포타슘 채널을 활성화시키는 CyPPA의 ERK/MITF 경로를 통한 멜라닌 생성 억제

다국어 초록 (Multilingual Abstract)

Background: The development of anti-melanogenesis agents is important for the treatment of hyperpigmentary disorders, such as lentigo and melisma. In the course of screening an ion channel-modulating chemical HTS library for inhibitors of melanogenesis in B16F10 cells, we found that CyPPA, a K+ activator, had strong anti-melanogenesis activity.

Objectives: In this study, we investigated whether CyPPA would affect melanogenesis in human melanocyte and mouse Mel-Ab cells.

Methods: The cell toxic effects of CyPPA on human melanocyte cells and mouse Mel-Ab cells were assessed by EZ-Cytox. To verify the effect of CyPPA on melanogenesis, human melanocytes and mouse Mel-Ab cells were treated with 0?10 μM of CyPPA for 72 h, and melanin content and tyrosinase (TYR) activity were determined. MelanoDerm™ was used to test the melanogenesis inhibitory effect of CyPPA. To investigate whether CyPPA decreases the expression levels of TYR and MITF, Mel-Ab cells were analyzed by western blot. Next, to determine whether MITF down-regulation by CyPPA is a result of the decrease in MITF mRNA expression or MITF degradation, RT-PCR assays using specific primers for MITF were performed. To understand the mechanism, we investigated whether ERK and CREB signaling pathways were involved in the inhibitory effect of CyPPA. We finally determined if the decreases in MITF and TYR levels were due to proteasome-mediated degradation induced by CyPPA treatment. Mel-Ab cells were incubated with CyPPA for 72 h in the absence or presence of the proteasome inhibitor MG132, and then melanin content was determined by western blot analysis.

Results: CyPPA did not show any cytotoxic effect within the tested concentration range. CyPPA treatment significantly decreased the melanin content in a concentration-dependent manner. In MelanoDerm™, the visual evaluations revealed a significantly lower melanin content in the CyPPA-treated human skin model than in the untreated control. TYR activity of CyPPA-treated cells was dose-dependently and time-dependently reduced. Treatment with CyPPA at a concentration of 10 μM decreased both the level of TYR protein and the level of MITF. However, there was no change in MITF mRNA expression following CyPPA treatment. CyPPA induced the phosphorylation of ERK with peak levels observed at 30 min after treatment. MG132 reversed the decreases in MITF and TYR protein levels induced by CyPPA.

Conclusion: CyPPA treatment suppressed melanogenesis by stimulating the degradation of MITF via its effect on the regulation of the ERK 1/2 signaling pathway. Our findings suggest that CyPPA is an ion channel modulator that antagonizes melanogenesis. It may have potential as a therapeutic agent for the treatment of hyperpigmentation.

목차 (Table of Contents)

• ABSTRCT i
• LIST OF FIGURE ⅳ
• INTRODUCTION 1
• MATIRIALS AND METHOD 3
• Chemicals and materials 3
• Cell culture and drug preparation 3
• Cell viability assay 3
• Measurement of melanin contents 4
• Tyrosinase activity assay 4
• Western immunoblot analysis 4
• Reverse transcription-polymerase chain reaction (RT-PCR) 5
• Human three-dimensional tissue model 5
• RESULT 7
• CyPPA has no effect on cell viability 7
• CyPPA inhibits melanin synthesis 7
• CyPPA inhibits melanogenesis in human three-dimensional tissue model 7
• CyPPA inhibits cellular tyrosinase activity 8
• CyPPA inhibits tyrosinase protein and MITF
• expression 8
• CyPPA inhibits melanogenesis via the ERK signaling pathways 8
• Down-regulation of MITF level is a result of MITF degradation 9
• DISSUSSION 10
• 국문초록 15
• REFERENCES 17