Korean J Physiol Pharmacol.  2021 Jan;25(1):15-26. 10.4196/kjpp.2021.25.1.15.

Whitening effect of novel peptide mixture by regulating melanosome biogenesis, transfer and degradation

• Lee EJ
• Kim J
• Jeong MK
• Lee YM
• Chung YJ
• Kim EM

• KMID: 2509651
• DOI: http://doi.org/10.4196/kjpp.2021.25.1.15

Abstract

Peptides are short chain of amino acids linked by peptide bonds. They are widely used as effective and biocompatible active ingredients in cosmetic industry. In this study, we developed novel peptide mixture and identified its anti-pigmentation effect on melanocytes and keratinocytes. Our results revealed that peptide mixture inhibited melanosome biogenesis through the regulation of microphthalmia-associated transcription factor, a key factor of melanogenesis in melanocytes. And we observed that peptide mixture inhibited melanosome uptake through the reduction of protease-activated receptor 2, a phagocytosis-related receptor in keratinocytes. Furthermore, peptide mixture activated autophagy system resulting in degradation of transferred melanosomes in keratinocytes. The anti-pigmentation effect of multi-targeting peptide mixture was assessed in a human skin equivalent model (MelanoDerm). Melanin contents in epidermal layer were significantly decreased by topical treatment of peptide mixture, suggesting that it can be applied as a novel cosmetics material having a whitening function.

Keyword

Autophagy; Melanosome; Microphthalmia-associated transcription factor PAR-2; Peptide

Figure

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  Fig. 1 Effects of the peptide mixture on cell viability and melanogenesis. (A) B16F10 melanoma cells and HaCaT keratinocytes were treated with indicated concentration of peptide mixture for 72 h. Cell viability was determined by MTT assay. (B) B16F10 cells were exposed to peptide mixture in the presence of 100 ng/ml α-MSH for 72 h. Melanin content and (C) cellular tyrosinase activity were measured. Two hundreds micromole of arbutin was used as a positive control. Each determination was made in triplicate, and data are shown as means ± standard deviation. MTT, 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide; α-MSH, alpha-melanocyte-stimulating hormone. *p < 0.05, **p < 0.01 compared to the α-MSH-treated control.

• 

  Fig. 2 Effects of the peptide mixture on the expression of melanogenesis-related factors and melanosome transport-related factors. B16F10 cells were exposed to peptide mixture in the presence of 100 ng/ml α-MSH for 72 h. Two hundreds micromole of arbutin was used as a positive control. (A) Expression levels of melanogenesis-related genes including MITF, TYR, TYRP1, and TYRP2 were analyzed by RT-PCR. (B) The protein levels of MITF and tyrosinase were detected using Western blotting. (C) Expression levels of melanosome transport-related genes including RAB27A, MLPH, and MYO5A were analyzed by RT-PCR. (D) The protein levels of Rab27a and melanophilin were detected using Western blotting. α-MSH, alpha-melanocyte-stimulating hormone; MITF, microphthalmia-associated transcription factor; TYRP, tyrosinase-related protein.

• 

  Fig. 3 Effects of the peptide mixture on the phosphorylation level of CREB and ERK. (A) B16F10 cells were exposed to peptide mixture in the presence of 100 ng/ml α-MSH for 30 min. Two hundreds micromole of arbutin was used as a positive control. Phosphorylation level of CREB, a transcriptional factor of MITF, was analyzed by Western blotting. (B) B16F10 cells were exposed to peptide mixture in the presence of 100 ng/ml α-MSH for 10 min. Two hundreds micromole of arbutin was used as a positive control. Phosphorylation level of ERK, a kinase regulates protein stability of MITF, was analyzed by Western blotting. CREB, cAMP responsive element binding protein; α-MSH, alpha-melanocyte-stimulating hormone; MITF, microphthalmia-associated transcription factor.

• 

  Fig. 4 Effects of the peptide mixture on the melanosome uptake of keratinocytes. HaCaT keratinocytes were exposed to peptide mixture for 1 h and additionally incubated with isolated melanosomes for 40 h. (A) After treatment, melanin content was analyzed. Each determination was made in triplicate, and data are shown as means ± standard deviation. *p < 0.05, **p < 0.01 compared to the melanosome-treated control. (B) Fontana-Masson staining was performed to observe the intracellular distribution of melanosomes. (C) HaCaT keratinocytes were pretreated with the peptide mixture for 1 h, cells were additionally incubated with 4U trypsin for 16 h. The expression level of F2RL1 gene was analyzed by RT-PCR.

• 

  Fig. 5 Effect of the peptide mixture on melanosome degradation in keratinocytes. HaCaT keratinocytes were exposed to isolated melanosomes for 48 h and additionally incubated with peptide mixture for 72 h. One micromole of rapamycin was used as a positive control. (A) After treatment, melanin content was analyzed. Each determination was made in triplicate, and data are shown as means ± standard deviation. *p < 0.05 compared to the melanosome-treated control. (B) Fontana-Masson staining was performed to observe the intracellular distribution of melanosomes. (C) HaCaT keratinocytes were treated with peptide mixture for 3 h and protein levels of Beclin-1, LC3, and p62 were analyzed by western blotting. 200 nM rapamycin was used as a positive control.

• 

  Fig. 6 The whitening effect of the peptide mixture on the skin equivalent. MelanoDerm (MEL-300-B) was topically treated with liposome containing 2,000 μg/ml of peptide mixture three times a week for 2 weeks. (A) Light microscopy was conducted to observe the cell morphology (B) and melanin content was analyzed using extract of equivalent. (C) Paraffin sections were stained with Fontana-Masson to observe the melanosome distribution of the epidermal layers (scale bar = 100 μm). Each determination was made in triplicate, and data shown are means ± standard deviation. *p < 0.05 compared to the untreated control.

• 

  Fig. 7 Schematic diagram showing the mechanism of action of peptide mixture. CREB, cAMP-response-element-binding protein; MITF, microphthalmia-associated transcription factor; TYRP1, tyrosinase-related protein 1; TYRP2, tyrosinase-related protein 2; ERK, extracellular signal regulated kinase; PAR-2, protease activated receptor 2; PE, phosphatidylethanolamine. Arrows indicate positive regulation while T-bars denote inhibitory effects.

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