The Protease-Activated Receptor 2 Regulates Pigmentation via Keratinocyte-Melanocyte Interactions

doi:10.1006/excr.1999.4692

Experimental Cell Research

Volume 254, Issue 1, 10 January 2000, Pages 25-32

https://doi.org/10.1006/excr.1999.4692 Get rights and content

Abstract

Close association exists between melanocytes, the pigment melanin-producing cells in the body, and their neighboring keratinocytes. Keratinocytes are the pigment recipients and skin pigmentation is the result of this interaction. While the chemical basis of melanin production (melanogenesis) is well documented, the molecular mechanism of melanosome transfer needs to be elucidated. We are now providing first evidence that the protease-activated receptor 2 (PAR-2) expressed on keratinocytes, but not on melanocytes, is involved in melanosome transfer and therefore may regulate pigmentation. Activation of PAR-2 with trypsin or with the peptide agonist SLIGRL induced pigmentation in both two- and three-dimensional cocultures of keratinocytes and melanocytes, but not in cocultures that were spatially separated, indicating the need for intimate cell-cell contact. Topical application of SLIGRL on human skin transplanted on SCID mice resulted in a visible skin darkening. Histological examination revealed increased deposits of melanin in the keratinocytes. Inhibition of PAR-2 activation by RWJ-50353, a serine protease inhibitor, resulted in depigmentation and changes in expression of melanogenic-specific genes. Keratinocyte-melanocyte contact was essential for this depigmenting effect. Topical application of this inhibitor induced lightening of the dark skin Yucatan swine, which was confirmed by histochemical analysis. The results presented here suggest a novel mechanism for the regulation of pigmentation, mediated by the activation or inhibition of the keratinocyte receptor PAR-2.

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Keratinocytes are recipients of melanosomes. Although the chemical basis of melanogenesis is well documented, the molecular mechanism of melanosome transfer must be elucidated. TRPA1 is a member of the transient receptor potential A subfamily. Previous studies have shown that inhibition of TRPA1 activity reduces melanin synthesis in human epidermal melanocytes; however, the mechanism remains unknown.
This study aimed to investigate the roles and mechanism(s) of action of TRPA1 in keratinocytes.
The correlation between TRPA1 expression levels and the ability of keratinocytes to phagocytize melanosomes was examined using melanin silver staining. TRPA1 depleted human epidermal keratinocytes and keratinocyte cell lines HaCaT were established using adenovirus-expressing shRNAs against TRPA1. The effects of TRPA1 on keratinocytes and HaCaT cells were determined using cell-based analyses, including light stimulation, calcium imaging, melanin phagocytosis, immunoblotting, and co-immunoprecipitation assays. The degree of epidermal pigmentation was determined in a guinea pig model.
TRPA1 mediated the phagocytic activity of keratinocytes. TRPA1 knockdown markedly suppressed melanosome transport to keratinocytes. Mechanistically, TRPA1 was required for PAR-2-induced melanosome phagocytosis in keratinocytes. Furthermore, TRPA1 activation indirectly stabilized microtubules by promoting the competitive binding of CYLD and acetylated α-tubulin. In addition, bortezomib (PS-341), a proteasome inhibitor, increased TRPA1 and CYLD expression and promoted phagocytic activity both in vitro and in vivo.
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TLR3 stimulation induces melanosome endo/phagocytosis through RHOA and CDC42 in human epidermal keratinocyte
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Keratinocytes and melanocytes in human epidermis express Toll-like receptors (TLR) and induce immune responses. We previously reported that TLR3 stimulation increases melanosome transport from perinuclear to cell membrane in melanocytes and enhanced release of melanosome from melanocytes, which were followed by increase in melanosome uptake into keratinocytes.
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Poly(I:C)-stimulated keratinocytes enhanced uptake of isolated melanosome-rich globules five-times as much as control. Poly(I:C) increases the RNA and protein expressions of RHOA and CDC42, which are small GTP-binding proteins inducing the endocytosis. Pull-down assay showed that Poly(I:C) increased the GTP-binding RHOA and CDC42, suggesting TLR3 stimulation activated RHOA and CDC42. The knockdown of TLR3 suppressed RHOA and CDC42 induction by Poly(I:C). Consistently, the knockdown of RHOA and CDC42 significantly suppressed the melanosome-rich globules uptake by Poly(I:C)-stimulated keratinocytes.
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¹: To whom correspondence and reprint requests should be addressed at Skin Research Center, J & J CPWW, 199 Grandview Rd., Skillman, NJ 08558. Fax: (908) 874-2323. E-mail: [email protected].

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Regular ArticleThe Protease-Activated Receptor 2 Regulates Pigmentation via Keratinocyte-Melanocyte Interactions

Abstract

Biochem. Biophys. Acta

Trends Genet.

FEBS Lett.

Exp. Cell Res.

Cell

J. Biol. Chem.

J. Biol. Chem.

J. Invest. Dermatol.

J. Invest. Dermatol.

Pigment Cell Res.

Cell. Mol. Biol. Res.

Keio J. Med.

Clin. Drug Invest.

Pigment Cell Res.

Pigment Cell Res.

Arch. Dermatol. Res.

Proc. Natl. Acad. Sci. USA

Regular Article
The Protease-Activated Receptor 2 Regulates Pigmentation via Keratinocyte-Melanocyte Interactions