Anti-melanogenic Activity of Demethoxycurcumin via Microphthalmia-associated Transcription Factor (MITF) in Melanocyte

학위논문 (석사)-- 서울대학교 대학원 : 약학대학 약학과 천연물과학전공, 2016. 2. 이상국.Natural products from plants, fungi, bacteria and other organisms continue to be used in pharmaceutical preparations. The anti-melanogenic activity of demethoxycurcumin (DC), a compound of C. aromatica, and its molecular mechanism was investigated in mouse melan-a cells. DC significantly inhibited melanin synthesis and down-regulated the expression 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 DC suppressed the SRY-related HMG-box 10 (SOX10), critical transcription factors of MITF. DC also effectively inhibited DNA-binding activity of phosphor-cAMP response element binding protein (p-CREB), an up-stream regulatory factor of MITF. DC exhibit the up-regulation of ERK and AKT activation through mitogen-activated protein kinases (MAP kinase) pathway but there is no effect of phosphorylation of MITF because MITF is already decreased by SOX10 and p-CREB-DNA binding. These findings suggest that DC might serve as a candidate for an effective melanogenic inhibitor in melanocytes via the regulation of MITF signaling pathway. The anti-inflammatory activity of pinosylvin (PS), a natural stilbenoid from the heartwood of Pinaceae, was investigated in human keratinocytes, HaCaT cells. PS inhibited the UV-induced production of prostaglandin E2 (PGE2) in HaCaT cells. The suppression of PGE2 productions by PS was correlated with the down-regulation of mRNA and protein expression of cyclooxygenase-2 (COX-2). Further analysis revealed that PS suppressed the activation of MAP kinases, JNK, c-FOS and c-JUN signaling. These findings suggest that PS might serve as a candidate for a potential UV-induced anti-inflammaging agent via the regulation of MAP kinase signaling pathway.Part I. Anti-melanogenic Activity of Demethoxycurcumin via Microphthalmia-associated Transcription Factor (MITF) in Melanocyte 1 I. Introduction 2 II. Materials and Methods 6 A. Materials 6 1. Reagents and antibodies 6 2. Compounds 7 3. Cell culture 7 B. Methods 9 1. Melanin content assay 9 2. MTT cell viability assay 9 3. Western blotting 10 4. RNA extraction and real-time RT PCR 11 5. Nuclear extraction 11 6. p-CREB-DNA binding assay 12 7. Statistical analysis 13 III. Results 15 A. Inhibition of melanin synthesis by DC in the mouse melan-a cells 15 B. Effect of DC on the expression of TYR, TRP-1 and TRP-2 18 C. Effects of DC on the expression of MITF protein and gene 22 D. Inhibition of DC on the expressions of PAX3 and SOX10, MITF promoter genes 24 E. Inhibition of DC on the DNA-binding activity of p-CREB 26 F. Effect of DC on ERK, AKT and MITF 28 G. Inhibition of melanin synthesis by DC in a reconstituted human skin model (Neoderm®) 30 IV. Discussion 33 Part II. Inhibitory Effect of Pinosylvin on UVB-induced Inflammation via MAPK Signaling Pathway in Human Keratinocytes 37 I. Introduction 38 II. Materials and Methods 41 A. Materials 41 1. Reagents and antibodies 41 2. Compounds 42 3. Cell culture 42 B. Methods 44 1. Cell proliferation assay 44 2. Prostaglandin E2 assay 44 3. Western blotting 44 4. RNA extraction and real-time RT PCR 45 5. Statistical analysis 46 III. Results 48 A. Inhibition of prostaglandin E2 production by PS in UVB irradiated HaCaT cells 48 B. Suppression of COX-2 protein and mRNA expression by PS in UVB irradiated HaCaT cells 51 C. Effects of PS on the expression of MAPK signaling pathway 54 IV. Discussion 56 References 59 Abstract in Korean 66Maste