HMGB1 deficiency reduces H2O2‐induced oxidative damage in human melanocytes via the Nrf2 pathway

Abstract Oxidative stress leads to melanocyte death and has been implicated in the pathogenesis of vitiligo. The nuclear factor, E2‐related factor 2 (Nrf2), is a critical transcription factor in protecting cells from oxidative damage. High‐mobility group box 1 (HMGB1) is a chromatin‐associated nuclear protein and an extracellular damage‐associated molecular pattern molecule. Extracellular HMGB1 released from activated immune cells, necrotic or injured cells, becomes a proinflammatory mediator through binding to cell‐surface receptors of responding cells. In this study, we investigated the role of HMGB1 from melanocytes in the response to oxidative stress and the mechanism involved. We showed that HMGB1 is expressed by primary normal human epidermal melanocytes (NHEMs). H2O2 treatment increased cytoplasmic translocation and extracellular release of HMGB1. HMGB1 knockdown by small interfering RNA (siRNA) led to decreased apoptosis of NHEMs. HMGB1 inhibition enhanced the expression of Nrf2 and its target genes. The expression of Nrf2 and its downstream antioxidant genes was downregulated after the supernatant of H2O2‐treated NHEMs was added to HMGB1‐deficient cells. HMGB1 knockdown by siRNA suppressed the expression of the autophagosome marker, LC3, and enhanced p62 expression. Coimmunoprecipitation with Keap1 showed a reduced Nrf2‐Keap1 interaction and an increased p62‐Keap1 interaction under oxidative stress. These data demonstrated that external stimuli (eg, oxidative stress) may trigger autocrine HMGB1 translocation and release by melanocytes, suppressing the expression of Nrf2 and downstream antioxidant genes to induce melanocyte apoptosis, and thereby participate in the pathological process of vitiligo.

and -independent mechanisms, in hormonal, auto-, para-, or intracrine fashion. Tyrosinase are the key elements in the regulation of melanogenesis. The presence of a large-binding site of the transcription factor family in the gene demonstrates the complexity and precise control of melanogenesis. Within the context of the skin as a stress organ, melanogenic activity serves as a unique molecular sensor and transducer of noxious signals and as regulator of local homoeostasis. 2 Vitiligo is a common acquired skin depigmentation disorder characterized by the death of melanocytes. 3 The aetiology of vitiligo and the causes of melanocyte death are not fully understood, yet. The oxidative stress hypothesis suggests an imbalanced redox state of the vitiliginous skin. This results in dramatic production of reactive oxygen species (ROS), such as H 2 O 2 . ROS oxidize cell components, leading to melanocyte destruction and creating depigmented macules. 4 Increased levels of H 2 O 2 were detected in the epidermis of vitiligo patients. 5,6 The nuclear factor, E2-related factor 2 (Nrf2), is a regulator of cellular resistance to oxidants. The Nrf2-kelch-like ECH-associated protein 1 (Keap1) system is one of the major cellular defence mechanisms against oxidative and electrophilic stresses. [7][8][9][10] Under quiescent conditions, the transcription factor, Nrf2, is constitutively degraded through the ubiquitin-proteasome pathway. Its binding partner, Keap1, is an adaptor of the ubiquitin ligase complex that targets Nrf2. Under oxidative stress, Nrf2 is rapidly released from Keap1 and translocates into the nucleus, where it binds to ARE and induces the phase II antioxidant genes. 11 These genes encode haem oxygenase-1 (HO-1), catalase, superoxide dismutase, glutathione-S-transferase, glutathione peroxidase, NADH quinone oxidoreductase 1 (NQO1), glutamate-cysteine ligase catalytic subunit (GCLC), and glutamyl cysteine ligase modulator subunit (GCLM), which are important antioxidants in melanocytes. 12 p62/ SQSTM1 (hereafter referred to as p62) interacts with the Nrf2binding site of Keap1 and competitively inhibits the Keap1-Nrf2 interaction, which is responsible for the expression of a battery of genes encoding antioxidant proteins and anti-inflammatory enzymes. 13,14 Nrf2 is a critical transcription factor in protecting melanocytes from oxidative damage. 15 Dysfunction of the Nrf2 signalling pathway may result in increased sensitivity of vitiligo melanocytes to H 2 O 2 -induced oxidative insult. 6 High-mobility group box 1 (HMGB1) is a highly conserved nuclear protein involved in transcriptional activation and DNA folding in eukaryotic cells. 16

| Skin specimens and ethics statement
Normal skin specimens (for primary human melanocyte culture) were obtained from six healthy individuals who underwent circumcision.
In this study, foreskin was from Asiatic-Chinese yellow race donors.
The study was performed in accordance with the Declaration of Helsinki (1964) and its later amendments, and the protocol was approved by the Clinic Research Ethics Board of the First Affiliated Hospital of Xi'an Jiaotong University. All participants gave written informed consent prior to inclusion in the study.

| NHEM cultures
The epidermis was separated from the dermis after an overnight incu- It must be noted that melanocytes used for the experiments were lightly pigmented and dendritic morphology did not change significantly during relatively short periods of incubation including 6 and 12 hours after 0.5 mM H 2 O 2 treatment ( Figure S1).

| Antibodies and reagents
In this study, we used rabbit anti-human HMGB1, anti-human p62,

| Small interfering RNA gene silencing
The

| Statistical analysis
Student's t test was used to determine significance of between-group differences. The dose-response analyses were conducted by one-way ANOVA with Dunnett's multiple comparison test. All values are expressed as means ± SD, and differences were considered statistically significant when P < 0.05. All statistical analysis were performed by the GraphPad Prism (GraphPad Software Inc, San Diego, CA, USA).

| HMGB1 protein expression in NHEMs
The expression of HMGB1 in NHEMs is shown in Figure 1.
Immunofluorescence staining with anti-HMGB1 showed that HMGB1 is predominantly located in the nucleus of NHEMs.

| Cell viability after H 2 O 2 treatment
To investigate the viability of NHEMs in the presence of oxidative stress, we used H 2 O 2 to induce oxidative stress. As shown in

| H 2 O 2 treatment promotes cytoplasmic translocation and release of HMGB1 from NHEMs
HMGB1 is an abundant nuclear protein with proinflammatory activity that depends on its extranuclear function. To investigate the distribution of HMGB1 under oxidative stress, immunofluorescence staining was used with specific anti-HMGB1 antibodies. As shown in Figure 3A, untreated NHEMs primarily displayed nuclear localization of HMGB1. However, in cells treated with exogenous H 2 O 2 , there was an increase in the percentage of HMGB1 localization at the cytoplasm. To confirm HMGB1 cytoplasmic translocation, cytoplasmic and nuclear fractions of NHEMs were isolated and immunoblotted with antibodies specific for HMGB1 ( Figure 3C). Consistently, levels of HMGB1 in the cytoplasmic fractions were increased dramatically after treatment with H 2 O 2 ( Figure 3D). Furthermore, the levels of HMGB1 released into the culture medium were measured by western blot analysis ( Figure 3E)

NHEM apoptosis
HMGB1 release is a common mediator of the response to oxidative stress; consequently, we examined the role of HMGB1 in NHEMs under oxidative stress. We used siRNA targeting HMGB1, and HMGB1 knockdown efficiency was evaluated by western blotting ( Figure 4A). In addition, using flow cytometry we examined apoptosis of cells transfected with scrambled siRNA or HMGB1 siRNA 24 and 48 hours after H 2 O 2 treatment. As shown in Figure 4B, decreased apoptosis was observed in HMGB1 siRNA-transfected cells compared with scrambled siRNA-transfected cells. Collectively, these data suggest that HMGB1 plays a major role in promoting NHEM apoptosis.
F I G U R E 1 HMGB1 expression in NHEMs. Detection of HMGB1 by immunofluorescence using rabbit anti-HMGB1 antibody followed by Alexa488-conjugated secondary antibody (HMGB1, green; nucleus, blue). Scale bar = 10 μm. Three independent assays were performed F I G U R E 2 NHEM viability decreases after H 2 O 2 treatment. Melanocytes were exposed to H 2 O 2 at the indicated concentrations for 24 hours. A significant decline in cell viability was detected when the H 2 O 2 concentration was ≥0.5 mM. Data are from three independent assays and are expressed as means ± SD. **P < 0.01

| HMGB1 knockdown enhances the expression of Nrf2 and its target genes, which can be reversed by addition of H 2 O 2 -treated melanocyte supernatant or recombinant HMGB1 protein
Nrf2-ARE, a major antioxidant pathway, regulates oxidative stressrelated cytoprotective genes in melanocytes. We investigated whether knocking down HMGB1 affects the expression of Nrf2 and its down-

| HMGB1 silencing inhibits NHEM autophagy and enhances p62 expression
It has been shown that HMGB1 translocation to the cytoplasm is closely associated with HMGB1 autophagy 20 and that HMGB1 activated an autophagic response to oxidative stress. 22 The selective autophagy substrate, p62, activates the stress-responsive transcription factor, Nrf2, through inactivation of Keap1. 13 To study the mechanism by which HMGB1 knockdown changes Nrf2 and its downstream genes' expression, we evaluated the expression of LC3 and p62, two characteristic markers of cell autophagy, in control and HMGB1-knockdown cells. As shown in Figure 6A and B, H 2 O 2 increased the expression levels of LC3II in NHEM control cells. However, the LC3II expression level in the HMGB1 siRNA group decreased significantly. In contrast, the p62 expression level in the HMGB1 siRNA group was significantly higher than that in the control group ( Figure 6C and D).

| HMGB1 knockdown suppresses Nrf2-Keap1 interaction and induces p62-Keap1 interaction under oxidative stress
To study the mechanism of the HMGB1 knockdown effect on the expression of Nrf2 and its downstream genes, we used co-IP techniques

| DISCUSSION
HMGB1 is a DNA-binding nonhistone protein that acts as a transcription regulator participating in DNA replication, recombination, in the development of vitiligo and helps understand the mechanism of melanocyte death.
We further studied the mechanism of HMGB1 affecting the expression of Nrf2 and its downstream genes. Our results suggest that HMGB1 can be transferred from the nucleus to the cytoplasm under oxidative stress (Figure 3). The literature indicates that HMGB1 can activate autophagy under oxidative stress.
HMGB1 plays a location-dependent role in inducing autophagy. 20 Consistently, our results showed that after HMGB1 knockdown, autophagy was suppressed and p62 expression was increased. The competitive interaction of p62 with Keap1 can promote Nrf2 activation to play its antioxidant role. It has been reported that ATG7 knockout in mice leads to p62 accumulation, and then to enhanced expression of Nrf2 target genes in melanocytes. As shown in Figure 7, in HMGB1-deficient NHEMs, SQSTM1/p62 competed with Nrf2 for binding to its cytoplasmic anchor Keap1, leading to enhanced Nrf2 nuclear localization and transcription of its target genes. These results explain the relationship between HMGB1 and Nrf2 through autophagy.
F I G U R E 7 Nrf2 and p62 co-IP with Keap1 after HMGB1 siRNA transfection. (A) Co-IP of Nrf2 and p62 from NHEM lysates. NHEMs transfected with HMGB1 siRNA or scrambled siRNA were stimulated with H 2 O 2 (0.5 mM) for 12 hours, and whole-cell lysates were immunoprecipitated with antibodies specific for Keap1. IgG was used as a control. The precipitated complexes were separately immunoblotted with Nrf2 or p62-specific antibodies. IP, immunoprecipitation; WB, western blotting. The blots are representative of three experiments with similar results It was reported that locally produced protective molecules such as melatonin also act on NRF2 in protection of melanocytes. 27 Melatonin and its metabolites protect melanocytes from UVB-induced DNA damage and oxidative stress through activation of NRF2dependent pathways; these actions are independent of an effect on the classic membrane melatonin receptors. 28 Melatonin and its derivatives counteract the ultraviolet B radiation-induced damage in human and porcine skin ex vivo. 29 We believe that the study of the relationship between HMGB1 and melatonin is also significant for melanocytes under oxidative stress.
In conclusion, our data showed that oxidative stress may trigger autocrine HMGB1 translocation and release by melanocytes, reducing the expression of Nrf2 and its downstream antioxidant genes to induce melanocyte apoptosis. We think that this is the mechanism by which HMGB1 participates in the pathological process of vitiligo.