158
J. Cosmet. Sci., 74.3, 158–172 (May/June 2023)
Hydrolyzed Conchiolin Protein Inhibits Dendrite Formation
of Melanocytes Via Wnt/β-catenin Signaling Pathway
KAI ZANG, ZIHENG XU, RUNXUAN WANG, XIYUN YE AND YONGYAN DANG
Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences,
East China Normal University, Shanghai, China (K.Z., Z.X., X.Y., Y.D.)
Accepted for publication July 12, 2023.
Synopsis
Soluble pearl extracts present the potential in inhibiting melanin synthesis in B16 melanoma cells. However,
the role of hydrolyzed conchiolin protein (HCP), the main protein ingredient in soluble pearl extracts, in
skin lightening remains unclear. In this study, we observed that HCP effectively inhibited endothelin-1
(ET-1) induced melanocyte dendrite formation in both PIG1 and MelanA cells, demonstrating its excellent
antagonistic properties against ET-1 in.melanosome transfer. HCP also exhibits the capability to reduce
ET-1-induced melanin secretion in MelanA cells. HCP also significantly suppressed the upregulation of
microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), Rab27a, and Rac1 gene expression.
Additionally, HCP treatments led to a notable reduction in the protein expression levels of p-GSK-3β,
β-catenin, as well as the nuclear translocation of β-catenin. Importantly, HCP exhibited low toxicity towards
MelanA and PIG1 cells. Our results indicate that HCP can effectively suppress melanin transfer in melanocytes
by antagonizing ET-1 and that HCP treatments can downregulate melanin synthesis or transfer-related gene
expression by inhibiting Wnt/β-catenin signaling pathway. HCP may emerge as a safe candidate for treating
skin pigmentation disorder in the future.
INTRODUCTION
Skin color mainly depends on the distribution of pigments in the epidermal layer.1 The
variations in skin color among individuals can be attributed to differences in the number,
size, composition, and distribution of melanosomes.2 Melanosome, a lysosome-like structure
in melanocytes, is responsible for the production of melanin.3 Mature melanosomes
undergo cytoskeletal transport towards the periphery of melanocytes to facilitate the
transfer of melanin content to the surrounding keratinocytes.4,5 A protein complex
consisting of Rab27A, Mlph/Slac2-a and myosin Va is thought to mediate actin-dependent
melanosome transportation in melanocytes.6 Dysfunction of Rab27A is known to cause
a hypopigmentation phenotype in human type 2 Griscelli syndrome patients, indicating
that Rab27A is essential in maintaining normal and well-organized pigmentation.7
Additionally, the activation of Rac1 is essential for the formation of melanocytic dendrites,
facilitating membrane ruffling, and lamellipodia formation.8 While several compounds
*Address all correspondence to Xiyun Ye, xyye@bio.ecnu.edu.cn and Yongyan Dang, yydang@bio.ecnu.cn

159 Hydrolyzed Conchiolin Protein Inhibits Melanin
have been employed for skin whitening by inhibiting melanin production, there is still a
lack of drugs that specifically target melanin transfer. Current whitening compounds, such
as hydroquinone and alpha arbutin, have been reported to be carcinogenic and can cause
redness or irritation when used in large amounts.9 Therefore, there has been an increasing
emphasis on the development of safer whitening products.
Pearl extract is rich in minerals and amino acids. Pearl extracts can enhance skin moisture, skin
repair, and UV protection without any unwanted side effects. It has been demonstrated that a
soluble pearl extract possesses the ability to inhibit melanin synthesis in B16 melanoma cells.10
In vitro studies have also shown that HCP exhibits anti–inflammatory and anti–apoptotic
properties.11 HCP is a hydrolyzing derivative of conchiolin protein extracted from pearls. As
a significant active component of pearl extract, HCP has been found to be biocompatible, but
the specific effects of HCP on melanin synthesis and transport remain unknown.
In this study, we aimed to investigate the effects of HCP on melanocyte morphology and
melanosome transport, and to further elucidate the underlying mechanism. We observed
that HCP effectively suppressed melanocyte dendrite extension induced by ET-1 and
downregulated the expression of genes involved in melanosome transport, including MITF,
Rab27A, and Rac1, suggesting a novel role for HCP in regulating melanosome transport.
Additionally, HCP was found to negatively regulate the activity of the Wnt/β-catenin
signaling pathway, leading to the decrease of pigmentation in zebrafish. Therefore, it seems
that HCP can inhibit melanin transfer by suppressing the activity of Wnt/β-Catenin
signaling pathway in melanocytes.
MATERIALS AND METHODS
REAGENTS
HCP was obtained from OSM Biology Co., Ltd. (Zhejiang, China). ET-1 was purchased
from MedChemExpress Co., Ltd. (Shanghai, China).
CELL CULTURE
PIG1 and MelanA cells were purchased from American Type Culture Collection (ATTC,
Virginia, USA) and cultured at 37°C with 5% CO
2 atmosphere in Dulbecco’s Modified
Eagle Medium (DMEM) containing 10% fetal bovine serum, 100 U/ml penicillin, and
100 µg/ml streptomycin.
CELL VIABILITY ASSAY
PIG1 and MelanA cells were cultured in 96-well plates at a density of 1.5 × 105/ml. Cells
were treated with HCP and ET-1 at various concentrations for 24 hours. For PIG1 cells, we
selected HCP treatment concentrations of 0, 0.01, 0.1, 5, 10, 20, 50, 80, and 100 µg/ml.
For MelanA cells, we selected HCP treatment concentrations of 0, 0.1, 1, 2, 4, 8, 10,
20, 40, 80 µg/ml. After a brief wash with medium, 3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) (0.5 mg/ml in DMEM)
was used for the quantification of metabolically active cells, and samples were measured
photometrically at 490 nm.