Whitening Effect Of Novel Peptide Mixture By Regulating Melanosome Biogenesis, Transfer And Degradation Part 2
Mar 30, 2023
Peptide mixture inhibits the expression of melanosome biogenesis/transportation-related factors
To investigate the mechanism of depigmenting action shown by peptide mixture, expression levels of melanogenesis-related factors and melanosome transportation-related factors were analyzed.
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As shown in Fig. 2A, transcription levels of MITF and its downstream genes, TYR, TYPR1, and TYRP2, were decreased by peptide mixture in B16F10 cells. Furthermore, α-MSH-induced protein levels of MITF and Tyrosinase were significantly decreased by peptide mixture in B16F10 cells (Fig. 2B).
These results suggest that the melanogenesis inhibition effect of the peptide mixture occurred by inhibiting the expression of melanogenesis-related factors.
It has been published that MITF also acts as a transcription factor that activates motor proteins which have a role in melanosome transportation [28,29]. To identify whether the peptide mixture, which has an inhibitory effect on MITF expression, decreased the expression of motor proteins, we analyzed mRNA and protein levels. As shown in Fig. 2C, transcription levels of RAB27A, MLPH, and MYO5A genes were significantly decreased by peptide mixture in B16F10 cells. Furthermore, α-MSH-induced protein levels of Rab27a and Melanophilin were decreased by peptide mixture in B16F10 cells (Fig. 2D).
These results suggest that inhibition of MITF expression by peptide mixture can result in inhibition of melanosome transportation.
Peptide mixture inhibits MITF activity through the regulation of CREB and ERK phosphorylation
To investigate the action mechanism of peptide mixture on MITF expression, we analyzed the phosphorylation level of transcriptional and post-translational regulatory factors of MITF.
As shown in Fig. 3A, phosphorylated CREB, a transcription factor activating MITF expression, was dose-dependently decreased by peptide mixture. Previously, it has been identified that the activity of MITF depends on its post-translational modifications and proteasome-mediated MITF protein degradation can be induced by phosphorylated ERK1/2 [30]. Peptide mixture significantly increased p-ERK1/2 level under the a-MSH-stimulated condition in B16F10 cells (Fig. 3B).
These results suggest that the expression and activity of MITF might be decreased through the regulation of CREB and ERK1/2 phosphorylations by peptide mixture.
Peptide mixture suppresses melanosome uptake of HaCaT keratinocytes
Melanosomes are known to be transferred from the dendrite tips of melanocytes to keratinocytes through the phagocytosis of keratinocytes. To identify whether the peptide mixture could inhibit the melanosome transfer to keratinocytes, HaCaT keratinocytes were treated with the peptide mixture at concentrations of 10, 50, 100, and 200 M and further treated with melanosomes isolated from B16F10 cells. The number of melanosomes transferred to the cells was dose-dependently decreased by peptide mixture (Fig. 4A). Image analysis through Fontana-Masson staining showed that the distribution of brown-stained melanosomes significantly decreased in the peptide mixture-treated group in a dose-dependent manner (Fig. 4B).
Furthermore, the expression of the F2RL1 (PAR-2) gene, a receptor that regulates the phagocytosis of keratinocytes, was significantly decreased by peptide mixture under the trypsin-stimulated condition (Fig. 4C).
These results suggest that the peptide mixture can reduce phagocytosis through the inhibition of PAR-2 expression in keratinocytes by the peptide mixture.
Peptide mixture induces melanosome degradation in HaCaT keratinocytes
Melanosomes transferred into keratinocytes are known to be gradually eliminated by the cornification of keratinocytes or degraded by the intracellular autophagy system. To identify whether the peptide mixture could promote melanosome degradation, we treated the peptide mixture to the melanosome containing HaCaT keratinocytes. When the amounts of melanosomes remaining were monitored, the dose-dependent decrease was observed from the cell lysate treated with peptide mixture (Fig. 5A). Image analysis through Fontana-Masson staining showed that the distribution of brown-stained melanosomes significantly decreased in the peptide mixture-treated group in a dose-dependent manner (Fig. 5B).
Furthermore, when the autophagic activity related-protein levels in keratinocytes were monitored, the protein levels of Beclin-1 and LC3-II about autophagosome formation were increased by peptide mixture treatment, while the protein level of p62 degraded by autophagolysosomes was decreased by peptide mixture treatment (Fig. 5C).
These results suggest that the peptide mixture induced autophagic activity to promote the degradation of the melanosomes in keratinocytes.
The peptide mixture showed an anti-pigmentation effect in the skin equivalent model
An experiment using MelanoDerm was conducted to identify whether the peptide mixture could result in anti-pigmentation of the skin. After topical treatment of liposome containing 2,000 ppm of the peptide mixture was conducted at doses of 50 and 100 g for 2 weeks, light microscopy was performed. The formation of dark melanosomes was induced entirely in the melanocyte bodies and dendrite tips of the control tissue, while the overall cell bodies were bright with a smaller number of dendrites because melanin production was inhibited in the tissue treated with the peptide mixture (Fig. 6A). Also, the melanin contents were dose-dependently decreased by peptide mixture, supporting the visual observation (Fig. 6B).
Moreover, histology was performed to observe the melanin distribution throughout the epidermis. According to the result, the control tissue showed a marked increase of melanogenesis in melanocytes of the basal layer, and brown-stained melanin was observed in keratinocytes due to the accompanied transfer to nearby keratinocytes. A considerable level of melanin was also observed in the upper cornified layers. On the other hand, the melanin contents of melanocytes were low in the tissues treated with the peptide mixture, and a significant decrease in melanin contents was observed throughout keratinocytes and cornified layers in comparison to the control group (Fig. 6C).
These results confirmed a significant anti-pigmentation effect of the peptide mixture on the skin equivalents.
DISCUSSION
The development of whitening materials has been targeted mainly to inhibit melanogenesis in melanocytes through the regulation of melanogenesis-related factors [31-35]. Recently, other targets of depigmentation, including melanosome migration, transfer, and degradation, have been studied and several agents have been identified [36-42]. Because pigmentation is an action involving multiple mechanisms, it might be efficient to use a combination of therapeutic agents which have regulatory effects for each target.
This study investigated the whitening activity of a peptide mixture containing four different peptides with the same molar ratio. The suggested action mechanism of peptide mixture against melanosome biogenesis, transfer, and degradation is illustrated in Fig. 7. In melanocytes, the peptide mixture inhibits CREB phosphorylation resulting in decreased MITF expression and induces phosphorylation of ERK1/2 results in MITF phosphorylation and proteasomal degradation. As a result, the expression of melanosome biogenesis and transport-related proteins, including Tyrosinase, TYRP1, TYRP2, Rab27A, Melanophilin, and MYO5A, is decreased. In keratinocytes, the peptide mixture decreases the expression of PAR-2 which mediates keratinocyte phagocytic capability correlated with actin filament reorganization. Furthermore, the peptide mixture activates autophagic flux, and transferred melanosomes are degraded in autophagolysosome.
Peptides have been extensively examined as active ingredients for cosmetics because of their high biocompatibility and protein-mimicking activity [43]. But they also have some disadvantages in that they can be easily degraded by proteases present in the skin and cannot easily penetrate the epidermis composed of lipids due to their hydrophilicity [44]. The studies for increasing peptide stability have suggested the methods of substituting amino acids at the predicted cleavage sites, acetylation of the N terminal, and amidation of the C terminal [45]. Furthermore, there are many studies to improve the transdermal penetration of peptides: methods involving the use of penetration inducers such as alcohols, azones, hexanoates, and unsaturated fatty acids; methods involving the use of specific peptide sequences that facilitate permeation; methods combining lipophilic derivatives; and methods encapsulating peptides such as liposomes, transfersomes, niosomes, and ethosomes [46-48]. In this regard, we prepared liposome encapsulating the peptide mixture to test its whitening effect in the skin equivalent model by topical treatment, and a significant reduction of melanin contents throughout the epidermal layers was shown in the test liposome treatment group (Fig. 6C).
In conclusion, the peptide mixture of this study exhibited a whitening effect through various actions, including inhibition of melanin synthesis and migration as well as inhibition of melanosome uptake to keratinocytes and promotion of melanosome degradation. The peptide mixture of this study could be used as a new whitening material and the liposome that facilitates the stability and skin penetration of peptides could be used to develop effective whitening products.
ACKNOWLEDGEMENTS
This study was conducted under the support of the World Class 300 R&D Project by the Ministry of Trade, Industry, and Energy (Project number: S2641452).
CONFLICTS OF INTEREST
The authors declare no conflicts of interest.
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