Effect And Mechanism Research Of Small Molecular Compound I942 On Melanogenesis In PIG1 Melanocyte

Background and Purpose:Vitiligo is an acquired depigmenting skin disorder caused by loss or dysfunction of epidermis melanocytes.With a global incidence ranging from 0.5% to 1%,vitiligo remains the leading cause of skin depigmentation worldwide.The exact pathogenesis of vitiligo is now unclear.Several possible mechanisms accounting for the disease include genetic factors,autoimmunity,neuropsychiatric factors and trace element deficiency.As a disfigurementing skin problem,vitiligo is frequently accompanied by depression,anxiety and ill social adaptation,which can severely undermine overall life quality of patients.Current treatments for vitiligo mainly refer to topical corticosteroids and phototherapies.For some drugresistant types of vitiligo,treatment remains tricky.To restore pigmentation,one of the most essential strategies is to facilitate melanogenesis in remaining melanocytes at the lesional area,or to complement the depigmenting area with functional melanocytes.Melanogenesis is a process in which tyrosine goes through a series of chemical reactions in the melanosome of melanocytes to produce melanin polymers,which are transported to the surface of surrounding keratinocytes' nuclear to resist internal and external stimulis.It is also a key process of skin photoprotection and the onset of many pigment-related skin diseases.Melanogenesis is regulated by many factors,among which are the external factors(mainly ultraviolet rays),the internal factors involving melanocytes themselves,the surrounding cells(mainly keratinocytes),endocrine,neural and immune factors.Signaling pathways regulating melanogenesis include c AMP/PKA,Wnt,and MAPK pathways.Exchange protein activated by c AMP(EPAC)is a family of c AMP-binding proteins with guanine nucleotide exchange factors(GEF)activity and is involved in a variety of pathophysiological processes,including glandular secretion,intercellular adhesion,wound healing,inflammation,fibrosis,and tumor cell metastasis,whose role is still being updated.EPAC is activated by the upstream c AMP,and is downstream associated with multiple members of the MAPK family that regulate melanogenesis.Some researchers have compared EPAC to a bridge between the c AMP pathway and the MAPK pathway.We suppose that EPAC may be involved in melanogenesis process.Moreover,EPAC-activated Rap proteins participate in the secretion of inflammatory cytokines in keratinocytes,which are also important components of paracrine factors that regulate melanogenesis.This also ignite our mind to think about EPAC's role in indirectly affecting the melanogenesis in melanocytes-keratinocyte co-culture system by changing the secretion characteristic of keratinocytes.Small molecular compound I942(I942 for short)is a novel selective agonist of EPAC1 and has been shown to alter gene expression associated with microtubule stability and cell cycle progression in human umbilical cord vascular endothelium.I942 was also demonstrated by our previous researches to have an impact on physical-pathological processes like wound healing and scar formation through c AMP EPAC pathway by stimulating the secretion of b-firoblast growth factor(b-FGF),epidermal growth factor(EGF),and interleukin 6(IL-6)in firboblasts,and promoting the keratin 19 secretion and cell migration of keratinocytes.Nevertheless,the effection of small molecular compound I942 on melanogenesis of melanocytes have not been tested so far.In this research,we aim to find out the effection of small molecular compound I942 on melanogenesis in PIG1 melanocytes and how.Furthermore,we investigated small molecular compound I942's effection and possible molecular mechainism on melanogenesis though regulating secretion activity of Ha Ca T cells.We hoped that our results would be of some use in bringing small molecular compound I942 into clinical use to treat depigmenting disorder like vitiligo or improving the quality of melanocytes culturing technique in vitro.Our research mainly includes following three parts:Section I: Effect of small molecular compound I942 on cell viability of PIG1 melanocytes and Ha Ca T keratinocytes.Methods: 1.The identification of PIG1 melanocytes was carried out by DOPA staining.2.The effection of small molecular compound I942 on PIG1 celanocytes and Ha Ca T cells'proliferation were performed through CCK8 assay.Results: 1.PIG1 melanocytes were dyed brown to black after being treated with 0.1% DOPA,whichshows that PIG1 melanocytes are functional melanocytes which has DOPA-oxidizationactivity.2.According to CCK8 results,small molecular compound I942 show no inhibiting effecton cell proliferation on both PIG1 melanocytes and Ha Ca T cells at concentration rangingfrom 0 to 50 ?mol/L.Section II: Effect of small molecular compound I942 on melanogenesis and its possible action mechanism in PIG1 melanocytes.Methods: 1.The effect of small molecular compound I942 and antagonist of EPAC ESi-09 of melanincontent in PIG1 melanocytes were measured by sodium hydroxide solubilization methodand their effects on tyrosinase activity were performed by measuring the rate of L-DOPAoxidation.2.The effect of small molecular compound I942 on m RNA expression levels ofMicrophthalmia-associated transcription factor(MITF),Tyrosinase(TYR),Tyrosinase-related protein 1(TRP-1),and Tyrosinase-related protein 2(TRP-2)in PIG1 melanocyteswere evaluated using q PCR analysis.3.The effect of small molecular compound I942 on protein expression level of TYR andTRP-2 was measured by western blot analysis.Results: 1 Small molecular compound I942 can significantly upregulate melanin content andtyrosinase activity in PIG1 melanocytes,and pretreatment of EPAC inhibitor ESi-09 cansignificantly inhibit I942's effection on both melanin content and tyrosinase activity.2 q RT-PCR analysis showed that small molecular compound I942 can upregulate MITF,TYR,TRP-1,TRP-2 m RNA expression level in PIG1 melanocytes.3 Western blot analysis showed that protein expression level of TYR and TRP-2 were notsignificantly influenced by the treatment of small molecular compound I942.Section III: Effect of small molecular compound I942 on melanogenesis in PIG1 melanocytes via Ha Ca T cells and its possible molecular mechainism.Methods: 1.The effect of small molecular compound I942 and antagonist of EPAC1 ESi-09 onmelanin content in PIG1 melanocytes through Ha Ca T cells were measured by sodiumhydroxide solubilization method and their effects on tyrosinase activity were performedby measuring the rate of L-DOPA oxidation.2.The effect of small molecular compound I942 on m RNA expression levels of MITF,TYR,TRP-1,TRP-2 in PIG1 melanocytes through Ha Ca T cells were evaluated usingq PCR analysis.3.The effection of small molecular compound I942 on protein expression level of TYRand TRP-2 via Ha Ca T cells were measured by western blot analysis.Results: 1.Small molecular compound I942 can significantly decrease melanin content andtyrosinase activity in PIG1 melanocytes via regulating secretion activity of Ha Ca T cells,and pretreatment of ESi-09 can inhibit I942's effection on both melanin content andtyrosinase activity.2.q RT-PCR analysis showed that small molecular compound I942 can significantlydownregulate MITF,TYR,TRP-1 m RNA expression level in PIG1 melanocytes viaregulating secretion activity of Ha Ca T cells.3.Western blot analysis showed that small molecular compound I942 can significantlydecrease protein expression level of TYR via regulating secretion activity of Ha Ca T cells,and pretreatment of ESi-09 can significantly inhibit I942's effection on TYR protein.However,no similar effection or trend was observed on TRP-2 protein expression levelin PIG1 melanocytes.Conclusion: 1.Small molecular compound I942 had no negative effect on proliferation activity of bothPIG1 melanocytes or Ha Ca T cells.2.Small molecular compound I942 can enhance tyrosinase activity thus increase melanincontent possibly through upregulating MITF,TYR,TRP-1,TRP-2 m RNA expressionlevel in PIG1 melanocytes.However,small molecular compound I942 showed no effecton protein expression level of TYR and TRP-2.3.Small molecular compound I942 can moderately inhibit tyrosinase activity thus decreasemelanin content through down-regulating MITF,TYR,TRP-1 m RNA expression levelin PIG1 melanocytes via regulating secretion activity of Ha Ca T cells and pretreatmentof ESi-09 can inhibit its effect on melanin content,tyrosinase activity,and m RNAexpression level.Small molecular compound I942 can decrease protein expression ofTYR via Ha Ca T cells and its effect can be inhibited by pretreatment of ESi-09.Smallmolecular compound cannot affect TRP-2 protein level in PIG1 melanocytes via Ha Ca Tcells.