R-Fluoxetine Increases Melanin Synthesis Through a 5-HT1A/2A Receptor and p38 MAPK Signaling Pathways
"> Figure 1
<p>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 (<b>a</b>) and s-fluoxetine (<b>b</b>). Tyrosinase activity (<b>c</b>) and melanin contents (<b>d</b>) were performed, as described in the Materials and Methods section. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001, compared with control.</p> "> Figure 2
<p>Effect of r-fluoxetine and s-fluoxetine on the expression of the tyrosinase (TYR) and the microphthalmia-associated transcription factor (MITF) in B16F10 cells. (<b>a</b>) Western Blot assays were performed to examine MITF and TYR expression levels. (<b>b</b>,<b>c</b>) 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. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001, compared vs. control.</p> "> Figure 3
<p>Effect of r/s-fluoxetine on tyrosinase activity and melanin synthesis in zebrafish. (<b>a</b>) Schematic representation for the schedule of pigmentation rescue study. (<b>b</b>) Mortality rate was calculated by calculating the normally developed embryos at various concentrations for 48 h. (<b>c</b>) The heart-beating rate was measured at 60 hpf (hours post-fertilization), under the stereomicroscope. (<b>d</b>) 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. (<b>e</b>) Tyrosinase activity and (<b>f</b>) 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 <span class="html-small-caps">l</span>-dopa, as described in <a href="#sec4-ijms-20-00080" class="html-sec">Section 4</a>. 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. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001, compared with the PTU35 (control).</p> "> Figure 4
<p>B16F10 cells were transfected with luciferase reporter constructs and treated with the r/s-fluoxetine 10 μM for 24 h. (<b>a</b>) pGL3-tyrp1a and (<b>b</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. ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001, compared with the control group.</p> "> Figure 5
<p>Effect of the r-fluoxetine and the s-fluoxetine on the gfp expression of the tyrp1a:eGFP and the mitfa:eGFP zebrafish. (<b>a</b>) tyrp1a:eGFP zebrafish and (<b>b</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.</p> "> Figure 6
<p>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. (<b>a</b>) Lateral view of embryos at 36 hpf, (<b>b</b>) dorsal view of embryos at 36 hpf. Scale bar, 200 μm. (<b>c</b>) Tyrosinase activity and (<b>d</b>) 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. *** <span class="html-italic">p</span> < 0.001, compared with the control; # <span class="html-italic">p</span> < 0.05, ## <span class="html-italic">p</span> < 0.01, compared with r-flu group.</p> "> Figure 7
<p>Effect of the r-fluoxetine on the expression of the MAPK signaling pathways in the B16F10 cells. (<b>a</b>) Western Blot assays were performed to examine the p38 MAPK, ERK, and JNK expression levels. (<b>b</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. ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001 vs. control group.</p> "> Figure 8
<p>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.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Effect of R/S-Fluoxetine on Tyrosinase Activity and Melanin Synthesis in B16F10 Cells
2.2. Effect of R/S-Fluoxetine on MITF and Tyrosinase Protein Expression in B16F10 Cells
2.3. Effect of R/S-Fluoxetine on Tyrosinase Activity and Melanin Synthesis in Zebrafish
2.4. Effect of R/S-Fluoxetine on the Promoter Activities of Mitfa and Tyrp1a in the Zebrafish
2.5. Effect of R/S-Fluoxetine on the GFP (Green Fluorescent Protein) Expression in the Tyrp1a:eGFP and Mitfa:eGFP Zebrafish
2.6. R-Fluoxetione Induce Melanin Synthesis through 5-HT1A Receptor and 5-HT2A Receptor
2.7. Effect of the R-Fluoxetine on Phosphorylation of the p38 MAPK, ERK1/2, and JNK in the B16F10 Cells
3. Discussion
4. Materials and Methods
4.1. Cell Culture and Materials
4.2. Cell Viability Assay
4.3. Melanin Measurement and Tyrosinase Assay
4.4. Western Blot Analysis
4.5. Zebrafish Maintenance
4.6. Measurement of the Pigmenting Activity in the Zebrafish
4.7. The Luciferase Assay
4.8. Measurement of the Heart-Beating Rate and the Mortality Rate
4.9. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
TYR | tyrosinase |
MITF | microphthalmia-associated transcription factor |
5-HT1AR | serotonin1A receptor |
5-HT2AR | serotonin 2A receptor |
PTU | 1-phenyl-2-thiourea |
PTU35 | 1-phenyl-2-thiourea 35 hpf |
PTU60 | 1-phenyl-2-thiourea 60 hpf |
MAPK | mitogen-activated protein kinase |
TBS | tris- buffered saline |
V/V | volume/volume |
DMSO | dimethyl sulfoxide |
hpf | hours post-fertilization |
MTT | 3-(4, 5-dimethylthiazol)-2, 5-diphenyl tetrazolium bromide |
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Liu, L.; Fu, M.; Pei, S.; Zhou, L.; Shang, J. R-Fluoxetine Increases Melanin Synthesis Through a 5-HT1A/2A Receptor and p38 MAPK Signaling Pathways. Int. J. Mol. Sci. 2019, 20, 80. https://doi.org/10.3390/ijms20010080
Liu L, Fu M, Pei S, Zhou L, Shang J. R-Fluoxetine Increases Melanin Synthesis Through a 5-HT1A/2A Receptor and p38 MAPK Signaling Pathways. International Journal of Molecular Sciences. 2019; 20(1):80. https://doi.org/10.3390/ijms20010080
Chicago/Turabian StyleLiu, Li, Mengsi Fu, Siran Pei, Liangliang Zhou, and Jing Shang. 2019. "R-Fluoxetine Increases Melanin Synthesis Through a 5-HT1A/2A Receptor and p38 MAPK Signaling Pathways" International Journal of Molecular Sciences 20, no. 1: 80. https://doi.org/10.3390/ijms20010080