R-Fluoxetine Increases Melanin Synthesis Through a 5-HT1A/2A Receptor and p38 MAPK Signaling Pathways

Abstract

Fluoxetine, a member of the class of selective serotonin reuptake inhibitors, is a racemic mixture and has an anxiolytic effect in rodents. Previously, we have shown that fluoxetine can up-regulate melanin synthesis in B16F10 melanoma cells and normal human melanocytes (NMHM). However, the role of r-fluoxetine and s-fluoxetine, in the regulation of melanin synthesis, is still unknown. Here, we show how r-fluoxetine plays a critical role in fluoxetine enhancing melanogenesis, both in vivo and vitro, by up-regulating tyrosinase (TYR) and the microphthalmia-associated transcription factor (MITF) expression, whereas, s-fluoxetine does not show any effect in the vivo and vitro systems. In addition, we found that r-fluoxetine induced melanin synthesis through the serotonin1A receptor (5-HT1A) and serotonin 2A receptor (5-HT2A). Furthermore, r-fluoxetine increased the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), without affecting the phosphorylation of extracellularly responsive kinase (ERK1/2) and c-Jun N-terminal kinase (JNK). These data suggest that r-fluoxetine may be used as a drug for skin hypopigmentation disorders.

Keywords: melanin; r-fluoxetine; zebrafish.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1

Effect of the r-fluoxetine and s-fluoxetine on the B16F10 cells viability, tyrosinase activity, and melanin content. B16F10 cells were incubated with r-fluoxetine and s-fluoxetine, at various concentrations, for 48 h, and the cell viability was examined by an MTT assay, r-fluoxetine (a) and s-fluoxetine (b). Tyrosinase activity (c) and melanin contents (d) were performed, as described in the Materials and Methods section. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with control.

Figure 2

Effect of r-fluoxetine and s-fluoxetine on the expression of the tyrosinase (TYR) and the microphthalmia-associated transcription factor (MITF) in B16F10 cells. (a) Western Blot assays were performed to examine MITF and TYR expression levels. (b,c) Densitometry scanning of the band densities were utilized to measure the expression of proteins by the Quantity One software. Bars indicate the means ± SEM of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, compared vs. control.

Figure 3

Effect of r/s-fluoxetine on tyrosinase activity and melanin synthesis in zebrafish. (a) Schematic representation for the schedule of pigmentation rescue study. (b) Mortality rate was calculated by calculating the normally developed embryos at various concentrations for 48 h. (c) The heart-beating rate was measured at 60 hpf (hours post-fertilization), under the stereomicroscope. (d) Synchronized embryos were treated with 0.2 mM 1-phenyl-2-thiourea (PTU) at 6 hpf. r-fluoxetine and s-fluoxetine was added and incubated for a further 25 h, after a PTU wash, at 35 hpf. Scale bar, 200 μm. (e) Tyrosinase activity and (f) melanin contents of about thirty synchronized embryos, collected and dissolved in cold lysis buffer. After centrifugation, 10 μg of the total protein was incubated with 0.1% of l-dopa, as described in Section 4. All experiments were repeated three times. Data were analyzed by one-way analysis of variance (ANOVA), followed by a post hoc Tukey test. Bars indicate the means ± SEM of the three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the PTU35 (control).

Figure 4

B16F10 cells were transfected with luciferase reporter constructs and treated with the r/s-fluoxetine 10 μM for 24 h. (a) pGL3-tyrp1a and (b) pGL3-mitfa. Results shown are means ± SEM and representative of three independent experiments. Data were analyzed by ANOVA, followed by a post hoc Tukey test. Bars indicate the means ± SEM of the three independent experiments. ** p < 0.01, *** p < 0.001, compared with the control group.

Figure 5

Effect of the r-fluoxetine and the s-fluoxetine on the gfp expression of the tyrp1a:eGFP and the mitfa:eGFP zebrafish. (a) tyrp1a:eGFP zebrafish and (b) mitfa:eGFP zebrafish Synchronized embryos were treated with 0.2 mM 1-phenyl-2-thiourea (PTU) at 6 hpf. r-fluoxetine (100 μM) and s-fluoxetine (100 μM) were added and incubated for a further 25 h, after a PTU wash at 35 hpf. Scale bar, 100 μm.

Figure 6

Effect of the WAY100635 and the ketanserin on the melanin contents in the r-fluoxetine-induced zebrafish pigmentation. Synchronized embryos were treated with r-flu (100 μm), WAY100635 (10 μM), and ketanserin (10 μM), at 6 hpf and incubated for further 30 hpf. The effect on the pigmentation of the zebrafish were photographed under the stereomicroscope. (a) Lateral view of embryos at 36 hpf, (b) dorsal view of embryos at 36 hpf. Scale bar, 200 μm. (c) Tyrosinase activity and (d) melanin contents were performed, as described in the Materials and Methods section. Data were analyzed by a one-way analysis of variance (ANOVA) followed by post hoc Tukey test. Bars indicate the means ± SEM of the three independent experiments. *** p < 0.001, compared with the control; # p < 0.05, ## p < 0.01, compared with r-flu group.

Figure 7

Effect of the r-fluoxetine on the expression of the MAPK signaling pathways in the B16F10 cells. (a) Western Blot assays were performed to examine the p38 MAPK, ERK, and JNK expression levels. (b) Densitometry scanning of the band densities of the p38 MAPK were utilized to measure the expression of proteins by Quantity One software. Bars indicate the means ± SEM of the three independent experiments. ** p < 0.01, *** p < 0.001 vs. control group.

Figure 8

Schematic description of the changes in melanin synthesis induced by r-fluoxetine. Red arrow define the activity of r-fluoxetine, black arrow define the direct stimulatory modification, dotted arrow define the tentative stimulatory modification.