A Study On The Molecular Mechanism Of Inhibition On Melanogenesis Induced By Paeonol And The Feedback Regulation On Melanin Synthesis By Cytokines In KC-MC Co-culture System

1 Paeonol,which is Chinese traditional medicine,has been proved to have a new function:inhibition of melanogenesis.Tyrosinase and its transcriptional regulator microphthalmia-associated transcription factor(MITF) play critical roles in regulation of melanogenesis,and are required for environmental cues or agents in modulation of melanin synthesis.In this report,we discovered that paeonol down-regulated melanin production via decreasing MITF expression and consequent mRNA and protein levels of tyrosinase.We also found that paeonol reduced phosphorylation of a cAMP responsive element binding protein (phospho-CREB),which binds and activates MITF.This research contributes to understanding the mechanism of inhibition by paeonol in B16F10 melamoma ceils.2 Identifying the signals mediating the effects of paeonol on melanogenesis is crucial to understanding how pigmentation responds to paeonol.Resultes showed that a selective inhibitor of c-jun N-terminal or stress-activated protein kinases(JNK/SAPK)-SP600125 significantly reversed paeonol-induced down-regulation of melanin synthesis.Inhibition of cAMP/PKA pathway intensified the hypopigmentation response to paeonol.These results identify a mechanism in which paeonol induces the down-regulation of melanogenesis through inhibition of CREB phosphorylation,leading to the expression reduction of MITF and subsequently tyrosinase.The key kinase mediating the effects of paeonol on melanogenesis in B16F10 cells is JNK/SAPK.Additionally,the cAMP/PKA pathway may take part in this process.Base on the two results,we conclude that melanogenesis inhibited by paeonol may be mediated by an unknown phosphorylase activated by JNK/SAPK pathway,and inhibiting phosphorylation of CREB and expression of MITF, consequent level of tyrosinase and melanin synthesis.The mediation of JNK/SAPK in pigmentation induced by chemicals has not been reported.The study on the key proteins,enzymes and signaling transduction pathways mediating the inhibition of melanogenesis by paeonol conduces to understanding the mechanism of paeonal on hypopigmentation,and contributes to the apply of the paeonol on pigmentary disorders in future.And it is also useful for the research on mechanism of pigmentation.3 In our prior researches,significant difference was found in the levels of inhibition on pigmentation by paeonol in MC culture and co-culture model.And the decreasing of melanogenesis in co-culture model was more obvious than that in MC culture cells.The phenomenon suggests that except for the direct effect on MC,paeonol may also act on KC,then influence on the melanogenesis indirectly. It is already kown that KC play a key role in regluating epidermal pigmentation by cytokines such as endothelin-1(ET-1) and stem cell factor(SCF) in paracrine way.Both of the two cytokines play important roles in melanin synthesis and proliferation of MC.We detected the levels of ET-1 and sSCF in KC and co-culture models after treated with classic regulators including melanogenesis agonists like HQ,and antagonists such as 8-MOP,in order to analysis the mechanism of different reactions to stimulators in single cell culture and co-culture model.Results showed that the vary effects on KC by different regulators is the main reason of different status of melanogenesis in two kinds of models.The effects on KC could be synergetic or antagonistic,which mainly dependes on the activity and/or dose of regulators.It shows that the KC-MC coculture model is more suitable for screening,studying the medicines applyed to clinical treatment.4 At the same time,significant difference was found in the levels of cytokines ET-1 and sSCF under stimulation of interferents in two models,although the number of cells were the same.For example,8-MOP in co-culture model inhibited the level of ET-1 with no effect on the level of ET-1 in KC model.We know 8-MOP is the antagonist of melanogenesis,and the finding suggests that the inhibition of ET-1 may result from the increasing of melanin induced by 8-MOP.What kind of interaction is it between the two cytes? Is it regulated by feedback mechanism?Because of close to the physiological environment of skin,the co-culture model is the suitable subject for us to study the interaction between KC and MC. Using co-culture model and specific regulators which affect melanin synthesis without effect on cytokines paracrined by KC,we set up an close loop system-feedback system,which is necessary for studying feedback mechanism. Negative correlation was proved between the melanin content and the concentration of ET-1 in co-culture model.And ET-1 is able to improve the melanin synthesis simultaneously.Base on the two results,we initially demonstrated that the feedback mechanism exists between MC and KC and regulates the pigmenattion to some extent.Summarily,the KC,MC,melnin and cytokines consist the total feedback system,in which the KC regulates the pigmentation by cytokines,when the MC is stimulated by environmental factors,the content of melanin transferred into KC changes,then KC regulates its cytokines' synthesis and secretion to adjust the pigmentation.Furthermore it suggests that in epidermal melanin unit,KC regulates the melanin synthesis by cytokines and MC regulates the cytokines secreted by melanogenesis,and it is an balancing feedback system.When there is a strong stimulators which exceeds the regulation of system,the feedback has no fuctions, so the balance is broken,abnormal pigmentation happens consequently.It is the first time to initiate and demonstrate the feedback mechanism existing in co-culture model.This research is an important part of the complete pigmentation theory,and it can help us to understand the pathogenesis of pigmentary diseases and to find new targets for treatment.