Inhibitory Mechanisms of MHY498 in Melanogenesis = MHY498의 멜라닌 생성 억제 기전

다국어 초록 (Multilingual Abstract)

Ultraviolet (UV)-mediated melanogenesis is a complex process that remains to be elucidated. α-Melanocyte stimulating hormone (α-MSH)-induced cyclic AMP (cAMP)-signaling pathway is the major melanogenic signal transduction leading to upregulation of tyrosinase. It is known that UV stimulates nitric oxide (NO) generation and that the NO-induced cyclic GMP (cGMP)-signaling pathway is also important in the expression of tyrosinase during melanogenesis. Because tyrosinase is a rate-limiting enzyme in melanin synthesis, it is an attractive target for research on the biochemistry of melanogenesis and management of hyperpigmentary disorders. In an attempt to find a novel anti-melanogenic agent, (Z)-5-(2,4-dihydroxybenzylidene)thiazolidine-2,4-dione (MHY498) was synthesized as a potential tyrosinase inhibitor. Current research is aimed to evaluate the inhibitory effects of MHY498 on melanogenesis and to elucidate its mechanism. First, MHY498 (IC50 = 3.55 μM) inhibited more potently mushroom tyrosinase activity than the well-known tyrosinase inhibitor, kojic acid (IC50 = 22.79 μM), in a concentration-dependent manner. Kinetic studies and docking simulation showed that MHY498 was a competitive inhibitor against tyrosinase with higher binding affinity than kojic acid. Second, MHY498 inhibited murine tyrosinase activity and decreased melanin production without cytotoxicity in B16F10 melanoma cells treated with α-MSH. Western blot analysis showed that MHY498 inhibited the expression of tyrosinase and its transcription factor, microphthalmia associated transcription factor (MITF), via cAMP-PKA-CREB signaling pathway. Third, MHY498 inhibited sodium nitroprusside (SNP)-induced NO levels, tyrosinase activity and melanin synthesis in B16F10 melanoma cells. The increased level of cGMP by SNP was reduced by pretreatment with MHY498 in a concentration-dependent manner. In Western blot analysis, MHY498 effectively suppressed the levels of pCREB, MITF, and tyrosinase in the SNP-stimulated melanogenesis. Collectively, MHY498 inhibited α-MSH-induced and NO-induced melanogenesis through the direct inhibition of tyrosinase activity and the suppression of the levels of MITF, CREB, and tyrosinase via the modulation of the two signaling pathways, cAMP-PKA and cGMP-PKG. These findings suggest that MHY498 can be a promising therapeutic agent for the prevention and treatment of skin hyperpigmentation.

국문 초록 (Abstract)

자외선에 의한 멜라닌 생성은 아직 규명되어져야 하는 복잡한 과정이다. α-Melanocyte stimulating hormone (α-MSH)에 의해 유도되는 cyclic AMP (cAMP) 신호전달 과정은 tyrosinase의 발현을 조절하는 중요한 멜라닌 생성 신호전달체계이다. 자외선이 nitric oxide (NO)의 생성을 자극하고, NO에 의해 유도되는 cyclic GMP (cGMP) 신호전달 과정 또한 멜라닌 생성 과정에서의 tyrosinase 발현에 아주 중요한 역할을 한다는 것이 알려져 있다. Tyrosinase는 멜라닌 생성과정의 율속단계의 효소이기 때문에 멜라닌 생성과 과색소침착 질환을 치료하는 연구나 생화학 분야에 있어서 매력적인 목표물질이 되어 왔다. 새로운 멜라닌 생성 억제물질을 찾기 위한 노력에 의해 (Z)-5-(2,4-dihydroxybenzylidene)thiazolidine-2,4-dione (MHY498)을 tyrosinase 억제제로 합성하였다. 본 연구는 멜라닌 생성과정에 있어서 MHY498의 억제효과를 평가하고 그 작용기전을 규명하는 것을 목표로 하고 있다. 첫번째, MHY498 (IC50 = 3.55 μM)은 기존에 잘 알려져 있는 tyrosinase 억제제인 kojic acid (IC50 = 22.79 μM) 보다 강하게 mushroom tyrosinase 활성을 농도의존적으로 억제하였다. 또한, 동력학 연구와 docking simulation을 통해서 MHY498이 kojic acid 보다 tyrosinase에 대해 더 높은 결합력을 가지는 경쟁적 길항제임을 확인하였다. 두번째, α-MSH를 처리한 B16F10 melanoma 세포에서 MHY498은 생쥐 tyrosinase 활성을 억제하였고, 세포독성 없이 멜라닌 생성을 감소시켰다. Western blot 분석에서, MHY498은 cAMP-PKA-CREB 신호전달 과정을 통하여 tyrosinase와 그의 전사인자인 microphthalmia associated transcription factor (MITF)의 발현을 억제하였다. 세번째, MHY498은 B16F10 melanoma 세포에서 sodium nitroprusside (SNP)에 의해 유도된 NO의 수치, tyrosinase의 활성, 그리고 멜라닌 합성을 억제하였다. SNP에 의해 증가된 cGMP의 양은 MHY498의 전처리에 의해 농도 의존적으로 감소하였다. Western blot 분석에서, MHY498은 SNP 자극에 의해 유도된 멜라닌 생성 과정에서 pCREB, MITF, 그리고 tyrosinase의 수치를 효과적으로 억제하였다. 종합하자면, MHY498은 직접적인 tyrosinase 활성 억제와 두가지 신호전달 과정, 즉 cAMP-PKA와 cGMP-PKG 신호전달 체계를 통한 CREB의 활성화와 MITF, 그리고 tyrosinase의 발현을 억제함으로써 α-MSH와 NO에 의해 유도된 멜라닌 생성 과정을 억제하였다. 따라서 본 연구는 MHY498이 피부색소 침착을 치료하거나 예방함에 있어서 치료학적 후보 물질이 될 수 있음을 제시하는 바이다.

목차 (Table of Contents)

• I. INTRODUCTION 1
• 1. Skin aging and melanogenesis 1
• (1) Skin aging 1
• (2) Photoaging 6
• (3) Melanogenesis 7
• (4) Tyrosinase and signaling pathways leading to tyrosinase expression 11
• 2. Skin-lightening agents 16
• (1) Tyrosinase inhibitors 16
• (2) Synthesis of (Z)-5-(2,4-dihydroxybenzylidene)thiazolidine-2,4-dione (MHY498) 17
• 3. Purpose of this study 18
• II. MATERIALS AND METHODS 22
• 1. Materials 22
• 2. Cell culture system 23
• 3. MTT assay 24
• 4. Assay of mushroom tyrosinase activity 24
• 5. Kinetics of tyrosinase inhibition 25
• 6. Griess assay 25
• 7. Quantitative assay of cGMP 26
• 8. Assay of murine tyrosinase activity 27
• 9. Assay of melanin contents 28
• 10. Cell lysis 28
• 11. Western blot analysis 29
• 12. Docking simulation 31
• 13. Pharmacophore prediction 31
• 14. Statistical analysis 31
• III. RESULTS 33
• Part I. Effect of MHY498 on mushroom tyrosinase activity 33
• 1. Inhibitory effect of MHY498 on mushroom tyrosinase activity 33
• 2. Kinetics of MHY498 in the inhibition of mushroom tyrosinase 33
• 3. Docking simulation of tyrosinase and binding residues that interact with MHY498 37
• Part II. Effects of MHY498 on cAMP-dependent signaling pathway leading to melanogenesis 40
• 1. Effect of MHY498 on B16F10 melanoma cell viability 40
• 2. Effects of MHY498 on the expression level of tyrosinase and on the cAMP-dependent signaling pathway 40
• 3. Inhibition of tyrosinase activity in B16F10 melanoma cells by MHY498 42
• 4. Inhibition of melanin production in B16F10 melanoma cells by MHY498 46
• Part III. Effect of MHY498 on NO levels and NO-induced signaling pathway related to melanogenesis 48
• 1. Inhibition of NO levels in SNP-treated B16F10 melanoma cells by MHY498 48
• 2. Inhibition of cGMP content in SNP-treated B16F10 melanoma cells by MHY498 48
• 3. Effect of MHY498 on the levels of CREB, MITF, and tyrosinase in SNP-treated B16F10 melanoma cells 52
• 4. Inhibitory effect of tyrosinase activity in SNP-treated B16F10 melanoma cells by MHY498 55
• 5. Effect of MHY498 on melanin contents in SNP-treated B16F10 melanoma cells 55
• IV. DISCUSSION AND CONCLUSION 59
• V. REFERENCES 73
• ABSTRACT (in Korean) 85