Abstract

Vitiligo is a depigmentation disorder that develops as a result of the progressive disappearance of epidermal melanocytes. The elevated level of amino acid metabolite homocysteine (Hcy) has been identified as circulating marker of oxidative stress and known as a risk factor for vitiligo. However, the mechanism underlying Hcy-regulated melanocytic destruction is currently unknown. The present study aims to elucidate the effect of Hcy on melanocytic destruction and its involvement in the pathogenesis of vitiligo. Our results showed that Hcy level was significantly elevated in the serum of progressive vitiligo patients. Notably, Hcy induced cell apoptosis in melanocytes via activating reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress protein kinase RNA-like ER kinase (PERK)–eukaryotic translation initiation factor 2α (eIF2α)–C/EBP homologous protein (CHOP) pathway. More importantly, folic acid, functioning in the transformation of Hcy, could lower the intracellular Hcy level and further reverse the apoptotic effect of Hcy on melanocytes. Additionally, Hcy disrupted melanogenesis whereas folic acid supplementation could reverse the melanogenesis defect induced by Hcy in melanocytes. Taken together, Hcy is highly increased in vitiligo patients at progressive stage, and our in vitro studies revealed that folic acid could protect melanocytes from Hcy-induced apoptosis and melanin synthesis inhibition, indicating folic acid as a potential benefit agent for patients with progressive vitiligo.

Introduction

Vitiligo is an acquired, chronic depigmentation disorder [1]. The primary physical symptom is patchy depigmentation on the skin, the mucous membranes and occasionally in some hair bulbs, which is characterized as loss of functioning melanocytes [2,3]. The etiology of vitiligo is complex, and metabolic alterations are central to current concepts in its pathophysiology [4].

Nonessential sulfhydryl-containing amino acid, homocysteine (Hcy) is a potential pathogenetic factor in a panel of diseases [5]. Hcy stands at the juncture of two crucial metabolic pathways. They are the one-carbon/folate cycle that provides one-carbon units for nucleotide and amino acid biosynthesis, and the metabolism of sulfur-containing amino acid that regenerates methionine and provides cysteine [6], respectively. In a recent hospital-based case–control study, we discovered that the serum levels of Hcy are significantly elevated in patients with vitiligo, and excessive Hcy increases the propensity for vitiligo [7]. Extensive researches have enrolled derailed Hcy metabolism as a critical mediator in many diseases including Alzheimer’s disease [8–10] and neural tube defects [11]. In Alzheimer’s disease, elevated Hcy level underlies neurons destruction [12,13]. It deserves noting that melanocytes have been suggested as a model for researches on diseases affecting neurons [14–16], as they share a multitude of common grounds, including their congruous origination, similar development, differentiation and even parallel signal transduction in mature tissue [17–20]. These studies underline the association between the Hcy perturbation and vitiligo, but the mechanism by which excessive Hcy dictates vitiligo development remains to be elucidated.

The endoplasmic reticulum (ER) is a specialized organelle orchestrating the synthesis, folding and transport of at least one-third of the proteins in eukaryotic cells [21], among which high-quality protein folding is essential for cell function and survival [22]. Altered ER homeostasis leads to ER stress, featured as an accumulation of unfolded or misfolded proteins in ER lumen. The ER stress elicits the unfolded protein response (UPR). The UPR adapts the cell to ER stress by activating three signaling pathways: protein kinase RNA-like ER kinase–eukaryotic translation initiation factor 2α (PERK–eIF2α), Inositol-requiring enzyme 1α–X-box binding protein 1 (IRE1α–XBP1) and activating transcription factor 6α (ATF6α) [23]. In spite of this, the UPR can also perpetuate cell death in the context of chronic or severe ER stress [24]. Previous studies have demonstrated that Hcy disrupts the formation of disulfide bonds in molecules due to its reactive sulfhydryl residue, thereby resulting in disturbance of ER function [25]. In addition, Zhang et al. also defined a critical role for Hcy in inducing apoptosis of human umbilical vein endothelial cells via ER stress [26]. Thus, we hypothesized that elevated Hcy could activate ER stress in melanocytes, which ultimately promotes melanocytes apoptosis in vitiligo.

Folic acid can be metabolized into 5-Methylenetetrahydrofolate (5-MTHF) to participate in the transformation of Hcy to methionine with methionine transferase as catalyst [27]. Hence Hcy and corresponding metabolites are bound to accumulate when folic acid is ingested deficiently or underutilized [28]. On the contrary, the supplement of folic acid facilitates Hcy diminution [29–31]. Folic acid has also reportedly viability-promoting and anti-apoptotic effects on neurons [32–34]. Accordingly, we speculated that folic acid might protect melanocytes from excessive Hcy-induced apoptosis via restoring disturbed UPR.

To test our hypothesis, we first tested the levels of serum Hcy in vitiligo and control subjects to evaluate the correlation between Hcy and vitiligo progression. After that, we determined the impact of Hcy on cell viability and apoptosis of melanocytes and further clarified the detailed signaling pathway of UPR that accounted for Hcy-induced cell apoptosis. Noteworthily, we validated folic acid could effectively inhibit Hcy-induced melanocyte apoptosis and restore melanin synthesis.

Materials and methods

Patients and samples

Peripheral blood was collected from 90 patients given a diagnosis of vitiligo according to clinical and Wood’s lamp test in the Department of Dermatology, Xijing Hospital. Only Han Chinese subjects (accounting for approximately 90% in China) were recruited to this research. All these patients recruited for this trial involved different disease activity, disease type, body surface area, family history, gender and age. Exclusion criteria included taking any systemic or topical vitiligo therapy for at least 3 months, receiving folic acid, vitamin B6, B12, hormonal therapy of drugs that might interfere with Hcy levels such as methotrexate, anticonvulsants, lipid lowering drugs, isotretinoin, penicillamine, metformin and insulin. After clinical examination, patients with non-segmental vitiligo were classified as mucosal, acral, facial, generalized, universal according to vitiligo global issues consensus conference classification [35]. Control blood samples were obtained from 90 age- and sex-matched healthy subjects during plastic surgery in Xijing Hospital. The frequency distributions of selected characteristics of the case and control are shown in Table 1. Perilesional skin samples were collected from five vitiligo patients at progressive stage by performing skin biopsy, and healthy skin samples were donated by five volunteers who received plastic surgery at our hospital. All these subjects are informed the destination and meaning of this trial and they consented by writing informed agreement. Before initiation of the present study, all samples were identified. The investigational protocol was approved by the Ethics Committee of the Fourth Military Medical University, according to the Declaration of Helsinki principles.

Table 1
Distribution of selected variables in case of vitiligo and controls
Case (n=90)Controls (n=90)
Average age (years, mean± SD) 27.92 ± 11.76 29.20 ± 10.41 
Female/male, n (%) 47 (52.2)/43 (47.8) 38 (42.2)/52 (57.8) 
With/without family history, n (%) 18 (20)/72 (80)  
Stable/progressive, n (%) 14 (15.6)/76 (84.4)  
Body surface area, n (%)   
<1% 50 (55.6)  
1–5% 22 (24.4)  
5–50% 12 (13.3)  
>50% 6 (6.7)  
Disease type, n (%)   
Mucosal 3 (3.3)  
Acral 8 (8.9)  
Facial 13 (14.5)  
Generalized 63 (70)  
Universal 3 (3.3)  
Case (n=90)Controls (n=90)
Average age (years, mean± SD) 27.92 ± 11.76 29.20 ± 10.41 
Female/male, n (%) 47 (52.2)/43 (47.8) 38 (42.2)/52 (57.8) 
With/without family history, n (%) 18 (20)/72 (80)  
Stable/progressive, n (%) 14 (15.6)/76 (84.4)  
Body surface area, n (%)   
<1% 50 (55.6)  
1–5% 22 (24.4)  
5–50% 12 (13.3)  
>50% 6 (6.7)  
Disease type, n (%)   
Mucosal 3 (3.3)  
Acral 8 (8.9)  
Facial 13 (14.5)  
Generalized 63 (70)  
Universal 3 (3.3)  

Measurement of serum Hcy level

The whole blood of each subject was allowed to coagulate before centrifugation (2000×g for 15 min at 4°C). The serum was collected and stored at −80°C until analysis. The Hcy concentration in serum was measured by enzyme-linked immunosorbent assay (ELISA) using an Hcy assay kit (Yaji Biosystems, China) according to the manufacturer’s instructions. The results were calculated from the standard concentration curve provided by the ELISA kit.

Cell culture and treatment

PIG1, an immortalized human epidermal melanocyte cell line (a gift from Dr Caroline Le Poole, Loyola University Chicago, Maywood, IL) and normal human melanocytes (NHMs) from foreskin tissues obtained during circumcision surgery were cultured in Medium 254 (Gibco, Grand Island, NY, U.S.A.) added with human melanocytes growth supplement (Gibco) at 37°C amid 5% CO2 as previously studied [36,37]. The samples were obtained with the approval of the Institutional Review Board of Fourth Military Medical University, Xi’an, China and the informed consents were written by all donors in the direction under the Declaration of Helsinki. The concentrations of Hcy applied to induce effective cell response ranged from 2.5 to 15 mM and the appropriate concentration used for following experiments was 7.5 mM. To determine the therapeutic effect of folic acid on Hcy detriment, folic acid was added to Hcy-pretreated melanocytes at indicated concentrations and time.

Investigation of cell viability and cell proliferation

The CCK8 assay kits (Beyotime Biotechnology, China) were used as a qualitative index of cell viability according to the manufacturer’s instructions. In general, cells were seeded in 96-well culture plates at a density of 3000 cells per well and incubated for 24 h. And then, cells were pretreated with (Hcy only, folic acid only, or folic acid treatment for 12 h prior to Hcy treatment) at the indicated time and concentration. Next, culture medium in each well was replaced by 90 μl fresh medium adding 10 μl of CCK8 solution for 2 h at 37°C and optical density (OD) was measured at 450 nm by Model 680 Microplate Reader (Bio-Rad, U.S.A.).

Western blot analysis

Thirty micrograms of total protein were subjected to electrophoresis on a 10% SDS/PAGE (Bio-Rad Laboratories) and transferred to polyvinylidene difluoride membranes (PVDF membranes) (Millipore, Billerica, MA). Then the blotted membranes were blocked in a solution of 5% fat-free dried milk diluted in Tris-buffered saline for 48 h. Sequentially, the membranes were incubated with respective primary antibodies at 4°C overnight. After washing using TBST in rotating apparatus for three times with an interval of 10 min, the membranes were incubated with horseradish–conjugated secondary antibodies. After washing, amounts of the protein expression were detected by chemiluminescence detection kit (KPL, Gaithersburg, MD).

Immunofluorescence analysis

Paraffin-embedded 5-μm skin sections were incubated with the primary antibodies (Supplementary Table S1) for 24 hours at 4°C after antigen retrieval with Tris-EDTA buffer (pH 9.0). After being washed, the secondary antibodies involving goat anti-rabbit Cy3 (CWBio, Beijing, China), goat anti-mouse Alexa Fluor 488 (CWBio, Beijing, China) were used for 60 min in dark. The nuclei were stained with Hoechst 33258 (1:1000, Sigma–Aldrich, Germany) for 10 min in dark. Fluorescence analysis was applied with laser scanning confocal microscopy (Olympus, Japan). For cell samples, to determine the therapeutic function of folic acid targeting the detriment of Hcy, we fixed cells disposed by matched stimulants with 4% paraformaldehyde for 10 min. After being washed, cells were incubated with 0.1% Triton X-100 for 10 min at room temperature. Then cells were incubated with primary antibodies (Supplementary Table S1) overnight at 4°C after being washed with phosphate buffer saline (PBS). The following procedures are in line with the aforementioned histological immunofluorescence methods.

Flow cytometry to measure apoptosis

After indicated treatment, cells were collected and washed with PBS. Then the apoptotic cells were measured by Annexin V-FITC/PI kit (7 Seabiotech, China) according to the manufacturer’s instructions. After incubation for indicated time, cells were placed in flow cytometry (Beckman Coulter, U.S.A.), and next Expo32 software (Beckman Coulter, U.S.A.) was used to analyze the results of apoptosis.

Reactive oxygen species measurements

After the indicated treatment, cells were trypsinized, resuspended in PBS containing 10 μM of 2,7-Dichlorodihydrofluorescein diacetate (Invitrogen, Carlsbad, U.S.A.), and incubated at 37°C for 30 min. The cells were washed twice and resuspended in 200 μl PBS. Levels of fluorescence were measured by flow cytometry analysis (Beckman Coulter, U.S.A.).

Short hairpin RNA knockdown

Cells were seeded at 2.5 × 105 cells per well for 24 h and then the human PERK short hairpin RNA (shRNA) plasmid, IRE1α shRNA plasmid and control shRNA plasmid were used to transfect PIG1 cells, as described by Li et al. [38]. Transfection were carried out using Lipofectamine 3000 and P3000 (Invitrogen, Carlsbad, U.S.A.) according to the manufacturer’s instructions.

Quantification of melanin

Quantification of melanin was evaluated as previous study by Park et al. [39]. Cells were washed twice with PBS, detached by incubation with trypsin/EDTA and collected by centrifugation. Thereafter, 5 × 105 cells were solubilized in 100 μl of 1 N NaOH-10% DMSO at 80°C for 2 h, and the melanin content of the solution was determined by measuring the absorbance at 405 nm and calculated from a standard concentration curve generated from synthetic melanin.

Tyrosinase activity assays

Tyrosinase (TYR) activity was evaluated by quantifying the 3,4-Dihydroxy-L-phenylalanine (L-DOPA) oxidation rate as reported earlier by Shi et al. [40]. Cells were washed with PBS for three times. Lysis buffer 100 μl containing 50 mM PBS (90 μl, pH 6.8), 0.1 mM PMSF (5 μl) and 1% Triton X-100 (5 μl) were added to the cells and the mixture was frozen at −80°C for 30 min to disintegrate cells completely. Cell lysates were centrifuged at 12000 rpm for 30 min at 4°C. The supernatant (80 μl) of the lysates was mixed with 20 μl of L-DOPA (10 mM) in a 96-well plate and incubated for 30 min at 37°C. Using a microplate reader (Thermo Fisher Scientific Inc, U.S.A.), the ODs of the solutions were measured at 500 nm.

Statistics analyses

The GraphPad Prism version 7.0 software (GraphPad Software, San Diego, CA, U.S.A.) was used to analyze all the data. Dual comparison was conducted with two-tailed Student’s unpaired t test. Groups of three or more were analyzed by one-way analysis of variance (ANOVA) with Dunnett’s post-test. P-values <0.05 were considered significant. The data represented the mean ± SD for at least three independent experiments.

Results

Increased expression of serum Hcy is closely related to vitiligo progression

To verify the relationship between Hcy and vitiligo, first, we detected serum Hcy levels in 90 Han Chinese vitiligo patients and 90 age- and sex-matched controls. The results indicated that serum levels of Hcy in vitiligo patients at progressive stage were significantly higher than those of control group (10.78 ± 1.82 vs. 8.90 ± 1.78 μmol.l−1; P<0.001) and slightly higher than stable group (10.78 ± 1.82 vs. 9.72 ± 1.23 μmol.l−1; P<0.05) (Figure 1A). In patients with stable vitiligo, there was no obvious difference compared with the control group (9.72 ± 1.23 vs. 8.90 ± 1.78 μmol.l−1; P>0.05) (Figure 1A).

Increased expression of Hcy is closely associated with vitiligo progression

Figure 1
Increased expression of Hcy is closely associated with vitiligo progression

(A) The level of Hcy in serum of healthy controls (n=90) and vitiligo patients (n=90) with different disease activity, analyzed by ELISA. (B) The level of Hcy in serum of healthy controls and vitiligo patients with the discrepant subtypes, analyzed by ELISA. (C) The level of Hcy in serum of vitiligo patients according to the body surface area, analyzed by ELISA. (D) The level of Hcy in serum of vitiligo patients according to the gender, analyzed by ELISA. (E) The level of Hcy in serum of vitiligo patients according to the family history, analyzed by ELISA. (F) The level of Hcy in serum of vitiligo patients according to the age, analyzed by ELISA. *P<0.05. **P<0.01. ***P<0.001. Abbreviation: ns, not significant.

Figure 1
Increased expression of Hcy is closely associated with vitiligo progression

(A) The level of Hcy in serum of healthy controls (n=90) and vitiligo patients (n=90) with different disease activity, analyzed by ELISA. (B) The level of Hcy in serum of healthy controls and vitiligo patients with the discrepant subtypes, analyzed by ELISA. (C) The level of Hcy in serum of vitiligo patients according to the body surface area, analyzed by ELISA. (D) The level of Hcy in serum of vitiligo patients according to the gender, analyzed by ELISA. (E) The level of Hcy in serum of vitiligo patients according to the family history, analyzed by ELISA. (F) The level of Hcy in serum of vitiligo patients according to the age, analyzed by ELISA. *P<0.05. **P<0.01. ***P<0.001. Abbreviation: ns, not significant.

Second, via analysis of the serum Hcy levels combined with clinical characteristics of subjects, we found the mucosal (11.66 ± 1.40 μmol.l−1), acral (10.52 ± 1.99 μmol.l−1) and generalized (10.77 ± 1.77 μmol.l−1) vitiligo patients exhibited elevated serum Hcy levels compared with the control (8.90 ± 1.78 μmol.l−1), but there is no significant discrepancy in facial (9.70 ± 1.62 μmol.l−1) and universal vitiligo (10.42 ± 2.17 μmol.l−1), respectively, in comparison with controls (Figure 1B). Moreover, serum Hcy levels might impact the lesional area when affected body surface area is less than 5% in vitiligo patient. But there is no above observation, when the body surface area is more than 5% (Figure 1C). Finally, no significant discrepancy of serum Hcy was observed in different clinical features such as male or female patients (10.69 ± 1.91 vs. 10.53 ± 1.65 μmol.l−1; P>0.05) (Figure 1D), presence or absence of family history (10.67 ± 1.47 vs. 10.60 ± 1.86 μmol.l−1; P>0.05) (Figure 1E), and age is irrelevant to serum Hcy levels in vitiligo patients (r = 0.049, P>0.05) (Figure 1F). Taken together, these results indicated that increased serum Hcy level is closely related to vitiligo progression.

Excessive Hcy constrains melanocyte proliferation and precipitates melanocyte apoptosis

We further explored the specific effects of excessive Hcy on melanocytes. We verified that Hcy drastically inhibited the growth of NHMs (Figure 2A) and 7.5 mM Hcy treatment for 24 h exerted apparent cytotoxicity on NHMs as presented by decreased cell viability (Figure 2B). After the same treatment, the human keratinocyte cell line (HaCaT) did not undergo obvious cytotoxicity from Hcy (Supplementary Figure S1). Moreover, Hcy treatment significantly promoted NHMs apoptosis in a dose-dependent manner (Figure 2C). In line with the cell viability assay, treatment with 7.5 mM Hcy for 24 h conspicuously elevated apoptotic ratio of melanocytes. These results suggested that Hcy could not only interfere with melanocyte proliferation but also promote melanocyte apoptosis. The treatment with Hcy lower than 10 mM did not significantly promote reactive oxygen species (ROS) generation despite causing NHMs apoptosis. Marked increment of ROS generation can be observed when Hcy concentration reaches 15 mM (Figure 2D). These results indicated that, there are other mechanisms may involve in Hcy-induced melanocyte apoptosis in addition to ROS-induced apoptosis.

Hcy inhibits melanocyte growth and promotes melanocyte apoptosis

Figure 2
Hcy inhibits melanocyte growth and promotes melanocyte apoptosis

(A) Cell proliferation capacity of NHMs disposed by Hcy in the indicated time and concentration, determined by CCK8 assay. (B) Cell viability of NHMs exposed to Hcy in different concentrations for 24 h, analyzed by CCK8 assay. (C) The apoptotic effect of NHMs after Hcy treatment for 24 h in indicated concentration, measured by flow cytometry and a statistical plot for apoptosis rate. (D) The intracellular ROS level of NHMs after Hcy treatment for 24 h in indicated concentration was quantized by flow cytometry assay and a statistical plot for ROS level. Data represent the mean ± SD of three independent experiments. *P<0.05. ***P<0.001. Abbreviation: ns, not significant.

Figure 2
Hcy inhibits melanocyte growth and promotes melanocyte apoptosis

(A) Cell proliferation capacity of NHMs disposed by Hcy in the indicated time and concentration, determined by CCK8 assay. (B) Cell viability of NHMs exposed to Hcy in different concentrations for 24 h, analyzed by CCK8 assay. (C) The apoptotic effect of NHMs after Hcy treatment for 24 h in indicated concentration, measured by flow cytometry and a statistical plot for apoptosis rate. (D) The intracellular ROS level of NHMs after Hcy treatment for 24 h in indicated concentration was quantized by flow cytometry assay and a statistical plot for ROS level. Data represent the mean ± SD of three independent experiments. *P<0.05. ***P<0.001. Abbreviation: ns, not significant.

Hcy instigates melanocyte apoptosis via ER stress PERK–eIF2α–CHOP pathway

To gain insight into the mechanism of Hcy-mediated melanocytes apoptosis, we first determined the activation of ER stress-related protein in NHMs treated with Hcy. We noted that Hcy up-regulated the ER stress response gene glucose-regulated protein 78 kDa (GRP78) as the concentration of Hcy rose, conforming activation of ER stress. Next, we found Hcy treatment augmented phospho-PERK and phospho-eIF2α expression rather than that of total PERK and eIF2α (Figure 3A). Besides, phospho-IRE1α and splicing of XBP1 also highly expressed in proportion to the increased Hcy (Figure 3B), and the expression of C/EBP homologous protein (CHOP) symbolizing apoptosis under ER stress was remarkably up-regulated (Figure 3A). Results mentioned above denote that Hcy activates PERK and IRE1α pathway.

Hcy promotes melanocyte apoptosis via ER stress PERK–eIF2α–CHOP pathway

Figure 3
Hcy promotes melanocyte apoptosis via ER stress PERK–eIF2α–CHOP pathway

(A) GRP78, PERK, phospho-PERK, eIF2α, phospho-eIF2α and CHOP expression in NHMs cells treated with Hcy in indicated concentration for 24 h, detected by Western blot. (B) IRE1α, phospho-IRE1α, XBP1, XBP1s expression in NHMs cells disposed with Hcy at indicated concentration for 24 h, determined by Western blot. (C) The apoptosis of PIG1 transfected with sh-PERK plasmid and sh-IRE1α plasmid for 24 h prior to the stimulation of Hcy for 24 h, determined by flow cytometry assay. Data represent the mean ± SD of three independent experiments. The corresponding statistical results are on the right. *P<0.05. **P<0.01. ***P<0.001. Abbreviation: ns, not significant. (D) The expression of phospho-PERK in melanocytes of perilesional skin from progressive vitiligo patients detected by immunofluorescence assay. Scale bar = 50 μm.

Figure 3
Hcy promotes melanocyte apoptosis via ER stress PERK–eIF2α–CHOP pathway

(A) GRP78, PERK, phospho-PERK, eIF2α, phospho-eIF2α and CHOP expression in NHMs cells treated with Hcy in indicated concentration for 24 h, detected by Western blot. (B) IRE1α, phospho-IRE1α, XBP1, XBP1s expression in NHMs cells disposed with Hcy at indicated concentration for 24 h, determined by Western blot. (C) The apoptosis of PIG1 transfected with sh-PERK plasmid and sh-IRE1α plasmid for 24 h prior to the stimulation of Hcy for 24 h, determined by flow cytometry assay. Data represent the mean ± SD of three independent experiments. The corresponding statistical results are on the right. *P<0.05. **P<0.01. ***P<0.001. Abbreviation: ns, not significant. (D) The expression of phospho-PERK in melanocytes of perilesional skin from progressive vitiligo patients detected by immunofluorescence assay. Scale bar = 50 μm.

Afterward, we found IRE1α pathway interference did not work out on melanocytes apoptosis, whereas the level of apoptosis declined remarkably after PERK pathway inhibition (Figure 3C). Moreover, we performed histological immunofluorescence assay to further confirm the expression of phospho-PERK in melanocytes of perilesional skin from progressive vitiligo patients, and data presented an elevated expression compared with that in healthy controls (Figure 3D). These results indicated that Hcy elicited ER stress in melanocytes of vitiligo and could mediate melanocyte apoptosis via PERK–eIF2α–CHOP pathway.

Folic acid treatment mitigates melanocyte apoptosis induced by Hcy

As mentioned above, folic acid serves as cofactor of Hcy methyltransferase for the conversion of methionine from Hcy. Accordingly, we detected whether folic acid application could effectively reduce intracellular Hcy levels thus mitigating ER stress and reducing apoptosis. According to the proliferation and viability assay, folic acid concentrations below 0.5 mM were labeled safe for NHMs (Figure 4A). Additionally, 0.25–0.5 mM folic acid treatment of NHMs for 72 h slightly increased melanocyte viability (Figure 4B).

Folic acid treatment reduces melanocyte apoptosis induced by Hcy

Figure 4
Folic acid treatment reduces melanocyte apoptosis induced by Hcy

(A) The proliferation capacity of NHMs treated with folic acid in indicated time and concentrations, analyzed by CCK8 assay. (B) Cell viability of NHMs treated with folic acid at different concentrations for 72 h, determined by CCK8 assay. (C) The contents of Hcy in Hcy-treated NHMs pretreated with folic acid for 12 h and the matched statistical results of fluorescence intensity. Scale bar = 10 μm. (D) The expression of PERK, phospho-PERK, eIF2α, phospho-eIF2α and CHOP in folic acid-pretreated NHMs prior to Hcy stimulation, detected by Western blot. The β-actin was detected as loading control. (E) The proliferation capacity of NHMs treated with Hcy for the indicated time after pretreated with folic acid for 12 h, detected by CCK8 assay. (F) The apoptosis in Hcy-treated NHMs with folic acid pretreatment, determined by flow cytometry. Data represent the mean ± SD of three independent experiments. The statistical result of apoptosis rate is on the right. *P<0.05. **P<0.01. ***P<0.001. Abbreviation: ns, not significant.

Figure 4
Folic acid treatment reduces melanocyte apoptosis induced by Hcy

(A) The proliferation capacity of NHMs treated with folic acid in indicated time and concentrations, analyzed by CCK8 assay. (B) Cell viability of NHMs treated with folic acid at different concentrations for 72 h, determined by CCK8 assay. (C) The contents of Hcy in Hcy-treated NHMs pretreated with folic acid for 12 h and the matched statistical results of fluorescence intensity. Scale bar = 10 μm. (D) The expression of PERK, phospho-PERK, eIF2α, phospho-eIF2α and CHOP in folic acid-pretreated NHMs prior to Hcy stimulation, detected by Western blot. The β-actin was detected as loading control. (E) The proliferation capacity of NHMs treated with Hcy for the indicated time after pretreated with folic acid for 12 h, detected by CCK8 assay. (F) The apoptosis in Hcy-treated NHMs with folic acid pretreatment, determined by flow cytometry. Data represent the mean ± SD of three independent experiments. The statistical result of apoptosis rate is on the right. *P<0.05. **P<0.01. ***P<0.001. Abbreviation: ns, not significant.

To investigate the effect of folic acid on Hcy-treated melanocytes, treatment of folic acid was administrated to NHMs before Hcy treatment. Of note, we confirmed folic acid pre-treatment attenuated the content of Hcy by immunofluorescence staining (Figure 4C). Following that, we determined whether folic acid could reduce Hcy-induced apoptosis mediated by PERK–eIF2α–CHOP pathway. Through Western blot assay, we found that pretreated with folic acid, melanocytes displayed markedly diminished expression of phospho-PERK, phospho-eIF2α and CHOP, which were elevated due to disposing of Hcy (Figure 4D). Moreover, treatment with 0.5 mM folic acid phenomenologically promoted the proliferation capacity of NHMs in response to 7.5 mM Hcy (Figure 4E). The same disposes also reduced the apoptosis rate of melanocytes (Figure 4F). These results indicated the positive effects of folic acid on reversing the undermining of Hcy in melanocytes, which was possibly attributed to hindering the PERK–eIF2α–CHOP pathway.

Folic acid restores Hcy-disrupted melanin synthesis

Apart from the apoptosis of melanocytes, vitiligo also features melanin synthesis defect. Accordingly, we studied the effect of excessive Hcy on melanogenesis in melanocytes. We found Hcy treatment reduced expression of melanin synthesis-related molecules such as microphthalmia-associated transcription factor (MITF), TYR and TYR-related protein 1 (TYRP1) in a dose-dependent manner (Figure 5A). Simultaneously, TYR activity (Figure 5B) and melanin content (Figure 5C) declined under the same circumstance. We proceeded to study the effect of folic acid on melanin synthesis-related molecules. Noteworthily, folic acid effectively enhanced melanin synthesis-related molecular expression (Figure 5D), restored TYR activity (Figure 5E) and increased the synthesis of melanin (Figure 5F). These results demonstrated that excessive Hcy could disrupt melanogenesis and supplementation of folic acid could restore melanin synthesis in melanocytes.

Folic acid has a protective effect on melanin synthesis by increasing the expression of melanin-related molecules under Hcy treatment

Figure 5
Folic acid has a protective effect on melanin synthesis by increasing the expression of melanin-related molecules under Hcy treatment

(A) The expression of MITF, TYR, TYRP1 and melan A in NHMs treated with Hcy at different concentrations, verified by Western blot. (B) TYR activity and (C) melanin content in NHMs treated with Hcy at different concentrations were measured by microplate reader. (D) The expression of MITF, TYR, TYRP1 and melan A in Hcy-treated NHMs with the pretreatment of folic acid for 12 h, verified by Western blot. (E) TYR activity and (F) melanin content in Hcy-treated NHMs with the pretreatment of folic acid for 12 h, measured by microplate reader. Data represent the mean ± SD of three independent experiments. *P<0.05. **P<0.01. ***P<0.001. Abbreviation: ns, not significant.

Figure 5
Folic acid has a protective effect on melanin synthesis by increasing the expression of melanin-related molecules under Hcy treatment

(A) The expression of MITF, TYR, TYRP1 and melan A in NHMs treated with Hcy at different concentrations, verified by Western blot. (B) TYR activity and (C) melanin content in NHMs treated with Hcy at different concentrations were measured by microplate reader. (D) The expression of MITF, TYR, TYRP1 and melan A in Hcy-treated NHMs with the pretreatment of folic acid for 12 h, verified by Western blot. (E) TYR activity and (F) melanin content in Hcy-treated NHMs with the pretreatment of folic acid for 12 h, measured by microplate reader. Data represent the mean ± SD of three independent experiments. *P<0.05. **P<0.01. ***P<0.001. Abbreviation: ns, not significant.

Discussion

Excessive Hcy confers risks for developing vitiligo, with the exact role of Hcy remaining under-recognized in vitiligo pathogenesis [7]. Our study demonstrated that Hcy not only induces melanocyte apoptosis through the PERK–eIF2α–CHOP signaling pathway, but also inhibits melanocyte melanin synthesis. Therapeutically, folic acid curtails Hcy-induced melanocytes apoptosis and restores melanin synthesis (Figure 6).

Potential mechanism of Hcy regulated melanocytes apoptosis and folic acid mitigated melanocyte apoptosis induced by Hcy

Figure 6
Potential mechanism of Hcy regulated melanocytes apoptosis and folic acid mitigated melanocyte apoptosis induced by Hcy

Excessive Hcy can induce ER stress and activate PERK–eIF2α–CHOP pathway, which leads to the dysfunction of melanocytes and facilitates melanocytes apoptosis. Folic acid supplementation can inhibit activation of the PERK–eIF2α–CHOP pathway, further promotes melanocyte survival and restores melanin synthesis.

Figure 6
Potential mechanism of Hcy regulated melanocytes apoptosis and folic acid mitigated melanocyte apoptosis induced by Hcy

Excessive Hcy can induce ER stress and activate PERK–eIF2α–CHOP pathway, which leads to the dysfunction of melanocytes and facilitates melanocytes apoptosis. Folic acid supplementation can inhibit activation of the PERK–eIF2α–CHOP pathway, further promotes melanocyte survival and restores melanin synthesis.

Based on the case–control study, we confirmed that Hcy is highly expressed in the serum of progressive vitiligo patients. These results reiterate conclusions from the pilot study of Tsai et al. and Shaker et al. [41,42]. We also found the mucosal, acral and generalized vitiligo patients exhibited elevated serum Hcy levels compared with the controls. Putative explanation of findings above might be that Hcy metabolism depends on folic acid and vitamin B12, and both decline in patients with vitiligo, which further elicits accumulation of Hcy [43]. In addition, some genes encoding the key enzymes in the metabolism of Hcy such as Methylenetetrahydrofolate reductase (MTHFR), methionine synthase reductase (MTRR) and cystathionine β-synthase enzyme (CBS) have been identified as the susceptible genes for vitiligo [44,45]. Defective enzymes-induced excessive accumulation of Hcy could possibly underlie vitiligo [44,45]. Consistently, previous studies unveiled that vitiligo patients showed lower activity of MTHFR and higher levels of Hcy than the controls [7]. The abovementioned conspicuously illustrated the tight relationship between Hcy and vitiligo.

Hcy-induced oxidative stress is substantiated to participate in a multitude of diseases pathogenesis, such as Alzheimer’s disease, Parkinson’s disease [46] and heart failure [47]. Oxidative stress is central to the event characterizing vitiligo, the melanocyte destruction. And most importantly, there exists a tight relationship between Hcy and vitiligo as shown in the last part. Insights gleaned from these studies prompted us to excavate the postulated relation between Hcy, oxidative stress and melanocyte destruction. Intriguingly, in higher Hcy concentration, stimulated melanocytes exhibited drastic intracellular oxidative stress and apoptosis. Under relatively low Hcy concentration, melanocyte apoptosis occurred independent of intracellular ROS level. Hence, apart from oxidative stress, other mechanisms might also be responsible for Hcy-associated melanocyte destruction in vitiligo skin.

To investigate the underlying mechanisms of Hcy detriment on melanocytes, we performed in vitro assay and validated the growth-inhibition and pro-apoptosis effects excessive Hcy exerts on melanocytes. Also, GRP78, as an ER-chaperone protein and recognized as an exclusive marker of ER stress in the process of modulating ER dynamic homeostasis, is up-regulated remarkably. It has been reported that PERK–eIF2α and IRE1α–XBP1 all exert apoptosis-inducing functions downstream GRP78 [48]. PERK, as a transmembrane protein containing a stress-sensing domain that faces the ER lumen, is an indispensable component of the UPR. The overload of misfolded proteins in ER lumen triggers the dissociation of PERK from GRP78, and the consequent oligomerization and autophosphorylation of PERK, which further yield the phosphorylation of eIF2α [49]. Sequentially, activation of CHOP, also known as growth-arrest and DNA-damage-inducible gene 153 in the downstream of eIF2α, contributes to apoptosis by down-regulating anti-apoptotic factor Bcl-2 [50,51]. Interestingly, we found that the PERK–eIF2α branch is of more significance than IRE1α–XBP1 branch in response to excessive Hcy, underpinning the predominant role of PERK–eIF2α–CHOP in Hcy-induced melanocyte apoptosis. In melanocytes, critical suppression of Hcy on melanin content has been observed, and repression of melanin-related molecules including MITF, TYR and TYRP1, confers an explainable mechanism for this phenomenon. Specifically, Reish et al. reported that Hcy could significantly inhibit TYR activity by directly binding to copper functioning in its active site [52].

Folic acid is an essential cofactor in Hcy metabolism. 5-MTHF is the circulating form of folic acid, serving as the methyl donor in the conversion of Hcy into methionine. Insufficient folic acid intake or utilization may cause Hcy accumulation and increase the risk of vitiligo. In the present study, we confirmed folic acid supplementation could attenuate the melanocyte apoptosis and restore melanin synthesis induced by Hcy. The previous study verified folic acid in a dose equal to recommended dietary allowances (400 μg/day) is associated with a 25–30% reduction in serum Hcy. A panel of researches focusing on the therapeutic effects of folic acid on vitiligo have been conducted. Juhlin et al. [53] and Don et al. [54] reported that folic acid supplementation to the vitiligo patient combined with broadband UVB treatment or sun exposure promoted repigmentation better than either of the two exclusively. Kim et al. analyzed the long-term benefits of childhood facial vitiligo patients who received folic acid supplementation, results being half patients exhibited >50% improvement [55]. Based on related clinical studies and our findings, we conclude that folic acid therapy holds promise in the treatment of vitiligo.

These results inform our understanding of vitiligo pathogenesis associated with Hcy, and discuss the feasibility of folic acid in progressive vitiligo treatment. Effect of low-dose folic acid treatment on progressive vitiligo warrants further clarification by large-scale clinical trials. Besides, the potential of folic acid fortification to decrease vitiligo risk in the general population merits more detailed analysis.

Clinical perspectives

  • Nonessential sulfhydryl-containing amino acid Hcy is higher expressed in serum of vitiligo patients and the excessive Hcy increases the susceptibility to suffering vitiligo. However, the mechanism of Hcy in the vitiligo pathogenesis is currently unknown.

  • We demonstrated that serum Hcy is highly increased in vitiligo patients at progressive stage, and in vitro, Hcy could inhibit the synthesis of melanin and aggravate apoptosis of melanocytes via the PERK–eIF2α–CHOP pathway.

  • Our in vitro study demonstrated that folic acid could protect melanocytes from Hcy-induced apoptosis and melanin synthesis inhibition, indicating folic acid as a potential benefit agent for progressive vitiligo patients with increased Hcy levels.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

This work was supported by National Natural Science Foundation of China [grant numbers 81930087, 81703096, 91742201, 81703128, 81773315]; the National Defense Science and Technology Excellence Youth Talent Foundation of China [grant number 2018-JCJQ-ZQ-006]; and the Academic Boost Plan of Xijing Hospital [grant number XJZT18Z07].

Author Contribution

Shuli Li and Chunying Li designed the study. Jiaxi Chen, Tongtian Zhuang, Jianru Chen, Shuli Li and Chunying Li wrote and revised the manuscript. Jiaxi Chen and Xiuli Yi performed most of the experiments. Yangzi Tian, Qingrong Ni and Weigang Zhang collected the samples from patients and controls. Pu Song, Zhe Jian and Ling Liu performed statistical analysis. Tingting Cui and Kai Li assembled the figures. Tianwen Gao reviewed and revised the design of the study.

Acknowledgements

We thank Dr. Sen Guo and Dr. Jingjing Ma in the Department of Dermatology, Xijing Hospital, Fourth Military Medical University for technical assistance.

Abbreviations

     
  • 5-MTHF

    5-methylenetetrahydrofolate

  •  
  • ATF6α

    activating transcription factor 6α

  •  
  • CBS

    cystathionine β-synthase

  •  
  • CCK8

    cell counting kit-8

  •  
  • CHOP

    C/EBP homologous protein

  •  
  • C/EBP

    CCAAT enhancer-binding protein

  •  
  • eIF2α

    eukaryotic translation initiation factor 2α

  •  
  • ELISA

    enzyme-linked immunosorbent assay

  •  
  • ER

    endoplasmic reticulum

  •  
  • GRP78

    glucose-regulated protein 78 kDa

  •  
  • Hcy

    homocysteine

  •  
  • IRE1α

    inositol-requiring enzyme 1α

  •  
  • L-DOPA

    3,4-Dihydroxy-L-phenylalanine

  •  
  • MITF

    microphthalmia-associated transcription factor

  •  
  • MTHFR

    methylenetetrahydrofolate reductase

  •  
  • MTRR

    methionine synthase reductase

  •  
  • NHM

    normal human melanocyte

  •  
  • OD

    optical density

  •  
  • PBS

    phosphate buffer saline

  •  
  • PERK

    protein kinase RNA-like ER kinase

  •  
  • ROS

    reactive oxygen species

  •  
  • TYR

    tyrosinase

  •  
  • TYRP1

    tyrosinase-related protein 1

  •  
  • UPR

    unfolded protein response

  •  
  • XBP1

    X-box binding protein 1

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Author notes

*

These authors contributed equally to this work.