On the role of melatonin in skin physiology and pathology

Abstract

Melatonin has been experimentally implicated in skin functions such as hair growth cycling, fur pigmentation, and melanoma control, and melatonin receptors are expressed in several skin cells including normal and malignant keratinocytes, melanocytes, and fibroblasts. Melatonin is also able to suppress ultraviolet (UV)-induced damage to skin cells and shows strong antioxidant activity in UV exposed cells. Moreover, we recently uncovered expression in the skin of the biochemical machinery involved in the sequential transformation of l-tryptophan to serotonin and melatonin. Existence of the biosynthetic pathway was confirmed by detection of the corresponding genes and proteins with actual demonstration of enzymatic activities for tryptophan hydroxylase, serotonin N-acetyl-transferase, and hydroxyindole-O-methyltransferase in extracts from skin and skin cells. Initial evidence for in vivo synthesis of melatonin and its metabolism was obtained in hamster skin organ culture and in one melanoma line. Therefore, we propose that melatonin (synthesized locally or delivered topically) could counteract or buffer external (environmental) or internal stresses to preserve the biological integrity of the organ and to maintain its home-ostasis. Furthermore, melatonin could have a role in protection against solar radiation or even in the management of skin diseases.

Fig. 1

Proposed pathway on melatonin synthesis in the skin. 1, tryptophan; 2,5-hydroxytryptophan; 3, serotonin(5-hydroxytryptamine); 4, N-acetylserotonin (N-acetyl-5-hydroxytryptamine); 5, melatonin.

Fig. 2

Proposed model of melatonin metabolism in the skin. 1, melatonin; 2, 5-methoxytryptamine; 3, 5-methoxyindoleacetaldehyde; 4, 5-methoxyindole acetic acid; 5, 5-methoxytryptophol; 6, N-acetylserotonin; 7, 6-hydroxymelatonin; 8, 2-hydroxymelatonin; 9, 2,3-dihydroxymelatonin; 10, N¹-acetyl-N²-formyl-5-methoxykynuramine.

Fig. 3

Expression of genes for membrane-bound melatonin receptors in human skin cells. (A) Nested RT-PCR for MT1 receptor. (B) Nested RT-PCR for MT2 receptor; 100 kbp DNA ladder (M), neonatal melanocytes (1), neonatal melanocytes after UVB treatment (2), immortalized keratinocytes (HaCaT) (3), immortalized keratinocytes (HaCaT) after UVB treatment (4), adult epidermal keratinocytes (5), adult epidermal keratinocytes after UVB treatment (6), adult dermal fibroblasts (7), adult dermal fibroblasts after UVB treatment (8), immortalized melanocytes (PIG-1) (9). (C) Nested RT-PCR for MT1 receptor in melanoma cells; 100 kbp DNA ladder (M), SKMEL188 (1), SKMEL188 after UVB treatment (2), WM164 (3), WM164 after UVB treatment (4), WM98 (5), WM98 after UV treatment (6). Sequences of primers and conditions for nested RT-PCR were as described (23).

Fig. 4

Predicted amino acid sequence for two new human iosforms of MT2 (MTNRb) receptor. Sequence of human MT2 (MTNRb) (gene accession #NP_005950.1) is shown in comparison to the predicted sequences of two newly discovered open reading frames (orf) detected after alternative splicing of MT2 mRNA. Alternative splicing was confirmed by sequencing the 209 bp PCR fragment of MT2 cDNA (gene accession #AY114100) (for gene structure cf. ref. 23). Both the stop codon for MT2b1 and the start codon for MT2b2 are coded by sequences generated after alternative splicing. There is a gap, 72 bp long, between the stop codon for MT2B1 and the start codon for MT2b2 (not shown). Splicing is marked with an arrow, and with A for MT2 or B for the splicing variant MT2b. Transmembrane regions (TM) are marked with # and with numbers 1–7 listed above the sequences. TMX is an alternative transmebrane fragment for MT2b2. MT2b2 has only five TM helices and lacks TM 1–3 helices, a portion partially substituted by a unique transmembrane helix (TMX), causing a shift of two amino acids to C-terminus in TM 4 helices (not show).

Fig. 5

Expression of genes coding for nuclear melatonin receptors and quinone reductase 2 (NQO2) in skin cells. RT-PCR for nuclear receptor izoforms: RORα receptor (common fragment for all isoforms, A), RORα1 (B), RORα2 (C), RORα3 (D), RZR1 (RORα4) (E), and NQO2 (F). 100 kB DNA ladder (M), neonatal melanocytes (1), neonatal melanocytes after UVB treatment (2), immortalized keratinocytes (HaCaT) (3), immortalized keratinocytes (HaCaT) after UVB treatment (4), adult epidermal keratinocytes (5), adult epidermal keratinocytes after UVB treatment (6), adult dermal fibroblasts (7), adult dermal fibroblasts after UVB treatment (8), immortalized melanocytes (PIG-1) (9). The RT-PCR primers conditions for common RORα fragment and RORα1–4 isoforms were as described (common fragment, ref. ; isoforms, ref. 45). RT-PCR to amplify NQO2 was preformed as described (49). UVB was used at dose of 100 mJ/cm² as described previously (94).

Fig. 6

Effect of UVB (35 mJ/cm²) on HaCaT keratinocytes with or without prior addition of 10⁻³ M melatonin for 30 min and collected 20 h after irradiation. RNA was extracted, cDNA produced, and microarray analysis performed as described (73). Differences between control (UVB-irradiated cells) and cells irradiated with UVB and treated with melatonin are presented as mean of the respective ratios (ratio 1 represents no difference) ± SEM, followed by analysis with Student’s t test (n = 4, *p < 0.05, **p < 0.005).

Fig. 7

Melatonin suppresses cell viability in squamous cell carcinoma cells. C_1–4 cells were incubated for 24 h in serum free medium in the presence of graded concentrations of melatonin. Cell viability was measured by MTT test (23). Data are presented as means ± SEM (n = 16 combined from two experiments), and the statistical analysis was performed with ANOVA (+p < 0.01; #p < 0.001).