Research Techniques Made Simple: Cell Biology Methods for the Analysis of Pigmentation

Benito-Martínez, Silvia; Zhu, Yueyao; Jani, Riddhi Atul; Harper, Dawn C.; Marks, Michael S.; Delevoye, Cédric

doi:10.1016/j.jid.2019.12.002

Cited by 27 publications

(24 citation statements)

References 97 publications

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“…We speculate that melanocore transfer through coupled exocytosis/phagocytosis occurs preferentially at basal levels of pigmentation and is the main mode of transfer, while the transfer of melanosome‐laden globules could be used in stress conditions, for instance during tanning to ensure a high throughput of pigment transfer 11 . Even though a potential caveat of our study is the use of mouse keratinocytes and melanin isolated from human MNT‐1 melanoma cells, it is important to mention that this in vitro melanin uptake assay was validated by others using human primary keratinocytes, 49 further supporting the relevance of our model and our observations. In conclusion, our current results and previously published studies suggest that the main mechanism of pigment transfer in the skin epidermis involves coupled exo/phagocytosis of melanocores, regulated by PAR‐2.…”

Section: Discussionmentioning

confidence: 98%

Melanocore uptake by keratinocytes occurs through phagocytosis and involves protease‐activated receptor‐2 internalization

Moreiras

Bento-Lopes

Neto

et al. 2022

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In the skin epidermis, melanin is produced and stored within melanosomes in melanocytes, and then transferred to keratinocytes. Different models have been proposed to explain the melanin transfer mechanism, which differ essentially in how melanin is transferred-either in a membrane-bound melanosome or as a melanosome core, that is, melanocore. Here, we investigated the endocytic route followed by melanocores and melanosomes during internalization by keratinocytes, by comparing the uptake of melanocores isolated from the supernatant of melanocyte cultures, with melanosomes isolated from melanocytes. We show that inhibition of actin dynamics impairs the uptake of both melanocores and melanosomes. Moreover, depletion of critical proteins involved in actin-dependent uptake mechanisms, namely Rac1, CtBP1/ BARS, Cdc42 or RhoA, together with inhibition of Rac1-dependent signaling pathways or macropinocytosis suggest that melanocores are internalized by phagocytosis, whereas melanosomes are internalized by macropinocytosis. Interestingly, we found that Rac1, Cdc42 and RhoA are differently activated by melanocore or melanosome stimulation, supporting the existence of two distinct routes of melanin internalization. Furthermore, we show that melanocore uptake induces protease-activated receptor-2 (PAR-2) internalization by keratinocytes to a higher extent than melanosomes. Because skin pigmentation was shown to be regulated by PAR-2 activation, our results further support the melanocore-based mechanism of melanin transfer and further refine this model, which can now be described as coupled melanocore exo/phagocytosis.

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Section: Discussionmentioning

confidence: 98%

Melanocore uptake by keratinocytes occurs through phagocytosis and involves protease‐activated receptor‐2 internalization

Moreiras

Bento-Lopes

Neto

et al. 2022

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“…Melanocytes and keratinocytes establish a complex intercellular dialog required for skin photoprotection. 2D co-culture systems, where these two cell types share the same medium, have been widely used to study intercellular communication and pigment transfer between epidermal cells 7,30 . To evaluate the distribution of caveolae within the epidermal unit in 2D, normal human melanocytes and keratinocytes were co-cultured and labeled for two main constituents of caveolae, Cav1 or Cavin1.…”

Section: Resultsmentioning

confidence: 99%

“…Different mechanisms have been proposed for melanin transfer to occur 5,54 and each one requires the local remodeling of the plasma membrane of melanocytes at the near vicinity of keratinocytes. To address the role of caveolae in melanin transfer, siCtrl-and siCav1-treated melanocytes were co-cultured with keratinocytes for 3 days, after which the cells were analyzed by immunofluorescence using HMB45 anti-PMEL antibody 30 (Fig. 4a).…”

Section: Caveolaementioning

confidence: 99%

Coupling of melanocyte signaling and mechanics by caveolae is required for human skin pigmentation

et al. 2020

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Tissue homeostasis requires regulation of cell-cell communication, which relies on signaling molecules and cell contacts. In skin epidermis, keratinocytes secrete factors transduced by melanocytes into signaling cues promoting their pigmentation and dendrite outgrowth, while melanocytes transfer melanin pigments to keratinocytes to convey skin photoprotection. How epidermal cells integrate these functions remains poorly characterized. Here, we show that caveolae are asymmetrically distributed in melanocytes and particularly abundant at the melanocyte-keratinocyte interface in epidermis. Caveolae in melanocytes are modulated by ultraviolet radiations and keratinocytes-released factors, like miRNAs. Preventing caveolae formation in melanocytes increases melanin pigment synthesis through upregulation of cAMP signaling and decreases cell protrusions, cell-cell contacts, pigment transfer and epidermis pigmentation. Altogether, we identify that caveolae serve as molecular hubs that couple signaling outputs from keratinocytes to mechanical plasticity of pigment cells. The coordination of intercellular communication and contacts by caveolae is thus crucial to skin pigmentation and tissue homeostasis.

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“…MNT1 and B16F0, which are rich in melanosomes, are widely used to study melanogenesis [ 38 ]. Consequently, these two cell lines were chosen for subsequent experiments.…”

Section: Methodsmentioning

confidence: 99%

Identification of Sitogluside as a Potential Skin‐Pigmentation‐Reducing Agent through Network Pharmacology

Guo

Zeng

et al. 2021

Oxidative Medicine and Cellular Longevity

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Many traditional Chinese medicines (TCMs) with skin-whitening properties have been recorded in the Ben-Cao-Gang-Mu and in folk prescriptions, and some literature confirms that their extracts do have the potential to inhibit pigmentation. However, no systematic studies have identified the specific regulatory mechanisms of the potential active ingredients. The aim of this study was to screen the ingredients in TCMs that inhibit skin pigmentation through a network pharmacology system and to explore underlying mechanisms. We identified 148 potential active ingredients from 14 TCMs, and based on the average “degree” of the topological parameters, the top five TCMs (Fructus Ligustri Lucidi, Hedysarum multijugum Maxim., Ampelopsis japonica, Pseudobulbus Cremastrae Seu Pleiones, and Paeoniae Radix Alba) that were most likely to cause skin-whitening through anti-inflammatory processes were selected. Sitogluside, the most common ingredient in the top five TCMs, inhibits melanogenesis in human melanoma cells (MNT1) and murine melanoma cells (B16F0) and decreases skin pigmentation in zebrafish. Furthermore, mechanistic research revealed that sitogluside is capable of downregulating tyrosinase (TYR) expression by inhibiting the ERK and p38 pathways and inhibiting TYR activity. These results demonstrate that network pharmacology is an effective tool for the discovery of natural compounds with skin-whitening properties and determination of their possible mechanisms. Sitogluside is a novel skin-whitening active ingredient with dual regulatory effects that inhibit TYR expression and activity.

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Research Techniques Made Simple: Cell Biology Methods for the Analysis of Pigmentation

Cited by 27 publications

References 97 publications

Melanocore uptake by keratinocytes occurs through phagocytosis and involves protease‐activated receptor‐2 internalization

Melanocore uptake by keratinocytes occurs through phagocytosis and involves protease‐activated receptor‐2 internalization

Coupling of melanocyte signaling and mechanics by caveolae is required for human skin pigmentation

Identification of Sitogluside as a Potential Skin‐Pigmentation‐Reducing Agent through Network Pharmacology

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