Dysregulated autophagy increased melanocyte sensitivity to H2O2-induced oxidative stress in vitiligo

In vitiligo, melanocytes are particularly vulnerable to oxidative stress owing to the pro-oxidant state generated during melanin synthesis and to the genetic antioxidant defects. Autophagy is a controlled self-digestion process which can protect cells against oxidative damage. However, the exact role of autophagy in vitiligo melanocytes in response to oxidative stress and the mechanism involved are still not clear. To determine the implications of autophagy for melanocyte survival in response to oxidative stress, we first detected the autophagic flux in normal melanocytes exposure to H2O2, and found that autophagy was significantly enhanced in normal melanocytes, for protecting cells against H2O2-induced oxidative damage. Nevertheless, vitiligo melanocytes exhibited dysregulated autophagy and hypersensitivity to H2O2-induced oxidative injury. In addition, we confirmed that the impairment of Nrf2-p62 pathway is responsible for the defects of autophagy in vitiligo melanocytes. Noteworthily, upregulation of the Nrf2-p62 pathway or p62 reduced H2O2-induced oxidative damage of vitiligo melanocytes. Therefore, our data demonstrated that dysregulated autophagy owing to the impairment of Nrf2-p62 pathway increase the sensitivity of vitiligo melanocytes to oxidative stress, thus promote the development of vitiligo. Upregulation of p62-dependent autophagy may be applied to vitiligo treatment in the future.

To test our hypothesis, we first detected the autophagy flux in normal and vitiligo melanocytes exposure to H 2 O 2 . Then, we confirmed the protective effects of autophagy on melanocytes against H 2 O 2 -induced oxidative stress. Furthermore, we elucidated whether H 2 O 2 -induced Nrf2 activation contributes to autophagic activity. The underlying mechanisms were investigated as well.

H 2 O 2 -induced oxidative stress increased autophagy flux in melanocytes.
We firstly examined the autophagy levels of primary normal human melanocyte (MC) and the human melanocyte cell line (PIG1) treated with H 2 O 2 . We found the microtubule associated protein 1 light-chain 3 (LC3)-II, the most widely used biomarker of autophagosome formation, was significantly increased in MC and PIG1 cells following H 2 O 2 treatment ( Fig. 1A-C). And the conversion of LC3-I to LC3-II reached the highest level after 12 hours of H 2 O 2 exposure in both groups ( Fig. 1A-C). Subsequent ultrastructural transmission electron microscopy (TEM) analysis also showed that after exposure to H 2 O 2 for 12 hours, MC and PIG1 cells displayed many cytoplasmic vesicles that had the typical single-membrane structure of autolysosome, whereas few autophagic vacuoles were observed in control cells (Fig. 1D). To further confirm the enhancement of autophagic flux induced by oxidative stress, mRFP-GFP-LC3 tandem construc was introduced. The results showed after exposure to H 2 O 2 for 12 hours, both autophagosome (yellow puncta) and autolysosomes (red puncta) were increased in MC and PIG1 cells, whereas no significant changes were observed in control cells (Fig. 1E-G). These data demonstrated that H 2 O 2 -induced oxidative stress can induce autophagy in normal melanocytes.
Autophagy protected melanocytes from oxidative stress-induced cell apoptosis, loss of mitochondrial membrane potential and ROS generation. We next identified the role of autophagy under oxidative stress in normal melanocytes. Cells were pretreated by rapamycin (RAP) to increase autophagic flux 19 , or chloroquine (CQ) to block autophagic activity 20 in MC and PIG1 prior to treatment with H 2 O 2 . The results showed that addition of RAP significantly reduced cell apoptosis induced by H 2 O 2 , whereas CQ further promoted H 2 O 2 -induced apoptosis in both MC and PIG1 cells ( Fig. 2A-C). Moreover, by using JC-1 dye and measuring the red/green ratio, a significant decrease in mitochondrial membrane potential (MMP) was detected in both MC and PIG1 cells following exposure to H 2 O 2 for 24 hours, which were further promoted by CQ pretreatment, while the RAP rescued the cells from the loss of MMP (Fig. 2D). In addition, we investigated whether autophagy could eliminate excessive ROS induced by H 2 O 2 . As shown in Fig. 2E-G, the level of intracellular ROS induced by H 2 O 2 was significantly attenuated when cells were pretreated with RAP, whereas CQ obviously increased H 2 O 2 -induced ROS generation. Taken together, these results confirmed that autophagy play a critical role in protecting normal melanocytes from oxidative stress-induced cell apoptosis, loss of MMP and intracellular ROS generation.
Vitiligo melanocytes displayed impaired autophagy and increased sensitivity to H 2 O 2 -induced oxidative stress. To evaluate whether autophagy was involved in the higher susceptibility to oxidative stress in vitiligo melanocytes, we first measured the level of autophagy in a vitiligo melanocyte cell line (PIG3V) exposed to H 2 O 2 . The results showed that after treatment with H 2 O 2 for 12 hours, both the level of LC3-II and the ratio of LC3-II/-I were relatively lower in the PIG3V cells compared to that in PIG1 cells ( Fig. 3A-C). According to mRFP-GFP-LC3 puncta formation assays, fewer autolysosomes dots in PIG3V cells were observed following H 2 O 2 treatment for 12 hours than those of PIG1 cells (Fig. 3D). These observations suggested that vitiligo melanocytes have faint autophagic induction in response to H 2 O 2 -induced oxidative stress.
We further evaluated the effect of decreased autophagy in vitiligo melanocytes under oxidative stress by determine cell apoptosis, MMP and ROS generation. Compared to PIG1 cells, treatment of H 2 O 2 caused more increased apoptosis (Fig. 3E,F), more reduced MMP (Fig. 3G,H), and more accumulated intracellular ROS (Fig. 3I,J) in PIG3V cells. Taken together, we concluded that vitiligo melanocytes with decreased autophagyic activity were more sensitive to H 2 O 2 -induced oxidative stress.
Aberrant Nrf2-p62 signaling pathway is responsible for impaired autophagy in vitiligo melanocytes under oxidative stress. Our previous study had ascertained that Nrf2 pathway has crucial role in protecting melanocytes from oxidative damage 14 . Meanwhile, recent studies have suggested that Nrf2 pathway protects cells from various pro-oxidant stimuli also via the induction of autophagy 18 . Therefore, we investigated whether Nrf2 pathway contributes to increase autophagy in normal melanocytes. Our results showed that the knockdown of Nrf2 by shRNA significantly attenuated the levels of LC3-II induced by H 2 O 2 (Fig. 4A,B). Considering that Nrf2 contributes to the induction of p62/sequestosome1 which functions as an autophagic adaptor 21 , we examined whether the expression of p62 would be affected in normal melanocytes under H 2 O 2 -induced oxidative stress. Since p62 could also be degraded by autophagy, we detected the basic protein levels of p62 by addition of chloroquine which can block autophagic flux. As shown in Fig. 4C, Nrf2 knockdown blocked the expression of p62 in normal melanocytes. Furthermore, we restored the protein level of p62 in Nrf2 knockdown cells under oxidative stress, and observed that the declined LC3-II protein levels were rescued (Fig. 4D,E). Consistently, mRFP-GFP-LC3 puncta formation assays detected attenuated autolysosome puncta in Nrf2-knockdowned cells, whereas the number of the puncta significantly increased in these cells after over-expression of p62 (Fig. 4F,G). These data indicated that H 2 O 2 -induced autophagy can be regulated by the Nrf2-p62 pathway in normal melanocytes.
We found both the nuclear Nrf2 translocation and basic protein levels of p62 failed to increase in response to H 2 O 2 -treated PIG3V cells compared to normal melanocytes (Fig. 4H,I). indicating an aberrant Nrf2-p62 signaling pathway in vitiligo melanocytes. Therefore, we further investigated whether the aberrant Nrf2-p62 pathway was the culprits for the damaged autophagy in vitiligo melanocytes under oxidative stress. Interestingly, upregulation Scientific RepoRts | 7:42394 | DOI: 10.1038/srep42394 of Nrf2 expression increased the protein levels of LC3-II in PIG3V cells after exposure to H 2 O 2 (Fig. 5A,B). The increased LC3-II was correspondingly detected in p62-overexpressed PIG3V cells during oxidative stress     (Fig. 5C,D). Moreover, increased autolysosome puncta were detected in both Nrf2-and p62-overexpressed PIG3V cells in response to H 2 O 2 (Fig. 5E,F). Taken together, these results proved that aberrant Nrf2-p62 signaling pathway is responsible for impaired autophagy in vitiligo melanocytes under oxidative stress.

Upregulation of p62 reduces cytotoxicity of vitiligo melanocytes under H 2 O 2 -induced oxidative stress.
Finally, we try to investigate whether upregulation of p62 expression could reduce the cytotoxicity only by promoting autophagy in vitiligo melanocytes under oxidative stress. Of note, our results showed that upregulation of p62 significantly reduced cell apoptosis (Fig. 6A,B), restored the loss of MMP (Fig. 6C,D), and eliminated excessive intracellular ROS (Fig. 6E,F) in PIG3V cells and in Nrf2-knockdown cells treated with H 2 O 2 . All the above data demonstrated that even upregulation of p62 can independently protect vitiligo melanocytes against H 2 O 2 -induced oxidative stress.

Discussion
In this study, we firstly found that H 2 O 2 -induced oxidative stress enhances autophagic flux in primary human melanocytes against oxidative damage, and then established that Nrf2-p62 is an essential pathway in protecting melanocytes against H 2 O 2 -induced oxidative damage. Moreover, we confirmed that vitiligo melanocytes under H 2 O 2 -induced oxidative stress showed lower autophagic flux owing to the dysfunction of Nrf2-p62 pathway, which resulted in increased susceptibility to excessive ROS (Fig. 7). Giving the first evidence, our data pointed that the enhancement of autophagic activity is sufficient to reduce the oxidative damage in vitiligo melanocytes. Modification of autophagy may provide a promising therapy for vitiligo. Autophagy is a key catabolic process that helps to maintain cell homeostasis under oxidative stress 19,22,23 . Because of the process of melanogenesis, excessive ROS is accumulated in melanocytes. Recently, Zhang et al. has reported their research on the autophagy process in melanocytes by detecting LC3, a classical protein marker for autophagy. Their study demonstrates that autophagy functions as a self-defense mechanism in mouse melanocytes against oxidative stress 13 . Since LC3 could be found in melanosomes, our study also used ultrastructural TEM to observe direct changes of autophagy flux in melanocytes, trying to exclude the interference of melanosomes when detecting autophagy using LC3 only. Noteworthily, the results of our TEM assay were always consistent with the ratio of LC3II/I detected by western blot in our study, indicating that using LC3 to detect autophagy in melanocytes is still feasible in spite of little expression of LC3 in melanosomes. Our results provide the first evidence that autophagy occurs to alleviate the oxidative damage as a part of cellular repair process in normal human melanocytes. Therefore, in addition to classical antioxidant system such as antioxidant genes or enzymes 7 , autophagy can protect melanocytes from oxidative stress, and consequently contribute to the cell homeostasis.
Our previous study has demonstrated that vitiligo melanocytes equipped with impaired antioxidant system, including the genetic defects of antioxidant enzymes 9 , dysfunctional Nrf2-HO1 antioxidant signaling pathway 10 , as well as aberrant expression of miRNAs 24 . In the current study, we confirmed that defects in autophagy could increase the apoptotic death and raise the intracellular ROS levels, as well as reducing the MMP in vitiligo melanocytes. These observations provide new evidences to explain why vitiligo melanocytes are more vulnerable to H 2 O 2 -induced oxidative damage compared to normal melanocytes.
Recently, several lines of evidence support an essential role for Nrf2 in the induction of autophagy, but the underlying mechanisms are still not fully understood 17,18 . It is well known that during autophagy, intracellular proteins and organelles can be banded to autophagosome by the adaptor protein p62 25 , which can be induced by the activation of Nrf2 under oxidative stress 21 . Moreover, Nrf2-mediated induction of p62 has been implicated in controlling Toll-like receptor-4 (TLR4)-driven selective autophagy structure formation and autophagic degradation 26 , as well as in cigarette smoke-induced autophagy in airway epithelial cells 17 . In this study, we also identified that Nrf2-mediated p62 upregulation is required for oxidative stress-induced autophagy in normal human melanocytes.
However, unlike normal melanocytes, both the activation of Nrf2 and the basic protein levels of p62 failed to increase in response to oxidative stress in vitiligo melanocytes. Notably, upregulation of Nrf2-p62 pathway increased the autophagic activity in vitiligo melanocytes, and therefore contributed to cell survival. These data suggested that the impairment of Nrf2-p62 pathway is responsible for the defects of autophagy in vitiligo melanocytes. Although several studies have reported that p62 could protect cells from oxidative damage via activation of Nrf2 27,28 , our current study demonstrated that even without activation of Nrf2, upregulation of autophagic flux by increasing p62 is sufficient to protect vitiligo melanocytes from H 2 O 2 -induced oxidative damage. These findings strengthen the importance of p62-dependent autophagy in melanocytes survival, providing a potential strategy for vitiligo treatment.
It has to be pointed out that only one vitiligo melanocyte cell line (PIG3V) was used in the current study. Considering that the genetic backgrounds vary among vitiligo patients, the cell line that we used can not completely represent the features of vitiligo melanocytes. Our findings need to be validated in other melanocyte lines extracted from vitiligo patients in future studies.
In conclusion, our study uncovers an essential role for autophagy in protecting normal human melanocytes from oxidative stress. Importantly, the dysregulation of Nrf2-p62 pathway causes the defects of autophagy under the condition of oxidative stress, which contributes to enhanced sensitivity of vitiligo melanocytes to oxidative stress. Our findings indicate a prospective treatment of upregulating p62-dependent autophagy for vitiligo.

Materials and Methods
Cell culture and reagents. Primary normal human melanocytes were isolated from human foreskin spec-  Western blot. Cells were washed with phosphate buffered saline (PBS) and lysed in RIPA buffer (Runde Biologicals, Xi'an, China) containing phenylmethanesulfonyl fluoride (protease inhibitor mix; Sigma-Aldrich). Equal amounts of the protein samples were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel and blotted onto a nitrocellulose membrane (Millipore, Bedford, MA, USA). After blocking by 5% non-fat dry milk, the membranes were washed and incubated with primary antibodies overnight at 4 °C. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. Bound antibodies were detected using a chemiluminescence detection kit (KPL, Gaithersburg, MD). Primary antibodies against LC3 I/II and p62 were purchased from Cell Signaling Technology, Inc., Danvers, MA; Antibodies against Nrf2 were purchased from Abcam, MA; Anti-Lamin and anti-β -actin were purchased from Life Science Products &Services, shanghai, China. Second antibodies including anti-mouse IgG or anti-rabbit IgG were purchased from Santa Cruz. Band intensities were determined by software (ImageJ; National Institutes of Health, Bethesda, MD). Measurement of mitochondrial membrane potential. After experimental treatments, cells were washed with PBS and loaded with JC-1 dye (Invitrogen) 5 mg/mL for 20 min at 37 °C. Then cells were washed twice in PBS, and fluorescence was measured by flow cytometer (Beckman Coulter) within an hour. Mean fluorescence intensity was quantized with Expo32 software (Beckman Coulter) and red/green ratio was calculated as an indicator of mitochondrial membrane potential.

Detection of apoptosis.
Transmission electron microscopy. After experimental treatments, cells were collected and washed in cold PBS, fixed in fixative buffer for 24 hours at 4 °C. Then the cell pellet was rinsed with Millonig's buffer and minced post-fixed in 1.0% OsO 4 , The pellet was stained with 2.0% aqueous uranyl acetate followed by ethanol dehydration, and embedded in Spurr's plasticresin. Ultrathin section analysis was visualized using a Tecnai ™ G2 Spirit Bio Twin (FEI, Hillsboro, OR).

Statistical analysis.
Each experiment was performed at least for three times, and statistical analyses of the data were performed using unpaired, two-tailed Student's t-tests or using one-way analysis of variance (ANOVA), followed by Newman-Keuls test built into GraphPad Prism (GraphPad Software 5.0; San Diego, CA). All the data were expressed as mean ± SD. Differences with P < 0.05 were considered statistically significant.