos and c-Jun) translocation was found to be inhibited by EGT treatments with the parallel inhibition of the collagenolytic matrix metalloproteinase- (MMP-) 1 activation and type I procollagen degradation. Moreover, EGT mitigated UVA-induced ROS generation. An increase in the amount of antioxidant genes (HO-1, NQO-1, and γ-GCLC) from EGT and were associated with upregulated Nrf2 expressions in a dose-dependent or time-dependent manner. We confirmed this from Nrf2 translocation and increased nuclear ARE promoter activity that underlie EGT dermatoprotective activities. Also, glutathione (GSH) levels (from γ-GCLC) were significantly increased. Moreover, we showed that mediated by ERK, JNK, and PKC, signaling cascades mediate Nrf2 translocation. We confirmed this phenomenon by the suppressed nuclear Nrf2 activation in cells that were treated with respective inhibitors (PD98059, SP600125, and GF109203X). However, antioxidant protein expressions were impaired in Nrf2 knockdown cells to confirm that ARE/Nrf2 pathways and the inhibition of AP-1 had significant roles in EGT-mediated protective effects. We can conclude that ergothioneine ameliorated UVA-induced skin aging and is a useful food supplement for skin care products.

Figure 1

EGT suppressed MMP-1 and IL1β expression but enhanced the procollagen expression in HSF cells. (a) Chemical structure of ergothioneine (EGT). (b, c) Different concentrations of EGT (0.125-1 μM) or vehicle (PBS) were treated with HSF cells for 24 h. (b) The percentage of cell viability was measured by the MTT colorimetric method. The formula used to calculate the percentage of viable cells was (A₅₇₀ of treated cells/A₅₇₀ of untreated cells) × 100. (c) EGT-mediated type I procollagen expression was measured by Western blot method. (d) HSF cells were pretreated with EGT (0.125-0.5 μM) for 24 h and then irradiated without or with 3 J/cm² UVA. The expression of MMP-1, IL1β proteins were measured by Western blot method against β-active as the internal control. Results were presented as mean ± SD of three or more assays. ^∗∗∗p < 0.001 compared with untreated control cells.

Figure 2

Inhibition of AP-1 activation by EGT pretreatment in UVA-irradiated HSF cells. HSF cells were pretreated with different concentrations of EGT (0.125-0.5 μM) for 24 h followed by irradiated with 3 J/cm² UVA. (a) EGT pretreatment suppressed the UVA-irradiated transactivation of nuclear c-Fos and c-Jun proteins in HSF cells. Western blot analysis of nuclear expression of phosphorylated c-Fos and c-Jun proteins was tested using their corresponding antibodies. Histone protein was used as an internal control. (b, c) Immunofluorescence staining indicates changes in AP-1 (p-c-Fos and p-c-Jun) expression. EGT pretreatment suppressed UVA-irradiated nuclear translocation of p-c-Fos and p-c-Jun in UVA-irradiated HSF cells. The percentage of fluorescence cell intensity of each experimental condition was quantified using Olympus Soft Imaging Solutions. Data were presented as mean ± SD of three or more assays. ^∗∗∗p < 0.001 compared with untreated control cells and ^##p < 0.01 and ^###p < 0.001 compared with UVA-irradiated cells.

Figure 3

EGT suppressed UVA-induced ROS production in HSF cells. Cells were pretreated with EGT (0.125-0.5 μM) for 24 h and then irradiated with 3 J/cm² UVA. DCF showed intracellular ROS levels and were measured by fluorescence microscopy (200x magnification). 30 min before each experiment was complete, the nonfluorescent, cell membrane-permeable probe DCFH₂-DA was added to the culture medium with a final concentration of 10 μM. The cells were penetrated by DCFH₂-DA and then reacted with cellular ROS for metabolization into fluorescent DCF. We quantified the percentage of the fluorescence intensity of the DCF-stained cells with Olympus Soft Imaging Solutions for each condition. Data were presented as mean ± SD of three or more assays. ^∗∗∗p < 0.001 compared with untreated control cells and ^##p < 0.01 and ^###p < 0.001 compared with UVA-irradiated cells.

Figure 4

EGT upregulated Nrf2 nuclear translocation in HSF cells. (a, b) EGT upregulated Nrf2 protein levels. HSF cells were treated with different concentrations of EGT (0.125-0.5 μM) for 1 h or 0.5 μM EGT for 1-4 h. Western blot results indicated the effect of EGT on total Nrf2 protein levels in whole cells. β-Actin was used as the internal control. Changes in Nrf2 bands were analyzed by densitometry. (c) EGT increased the nuclear translocation of endogenous Nrf2. HSF cells were treated with 0.5 μM EGT for 1-4 h. Western blot results indicated the effect of EGT on Nrf2 protein levels in the nucleus and cytoplasm. For internal controls, histone and β-actin proteins were used.

Figure 5

Effect of EGT on ARE promoter activation and subsequent expression of HO-1, NQO-1, and γ-GCLC proteins in HSF cells. (a) EGT stimulates Nrf2-mediated ARE activity. HSF cells were cotransfected with pGL3-ARE and treated with different concentrations of EGT (0.125-0.5 μM) for 2 h to measure the percentage of ARE promoter activity. Data were presented as fold over increase in the percentage of ARE promoter activity. (b, c) Effect of EGT concentration and the time of EGT exposure to HSF cells in the induction of antioxidant proteins. HSF cells were treated with different concentrations of EGT for 6 h (b) or 0.5 μM EGT for 1-8 h (c). These cells were harvested, and the expressions of HO-1, NQO-1, and γ-GCLC antioxidant proteins were determined by Western blot analysis. In these conditions, β-actin was used as an internal control. Relative changes in protein bands were measured by densitometry. (d) EGT upregulated the GSH production. HSF cells were incubated with different concentrations of EGT (0.125-0.5 μM) for 24 h. Intracellular total GSH content was measured by a commercially available ELISA kit, as described in Materials and Methods and was expressed in micromolar concentrations as compared to the untreated cells. Data were presented as mean ± SD of three or more experiments. Statistical significance was considered as ^∗∗∗p < 0.001 as compared to untreated control cells.

Figure 6

EGT pretreatment facilitated the nuclear translocation of Nrf2 to induce downstream antioxidant protein expression in UVA-irradiated HSF cells. (a) Effect of EGT concentration on total Nrf2, HO-1, γ-GCLC, and NQO-1 expression in UVA-irradiated HSF cells. HSF cells were pretreated with EGT (0.125-0.5 μM) for 24 h followed by irradiated without or with 3 J/cm² UVA. Western blot results showing that EGT dose-dependently upregulated total Nrf2, HO-1, γ-GCLC, and NQO-1 levels. β-Actin as an internal control using densitometry. (b) Immunofluorescence staining of subcellular localization of Nrf2 in EGT-treated and UVA-irradiated cells. HSF cells were pretreated with 0.5 μM EGT for 24 h and then irradiated without or with 3 J/cm² UVA. The percentage of fluorescence cell intensity of each experimental condition was quantified using Olympus Soft Imaging Solutions. (c) Effect of EGT on nuclear translocation of endogenous Nrf2 in UVA-irradiated cells. HSF cells were pretreated with 0.5 μM EGT for 24 h and then irradiated without or with 3 J/cm² UVA. Western blot results showing the effect of EGT on the protein expressions of nuclear as well as the cytosolic Nrf2 levels. Changes in protein expressions were analyzed using densitometry against histone and β-actin as the internal controls. Changes in protein expressions were analyzed against β-actin as an internal control using densitometry. Data were presented as mean ± SD of three or more assays. ^∗∗p < 0.01 and ^∗∗∗p < 0.001 compared with untreated control cells and ^##p < 0.01 compared with UVA-irradiated cells.

Figure 7

EGT-mediated Nrf2 activation by ERK, JNK, and PKC signaling pathways in HSF cells. (a) Cells were pretreated with pharmacological inhibitors of ERK (PD98059, 30 μM), JNK (SP600125, 25 μM), MAPK p38 (SB203580, 20 μM), PI3K/AKT (LY294002, 30 μM), or PKC (GF109203X, 2.5 μM) for 30 min followed by EGT (0.5 μM) for 1 h. Western blot results showed the nuclear Nrf2 expression with response to inhibitors in the presence of EGT. (b) The protein levels of HO-1, γ-GCLC, and NQO-1 were estimated by immunoblot analysis. Cells were pretreated with inhibitors of ERK (PD98059, 30 μM), JNK (SP600125, 25 μM), or PKC (GF109203X, 2.5 μM) for 30 min followed by EGT (0.5 μM) treatment for 6 h. Protein levels of respective markers are significant compared to EGT alone (0.5 μM) treated cells (without inhibitors). (c) EGT activated ERK, JNK, and PKC signaling pathways. Cells treated with 0.5 μM EGT for 15-120 min and the protein levels of activated forms of ERK, JNK, and PKC were evaluated using a specific antibody to p-ERK, ERK, p-JNK, JNK, and PKC by immunoblot analysis. Data were presented as mean ± SD of three or more experiments. Statistical significance was considered as ^∗∗∗p < 0.001 as compared to untreated control cells and ^#p < 0.05 and ^##p < 0.01 as compared to the EGT alone treated cells.

Figure 8

Nrf2 knockdown attenuated the antioxidant protein expression-mediated protective effects of EGT in HSF cells. Cells were transfected with a specific siRNA against Nrf2 or a nonsilencing control. Following the transfection, cells were incubated with EGT (0.5 μM) for 1 or 6 h. The protein levels of Nrf2 (1 h) or HO-1 and γ-GCLC (6 h) in control siRNA-transfected and siNRf2-transfected cells were measured by Western blot analysis.