Role Of Vasoactive Intestinal Peptide In The Regulation Of Melanogenesis And The Underlying Mechanisms

Vasoactive intestinal peptide(VIP)is one of the major skin neuropeptides released from sensory and autonomic nerve endings distributed in the epidermis,basal layer,dermis,sweat glands,and hair follicles in a variety of biological· conditions.VIP belongs to the gastrin/secretin/glucagon family of secretory peptides,is composed of 28 amino acids,and binds to structurally distinct G protein-coupled receptors,VIP receptor 1(VIPR1)and VIPR2.Activation of the VIP/VIPR system mediates various physiological processes,including vasodilatation,plasma extravasation,mast cell degranulation,and immunomodulation.Previous studies suggest active roles of VIP in the pathogenesis of inflammatory skin disorders such as atopic dermatitis and psoriasis,which can commonly cause post-inflammatory hyperpigmentation.However,the effect of VIP on melanogenesis remains unknown.Objectives:To investigate the effect of VIP,an important skin neuropeptide,on melanogenesis and the underlying mechanisms.Methods:1)B16F10 mouse melanoma cells were treated with VIP at the concentrations of 1 to 100 nM,and collected at the indicated time point for analyzing cell viability and melanin contents.Then intracellular tyrosinase activity was measured at different time points at the VIP concentration of 100 nM;2)After treatment with VIP(100 nM),the mRNA and protein expression of tyrosinase and microphthalmia associated transcription factor(MITF)were investigated using reverse transcription,real-time quantitative polymerase chain reaction(RT-qPCR)and Western blotting analysis,respectively;3)Effects of VIP on the expression of phosphorylated CREB,PKA,p38 MAPK,Akt,and Erkl/2 were analyzed in B16F10 cells by western blotting regarding as potent VIP-induced signaling pathways in the regulation of melanogenesis;4)The cAMP-PKA-CREB signaling pathway was inhibited by H89(a selective PKA inhibitor)to confirm its positive role in the VIP-induced stimulation of melanogenesis in B16F10 cells by investigating relative melanin contents and protein expression of MITF and tyrosinase;5)Expression of VIP receptors were investigated by Western blotting in B16F10 cells;6)B16F10 cells were transfected with 300 nM of scrambled negative control or VIPR1/2 small interfering RNAs(siRNAs)to investigate which receptor exerts potentially positive role in VIP-induced melanogenesis;7)VIP-induced stimulatory melanogenic effect was further investigated in human epidermal melanocytes(HEMns)by assessing melanin production and the protein expression of tyrosinase and MITF by Western blotting analysis.Results:1)VIP enhanced melanin synthesis in a concentration-dependent manner(1-100 nM)in B16F10 cells and HEMns without cytotoxicity;2)VIP significantly increased intracellular tyrosinase activity in B16F10 cells;3)VIP significantly increased protein expression of tyrosinase and MITF in B16F10 cells;4)VIP increased mRNA expression of MITF and tyrosinase in a time-dependent manner in B16F10 cells;5)VIP induced activation of CREB and PKA,but not p38 MAPK,Akt,or ERK phosphorylation in B16F10 cells;6)Suppression of the PKA-CREB pathway attenuated VIP-induced stimulation of melanogenesis in B16F10 cells by decreasing melanin production and protein expression of tyrosinase and MITF;7)Both the VIP receptors VIPR1 and VIPR2 were expressed in B16F10 cells;8)Knockdown of the gene expression of VIPR1 or VIPR2 inhibited VIP-induced increased melanin synthesis and tyrosinase expression in B16F10 cells;9)VIP increased melanin production in a dose-dependent manner(1-100 nM)in HEMns;10)VIP markedly increased protein expression of tyrosinase and MITF in HEMns.Conclusion:1)VIP increases melanin production in B16F10 cells and HEMns;2)VIP increases tyrosinase activity and gene expression in mRNA and protein levels;3)VIP increases MITF expression both in mRNA and protein levels;4)VIP induces melanogenesis via PKA-CERB-MITF-Tyrosinase signaling pathway.