Korean J Physiol Pharmacol.  2021 Jan;25(1):87-94. 10.4196/kjpp.2021.25.1.87.

Nootkatol prevents ultraviolet radiation-induced photoaging via ORAI1 and TRPV1 inhibition in melanocytes and keratinocytes

• Woo JH
• Nam DY
• Kim HJ
• Hong PTL
• Kim WK
• Nam JH

• KMID: 2509658
• DOI: http://doi.org/10.4196/kjpp.2021.25.1.87

Abstract

Skin photoaging occurs due to chronic exposure to solar ultraviolet radiation (UV), the main factor contributing to extrinsic skin aging. Clinical signs of photoaging include the formation of deep, coarse skin wrinkles and hyperpigmentation. Although melanogenesis and skin wrinkling occur in different skin cells and have different underlying mechanisms, their initiation involves intracellular calcium signaling via calcium ion channels. The ORAI1 channel initiates melanogenesis in melanocytes, and the TRPV1 channel initiates MMP-1 production in keratinocytes in response to UV stimulation. We aimed to develop a drug that may simultaneously inhibit ORAI1 and TRPV1 activity to help prevent photoaging. We synthesized nootkatol, a chemical derivative of valencene. TRPV1 and ORAI1 activities were measured using the whole-cell patch-clamp technique. Intracellular calcium concentration [Ca²⁺ ] i was measured using calcium-sensitive fluorescent dye (Fura-2 AM). UV-induced melanin formation and MMP-1 production were quantified in B16F10 melanoma cells and HaCaT cells, respectively. Our results indicate that nootkatol (90 μM) reduced TRPV1 current by 94% ± 2% at –60 mV and ORAI1 current by 97% ± 1% at –120 mV. Intracellular calcium signaling was significantly inhibited by nootkatol in response to ORAI1 activation in human primary melanocytes (51.6% ± 0.98% at 100 μM). Additionally, UV-induced melanin synthesis was reduced by 76.38% ± 5.90% in B16F10 melanoma cells, and UV-induced MMP-1 production was reduced by 59.33% ± 1.49% in HaCaT cells. In conclusion, nootkatol inhibits both TRPV1 and ORAI1 to prevent photoaging, and targeting ion channels may be a promising strategy for preventing photoaging.

Keyword

Ion channel; Nootkatol; ORAI1 protein; Skin aging

Figure

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  Fig. 1 Inhibitory effects of nootkatol on the human TRPV1 (hTRPV1) and human ORAI1 (hORAI1) currents. (A) Representative chart trace recording of hTRPV1 inhibited by nootkatol in hTRPV1-overexpressing HEK293T cells. The solid line indicates the holding current, whereas solid bars indicate the corresponding current induced by ramp-like pulse. After hTRPV1 activation by 1 µM capsaicin, the marked states indicate the steady-state currents of each condition, namely control (1) and treatment with 10 (2), 30 (3), and 90 µM (4) nootkatol. (B) Related current-voltage (I–V) relationship curve at the steady-state of the control (1) and cells treated with 10 (2), 30 (3), and 90 µM (4) nootkatol. (C) Summary of current inhibition by nootkatol treatment. Current amplitude is normalized to control current at –60 mV (mean ± standard error of the mean [SEM], n = 7, 4, 4, 7, respectively) (***p < 0.001). (D) Representative chart trace recording of hORAI1 inhibited by nootkatol in hORAI1-overexpressing HEK293T cells. (E) Related current-voltage (I–V) relationship curve at the steady-state of the control (1) and cells treated with 10 (2), 30 (3), and 90 µM (4) nootkatol. (F) Summary of the current level of hORAI1 upon nootkatol treatment. Current amplitude is normalized to control current at –120 mV (mean ± SEM, n = 7, 6, 6, 4, respectively) (***p < 0.001).

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  Fig. 2 Inhibition of thapsigargin-induced store-operated Ca2+ entry (SOCE) by nootkatol in primary human melanocytes. (A) Representative trace for intracellular Ca2+ measurement by Fura-2 in primary human melanocytes. Treatment with 10 and 100 µM nootkatol after the 2 mM Ca2+ add-back procedure partially inhibited [Ca2+]i. To confirm the basal [Ca2+]i, 10 µM BTP2, a potent ORAI1 inhibitor, was used. Asterisks (*) indicate the “time point” for each chemical treatment. (B) Summary of the inhibitory effects of 10 and 100 µM nootkatol and 10 µM BTP2 on [Ca2+]i in primary human melanocytes. ***p < 0.001 vs. the control (mean ± standard error of the mean, n = 8).

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  Fig. 3 Inhibitory effect of nootkatol on ultraviolet radiation (UV)-induced melanogenesis. (A) In the cytotoxicity assay performed with B16F10 cells, nootkatol-treated cells showed 99.22% ± 0.70%, 98.36% ± 1.65%, 99.20% ± 0.52%, 97.80% ± 1.07%, 62.29% ± 2.45%, and 22.06% ± 1.26% cell survival rate at 7.5, 15, 30, 60, 120, and 200 µg/ml, respectively (mean ± standard error of the mean [SEM], n = 3). (B) The inhibitory effect on UV-induced melanogenesis in B16F10 cells was 84.14% ± 1.54% and 76.38% ± 5.90% at 7.5 µg/ml and 15 µg/ml nootkatol, respectively. Kojic acid showed 78.12% ± 7.29% melanin inhibition at 15 µg/ml (mean ± SEM, n = 3) (***p < 0.001). (C) Direct tyrosinase inhibitory activity of 200 µM nootkatol and kojic acid (positive control) (mean ± SEM, n = 3) (****p < 0.0001).

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  Fig. 4 Effects of nootkatol on ultraviolet radiation (UV)-induced matrix metalloproteinase-1 (MMP-1) production. (A) Treatment with 7.5, 15, 30, 60, 120, and 240 µg/ml nootkatol showed 111.37% ± 2.88%, 91.33% ± 2.74%, 27.24% ± 3.73%, 21.59% ± 0.51%, 21.50% ± 0.75%, and 20.65% ± 0.23% cell viability, respectively (n = 3). (B) MMP-1 level, expressed as % of control, following exposure to UVB irradiation (16 mJ/cm2) alone and with nootkatol pretreatment (7.5 µg/ml, 59.33% ± 1.49%) (mean ± standard error of the mean, n = 3) (****p < 0.0001).

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