2023 Impact Factor
2023 Impact Factor
Vitiligo is characterized by the death or loss of melanocytes, resulting in reduced melanin pigmentation (Rodrigues et al., 2017; Bergqvist and Ezzedine, 2020). The pathogenesis of vitiligo remains unclear, as it is a complex, multifactorial, and polygenic disease. Growing evidence indicates that oxidative stress linked to the immune system plays a critical role in the pathogenesis of vitiligo (Laddha et al., 2013; Delmas and Larue, 2019; Wang et al., 2019; Bergqvist and Ezzedine, 2021; Seneschal et al., 2021; Xuan et al., 2022; Chang and Ko, 2023). Whenever the cellular accumulation of reactive oxygen species (ROS), a family of oxygen-based free radicals including hydrogen peroxide (H2O2), overcomes cellular antioxidant capacity, oxidative stress develops. ROS are generated by exogenous stimuli, such as ultraviolet (UV) irradiation (Zhang et al., 1997; Laddha et al., 2013; Chen et al., 2019) and endogenous stimuli, including cellular metabolism. Nuclear factor E2-related factor 2 (Nrf2) and its primary targets have been identified as enzymatic and non-enzymatic antioxidants (Motohashi and Yamamoto, 2004; Xie et al., 2016). Heme oxygenase-1 (HO-1) and NADPH-Quinone-Oxidoreductase 1 (NQO-1) are the main targets of Nrf2 in melanocytes and keratinocytes, respectively (Marrot et al., 2008), although both targets are detectable in keratinocytes (Lee et al., 2021). Levels of Nrf2 in melanocytes derived from vitiligo-affected individuals have been found to be lower compared to melanocytes from normal healthy controls, resulting in an inability to overcome oxidative stress in vitiligo (Schallreuter et al., 1991; Maresca et al., 1997; Jian et al., 2014; Lin et al., 2020; Romano-Lozano et al., 2022).
Aquaporin-3 (AQP3), a water and glycerol channel protein, regulates redox signaling by promoting uptake of H2O2 through the cell membrane (Miller et al., 2010; da Silva et al., 2021; Milković and Čipak Gašparović, 2021; Wang et al., 2021). We previously identified that AQP3 is downregulated in the lesional epidermis of patients with vitiligo, which reduces the survival of keratinocytes and leads to the passive death of melanocytes (Kim and Lee, 2010). However, the role of AQP3 in oxidative stress under vitiligo conditions remains unclear, and our study aimed to better understand the function of AQP3 under such circumstances. Because the main AQP3 source in the skin is keratinocytes (Kim and Lee, 2010), it should also be determined whether oxidative stress derived from keratinocytes directly affects neighboring melanocytes. Compared with traditional genome editing technology, clustered regularly interspaced short palindromic repeat (CRISPR) systems provide an unprecedented degree of specificity, effectiveness, and versatility in genetic manipulation (White et al., 2017). Thus, the CRISPR system was used to examine the role of AQP3 in oxidative stress in primary cultured adult human epidermal keratinocytes, with and without AQP3 knockdown, in the presence or absence of H2O2 treatment. The role of AQP3 in oxidative stress was also examined using lesional and non-lesional skin sets from vitiligo patients with AQP3 downregulation.
Adult skin specimens obtained from Cesarean sections and circumcisions were used to establish cells for the cell culture. The epidermis was separated from the dermis and individual epidermal cell suspensions were prepared as previously described. Keratinocytes were suspended in EpiLife Medium (Invitrogen, Carlsbad, CA, USA) supplemented with bovine pituitary extract (BPE), bovine insulin (BI), hydrocortisone, human epidermal growth factor, and bovine transferrin (BT) (Invitrogen). Melanocytes were suspended in Medium 254 (Invitrogen) supplemented with BPE, fetal bovine serum, BI, hydrocortisone, bFGF, BT, heparin, and phorbol 12-myristate 13-acetate (Invitrogen). For keratinocytes, subculture passages 3-5 were used for the experiments. Keratinocytes were seeded at 2×105 cells/well in six-well plates and incubated for 24 h. Keratinocytes were transfected with the indicated genes and cultured in the presence or absence of H2O2. After 24 or 48 h, cells and supernatants were harvested for subsequent experiments. All the collected cells and supernatants were used for western blot analysis, ROS assays, and immunohistochemistry.
Keratinocytes were transfected with 25 nM CRISPR-CAS9 sgRNA for human AQP3, NRF2, or negative control sgRNA (Integrated DNA Technologies, San Diego, CA, USA) using CRISPRMAX transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA). For AQP3 overexpression, the cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. The cells were used for experiments 24 or 48 h after transfection.
Equal amounts of extracted proteins were resolved and transferred onto nitrocellulose membranes. The membranes were then incubated with antibodies against NQO1 (mouse monoclonal; Santa Cruz Biotechnology, Dallas, TX, USA), AQP3, NRF2 (rabbit polyclonal; Cell Signaling Technology, Beverly, MA, USA), and ß-actin (mouse monoclonal; Sigma Aldrich, St. Louis, MO, USA). After incubation with the appropriate anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies (Thermo Fisher Scientific) or with anti-goat horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology), an enhanced chemiluminescence solution (Thermo Fisher Scientific) was applied, and signals were captured using an image reader (LAS-3000; Fuji Photo Film, Tokyo, Japan). Protein bands were analyzed by densitometry.
After deparaffinization and rehydration, the sections were pre-incubated with 3% bovine serum albumin. These sections were reacted sequentially with an anti-AQP3 antibody and 1:200 Alexa Fluor-labeled goat anti-rabbit IgG (594; Molecular Probes, Eugene, OR, USA), anti-HO and Alexa Fluor-labeled goat anti-mouse IgG (488; Molecular Probes), or anti-NQO-1 and Alexa Fluor-labeled goat anti-mouse IgG (594; Molecular Probes) antibodies. The cultured cells were fixed with 2% paraformaldehyde at room temperature for 20 min. Fixed cells were sequentially incubated with anti-AQP3 antibody and 1:200 Alexa Fluor-labeled goat anti-rabbit IgG, anti-NRF2 or anti-NQO1 antibody and Alexa Fluor-labeled goat anti-mouse IgG. The nuclei were counterstained with Hoechst 33258 (Sigma-Aldrich). Fluorescence images were evaluated using an image analysis system (Dp Manager 2.1; Olympus Optical Co., Tokyo, Japan) and Wright Cell Imaging Facility (WCIF) ImageJ 1.54d software (http://www.uhnresearch.ca/facilities/wcif/imagej).
ROS were detected using the Total ROS Detection Kit (Enzo Life, Farmingdale, NY, USA) according to the manufacturer’s instructions. Keratinocytes were treated with 250 μM of H2O2 for 30 min, and then an ROS assay was performed using supernatants and cells which were washed twice with wash buffer. After the supernatants and cells were incubated with the ROS detection solution for 1 h at 37°C in the dark, their values were immediately measured using a fluorescence microplate reader (Spark; TECAN, Männedorf, Switzerland).
Culture supernatants from keratinocytes treated with H2O2 were added to melanocytes for 24 h. The degree of viability or cytotoxicity of melanocytes was measured by MTT and lactate dehydrogenase (LDH) assays, respectively. To measure cell viability, the cells were incubated with 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for 4 h, and the precipitated formazan was dissolved in dimethyl sulfoxide (DMSO). Optical density was measured at 570 nm with background subtraction at 630 nm using a spectrophotometer.
For cytotoxicity, LDH activity was measured in the culture medium using a cytotoxicity detection kit (Roche, Penzberg, Germany) according to the manufacturer’s instructions. LDH release was determined by measuring the optical density at 490 nm and subtracting the optical density at 620 nm from the supernatant of the experimental cultures. The effects of the chemicals on cytotoxicity were calculated from the percentage of LDH released by each chemical relative to the appropriate solvent.
Statistical analyses of the experimental data were performed using Student’s t-test. Results are expressed as means ± SD. A p-value <0.05 was considered statistically significant. For human sample data, differences between the non-lesions and lesions were assessed by the Mann–Whitney U test and expressed as mean ± standard error of the mean (SEM).
To identify the role of AQP3 in oxidative stress, it was necessary to examine whether the expression of NRF2 and its main target in keratinocytes, NQO-1 (Marrot et al., 2008; Lee et al., 2021), were regulated by AQP3. Keratinocytes are the main source of AQP3 in the skin (Kim and Lee, 2010). Thus, primary cultured adult human keratinocytes with or without AQP3 knockdown and overexpression were examined. Western blot analysis showed that the expression of NRF2 and NQO-1 was reduced in cultured keratinocytes with AQP3 knockdown (Fig. 1A). Conversely, AQP3 overexpression upregulated NRF2 and NQO-1 expression in cultured keratinocytes (Fig. 1B). However, AQP3 expression was not reduced by the NRF2 knockdown (Fig. 1C). Immunofluorescence staining reveals that the relative expression levels of NRF2 (Fig. 1D) and NQO-1 (Fig. 1E) decreased in cultured keratinocytes with AQP3 knockdown. The expression levels after immunofluorescence staining with anti-NQO1 antibody were weaker in the lesional epidermis than non-lesional epidermis in vitiligo patients with AQP3 downregulation (Fig. 1F). After immunofluorescence staining, the expression levels of HO-1, another target of Nrf2 (Chen et al., 2019; Saha et al., 2020; Lee et al., 2021; Pang et al., 2021), were also weaker in the lesional epidermis (Fig. 1G).
Figure 1. Expression levels of NRF2 and NQO-1 are regulated by AQP3. (A, B) Representative western blotting using anti-NRF2 and anti-NQO-1 antibodies in the primary cultured adult epidermal keratinocytes, with or without AQP3 knockdown (A) or AQP3 overexpression. AQP3 overexpression (B). (C) Representative western blotting using anti-AQP3 antibodies in the primary cultured adult epidermal keratinocytes, with or without NRF2 knockdown. For the western blot analysis, β-actin was used as an internal control. The data represent the means ± SD from four independent experiments. *p<0.05, **p<0.01. Con (-): control RNA knockdown, AQP3 (-): AQP3 RNA knockdown. (D-E) Representative immunofluorescence staining using anti-NRF2 (D) or anti-NQO-1 antibody (E) in cultured keratinocytes, with or without AQP3 knockdown. (F-G) Representative immunofluorescence staining using anti-NQO1 (F) or anti-HO antibody (G) in the lesional (L) and non-lesional (N) epidermis of four vitiligo patients with AQP3 downregulation. For the immunofluorescence staining, the nuclei were counterstained with Hoechst 33258 (Bar=0.05 mm), and the intensities were measured using ImageJ software. *p<0.05, **p<0.01.
The expression levels of NRF2 and NQO-1 were regulated by AQP3 (Fig. 1A-1D). This result suggests that AQP3 plays a role in oxidative stress. Thus, their association under H2O2-induced oxidative stress was examined. The reduced ratios of AQP3 expression between control keratinocytes and AQP3-knockdown keratinocytes were similar regardless of H2O2 treatment (Fig. 2A). The reduced ratios of total NRF2 expression levels between control and AQP3-knockdown keratinocytes were not altered by H2O2 treatment (Fig. 2A). However, the ratio of nuclear NRF2 was increased by H2O2 treatment (Fig. 2B). The ratio of NQO-1 expression was also further increased in the control keratinocytes than the AQP3-knockdown keratinocytes after H2O2 treatment (Fig. 2C).
Figure 2. Ratios of NRF2 nuclear translocation and NQO-1 expression are further reduced in AQP3-knockdown keratinocytes after H2O2 treatment. (A-C) Representative western blotting using anti-AQP3 (A), anti-NRF2 (B), and anti-NQO-1 antibodies (C) in cultured keratinocytes (total cell extracts or nuclear fractions), with or without AQP3 knockdown in the presence or absence of H2O2 treatment. β-actin was used as an internal control. The data represent the means ± SD from four independent experiments. *p<0.05 vs control keratinocytes [con (-)], #p<0.05 vs relative ratios in AQP3-knockdown keratinocytes [AQP3 (-)] without H2O2 treatment.
NQO-1 synthesis via NRF2 nuclear translocation was reduced under H2O2-induced oxidative stress in AQP3-knockdown keratinocytes (Fig 2A-2C). This result indicates that AQP3-knockdown keratinocytes as in vitiligo may be unable to overcome oxidative stress. Because vitiligo involves melanocyte death (Rodrigues et al., 2017; Bergqvist and Ezzedine, 2020), how the result impacts melanocyte survival should be determinded. To examine this topic, intracellular and extracellular (culture supernatants) ROS concentrations were measured sequentially in AQP3-knockdown keratinocytes and control keratinocytes after H2O2 treatment. Intracellular concentrations were lower in the AQP3-knockdown keratinocytes than in control keratinocytes (Fig. 3A). ROS concentrations in culture supernatants were similar regardless of AQP3 knockdown in the absence of H2O2. After H2O2 treatment, ROS concentrations increased in control and AQP3-knockdown keratinocytes. However, the ROS concentration steeply increased in AQP3-knockdown keratinocytes (Fig. 3B). After melanocyte culturing using these keratinocyte culture supernatants, the MTT assay revealed that the relative number of viable melanocytes was decreased by the conditioned media from AQP3-knockdown keratinocytes (Fig. 3C). Conversely, the LDH assay suggested greater cytotoxicity to melanocytes by the conditioned media from AQP3-knockdown keratinocytes (Fig. 3D).
Figure 3. AQP3-knockdown keratinocytes elevate extracellular ROS concentrations under H2O2 treatment, leading to melanocyte apoptosis. (A-B) Intracellular (A) and extracellular (culture supernatant) (B) ROS concentrations in cultured keratinocytes, with or without AQP3 knockdown after H2O2 treatment. (C-D) MTT assay for cell survival (C) and LDH release for cytotoxicity of primary cultured adult human melanocytes using culture supernatants obtained from AQP3-knockdown keratinocytes cultured in the presence of H2O2. The data represent the means ± SD from four independent experiments. *p<0.05 vs control keratinocytes [con (-)], #p<0.05 vs relative ratios in AQP3-knockdown keratinocytes [AQP3 (-)] without H2O2 treatment.
Oxidative stress is a major factor in vitiligo pathogenesis (Laddha et al., 2013; Wang et al., 2019; Bergqvist and Ezzedine, 2021; Xuan et al., 2022; Chang and Ko, 2023). The regulatory role of AQP3 in redox signaling (Miller et al., 2010; da Silva et al., 2021; Milković and Čipak Gašparović, 2021; Wang et al., 2021) and the downregulation of AQP3 in vitiligo lesional skin (Kim and Lee, 2010) prompted us to examine the role in vitiligo of AQP3 in oxidative stress. This study aimed to sequentially examine the relationship between AQP3 and NRF2 and their main targets, the effect of AQP3 on NRF2-NQO-1 signaling (with or without H2O2 treatment) in culture media, and the effect of oxidative stress induced by AQP3-knockdown keratinocytes on melanocyte survival and death.
Western blot analysis and immunofluorescence staining of cultured keratinocytes showed that NRF2 and NQO-1 were downregulated by AQP3 knockdown and upregulated by AQP3 overexpression (Fig 1A, 1B, 1D, 1E). Reduced NQO-1 expression was also examined in the lesional skin of patients with AQP3 downregulation (Fig. 1F). These results suggest a clear connection between AQP3 and NRF2-NQO-1 signaling. Although HO-1 is the main target of Nrf2 in melanocytes (Marrot et al., 2008), a reduction similar to that observed for NQO-1 was shown in lesional keratinocytes (Fig. 1G). There was no change in AQP3 expression levels due to NRF2 downregulation (Fig. 1C), indicating that AQP3 is an upstream molecule in the regulation of NRF2 expression.
To identify the role of AQP3 in oxidative stress, concentrations from 100 μM to 1 mM of H2O2 was added to media for keratinocyte cultures (data not shown), with 250 μM used in the current study. H2O2 treatment increased the expression of AQP3 along with nuclear NRF2 and NQO-1 except when AQP3 expression was reduced (Fig. 2A-2C). These results suggest that AQP3 upregulation is involved in overcoming oxidative stress in keratinocytes at the cellular level. Impaired cellular antioxidant responses due to AQP3 downregulation may trigger oxidative stress in patients with vitiligo.
Keratinocytes are the main source of AQP3 in skin (Kim and Lee, 2010). Therefore, it needs to be clarified how the inadequate handling of oxidative stress in AQP3-knockdown keratinocytes could be linked to melanocyte death. Keratinocyte death can also induce passive melanocyte death (Lee et al., 2005; Lee, 2012). However, melanocytes are vulnerable to oxidative stress during vitiligo (Denat et al., 2014; Kim et al., 2017). Thus, we examined whether inadequate handling of ROS in AQP3-knockdown keratinocytes increased the extracellular concentrations of ROS, leading to melanocyte death. This study showed that ROS concentrations were lower within cells but higher in culture media when AQP3-knockdown keratinocytes were cultured in media containing H2O2 (Fig. 3A, 3B). This suggests the handling of ROS in AQP3-knockdown keratinocytes is impaired. AQP3 promotes uptake of H2O2 through the cell membrane (Miller et al., 2010; da Silva et al., 2021; Milković and Čipak Gašparović, 2021; Wang et al., 2021). A decline in H2O2 uptake by AQP3-knockdown keratinocytes could increase the concentration of ROS in the surrounding environment. Moreover, the culture supernatants from AQP3-knockdown keratinocytes reduced the viable number of melanocytes (Fig. 3C), but increased their cytotoxicity (Fig. 3D). These results verified that AQP3-knockdown-induced oxidative stress in keratinocytes in the surrounding environment (where melanocytes reside) played a role in melanocyte death. Additionally, impaired activation of the Nrf2-ARE signaling pathway has been suggested as a possible mechanism for melanocyte degeneration in vitiligo (Jian et al., 2014; Lin et al., 2020; Romano-Lozano et al., 2022). The role of oxidative stress linked to the immune system is emphasized in the pathogenesis of vitiligo (Laddha et al., 2013; Delmas and Larue, 2019; Wang et al., 2019; Bergqvist and Ezzedine, 2021; Seneschal et al., 2021; Xuan et al., 2022; Chang and Ko, 2023). Although AQP3 is not the only way to activate the Nrf2-ARE signaling pathway, our results suggest that AQP3 may be a possible therapeutic target for vitiligo.
Overall, this study showed that AQP3 downregulation in vitiligo keratinocytes impairs cellular antioxidant responses and H2O2 uptake under oxidative stress, leading to neighboring melanocyte death (Fig. 4).
Figure 4. Schematic view of melanocyte death induced by AQP3-downregulated keratinocytes in vitiligo under oxidative stress. AQP3 downregulation in vitiligo keratinocytes impairs cellular antioxidant responses and H2O2 uptake under oxidative stress, increasing ROS concentrations in the surrounding environment, leading to neighboring melanocyte death.
This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HP20C0131).
NH Kim, HJ Kim, and AY Lee declare no conflict of interest.
Funding Information
Korea Health Industry Development Institute Ministry of Health and Welfare |
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