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* Department of Pathology, University of Tennessee, Memphis, Tennessee, USA;
Department of Medicine, Southern Illinois University, Springfield, Illinois, USA; and
Department of Biomedical Sciences, University of Bradford, Bradford, West Yorkshire, England
1Correspondence: Department of Pathology, Suite 599, University of Tennessee Health Science Center, 930 Madison Ave., Memphis, TN 38163, USA. E-mail: aslominski{at}utmem.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONBIOLOGY OF SEROTONIN AND...SKIN SEROTONIN: SYNTHESIS AND...SKIN BIOREGULATION BY SEROTONINSKIN BIOREGULATION BY MELATONINFUNCTIONAL PROJECTION OF THE...CONCLUSIONSREFERENCES |
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Key Words: skin • melatonin • serotonin • N-acetylserotonin • endocrinology of the skin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONBIOLOGY OF SEROTONIN AND...SKIN SEROTONIN: SYNTHESIS AND...SKIN BIOREGULATION BY SEROTONINSKIN BIOREGULATION BY MELATONINFUNCTIONAL PROJECTION OF THE...CONCLUSIONSREFERENCES |
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, 2)
. In fact, environmental factors are thought to participate in the pathogenesis of skin diseases including inflammatory dermatoses and benign hyperproliferative disorders, defects in pigmentation (vitiligo), hair growth disorders, and premalignant and malignant processes. Maintenance of its cutaneous integrity is critical, however, for maintenance of systemic homeostasis. Therefore, an ability to focally recognize, discriminate, and integrate specific signals within a highly heterogeneous environment must be a widespread property and a basic cutaneous attribute (1)
. To some extent, those capabilities are integrated into the less localized responses of the skin immune and pigmentary systems (3
, 4)
and in the cyclic activity of the hair follicle in fury animals (5)
. However, the high-level barrier function of the skin demands stringent specificity in such responses, and therefore full recognition of the damage potential of a stressor would require a more complex organization; we have proposed that such a role might be served by a local neuroendocrine system (1
, 2
, 6)
.
Expression of neuroendocrine activities in the skin is substantial since the organ can locally produce stress mediators ACTH, MSH, and ß-endorphin, along with corticotrophin-releasing hormone (CRH) and urocortin (reviewed in refs 2
, 6
, 7
). Skin involvement in the synthesis, activation, or metabolism of steroid hormones (1
, 8)
, and of vitamin D is well known (9)
, as is local epidermal production of catecholamines (10)
and acetylcholine (11)
. Of great interest is the cutaneous expression of enzymatic systems: epidermal expression of phenylalanine hydroxylase (PAH) and tyrosine hydroxylase (TH), as well as the 6-tetrahydrobiopterin (6BH4) generating system, which acts as an essential cofactor for these enzymes (10
, 12
13
14)
; 6BH4 is also a cofactor for tryptophan hydroxylase (TPH, EC 1.14.16.4), important for the synthesis of serotonin and melatonin (15
, 16)
.
In this review we summarize data on a skin serotoninergic/melatoninergic system generated in vitro and in vivo. Then we examine a potential role for this system in skin’s physiology and pathology. Last, we present a unified concept that integrates the involvement of the local serotoninergic/melatoninergic system in the skin response to stresses (environmental and endogenous) and possible global implications.
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BIOLOGY OF SEROTONIN AND MELATONIN: AN OVERVIEW |
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TOPABSTRACTINTRODUCTIONBIOLOGY OF SEROTONIN AND...SKIN SEROTONIN: SYNTHESIS AND...SKIN BIOREGULATION BY SEROTONINSKIN BIOREGULATION BY MELATONINFUNCTIONAL PROJECTION OF THE...CONCLUSIONSREFERENCES |
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, 17)
. The genes for human and mouse TPH (TPH-1) are composed of 10 coding exons and at least one noncoding exon (15
, 18
, 19)
. The actual coding sequence for human TPH has 1332 bp, which encodes a protein of 444 amino acids (aa) with a predicted molecular mass of 51 kDa (15)
. Post-translational modifications generate species of 50–60 kDa; the most frequent form is 53 kDa (15
, 18
, 19)
. Alternative splicing of the gene can yield isoforms with variable spliced 5'-untranslated regions and alternative splicing of intron 11 produces a protein 22 aa longer (18
, 19)
. TPH monomers are organized into regulatory (N-terminal) and catalytic (C-terminal) domains; the latter includes a leucine zipper involved in the formation of the tetrameric holoenzyme of 220 kDa. Deletional studies show that removal of up to 106 aa fragments from the regulatory domain generates truncated proteins with TPH activity compared with the unmodified variant (15
, 20)
. A second enzyme that can hydroxylate L-tryptophan (TPH2) was cloned recently and found to be expressed predominantly in the central nervous system (CNS) (21)
. It is now accepted that TPH1 (TPH) is the predominant enzyme expressed in peripheral tissues whereas TPH2 predominates in the CNS (21
, 22)
.
Hydroxytryptophan is decarboxylated to generate serotonin (17)
. Although decarboxylase activity is ubiquitous in peripheral tissues, serotonin is predominantly synthesized by intestinal enterochromafin cells, with smaller quantities produced by the CNS, rectum (cylindrical epithelium), bronchial cells (epithelium), thyroid parafollicular cells, ovaries, thymus, pancreas (17
, 22)
, breast, and skin (23
, 24)
. In rodents, mast cells are also an important source of serotonin. Serotonin released into the blood is actively taken up by platelets and stored in their solid granules; there is active uptake of serotonin by lymphocytes via serotonin transporters (17
, 25)
. Plasma serotonin is cleared by the liver and lung endothelial cells, where serotonin is stored in vesicles until released or converted (by deamination) into 5-hydroxyindoleacetaldehyde through the action of mitochondrial monoaminooxidase (MAO) (EC 1.4.3.4) (17
, 25)
. In turn, 5-hydroxyindoleacetaldehyde may be oxidized to 5-hydroxyindole acetic acid (by aldehyde dehydrogenase; EC 1.2.1.3), or reduced to 5-hydroxytryptophol (by alcohol dehydrogenase; EC 1.1.1.1).
Melatonin
Melatonin is widely distributed in nature, where it is detected not only in vertebrates and invertebrates but even in plants, bacteria, unicellular eukaryotes, and algae (26
27
28
29)
. In mammals, melatonin is produced predominantly in the pineal gland, retina, and, in lesser amounts, in the brain and extracranial sites (gastrointestinal tract, the eye, the immune system, and ovaries) (16
, 30
31
32
33
34
35
36
37
38
39)
. Pineal production and release of melatonin is controlled by the biologic clock in the suprachiasmatic nuclei, but responds to light changes (16
, 30)
. Melatonin production is positively regulated by ß-adrenergic receptors via subsequent activation of adenylate cyclase (16
, 40)
. The rate-limiting step in melatonin synthesis is serotonin (5-HT) acetylation catalyzed by arylalkylamine N-acetyltransferase (AANAT, EC 2.3.1.87) in a reaction that generates N-acetylserotonin (NAS) (41)
. NAS is then methylated into melatonin by hydroxyindole-O-methyltransferase (HIOMT, EC 2.1.1.4) (16)
(42)
. The AANAT gene is composed of 4 exons and 3 introns that introduce different isoforms through alternative splicing (43)
. The human, but not rat, HIOMT gene contains an additional exon (6th) corresponding to the line-1 repetitive element (44)
. Human HIOMT mRNA is alternatively spliced to generate various isoforms of undetermined activity (45)
.
Melatonin metabolism yields 6-hydroxymelatonin (6-OHM), detectable in blood and urine as the major systemic product (30
, 46
, 47)
. Minor metabolic products are cyclic 2-hydroxymelatonin and cyclic 3-hydroxymelatonin (48
, 49)
. Melatonin metabolites can be conjugated with either sulfate or glucuronide (50)
. The most extensive degradation of melatonin occurs in the liver, where it undergoes cytochrome P-450-mediated O-demethylation and 6-hydroxylation to yield N-acetyl-5-hydroxytryptamine and 6-OHM, respectively (51)
. Accessory pathways can transform melatonin, generating 5-methoxyindole acetic acid (5MIAA) or 5-methoxytryptophol (5MTOL) (52
, 53)
. Cleavage of the melatonin pyrrole ring by indoleamine 2,3-dioxygenase yields N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), further degraded by arylamine formamidase to N1-acetyl-5-methoxykynuramine (AMK) (54)
. Alternately, the hemoproteins horseradish peroxidase, myeloperoxidase, and oxyferrylhemoglobin can oxidize melatonin to AFMK (55
, 56)
; under the action of catalase, at least in vitro, AFMK is deformylated to AMK (57)
. In frog skin and retina melatonin is first deacetylated to 5-methoxytryptamine (5MT), then deaminated to produce 5MIAA and 5MTOL (53)
.
Phenotypic actions
Serotonin
Serotonin is a neurotransmitter involved in cognition, regulation of feeding behavior, mood, anxiety, aggression and pain, sexual activity, sleep, and other body rhythms (25
, 58
59
60)
. It also serves as a hormone, cytokine, biological modifier, growth factor, and as a regulator of vascular tone and intestinal activity (25
, 58
, 59
, 61
62
63)
. These actions are mediated through interaction with membrane-bound receptors, which are categorized into 7 families (5HT1-7) with at least 21 subtypes (58
, 59)
. Overall, serotonin receptors are coupled to G-proteins with the exception of 5HT3, which is an ionotropic receptor (58
, 59)
. The relationship between serotonin receptor types and function is complex: they differ in the signal transduction system used and the physiological processes affected. This results in the simultaneous expression of multiple type receptors that are widely distributed in the CNS, endocrine organs, and peripheral and define the selectivity of serotonin regulatory functions (25
, 58
, 59)
. Serotonin transporters may have direct signaling consequences of their own (25)
.
In peripheral tissues, serotonin plays important roles in the regulation of the immune inflammatory axis, gastrointestinal physiology, cardiovascular system, coagulation and fibrinolysis, adrenal cortex, sexual functions, mammary gland development, cell proliferation (negative and positive), migration, and differentiation (24
, 25
, 61
62
63
64
65
66
67
68)
. Even during development serotonin plays embryogenic and morphogenic functions (62
, 69)
. In fact, both early and late serotonin embryogenic functions as well as its morphogenic activities are evolutionarily conserved from lower invertebrates to mammals (62
, 70)
. Most of these peripheral and systemic actions are mediated through membrane-bound receptors (58
, 59
, 71)
. These include the recently described serotonin anti-apoptotic effect that targets the mitochondria and appears to be mediated by 5-HT2B (72)
. Growth factor activity may be mediated by any of the following receptor subtypes: 5HT1A, 5HT1B, 5HT2A, 5HT2B, and 5HT2C (61
, 65
, 71)
. Notwithstanding the above, nonreceptor-mediated serotonin mechanisms of action have been described. For example, in neuronal cells oxidative metabolism of the monoamines has been implicated in apoptosis (73)
. In immune cells, a novel mechanism for serotonin-associated induction of apoptosis may depend on the uptake of serotonin (via 5-HT transporters) independent of intracellular oxidative transformation (74)
.
Melatonin
Melatonin bioactivity encompasses numerous behavioral, endocrinological, and immune processes (16
, 30
, 75)
. These actions are mediated by receptor-dependent and -independent regulation/modulation (28
, 76
77
78
79
80)
. Because of its small size and amphiphilic nature, melatonin can easily reach all cellular compartments, being localized ubiquitously in cytosolic, membrane, mitochondrial, and nuclear structures (26
, 81)
. Intracellular melatonin may exert a protective role related to its potent anti-oxidative capacity (directly or through its metabolites) by scavenging reactive oxygen species and nitrogen-based reactants (81
, 82)
. Alternately, these anti-oxidative actions may be mediated by the active stimulation or synthesis of enzymes that metabolize toxic reactants e.g., superoxide dismutase, gluthathione peroxidase, glutathione reductase, and catalase (26
, 83
, 84)
. Melatonin contributes to the maintenance of a high GSH/GSSG ratio, promoting the synthesis of glutathione (85)
. Additional activities of melatonin/melatonin metabolites include their inhibition of nitric oxide production and interaction with calmodulin and protein kinase C (26)
. Melatonin contributes to the preservation of mitochondrial integrity by increasing the efficiency of the electron transfer chain and promoting ATP synthesis (26)
. Finally, melatonin (or its metabolites) has anti-apoptotic and anti-carcinogenic effects by protecting nuclear and/or mitochondrial DNA from oxidative damage or acting as electrons donor(s) to selected proteins (26
, 86)
. Thus, nonreceptor-mediated actions of melatonin (or its metabolites) may play a crucial role in preserving cell function/survival in maintaining tissue homeostasis after its disruption by external or internal stressors and counteracting pathology at local or systemic levels (26
, 75
, 76
, 86)
.
The main systemic activities of melatonin are in the areas of regulation/modulation of circadian rhythm, seasonal reproduction, and retinal function (16)
. Depending on the production site and target organ, melatonin can act as a hormone, neurotransmitter, cytokine, growth factor, or biological modifier. An immunomodulatory (stimulatory) action of melatonin has been documented (16
, 87)
, whereas an oncostatic effect of melatonin appears to be possible for several types of human tumors, including breast cancer (88
, 89)
. Many melatonin effects are mediated through interactions with high-affinity, membrane-bound or nuclear melatonin receptors (77
78
79
80)
. In mammals two distinct seven-transmembrane domain G-protein-coupled melatonin receptors have been cloned and named MT1 (Mel1a) and MT2 (Mel1b) (77
, 78
, 90
, 91)
. Their gene structure of the coding region is similar and is composed of 2 exons and 1 intron. The two receptor subtypes show 60% homology at the amino acid level (77
, 78
, 90
, 91)
and were distinguished pharmacologically with agonists (77
, 78
, 92
93
94)
. Activation of both receptors inhibits adenylate cyclase via pertussin-sensitive Gi proteins, but MT1 receptors can control calcium mobilization through pertussin toxin-insensitive Gq/11 proteins, whereas MT2 receptors can be coupled to cGMP inhibition (77
, 78
, 92
93
94
95
96)
. MT1 and MT2 are expressed in the CNS and in retina, but MT1 predominates here and in peripheral tissues. Moreover, the presence of functional nuclear receptors for melatonin, RZR/ROR
, or RZRß have been reported (97
, 98)
, although the nuclear signaling pathway required for the functioning has yet to be characterized (79)
. A quinone reductase enzyme family has been reported to serve as a unique membrane-bound MT3 receptor (80
, 99)
. To conclude, the numerous phenotypic effects of melatonin and/or its metabolites are mediated by heterogeneous mechanisms including membrane-bound or nuclear receptors coupled to multiple and distinct signal transduction systems or by receptor independent means, and involve a diversity of targets at cellular, organ, and systemic levels.
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SKIN SEROTONIN: SYNTHESIS AND METABOLISM |
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TOPABSTRACTINTRODUCTIONBIOLOGY OF SEROTONIN AND...SKIN SEROTONIN: SYNTHESIS AND...SKIN BIOREGULATION BY SEROTONINSKIN BIOREGULATION BY MELATONINFUNCTIONAL PROJECTION OF THE...CONCLUSIONSREFERENCES |
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) (23
, 100)
. The widely used experimental model of immortalized HaCaT keratinocytes was found to express the aberrant TPH transcript (100)
containing several mutations (Genebank accession AY196344 and AY196345). These would result in amino acid substitutions in the regulatory domain (F77L), the catalytic domain (F377P), and probably the C terminus. Another aberrantly spliced form of TPH detected in melanomas (Genebank accession AY196346) contained a stop codon, thus coding for a protein without enzymatic activity (100)
. Hence, the correct TPH transcript and aberrant TPH isoforms are both expressed in skin cells. In addition, we found that ultraviolet radiation (UVR) inhibits the expression of the TPH gene in squamous cell carcinoma C1-4 cells and human melanoma cells, suggesting potential environmental regulation of the TPH pathway (23)
. We detected the expression of the TPH gene in the pituitary, adrenal gland, and myometrium (23)
, consistent with a predominant extrabrain expression of the TPH (TPH1) gene (21)
. Others have reported TPH gene expression in human breast and breast epithelial cells in vitro (24)
, and transformation of tryptophan to serotonin has been observed in cultured peripheral blood cells (33
, 101)
. Expression of the TPH2 gene has not yet been tested in human skin.
|
Actual TPH protein has been identified by Western blot analyses in extracts of whole skin, cultured normal epidermal keratinocytes and dermal fibroblasts, normal epidermal melanocytes, immortalized HaCaT keratinocytes, squamous cell carcinoma, and melanoma cells (100)
. TPH immunoreactivities detected include forms with the size expected for the intact enzyme, as well as higher or lower molecular weight forms. These findings indicate extensive turnover of the enzyme in skin, as described in other models (102)
. It is thought that high molecular weight TPH-like species may represent ubiquitinated forms of TPH whereas low molecular weight TPH-like species may represent degradation products (102)
. TPH enzymatic activity has been detected in extracts of cutaneous melanoma cells (23)
and in skin melanocytes, keratinocytes, and fibroblasts (Slominski et al., unpublished results), while metabolic products hydroxytryptophan and serotonin were identified in melanoma cells by liquid chromatography-mass spectrometry (LS/MS). RP-HPLC analysis of products of the TPH-mediated pathway in whole skin revealed fluorescent species with the retention times of serotonin and NAS (23
, 100)
.
Since immunofluorescence of skin biopsies tended to localize TPH protein and serotonin immunoreactivity to normal and malignant melanocytes in vivo, the cutaneous pathway for local synthesis of serotonin may be expressed predominantly in melanocytic cells (100
, 103)
. Nevertheless, immunohistochemistry studies of scalp biopsies performed with more sensitive methods detected TPH immunoreactivity in additional skin cell types that include epithelial cells and adnexal structures (Slominski and Tobin, unpublished results). Overall, these studies are consistent with the widespread distribution of TPH in related tissues, breast epithelial cells that embryologically arise as an appendage of the skin (24)
. Aromatic amino acid decarboxylase (AAD) transcript was detected in epidermal keratinocytes and melanocytes with actual protein expression in melanocytes (13)
.
Serotonin itself was detected by immunocytochemistry in immortalized HaCaT keratinocytes and confirmed by RP-HPLC using electrochemical detection, which also detected 5-hydroxyindole-acetic acid (5HIAA) (23)
. Since TPH activity was low to absent in HaCaT keratinocytes, serotonin must be transported into the cells by the serotonin transporter, also detected by immunocytochemistry (Slominski et al., unpublished), followed by degradation by monoamine oxidase and aldehyde dehydrogenase (104
, 105)
. Immunocytochemistry studies of human scalp showed serotonin immunoreactivity in the epidermal and adnexal structures as well as in cells of dermal compartments (Slominski and Tobin, unpublished results). Marked expression of serotonin immunoreactivity was detected in cutaneous mast cells (Slominski and Tobin, unpublished results), which is consistent with immunodetection of serotonin in perivascular human mast cells of adrenal cortex (67)
.
Melatonin
Transcripts of AANAT (gene coding rate-limiting step in melatonin synthesis) and HIOMT have been detected in normal and pathological skin and in most skin cell populations (23)
(Fig. 1)
. The latter included normal keratinocytes (neonatal and adult, epidermal, and follicular), immortalized HaCaT keratinocytes, fibroblasts (dermal and hair follicle papilla), normal melanocytes, several melanoma cell lines, and squamous cell carcinoma cells. With regard to AANAT isoforms, it was interesting to detect a previously unreported aberrant isoform in normal and pathological skin (involved by basal cell carcinoma cells) and in neonatal keratinocytes. This isoform had an insertion of a 59 bp segment from intron 2 (Genebank accession AY055827). Notably, the isoform lacking exons 6 and 7 was most prevalent (23)
. As in the case of TPH, AANAT and HIOMT transcripts were detected in human pituitary, adrenal, and myometrium (23)
, consistent with the peripheral expression of both genes as reported in human lymphocytes (36)
.
The AANAT protein was detected in vitro in cultured skin cells (Fig. 2
) whereas enzymatic activity was detected with RP-HPLC in human skin in all melanoma lines tested, immortalized normal melanocytes and keratinocytes (23)
. Using serotonin as a substrate, analysis of the acetylation kinetics showed a Km and Vmax for human skin of 0.69±0.08 mM and 36.64 pmol/h, respectively; in immortalized melanocytes and HaCaT keratinocytes, the calculated Km for the same reaction were 3.96±0.6 and 2.75±0.57 mM and Vmax 40.64 and 44.3 pmol/h, respectively (23)
. Enzyme activity was therefore higher in melanoma lines than in whole-skin, immortalized keratinocytes, or immortalized melanocytes. According to the Km values, keratinocyte and melanocyte AANAT is similar to human AANAT expressed in COS-7 cells (2.6 mM) but higher than in bacterially expressed human (1.3 mM) or ovine (0.31 mM) AANAT (106
, 107)
. In the ovary, the apparent AANAT Km calculated as 0.15 mM, 
one-fourth that found in the skin (35)
. Thus, serotonin metabolism may be determined locally, being presumably dependent on conditions that include cellular local environment and anatomical location. Indeed, when AANAT activity was calculated for tryptamine and serotonin, activity ratios were close to 1 for all melanoma lines and for HaCaT keratinocytes, but ranged from 2.5 to 6 for whole skin from three white subjects and zero in immortalized normal melanocytes and in whole skin from a black subject whose AANAT activity toward tryptamine was below detectability (23)
. Since AANAT activity for serotonin was higher in melanoma cells than in whole-skin or immortalized normal keratinocytes and melanocytes, skin racial pigmentation and type of cutaneous pathology (melanoma) may both be important determinants of reaction rate and specificity of serotonin acetylation.
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Serotonin N-acetyltransferase immunoreactivity can be detected in suprabasal differentiating keratinocytes in human scalp epidermis (Fig. 3
a). Occasional singly scattered cells with a melanocyte distribution exhibit immunoreactivity for this enzyme. Serotonin N-acetyltransferase immunoreactivity is low in skin compartments with very high proliferative activity (e.g., basal layer of the epidermis and hair bulb matrix) (Fig. 3a, b, d
). Intense expression of the enzyme was observed in the outer peripheral epithelial layers of the anagen hair follicle (Fig. 3c, d
) and in basal cells of the sebaceous and eccrine glands. Enzyme expression could be detected in sensory nerve endings abutting the epidermal layers (Fig. 3b
).
|
Immunolocalization of melatonin in skin has not yet been reported, perhaps due to the difficulty of retaining such a small molecule in sections. However, using a sheep polyclonal antibody raised to N-acetyl-5-methoxytryptamine conjugated to bovine thyroglobulin, we have detected melatonin immunoreactivity in the human scalp (Fig. 4
). This antibody reacted with an epitope expressed in a highly restricted manner on keratinocytes in the differentiating layers of the epidermis, including strata spinosum and granulosum, where the epitope appears to be enriched close/at the plasma membrane (Fig. 4a
). Little or no expression was detected in the proliferative basal layer of the epidermis except in singly scattered cells (possibly melanocytes). By contract, melatonin immunoreactivity was detected throughout the hair follicle epithelium (Fig. 4b, c
) and intensely in blood vessels (Fig. 4a
), including capillary loops in the follicular dermal papilla. A marked expression of melatonin immunoreactivity was detected in cutaneous mast cells (Fig. 4d
).
|
Actual HIOMT activity was detected in whole skin of Caucasian and African-American subjects and in cultured immortalized keratinocytes and melanoma cells (23)
. NAS, the substrate for HIOMT, was detected by RP-HPLC in skin extracts, whereas tandem LC/MS detected NAS and melatonin in immortalized keratinocytes and melanoma cells (23
, 108)
. These findings indicate that the human skin, like the pineal gland and retina, is another peripheral organ possessing the intrinsic ability to synthesize melatonin (32
, 33
, 35
, 36)
. Moreover, cutaneous melatoninergic pathways may operate in a compartment-specific manner topographically restricted to the epidermal, adnexal and dermal cell populations. Further selectivity and specificity of melatonin effects in skin may be provided by melatonin and NAS entering alternative metabolic pathways with or without rapid degradation. Detection of the melatonin metabolites 5MTT and 5MTOL in skin cells by LC/MS (108)
is consistent with the finding of similar end products in frog skin and retina (53
, 109)
. We have detected 6-hydroxymelatonin in HaCaT keratinocytes (Fischer et al., unpublished results) suggesting extensive melatonin metabolism in these cells to include pathways known to be operative in the liver and kidney.
Animal skin
Serotonin
Pathways transforming tryptophan to serotonin and melatonin organized in a manner similar to those in human skin have been described in the skin of rodents (Fig. 5
). Thus, in hamsters we documented TPH expression in the skin and in transplantable and established in vitro melanoma lines, as well as in spleen and liver, when probing for TPH gene cloned from the pituitary (Genebank accession AY034600) (110)
. Testing for expression of the TPH gene in the mouse (accession no. NM_009414), we found continuous expression in the skin throughout all phases of the hair cycle (lowest expression in telogen) and in cultured follicular melanocytes, melanoma cells, and the spleen (105)
. By in situ hybridization, the TPH gene was randomly detected in mouse breast epithelial cells and in mast cells of the breast stroma (24)
. In cutaneous TPH, gene expression was accompanied by production of the actual protein of expected 53–55 kDa, although TPH-like immunoreactivies of higher and lower molecular weights were detected (105)
. By analogy with the TPH species detected from rapid metabolism of TPH in mastocytoma cells, the high molecular weight TPH-like species could represent ubiquitinated TPH and the low molecular weight TPH-like species could represent degradation products (111)
. On direct biochemical assays, the enzymatic conversion of tryptophan to hydroxytryptophan in hamster melanoma cells proceeded at high levels comparable to those of a rat brain (110)
. In fact, serotonin itself was subsequently detected by RP-HPLC and LC/MS in extracts from hamster melanoma cells (110)
. These observations are concordant with the known immunolocalization of serotonin to epithelial cells of the breast (24)
and with the production of serotonin by rodent skin mast cells (4
, 112)
.
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Monoaminoxidase can deaminate serotonin, followed by oxidation or reduction of the resultant 5-hydroxyindole acetaldehyde to 5HIAA and 5-HTPOL, as demonstrated by LC/MS analyses of mouse skin extracts (105)
. Experiments using pargyline (specific MAO inhibitor) confirmed MAO involvement in this process by its attenuation of 5HIAA and 5-HTPOL production. These metabolites were uncovered in mouse and rat skin, although in the former 5HIAA was the main degradation product and 5-HTPOL remained below the limit of detectability (104)
. MAO activity toward serotonin was detected in guinea pig skin (113)
.
Melatonin
Transcripts of AANAT coding for the active enzyme were documented in normal hamster skin and melanomas and in murine S91 melanoma (DBA mouse genotype), although not in skin of the C57BL6 mouse (105)
. AANAT gene expression was accompanied by expression of AANAT protein of the expected size for the functional enzyme in hamster and mouse S91 melanoma cells (105)
. Enzymatic activity for the conversion of serotonin and tryptamine to NAS and N-acetyltryptamine, respectively, has been detected in hamster and rat skin and in hamster and mouse (DBA) melanomas (104
, 105
, 110)
. There is marked regional variation in serotonin N-acetyltransferase activity, being higher in ear skin than in corpus skin and lower in melanomas than in normal skin. Serotonin N-acetyltransferase activity was in turn significantly inhibited by low concentrations of CoA-S-N-acetyltryptamine (Cole bisubstrate; BSI). This is a specific inhibitor of arylalkylamine N-acetyltransferase that covalently binds structural components of serotonin and acetyl CoA (114
, 115)
. Taken together, the evidence indicates that AANAT indeed produces NAS in hamster and rat skin (Fig. 6
). There was, however, residual enzymatic activity that was resistant to BSI suppression even at concentrations of 5–100 µM, raising the possibility that arylamine activity (NAT) resistant to BSI can participate in the acetylation of serotonin in the skin (Fig. 6)
(104
, 110)
. We previously identified and characterized two N-acetyltransferase activities in hamster skin, one representing NAT and the other (previously named NAT-2) showing similarities to pineal AANAT (116)
. Recent data suggest that at least part of this activity must represent native AANAT (110)
. Of further interest, hamster skin acetylated dopamine (a negative regulator of melatonin synthesis) to N-acetyldopamine (116)
. Serotonin N-acetyltransferase activity was detected in guinea pig skin (113)
.
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Serotonin undergoes extensive metabolic conversion in the skin. Tracer studies with radioactive serotonin in rodent skin culture yielded radio derivatives that eluted with retention times identical to NAS, melatonin, and 5MTT standards; NAS identity was verified by GC/MS analysis (117)
. NAS has been identified by LC/MS in cultured hamster melanoma cells (110)
; HIOMT activity was detected biochemically in mouse S91 melanoma cells and mouse ears (105)
.
Production of N-acetylserotonin in the C57/BL6 mouse
The C57BL/6 mouse strain has been proposed as a natural melatonin "knockdown" because of a genetic defect in AANAT function (40
, 118)
. This is represented by a mutated gene producing an aberrantly spliced transcript, where insertion of a 102 bp fragment had produced a frame-shift. Subsequent translation of this mRNA should generate an inactive enzyme (118)
. Indeed, C57Bl/6 mice have undetectable production of melatonin in the pineal gland and very low to undetectable concentrations in plasma (40)
. Surprisingly, significant production of melatonin has been reported in peripheral organs of this species, most notably in bone marrow-derived cells (30
, 32)
. When we performed tests to probe the expression of a melatoninergic system in the skin of C57BL/6 mice, we confirmed that the C57BL6 produced an aberrant AANAT isoform with the insertion of the 102 bp fragment (105)
; this was the predominant species in brain, pituitary, and anagen IV skin of the C57BL/6 mouse. We detected two additional alternatively spliced AANAT isoforms, one of which, expressed in brain, pituitary, and anagen IV skin, had an insertion of 89 bp from an intron and resulted in a frame shift after the first exon (105)
. Translation of this transcript would produce a protein of 59 aa with molecular mass of 6.5 kDa, devoid of enzymatic activity. A second AANAT isoform was detected solely in the spleen; this had a deletion of 69 bp, 24 bp from exon 3 and 45 bp from exon 4, but the reading frame was preserved (105)
. This transcript would produce a protein with deletion of 23 aa and an apparent molecular mass of 20.4 kDa. Extensive testing was performed on skin samples obtained at different phases of the hair growth cycle and on immortalized follicular melanocytes that showed expression of aberrant AANAT transcripts only, although low expression levels of the correct transcript were noted in anagen IV skin, brain, and pituitary (105)
.
Despite the lack of expression of the correct AANAT enzyme in the C57BL/6 mouse, extracts of skin nevertheless transformed serotonin to N-acetylserotonin (with Km=0.56 mM and Vmax=174 pmol/h) and acetylated tryptamine, but with a lower efficiency than for serotonin (105)
. Identity of the reaction products was confirmed by LC/MS. Enzymatic activity was inhibited by low concentrations of the specific arylalkylamine inhibitor BIS (by 65%) (105)
. Thus, the C57BL/6 mouse skin does acetylate serotonin to NAS in a reaction mediated by an enzyme different from conventional AANAT, most likely NAT-1 (105)
. The latter enzyme is ubiquitously expressed and with the capability, at least in vitro, of using aromatic amines as substrates (119
120
121)
. These findings provide the biochemical explanation for the finding of melatonin production in C57BL/6 mice at selected extracranial sites expressing HIOMT (32)
. These may use NAS substrate originating in the skin and delivered through the systemic circulation (105)
. It is nonetheless possible that additional peripheral sites could contribute NAS produced through AANAT-independent pathways. Our enzymatic studies did exclude corporal skin of the C57BL/6 mouse as a site of melatonin production, although we did detect HIOMT activity (at low levels) in mouse ears.
Serotonin (but not tryptamine) acetylation oscillates according to hair cycle phase and varies with anatomic location. NAS metabolism was extensive, with product nature being hair cycle dependent (in mouse back skin); some metabolites of the indoleamine remain to be identified (105)
. These metabolites could be products of NAS oxidation by skin hemoproteins, since NAS has been shown to be a substrate for horseradish peroxidase (122)
. NAS oxidative mechanisms could therefore include metabolites such as kynuramines (N1-acetyl-N2-formyl-5-methoxykynuramine and N-acetyl-5-methoxykynuramine), similar to the oxidation of melatonin (56
, 123)
.
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TOPABSTRACTINTRODUCTIONBIOLOGY OF SEROTONIN AND...SKIN SEROTONIN: SYNTHESIS AND...SKIN BIOREGULATION BY SEROTONINSKIN BIOREGULATION BY MELATONINFUNCTIONAL PROJECTION OF THE...CONCLUSIONSREFERENCES |
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, 125)
. This is supported by detailed molecular analyses that identified expression of mRNA encoding receptors 5-HT1A, 1B, 2A, 2B, 2C, and 7 in human skin (125)
. Further, in situ studies demonstrated 5-HT1A protein expression in basal epidermal melanocytes; 5-HT2A was found in the upper epidermis of eczematous skin, where its distribution was more homogeneous than in normal control skin (124)
. 5HT-3 was expressed in the proliferative basal epidermal layer of control and eczematous skin. In general, the in situ data were consistent with molecular analyses with the exception of the immunocytochemical detection of 5-HT3. Whether the latter is due to biological properties of the skin cells in situ or to the degree of specificity of the antibodies used is not clear. Pharmacological studies indicate that HT3 receptors are expressed on sensory nerve endings (126)
.
Serotonin shows variable effects on cell proliferation (125)
. It stimulates cell growth of dermal fibroblasts in a dose-dependent manner, consistent with its mitogenic activity in fibroblasts from nonskin fibroblast areas (61
, 71)
. Immortalized epidermal melanocytes exhibit serotonin-stimulated growth, but only when the cells had been incubated in medium deprived of melanocyte growth supplements (125)
. When media had been supplemented with growth factors, serotonin instead inhibited cell growth. Thus, depending on culture conditions, serotonin either supported melanocyte proliferation or inhibited their growth apparently acting through receptor-mediated regulation of apoptosis and proliferation (71)
. In SK-MEL188 human melanoma cells, serotonin inhibited DMEM-induced melanogenesis in a dose-dependent manner (3)
. Other studies have shown that serotonin-uptake inhibitors inhibited melanization in human melanoma cells (127)
. These results indicate a presumptive role of serotonin on melanogenesis.
Serotonin involvement in skin physiology and pathology is based on its vasoactive and immune modulator effects leading to its participation in the development of allergic reactions (4
, 128
, 129)
. Moreover, serotonin receptor antagonists (e.g., ketanserin) improve skin microcirculation by inhibiting serotonin-induced vasoconstriction and platelet activation (130)
. However, serotonin induces enhanced contractility of the small arteries in the skin of individuals with primary hypertension (130)
. Serotonin may be involved in the pathogenesis of some manifestations of skin dermatoses (4
, 129)
, including cholestatic and uremic pruritus (126
, 131)
, urticaria, and itch reaction (132)
. Although anti-histamines are the first choice for treatment of pruritis, serotonin antagonists may offer an attractive alternative (126
, 133)
.
Experimental animals
Early animal studies suggested that serotonin might play a pathogenic role in skin disease; serotonin has been implicated in the Arthus reaction (113)
. Its intradermal injection into rats decreased ATP skin content, reducing glycolytic enzyme activity and the levels of regulators of glucose metabolism (e.g., glucose 1,6-bisphosphate) (134)
. Calmodulin antagonists were found to prevent those serotonin effects, supporting the evaluation of calmodulin antagonists in dermatoses associated with increased serotonin levels ((134). Serotonin treatment has been reported to induce sustained vascular permeability in the skin of sheep and mouse (135
, 136)
, probably mediated through activation of 5-HT1 and 5-HT2A (136)
. Using a blister model of inflammation in the rat hind footpad, it was demonstrated that serotonin extends the plasma extravasation and vasodilatation responses, modulating the inflammatory response to substance P via involvement of capsaicin-sensitive sensory fibers in skin (137)
. Furthermore, serotonin levels within mast cell granules steadily diminish throughout anagen and increase, again during catagen and telogen. It must be noted that quantitative changes in mast cells numbers have long been known to correlate with murine hair cycling (cf. ref 138
). In several species, which include rat (139)
and pig (140)
, serotonin immunoreactivity has been detected in Merkel cells located in epidermal rete ridges and upper hair follicle. Immune staining was present throughout the entire cell or, more commonly, localized to the basal side where dense-core granules are more numerous and where the cell is adjacent to nerve terminals. In vitro studies have shown a modulatory effect of serotonin on epidermal keratinocytes proliferation (141)
, whereas the serotonin metabolites NAS and 5MT could, respectively, stimulate or inhibit melanoma cell proliferation at very high ligand concentrations (close to macromolar) (142)
.
Molecular analyses have identified expression of 5HT2B and 5HT7 in mouse skin samples and hamster melanomas (Slominski et al., unpublished results). Expression of the gene coding for the serotonin receptor 5HT2B in mouse skin was modulated by the hair growth cycle, being expressed in anagen but not telogen skin. The same gene was expressed in S91 melanoma in immortalized follicular melanocytes and in hamster transplantable melanomas. The transcript for the serotonin receptor 5HT7 was detected in mouse anagen skin, being absent in telogen skin and cultured melanocytes and melanoma cells. 5HT7 was detected in hamster melanomas. The pattern of detection of 5HT2B and 5HT7 is therefore similar to the findings in human skin, where the same types of 5HT receptors represent the predominant species expressed (125)
. 5-HT2A receptors were detected in unmyelinated sensory axons at the dermal-epidermal junction and the nerve endings of Pacinian corpuscles of rat glabrous skin (143)
. In situ autoradiography demonstrated presence of 5-HT1 in the dermis of rabbits, predominantly around hair follicles and sebaceous glands (144)
. The authors suggested that the receptors are expressed on primary afferent nerve fibers.
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. It lightens frog skin by inducing melanosomes aggregation around the nucleus in melanophores (reviewed in ref 145
). The skin lightening response to melatonin depends on the species and source tested (145
146
147)
. Melatonin reverses the pigmentary response to -MSH (145)
.
Mammalian system (in vivo effects)
Melatonin can modify hair growth and pigmentation. For example, in fury animals of subarctic or northern latitudes, circulating or local melatonin levels appear to participate in the seasonal changes in pelage color (147)
, whereas changes in photoperiod affecting melatonin levels could influence fur color and body weight of the Djungarian hamsters (148)
. Subcutaneous melatonin implants inhibit pigmentation of regrowing hairs (after depilation) in the short-tailed weasel and during molting in white-footed deer mice and Syberian hamsters, whereas melatonin may attenuate melanosis in dogs (147)
.
Experimental interference with photoperiodic activities and with mechanisms of hormonal control can affect hair growth and reproductive cycles of some mammals for several months. Implants of melatonin, which have been used to mimic the effect of short days, induce an advance and a synchronization of seasonal hair growth, causing suppression of plasma prolactin molting and the early formation of a winter coat (149)
. Constant-release melatonin implants affected molting in raccoon dogs; this treatment induced a more rapid shedding of mature underfur hairs and stimulated entry into anagen by increasing the growth of new underfur hairs of treated animals several weeks earlier than in the control untreated animals (150)
. Induction of early anagen by melatonin implants was observed in goats (151)
Similar effects were seen in the female mink, where melatonin implants induced an early molt, followed by an early telogen-to-anagen transition in resting hair follicles (152
, 153)
. This effect is not completely reproducible across different species (154)
.
Melatonin has a potent anti-oxidant effect that could be used to improve viability of tissues such as the skin during surgical procedures (155)
. Thus, melatonin reduced surgery-associated cell necrosis apparently through its ability to scavenge free radicals and to reduce malondialdehyde and nitric oxide levels and increase glutathione levels and activities while augmenting the activities of anti-oxidant enzymes such as glutathione peroxidase and superoxide dismutase. A similar protective melatonin capacity against UVB-induced oxidative stress was demonstrated in rat lens (112)
. Anti-mutagenic and oncostatic actions of melatonin were also reported. Thus, melatonin decreases not only the initiation but the promotion stages of skin carcinogenesis (156)
. Melatonin-treated animals exhibit lower lipid peroxide levels and there is some evidence melatonin can prevent the binding of chemical mutagens and/or their metabolites to DNA. Melatonin has been reported to exhibit tumorostatic properties in some rodent melanomas whereas pinealectomy stimulated growth of those tumors (89)
.
Cell culture
Melatonin inhibits basal, MSH, or cAMP-stimulated melanogenesis through post-tyrosinase mechanisms in cultured hair follicles from Siberian hamsters (157
, 158)
. High concentrations of melatonin inhibited tyrosinase activity in histocultured skin from C57BL/6 mouse with hair follicles at anagen stage, whereas NAS or its metabolite 5MT were without effect (159)
. Similar results were observed in Bomirski hamster amelanotic melanoma cells and Cloudman mouse melanoma cells (142)
. These actions represent antagonistic activity of melatonin against melanogenesis inducers (L-tyrosine or MSH), since noninduced or already melanized cells did not react to melatonin (142)
. The requirement for high concentrations of melatonin to elicit its anti-melanogenic effect suggests that melatonin is not acting through the melatonin receptor, e.g., this is either a metabolic effect or melatonin interacts with a receptor for an unrelated ligand. However, the inhibitory effect of melatonin on cell proliferation achieved at low ligand concentrations indicates a receptor-mediated process, particularly since these cells express membrane-bound receptors. A similar inhibitory but modest effect of melatonin on MSH-stimulated melanogenesis was found in B16 melanoma at relatively low concentrations (160)
. In this cell system, melatonin inhibited tyrosinase activity while slightly stimulating dopachrome tautomerase activity in a dose- and time-dependent manner. The expression of -MSH binding sites was markedly decreased by pharmacological concentrations of melatonin whereas physiological melatonin concentrations interfered with cAMP signaling in B16 melanoma (160)
. Anti-proliferative effects for melatonin, apparently through nonreceptor-mediated mechanisms, were reported in S91 melanoma cells (161)
.
A possible regulatory role for melatonin in rodent skin is further substantiated by the observation of its stimulation of epidermal keratinocyte proliferation (at physiological concentrations) and inhibition (at pharmacological doses) of anagen-coupled melanogenesis in skin organ culture (159)
. Melatonin has been reported to stimulate shaft elongation in cultured secondary hair follicles isolated from Cashmere goat. In this model system, melatonin treatment was associated with increased incorporation of [methyl-3H]-thymidine in hair follicle matrix cells, but only during the first 24 h of culture. Melatonin, however, markedly reduced hair follicle viability after 4 days of continuous culture (162)
. Melatonin directly affected the pathophysiology of rat dermis by its ability to inhibit skin collagen production in
