Endocannabinoids stimulate human melanogenesis via type-1 cannabinoid receptor

Abstract

We show that a fully functional endocannabinoid system is present in primary human melanocytes (normal human epidermal melanocyte cells), including anandamide (AEA), 2-arachidonoylglycerol, the respective target receptors (CB(1), CB(2), and TRPV1), and their metabolic enzymes. We also show that at higher concentrations AEA induces normal human epidermal melanocyte apoptosis (∼3-fold over controls at 5 μM) through a TRPV1-mediated pathway that increases DNA fragmentation and p53 expression. However, at lower concentrations, AEA and other CB(1)-binding endocannabinoids dose-dependently stimulate melanin synthesis and enhance tyrosinase gene expression and activity (∼3- and ∼2-fold over controls at 1 μM). This CB(1)-dependent activity was fully abolished by the selective CB(1) antagonist SR141716 or by RNA interference of the receptor. CB(1) signaling engaged p38 and p42/44 mitogen-activated protein kinases, which in turn activated the cyclic AMP response element-binding protein and the microphthalmia-associated transcription factor. Silencing of tyrosinase or microphthalmia-associated transcription factor further demonstrated the involvement of these proteins in AEA-induced melanogenesis. In addition, CB(1) activation did not engage the key regulator of skin pigmentation, cyclic AMP, showing a major difference compared with the regulation of melanogenesis by α-melanocyte-stimulating hormone through melanocortin 1 receptor.

FIGURE 1.

Characterization of ECS in primary human melanocytes. The Western blot analysis of the major ECS elements in NHEM cells included homogenates of mouse brain, mouse spleen, and human HeLa cells as positive controls.

FIGURE 2.

Induction of NHEM cell apoptosis by mAEA and related compounds. The effect of different doses of mAEA on cell viability (A) and apoptotic cell death (B) is shown. C, effect of mAEA, AEA, ACEA, 2-AG, JWH133, or CPS (each used at 5 μm) and of mAEA (5 μm) in combination with SR141716 (SR1; 0.1 μm), SR144528 (SR2; 0.1 μm), or I-RTX (1 μm) on p53 mRNA expression. SR141716, SR144528, and I-RTX were ineffective when used alone (not shown for the sake of clarity). Error bars represent S.E. values. *, p < 0.05; **, p < 0.01; ***, p <0.001 versus control (Ctrl). ###, p < 0.001 versus mAEA.

FIGURE 3.

Induction of melanogenesis by mAEA and related compounds. A, effect of different non-toxic doses of mAEA on melanin synthesis. B, effect of mAEA, AEA, ACEA, 2-AG, JWH133, or CPS (each used at 1 μm) and of mAEA (1 μm) in combination with SR141716 (SR1; 0.1 μm), SR144528 (SR2; 0.1 μm) or I-RTX (1 μm). C, effect of CB₁ silencing (siCB₁) on melanin content and CB₁ mRNA and protein expression (inset). D, effect of mAEA (1 μm) alone or in combination with SB203580 (10 μm) or PD98059 (10 μm) on melanin synthesis. In B, SR141716, SR144528, and I-RTX were ineffective when used alone (not shown for the sake of clarity). Error bars represent S.E. values. ***, p < 0.001 versus control (Ctrl). ###, p < 0.001 versus mAEA.

FIGURE 4.

Induction of tyrosinase expression and activity by mAEA and related compounds. A, induction of tyrosinase mRNA levels by mAEA or CPS (each used at 1 μm) and of mAEA (1 μm) in combination with SR141716 (SR1; 0.1 μm), SR144528 (SR2; 0.1 μm), I-RTX (1 μm), SB203580 (10 μm), or PD98059 (10 μm). Induction of tyrosinase protein expression (B) and of tyrosinase activity (C) by mAEA (1 μm) alone or in combination with SR141716 (SR1), SB203580, or PD98059 as detailed in A. In all panels, SR141716, SR144528, I-RTX, SB203580, and PD98059 were ineffective when used alone (not shown for the sake of clarity). Error bars represent S.E. ***, p < 0.001 versus control (Ctrl). ##, p < 0.05; ###, p < 0.001 versus mAEA.

FIGURE 5.

Effect of tyrosinase and MITF silencing on mAEA activity. A, RNA interference of tyrosinase (siTYR) or MITF (siMITF) reduced tyrosinase mRNA and abrogated the ability of mAEA to induce its expression. B, RNA interference of tyrosinase (siTYR) did not reduce MITF mRNA nor did it abrogate the ability of mAEA to induce its expression. However, RNA interference of MITF (siMITF) had both effects. C, RNA interference of tyrosinase (siTYR) or MITF (siMITF) abrogated the ability of mAEA to induce melanin synthesis. Error bars represent S.E. values. *, p < 0.05; ***, p < 0.001 versus control (Ctrl). ###, p < 0.001 versus controls treated with 1 μm mAEA.

FIGURE 6.

Induction of melanogenesis by α-MSH. The effect of α-MSH (100 nm) alone or in combination with Agouti-related signaling protein (ASP; 10 nm), SB203580 (10 μm), and PD98059 (10 μm) on melanin content (A) and tyrosinase mRNA expression (B) is shown. C, effect of mAEA (1 μm), α-MSH (100 nm), and their combination on tyrosinase mRNA. Error bars represent S.E. values. ***, p < 0.001 versus control (Ctrl). ##, p < 0.05; ###, p < 0.001 versus α-MSH.

FIGURE 7.

Overall scheme of hypothetical effect of endocannabinoid signaling in human melanocytes. A, low doses of AEA (∼1 μm) and other CB₁-binding endocannabinoids stimulate melanogenesis through a p38- and p42/44-mediated pathway, which activates CREB and hence tyrosinase expression through the master regulator MITF. However, at higher doses (∼5 μm or above), AEA promotes programmed cell death through a TRPV1-dependent pathway that engages p53. B, stimulation of melanogenesis by endocannabinoids (triangles) via CB₁ receptors can be a faster alternative compared with the classical route activated by α-MSH. In fact, the latter pathway requires proopiomelanocortin (POMC) expression and cleavage into an ACTH intermediate, which then matures into the final signaling molecule, α-MSH. Both routes might be triggered in keratinocytes by UV light or other stress factors to stimulate production by melanocytes of the protective agent melanin. β-LPH, β-lipotropic hormone.