Effect Of α-melanocyte Stimulating Hormone And Cyclic Adenosine Monophosphate On Proliferation And Melanin Synthesis Of Melanocytes From Taihe Silky Fowls In Vitro

Taihe silky fowls with the outstanding characteristic of higer pigmentation thanordinary chicken, are both consumed as a healthy food and used particularly as a kind oftraditional Chinese medicine. Melanin is the key component in silky fowl by which silkyfowl exerts its medicinal tonic effect. It has been proved in mammals and humans thatα-MSH (α-melanocyte stimulating hormone)－MC1R (melanocortin1receptor)－cAMP－TRP (tyrosinase-related protein) signaling pathway plays an important role in melaninbiosynthesis, in which α-MSH is the key factor in regulating synthesis of melanin, MC1Rbeing the key gene in the regulation of pigmentation and with the cAMP signaling pathwaybeing the key pathway in the pigmentation. No work has been conducted to illustrate theroles of related signaling molecules in the α-MSH－MC1R－cAMP－TRP pathway in themelanin synthesis in Taihe silky fowls. Melanocytes were isolated and purified from skinof Taihe silky fowl embryo and the effects of α-MSH and cAMP as the downstreamsignaling molecule on proliferation and melanin synthesis of silky fowl skin melanocyteswere investigated using in vitro cultured melanocytes in this study. The results showed asfollows.(1) Dispase II-trypsin degestion method provied melanocytes cultures with higher cellyields, good viability and rapid growth rate. Basic fibroblast growth factor (bFGF) andcholera toxin (CT) synergisticly stimulated the growth of silky fowl skin melanocytes. Thecultured cells isolated and purified from the embyro skin were identified as melanocytes bymorphological obervation, L-Dopa and anti-S-100protein staining andimmunohistochemical characterization.(2) α-MSH at various concentrations ranged from2.5to10μg/ml promoted theproliferation, tyrosinase (TYR) activity, cAMP content and thus stimulated melanogenesiswith different extents in Taihe silky fowl skin melanocytes compared with control. Theoptimum concentration of α-MSH to stimulate proliferation and melanogenesis in these invitro skin melanocytes was2.5μg/ml (P<0.05or P<0.01). α-MSH upregulated mRNAexpression of rne1anoeortin-1receptor (MC1R) in a dose-dependent manner. α-MSHantagonist (D-Trp7,Ala8,D-Phe10] α-MSH (6-11)-amide) significantly inhibited thestimulatory effects of α-MSH on TYR activities, cAMP contents, MC1R mRNAexpression (P<0.05). α-MSH plays a critical role in melanogenesis in these in vitro skinmelanocytes.(3) cAMP at various concentrations ranged from1×10-5to1×10-3μg/ml significantlyincreased TYR activities and stimulated pigmentation in in vitro silky fowl skinmelanocytes with the optimum concentration of cAMP being1×10-4mol/L (P<0.05).cAMP inhibitor RP-c-AMPS significantly inhibited cAMP (10-4mol/l)-induced upregulation of TYR activity (P<0.01) and melanogenesis (P<0.01). Furthermore, cAMPinhibitor RP-c-AMPS and adenylate cyclase (AC) inhibitor NKY80significantly inhibitedstimulatory effects of α-MSH (2.5μg/ml) on TYR activities, cAMP contents andpigmentation in silky fowl skin melanocytes (P<0.05or P<0.01). cAMP as thedownstream signaling molecule in the α-MSH—MC1R signaling pathway plays animmportant role in the regulation of melanin synthesis in silky fowl skin melanocytes.In conclusion, α-MSH plays a critical role in proliferation and melanogenesis inskin melanocytes, and cAMP as the downstream signaling molecule in the α-MSH—MC1R signaling pathway plays an immportant modulatory role in melanogenesis of Taihesilky fowl skin melanocytes.