Skin pigmentation in the epidermis consists mainly of the synthesis and transfer of melanin, regulated by keratinocytes and melanocytes. Ultraviolet (UV) irradiation stimulates the production of proopiomelanocortin (POMC) and its derived peptides, such as α-melanocyte stimulating hormone (α-MSH), leading to increased melanogenesis in melanocytes. Melanin is processed in melanosomes, transported to surrounding keratinocytes, and directed to the perinuclear region, forming a melanin cap for photoprotection. The cholinergic system is involved in the signal transmission in the neuronal system, forming the cholinergic synapse, which is compartmentalized mainly by acetylcholine (ACh), acetylcholinesterase (AChE), and cholinergic receptors. The idea of "skin synapse" describes the interplay be...[ Read more ]

Skin pigmentation in the epidermis consists mainly of the synthesis and transfer of melanin, regulated by keratinocytes and melanocytes. Ultraviolet (UV) irradiation stimulates the production of proopiomelanocortin (POMC) and its derived peptides, such as α-melanocyte stimulating hormone (α-MSH), leading to increased melanogenesis in melanocytes. Melanin is processed in melanosomes, transported to surrounding keratinocytes, and directed to the perinuclear region, forming a melanin cap for photoprotection. The cholinergic system is involved in the signal transmission in the neuronal system, forming the cholinergic synapse, which is compartmentalized mainly by acetylcholine (ACh), acetylcholinesterase (AChE), and cholinergic receptors. The idea of "skin synapse" describes the interplay between keratinocytes and melanocytes modulated by cholinergic signaling. Previously, we unraveled the regulatory role of UV irradiation on ACh release by keratinocytes. Here, we aimed to investigate the involvement of cholinergic signaling in UV-mediated pigmentation, including the uptake and release of melanosomes and melanogenesis.

HaCaT keratinocytes and B16F10 melanoma cell lines were used in this study. Fluorescent beads in sizes corresponding to different forms of melanosomes, isolated melanosomes added to HaCaT cells, and HaCaT-B16F10 co-culture were established to study melanosome uptake. The UV-induced phagocytosis in keratinocytes was markedly increased by applied ACh or α7 nicotinic ACh receptor (α7 nAChR) agonist, and fully blocked by the α7 nAChR antagonist. Ca²⁺ influx, measured by the Ca²⁺ indicator Fluo4-AM, was observed during the event. Besides, cofilin phosphorylation, as well as the expression and activation of RhoA, accounting for phagocytosis, was induced by UVB: the phosphorylation was blocked by Ca²⁺ chelator BAPTA-AM or α7 nAChR antagonist. The results uncovered the role of α7 nAChR in mediating melanosome phagocytosis induced by UVB through regulation of actin dynamics.

In B16F10 melanoma cells, the extracellular melanosomes were isolated and quantified. UVB could elicit melanosome release, which was further enhanced by the muscarinic AChR (mAChR) agonist bethanechol. The antagonists corresponding to M1/M3 mAChRs could inhibit the event. The mobilization of Ca²⁺ and phosphorylation of Protein kinase C (PKC) were assessed, which could be triggered by UVB and bethanechol and suppressed by the M1/M3 mAChR antagonists. The expressions of the tethering exocyst subunits, i.e., Sec8, Exo70, and Rab11b, as well as the Ca²⁺ sensor synaptotagmin, were increased under UVB exposure together with mAChR agonist treatment, which were fully abolished by M1/M3 mAChR antagonists. Thus, the role of M1/M3 mAChR in modulating melanosome exocytosis by recruiting the exocyst tethering complex was proposed.

The role of the cholinergic system in regulating α-MSH production was probed by performing luciferase reporter assay, RT-qPCR, and Western blotting. Inhibition of α7 nAChR by its antagonist or Ca²⁺ influx by BAPTA-AM suppressed POMC expression. The production of α-MSH was consistent with POMC expression. Silencing α7 nAChR with shRNA prevented UVB-induced POMC expression. Besides, the conditioned medium collected from HaCaT cells treated with α7 nAChR antagonist and UVB was subjected to B16F10 cells for the assessment of melanogenesis. Melanogenesis could be downregulated, as implied by the expression of melanogenic enzymes, indicating the suppressing effect of α7 nAChR antagonist on keratinocytes in the production of α-MSH.

The current results support the role of non-neuronal cholinergic signaling in the skin epidermis in regulating pigmentation, and additionally, the intrinsic mechanism was explored. The outcomes provide insights into the development of novel therapeutic strategies for photoprotection and pigmentation disorders, such as hyperpigmentation and melasma. By targeting the cholinergic signaling, agents such as inhibitors of AChE or agonists/antagonists of AChRs may be developed to address pigmentation disorders.