Inhibitors of intracellular signaling pathways that lead to stimulated epidermal pigmentation: perspective of anti-pigmenting agents

Abstract

Few anti-pigmenting agents have been designed and developed according to their known hyperpigmentation mechanisms and corresponding intracellular signaling cascades. Most anti-pigmenting agents developed so far are mechanistically involved in the interruption of constitutional melanogenic mechanisms by which skin color is maintained at a normal and unstimulated level. Thus, owing to the difficulty of confining topical application to a specific hyperpigmented skin area, potent anti-pigmenting agents capable of attenuating the natural unstimulated pigmentation process have the risk of leading to hypopigmentation. Since intracellular signaling pathways within melanocytes do not function substantially in maintaining normal skin color and are activated only by environmental stimuli such as UV radiation, specifically down-regulating the activation of melanogenesis to the constitutive level would be an appropriate strategy to develop new potent anti-pigmenting agents with a low risk of hypopigmentation. In this article, we review the hyperpigmentation mechanisms and intracellular signaling pathways that lead to the stimulation of melanogenesis. We also discuss a screening and evaluation system to select candidates for new anti-melanogenic substances by focusing on inhibitors of endothelin-1 or stem cell factor-triggered intracellular signaling cascades. From this viewpoint, we show that extracts of the herbs Withania somnifera and Melia toosendan and the natural chemicals Withaferin A and Astaxanthin are new candidates for potent anti-pigmenting substances that avoid the risk of hypopigmentation.

Figure 1.

Paracrine cytokine mechanisms underlying hyperpigmentation in UVB-melanosis, solar lentigo and melasma. TNF, tumor necrosis factor; IL-1, interleukin-1; EDN1, endothelin-1; mSCF, membrane-bound stem cell factor; sSCF, soluble stem cell factor.

Figure 2.

Intracellular signaling mechanisms associated with EDN1 and SCF. EDN1, endothelin-1; EDNRB, endothelin B receptor; MITF, microphthalmia associated transcription factor; PKA, protein kinase A; PKC, protein kinase C; SCF, stem cell factor; TYRP-1, tyrosinase-related protein-1; DCT, dopachrome tautomerase; TYK, tyrosine kinase; αMSH, alpha melanocyte stimulating hormone.

Figure 3.

Effects of specific signaling inhibitors on ERK (A); CREB (B); MITF (C) phosphorylation during SCF signaling.

Figure 3.

Effects of specific signaling inhibitors on ERK (A); CREB (B); MITF (C) phosphorylation during SCF signaling.

Figure 4.

(A) Experimental procedure for human epidermal equivalents (HEEs) using the J-TEC Melano-Model [–42]; (B) Effects of EDN1 on the pigmentation of HEEs during 14 days of culture; (C) Time course of expression of melanocyte-specific genes in the EDN1-stimulated pigmentation of HEEs. * p < 0.05; ** p < 0.01. EDN1, endothelin-1; SCF, stem cell factor, PTCA, pyrrole-2,3,5-tricarboxylic acid.

Figure 4.

(A) Experimental procedure for human epidermal equivalents (HEEs) using the J-TEC Melano-Model [–42]; (B) Effects of EDN1 on the pigmentation of HEEs during 14 days of culture; (C) Time course of expression of melanocyte-specific genes in the EDN1-stimulated pigmentation of HEEs. * p < 0.05; ** p < 0.01. EDN1, endothelin-1; SCF, stem cell factor, PTCA, pyrrole-2,3,5-tricarboxylic acid.

Figure 5.

Inhibitory effects of signaling inhibitors on the EDN1-stimulated pigmentation/Hematoxylin and eosin stain (HE)/Fontana Masson (FM) staining (a); eumelanin content (PTCA) (b) of HEEs at day 14. ** p < 0.01; Bar = 100 μm.

Figure 6.

Effects of the Withania somnifera extract (WSE) on tyrosinase activity. (a) Addition 3 h before EDN1 stimulation; (b) Direct addition to cell lysate 72 h after EDN1 stimulation, EDN1: 10 nM, WSE: 10 μg/mL. ** p < 0.01.

Figure 6.

Effects of the Withania somnifera extract (WSE) on tyrosinase activity. (a) Addition 3 h before EDN1 stimulation; (b) Direct addition to cell lysate 72 h after EDN1 stimulation, EDN1: 10 nM, WSE: 10 μg/mL. ** p < 0.01.

Figure 7.

(A) Effect of the WSE on the EDN1-stimulated phosphorylation of ERK (a), CREB (b) and MITF (c) in ALM melanoma cells; (B) Effect of the WSE on the EDN1-stimulated phosphorylation of MEK (a) and Raf-1 (b) in ALM melanoma cells; (C) Effect of the WSE on the EDN1-induced mobilization of intracellular calcium in NHMs, (a) control; (b) EDN1 (10 nM); (c) WSE 10 mg/mL + EDN1 (10 nM). * p < 0.05; ** p < 0.01.

Figure 7.

(A) Effect of the WSE on the EDN1-stimulated phosphorylation of ERK (a), CREB (b) and MITF (c) in ALM melanoma cells; (B) Effect of the WSE on the EDN1-stimulated phosphorylation of MEK (a) and Raf-1 (b) in ALM melanoma cells; (C) Effect of the WSE on the EDN1-induced mobilization of intracellular calcium in NHMs, (a) control; (b) EDN1 (10 nM); (c) WSE 10 mg/mL + EDN1 (10 nM). * p < 0.05; ** p < 0.01.

Figure 8.

EDN1-activated intracellular signaling pathways leading to the expression of melanocyte-specific proteins and the signaling step affected by the WSE.

Figure 9.

(A) Inhibitory effect of the WSE on the EDN1-stimulated pigmentation of HEEs: (a) Pigmentation; (b) HE staining and Fontana Masson (FM) staining of WSE-treated HEEs at day 14; Bar = 100 μm; (c) HPLC analysis of eumelanin content in WSE-treated HEEs at day 14; (B) Immunohistochemistry of S-100 staining at day 14: (a) Immunostaining with anti-S-100 following treatment with EDN1 (10 nM); (b) Immunostaining with anti-S-100 following treatment with EDN1 (10 nM) + WSE (10 μg/mL); (c) Immunostaining with anti-S-100 + DRAQ5 following treatment with EDN1 (10 nM); (d) Immunostaining with anti-S-100 + DRAQ5 following treatment with EDN1 (10 nM) + WSE (10 μg/mL). * p < 0.05; Bar = 100 μm.

Figure 9.

(A) Inhibitory effect of the WSE on the EDN1-stimulated pigmentation of HEEs: (a) Pigmentation; (b) HE staining and Fontana Masson (FM) staining of WSE-treated HEEs at day 14; Bar = 100 μm; (c) HPLC analysis of eumelanin content in WSE-treated HEEs at day 14; (B) Immunohistochemistry of S-100 staining at day 14: (a) Immunostaining with anti-S-100 following treatment with EDN1 (10 nM); (b) Immunostaining with anti-S-100 following treatment with EDN1 (10 nM) + WSE (10 μg/mL); (c) Immunostaining with anti-S-100 + DRAQ5 following treatment with EDN1 (10 nM); (d) Immunostaining with anti-S-100 + DRAQ5 following treatment with EDN1 (10 nM) + WSE (10 μg/mL). * p < 0.05; Bar = 100 μm.

Figure 10.

(A) Inhibitory effects of the WSE on EDN1-stimulated gene and protein expression at days 7 and 10, respectively: (a) Tyrosinase; (b) TYRP1; (c) DCT; (d) PMEL17; n = 3; (B) Inhibitory effects of the WSE on EDN1-stimulated gene and protein expression of MITF at days 7 and 10, respectively. * p < 0.05; ** p < 0.01.