Dataset Information

Induction of retinal-dependent calcium influx in human melanocytes by UVA or UVB radiation contributes to the stimulation of melanosome transfer.

ABSTRACT:

Objectives

The transfer of melanosomes from melanocytes to neighbouring keratinocytes is critical to protect the skin from the deleterious effects of ultraviolet A (UVA) and ultraviolet B (UVB) irradiation; however, the initial factor(s) that stimulates melanosome transfer remains unclear. In this study, we investigated the induction of retinal-dependent calcium (Ca²⁺ ) influx in melanocytes (MCs) by UVA or UVB irradiation and the effect of transient receptor potential cation channel subfamily M member 1 (TRPM1) (melastatin1)-related Ca²⁺ influx on melanosome transfer.

Materials and methods

Primary human epidermal MCs were exposed to physiological doses of UVB or UVA light and loaded with a calcium indicator Fluo-4 dye. The change of intracellular calcium of MCs was monitored using a two-photon confocal fluorescence microscopy. MCs were co-cultured with human epidermal keratinocytes (KCs) in the absence or presence of voriconazole (a TRPM1 blocker) or calcium chelators. MCs were also transfected with TRPM1 siRNA for silencing the expression of TRPM1 gene. The melanosome transfer in the co-cultured cells was quantitatively analysed using flow cytometry and was further confirmed by immunofluorescent double-staining. The protein levels and distributions of TRPM1, OPN3 and OPN5 in MCs were measured by Western blotting or immunofluorescent staining.

Results

The retinal-dependent Ca²⁺ influx of UVA-exposed melanocytes differed greatly from that of UVB-exposed melanocytes in the timing-phase. The protein expression of TRPM1 in mono- and co-cultured MCs was dose-dependently up-regulated by UVA and UVB. TRPM1 siRNA-mediated knockdown and the blockage of TRPM1 channel using a putative antagonist (voriconazole) significantly inhibited melanosome transfer in co-cultures following UVA or UVB exposure.

Conclusions

The distinct time-phases of Ca²⁺ influx in MCs induced by UVA or UVB contribute to the consecutive stimulation of melanosome transfer, thereby providing a potent photoprotection against harmful UV radiation.

SUBMITTER: Hu QM

PROVIDER: S-EPMC6529151 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Publications

Induction of retinal-dependent calcium influx in human melanocytes by UVA or UVB radiation contributes to the stimulation of melanosome transfer.

Hu Qing-Mei QM Yi Wen-Juan WJ Su Meng-Yun MY Jiang Shan S Xu Shi-Zheng SZ Lei Tie-Chi TC

Cell proliferation 20170823 6

<h4>Objectives</h4>The transfer of melanosomes from melanocytes to neighbouring keratinocytes is critical to protect the skin from the deleterious effects of ultraviolet A (UVA) and ultraviolet B (UVB) irradiation; however, the initial factor(s) that stimulates melanosome transfer remains unclear. In this study, we investigated the induction of retinal-dependent calcium (Ca<sup>2+</sup> ) influx in melanocytes (MCs) by UVA or UVB irradiation and the effect of transient receptor potential cation ...[more]

PMID: 28833830

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Project description:BACKGROUND:Age-related macular degeneration (AMD) is the primary cause of blindness and severe vision loss in developed countries and is responsible for 8.7% of blindness globally. Ultraviolet radiation can induce DNA breakdown, produce reactive oxygen species, and has been implicated as a risk factor for AMD. This study investigated the effects of UVA radiation on Human retinal pigment epithelial cell (ARPE-19) growth and protein expression. METHODS:ARPE-19 cells were irradiated with a UVA lamp at different doses (5, 10, 20, 30 and 40?J/cm2) from 10?cm. Cell viability was determined by MTT assay. Visual inspection was first achieved with inverted light microscopy and then the DeadEnd™ Fluorometric TUNEL System was used to observe nuclear DNA fragmentation. Flow cytometry based-Annexin V-FITC/PI double-staining was used to further quantify cellular viability. Mitochondrial membrane potential was assessed with JC-1 staining. 2D electrophoresis maps of exposed cells were compared to nonexposed cells and gel images analyzed with PDQuest 2-D Analysis Software. Spots with greater than a 1.5-fold difference were selected for LC-MS/MS analysis and some confirmed by western blot. We further investigated whether caspase activation, apoptotic-related mitochondrial proteins, and regulators of ER stress sensors were involved in UVA-induced apoptosis. RESULTS:We detected 29 differentially expressed proteins (9 up-regulated and 20 down-regulated) in the exposed cells. Some of these proteins such as CALR, GRP78, NPM, Hsp27, PDI, ATP synthase subunit alpha, PRDX1, and GAPDH are associated with anti-proliferation, induction of apoptosis, and oxidative-stress protection. We also detected altered protein expression levels among caspases (caspase 3 and 9) and in the mitochondrial (cytosolic cytochrome C, AIF, Mcl-1, Bcl-2, Bcl-xl, Bax, Bad, and p-Bad) and ER stress-related (p-PERK, p-eIF2?, ATF4 and CHOP) apoptotic pathways. CONCLUSIONS:UVA irradiation suppressed the proliferation of ARPE-19 cells in a dose-dependent manner, caused quantitative loses in transmembrane potential (??m), and induced both early and late apoptosis.

Dataset Information

Induction of retinal-dependent calcium influx in human melanocytes by UVA or UVB radiation contributes to the stimulation of melanosome transfer.

Objectives

Materials and methods

Results

Conclusions

Publications

Induction of retinal-dependent calcium influx in human melanocytes by UVA or UVB radiation contributes to the stimulation of melanosome transfer.

Similar Datasets

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