Anti-melanogenic activity of phytosphingosine via modulation of microphthalmia-associated transcription factor signaling pathway = 멜라닌 생성 전사인자 조절을 통한 phytosphingosine의 미백 효능 기전 연구

국문 초록 (Abstract)

사람의 피부색은 유전적, 외적 요인에 의해 차이가 있으며 그 중 피부색을 결정하는 주요 요인은 멜라닌이라는 색소이다. 멜라닌은 표피의 기저층에 존재는 melanocytes에서 여러 단계에 걸쳐 합성되어지는데 이 모든 과정을 melanogenesis라고 한다. 본 연구의 대상 물질인 phytosphingosine은 세라마이드를 구성하는 스핑고이드의 일종으로 항염증, 보습 효과, 각질 강화 효능 등으로 이미 잘 알려진 물질이지만 미백 효능 및 그 기전 연구는 보고되어 있지 않다. 따라서 본 연구에서는 phytosphingosine을 이용하여 멜라닌 생성 첫 단계인 tyrosinase 및 멜라닌 생성 억제효능을 분석한 결과, 농도가 증가함에 따라 유의적으로 tyrosinase 활성과 melanocytes에서의 멜라닌 생성이 억제되었다. 또한 microphthalmia-associated transcription factor (MITF)는 멜라닌 생성시 중요한 역할을 하는 전사인자로 phytosphingosine의 MITF에 대한 저해 활성을 확인한 결과 MITF 뿐만 아니라 melanogenesis 관련 효소계인 tyrosinase, tyrosinase-related protein-1, and -2 (TRP-1, -2)의 발현을 모두 억제하였다. 더 나아가 MITF의 중요한 전사 인자인 paired box 3 (PAX3), SRY-related HMG-box 10 (SOX10), β-catenin, phospho cAMP response element binding protein (p-CREB)의 단백질 발현을 억제함으로서, MITF의 유전자 발현을 저해시킨다는 것을 확인할 수 있었다.Phytosphingosine을 12시간 이상 melanocytes에 처리하였을 때, MITF의 전사 인자들에 대한 변화 없이 MITF의 단백질 발현을 감소시켰다. 반면, MITF의 ubiquitination과 관련 있는 extracellular signal-regulated kinase (ERK)의 단백질 발현은 증가되었다. 따라서, phytosphingosine을 12시간 이상 처리시의 MITF 감소는 MAPK kinase의 활성화와 연관되어 있음을 알 수 있었다. 본 연구를 통해 phytosphingosine에 의한 MITF의 감소는 MITF의 유전자 발현을 억제시키거나 직접적으로 MITF 단백질 발현을 제어함으로서 영향을 받는 것으로 나타났다. 따라서 phytosphingosine은 melanocytes에서 MITF를 조절하여 melanogenesis를 억제하는 새로운 미백 후보물질이 될 가능성이 높을 것으로 여겨진다.

다국어 초록 (Multilingual Abstract)

Sphingolipids are ubiquitous components of the plasma membrane in animals, plants, and fungi. Of the structural analogues of sphingolipds, phytosphingosine (PSX) is well-known to be involved in many significant cellular responses including apoptosis, differentiation, and migration. Moreover, PSX is commonly used as acne treatments because of its anti-bacterial and anti-inflammatory activities. PSX also has use in cosmetic compositions, and can be found in a variety of cosmetic products as skin and hair conditioning agents. Nevertheless, its mechanism of action remains unclear. In this present study, we investigated anti-melanogenic activity of PSX and its molecular mechanism in mouse originated melan-a cells. The present findings showed that PSX significantly inhibits melanin synthesis in a concentration-dependent manner. One Plausible mechanism of the anti-melanogenic activity of PSX seems to be associated with the modulation of the microphthalmia-associated transcription factor (MITF) which suppresses the expression of tyrosinase, tyrosinase-related protein-1, and -2 (TRP-1, -2). Further analysis revealed that PSX suppressed the paired box 3 (PAX3) and SRY-related HMG-box 10 (SOX10), critical transcription factors of MITF. PSX also effectively down-regulated protein level of ?-catenin and phospho cAMP response element binding protein (p-CREB), an up-stream regulatory factor of MITF. These results were closely related with the suppression of MITF gene expression. In addition, the treatment of PSX for over 12 hours, relatively long period of time, did not directly suppressed these MITF transcriptional factors. Instead, PSX induced ERK activation which led to the MITF phosphorylation and subsequently its degradation. Therefore, the down-regulation of the MITF protein level in a long period of time exposure of PSX is in part associated with the MITF protein degradation through MAPK kinase activation pathway. In conclusion, the modulation of MITF by PSX is correlated with the signaling pathways such as the suppression of MITF gene expression and the degradation of MITF protein depending on a period of treatment time. These results suggest that PSX might serve as a candidate for an effective melanogenesis inhibitor in melanocytes via the regulation of MITF signaling pathway.

목차 (Table of Contents)

• Abstract i
• Table of Contents iv
• List of Figures vii
• List of Tables ix
• I. Introduction 1
• II. Materials and Methods 5
• A. Materials 5
• 1. Reagents and antibodies 5
• 2. Compounds 6
• 3. Cell culture 6
• B. Methods 8
• 1. Melanin content assay 8
• 2. MTT cell viability assay 8
• 3. Western blotting 8
• 4. RNA extraction and real-time RT PCR 9
• 5. Nuclear extraction 10
• 6. p-CREB-DNA binding assay 10
• 7. Cellular tyrosinase activity assay 11
• 8. Mushroom tyrosinase activity assay 11
• 9. Transient transfection and dual luciferase assay 12
• 10. Immunocytochemistry 12
• III. Results 15
• A. Inhibition of melanin biosynthesis by phytosphingosine in melan-a cells 15
• B. Inhibitory effects of phytosphingosine on both cellular and non-cellular tyrosinase activities 18
• C. Effects of phytosphingosine on the expression of melanogenic proteins and genes 21
• D. Function of phytosphingosine as the regulator of the intracellular level of MITF changes over time 24
• E. Suppression of MITF transcriptional factors after 30 minute treatment of phytosphingosine 26
• F. Reduction in the p-CREB level by phytosphingosine was correlated with the inhibition of the AKT and ERK levels 28
• G. Inhibition of phytosphingosine on the DNA binding activity of p-CREB 30
• H. Inhibition of phytosphingosine on the expressions of Pax3 and Sox10 genes 32
• I. Induction of ERK activation after 12 hours treatment by phytosphingosine 34
• J. Induction of MITF phosphorylation by phytosphingosine 36
• K. Reduction in the tyrosinase level was correlated with the inhibition of the MITF levels 38
• L. Suppression of gene expression and promoter activities of MITF and tyrosinase by phytosphingosine 40
• IV. Discussion 44
• References 49
• Abstract in Korean 54