Neural stem cells inhibit melanin production by activation of Wnt inhibitors

Abstract

Background: Melanin for skin pigmentation is synthesized from tyrosine via an enzymatic cascade that is controlled by tyrosinase (TYR), tyrosinase-related protein 1 (TRP1), and dopachrome tautomerase/tyrosinase related protein 2 (Dct/TRP2), which are the targets of microphthalmia-associated transcription factor (MITF). MITF is a master regulator of pigmentation and a target of β-catenin in Wnt/β-catenin signaling during melanocyte differentiation. Stem cells have been used in skin pigmentation studies, but the mechanisms were not determined for the conditioned medium (CM)-mediated effects.

Objectives: In this study, the inhibition and mechanisms of melanin synthesis were elucidated in B16 melanoma cells and UV-B irradiated C57/BL-6 mice that were treated with human neural stem cell-conditioned medium (NSC-CM).

Methods: B16-F10 melanoma cells (1.5×10(4)cells/well) and the shaved dorsal skin of mice were pretreated with various amount (5, 10, 20, 50, and 100%) of NSC-CM. Melanin contents and TYR activity were measured by a Spectramax spectrophotometer. The expression of TYR, TRP1, Dct/TRP2, MITF, β-catenin and Wnt inhibitors were evaluated by RT-PCR and western blot. The dorsal skin samples were analyzed by immunofluorescence with various antibodies and compared with that control of tissues.

Results: Marked decreases were evident in melanin content and TYR, TRP1, DCT/TRP2, MITF, and β-catenin expression in B16 cells and C57/BL-6 mice. NSC-CM negatively regulated Wnt/β-catenin signaling by decreasing the expression of β-catenin protein, which resulted from robust expression of Wnt inhibitors Dickkopf-1 (DKK1) and secreted frizzled-related protein 2 (sFRP2).

Conclusions: These results demonstrate that NSC-CM suppresses melanin production in vitro and in vivo, suggesting that factors in NSC-CM may play an important role in deregulation of epidermal melanogenesis.

Keywords: (2′Z, 3′E)-6-bromoindirubin-3′-oxime; APC; BIO; CK1α; DKK1,2,3; Dct/TRP2; Dickkopf-1,2,3; Dishevelled; Dvl; GSK-3β; MITF; Melanin; NSC culture medium; NSC-CM; Neural stem cell-conditioned medium; PMSF; SCF; T-cell factor/lymphoid enhancer factor; TCF/LEF; TRP1; TYR; Tyrosinase; WIF1; Wnt inhibitors; Wnt inhibitory factor1; adenomatous polyposis coli; casein kinase1α; dopachrome tautomerase/tyrosinase related protein 2; glycogen synthase kinase 3β; microphthalmia-associated transcription factor; neural stem cell conditioned medium; non-CM; phenylmethyl sulfonyl fluoride; sFRP1,2,4,5; secreted frizzled-related protein; stem cell factor; tyrosinase; tyrosinase-related protein 1; α-MSH; α-melanocyte-stimulating hormone.