Decreased Methionine Sulfoxide Reductase A Expression Renders Melanocytes More Sensitive To Oxidative Stress: A Possible Cause For Melanocytes Loss In Vitiligo

Vitiligo is a depigmenting skin disorder with yet undefined mechanisms of epidermal melanocytes loss. The underlying pathogenesis includes genetics, autoimmunity, etc. In recent years, the relationship between oxidative stress and vitiligo has been extensively studied and oxidative stress now is recognized as an integral part contributing to vitiligo.In normal conditions, the generation and removal of ROS is under delicate balance, whereas in vitiligo such balance is lost. Several exogenous and endogenous sources for constant and excessive ROS production have been confirmed. The most convincing evidence is the detection of H2O2 accumulation in the epidermis of vitiligo patients with a concentration range of 10-3 M, far exceeding the physiological range of 10^-7-10^-6 M.Amino acid residues in proteins are one of the major targets of ROS. Among them sulfur-containing methionine is highly susceptible to oxidation, yielding two enantiomers: methionine-S-sulfoxide and methionine-R-sulfoxide, which can be catalytically reduced by methionine sulfoxide reductase A (MSRA) and methionine sulfoxide reductase B (MSRB), respectively. MSR is unique in that it serves both as a protein repairer by reversing methionine oxidation and a ROS scavenger by regenerating methionine. MSR system plays an important role in longevity, antioxidant defense and regulation of protein function.MSR is a crucial member of skin antioxidant network. Besides, both keratinocytes and melanocytes express functioning MSR. However, MSR expression as well as enzyme activity is significantly reduced in the epidermis of vitiligo patients. Recombinant human MSRA are deactivated by 10-3 M H₂O₂ by 85%. MSRA is deactivated due to H₂O₂-mediated oxidation of methionine, tryptophan and cysteine residues in its active site.The loss of MSR means the loss of repair capacity for melanocytes when they are confronted with oxidative stress in vitiligo. We here asked the question whether inadequate protection from MSRA is one of the reasons why melanocytes are vulnerable to excessive H₂O₂ accumulation in vitiligo. In consideration of the therapeutic potential of MSRA, this question should be resolved first.Partâ… : Screening for the most efficient MSRA-specific siRNA1. Main methodsTransient transfection of Hela cell (human cervical cancer cell lines) was carried out using three pairs of Pre-designed MSRA-specific siRNA. Mock transfection control (without siRNA) and negative control transfection (using a Non-trargeting siRNA) were performed in each experiment. The effect of MSRA suppression was checked 24h and 48h posttransfection by RT-PCR and Western blot, respectively.2. Main results #111798 MSRA siRNA was turned out to be the most efficient one according the results of RT-PCR and Western blot. Compared with negative controls with no difference in the expression of internal standard GAPDH, MSRA expression was remarkably decreased in cells transfectd with#111798 MSRA siRNA.3. Main conclusionsThe technology of RNAi enables us to study the consequences of low MSRA expression for melanocytes survival under oxidative stress. The most efficient MSRA-specific siRNA has been chosen, which serves as the foundation for our following experiments.Partâ…¡: Decreased MSRA expression renders melanocytes more sensitive to oxidative stress1. Main methodsImmortalized human epidermal melanocytes cell line PIG1 was transfected with #111798 MSRA siRNA. MSRA expression was checked 48h and 72h posttransfection by RT-PCR and Western blot, respectively. Mock transfected ells, negative control cells and MSRA-silenced cells were all subjected to 1mM H₂O₂ for 24h and then cell viabilities were investigated. The data was analyzed by unpaired two-tailed Student t test.2. Main resultsThe siRNA specific for MSRA successfully suppressed MSRA expression in melanocytes. The declined MSRA expression in melanocytes leads to an increased sensitivity to oxidative stress, resulting in more cell death. Furthermore, a remarkable loss of viable cells is found in MSRA-silenced melanocytes even in the absence of exogenously added oxidative stress.3. Main conclusionsDecreased MSRA expression renders melanocytes more sensitive to H₂O₂-mediated oxidative stress, which may be one of the mechanisms of melanocyte loss in vitiligo. We postulate that there exists a vicious circle in vitiligo. Excessive H₂O₂ disturbs normal pathways such as antioxidant system, pigmentation, synthesis/recycling of the essential cofactor 6BH4, etc by oxidizing amino acid residues in the active sites of enzymes. Dysfunctioning pathways lead to compromised ability to remove ROS and abnormal endogenous ROS generation. H₂O₂-mediated oxidative stress not only causes decreased MSRA expression but also deactivates MSRA. The consequences of it include insufficient repair to functionally critical methionine, loss of mitochondrial membrane potential and an increase of intracellular ROS level. All those further exacerbate oxidative stress and ultimately result in loss of function, even melanocytes death. Overexpression of MSRA has been proved to be beneficial for cell survival under oxidative stress, suggesting its preventive and therapeutic potential.