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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 23, 2002 as doi:10.1096/fj.01-0952fje. |
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Departments of Pathology,
Pharmacology, and
|| Surgery, University of Tennessee Health Science Center, Memphis, Tennessee, USA;
* Department of Biochemistry, Belarus State University, Minsk, Belarus;
Department of Internal Medicine, Southern Illinois University, Springfield, Illinois, USA;
Agilent Technologies, Inc., Schaumburg, Illinois, USA;
Department of Cell Biology, Neurobiology and Anatomy, Loyola Medical Center, Maywood, Illinois, USA;
Department of Biomedical Sciences, University of Bradford, England; and Experimental Dermatology Unit, Department of Neuroscience, Karolinska Institute, Stockholm, Sweden
2Correspondence: Department of Pathology, 899 Madison Ave., Room 576M, Memphis, TN 38163, USA. E-mail: aslominski{at}utmem.edu
SPECIFIC AIMS
The skin can metabolize serotonin to N-acetylserotonin (NAS) and possibly melatonin as recently shown in the hamster. We investigated these biosynthetic pathways in normal and pathological human skin, cultured normal and malignant keratinocytes and melanocytes derived from epidermal or follicular compartments, and follicular and dermal fibroblasts.
PRINCIPAL FINDINGS
1. RT-PCR detection of TPH, AANAT, and HIOMT mRNAs
The 380 bp TPH transcript was present in pituitary, adrenal gland, myometrium, and all samples of normal skin and skin containing basal cell carcinoma; it was also present in cultured normal epidermal and follicular melanocytes, all melanoma cell lines, normal neonatal and adult epidermal and follicular keratinocytes, squamous cell carcinoma cells, and dermal and dermal follicular fibroblasts; the only cells that tested negative for the transcript were HaCaT immortalized keratinocytes. Transcripts of AANAT showed the expected 176 bp fragment spanning exon 2 and 3, and an aberrant 220 bp cDNA fragment corresponding to an isoform with an inserted 59 bp segment from intron 2 (GeneBank accession no. AY055827). The 176 bp fragment was expressed in the majority of tissues tested with the exception of a single biopsy specimen of basal cell carcinoma, a single melanoma line, human follicular melanocytes, and immortalized melanocytes. Human pituitary and adrenal glands contained, besides the 176 bp fragment, an additional 220 bp aberrantly spliced isoform; the latter isoform was the sole transcript in a single biopsy specimen of basal cell carcinoma. Human skin contained the HIOMT cDNA species of 312 and 171 bp corresponding to isoforms lacking exon 6 or exons 6 and 7, respectively. The 171 kb HIOMT transcript was expressed in all tissues and cell lines tested; the 312 bp transcript was expressed in the majority of tissues tested except for a single skin biopsy specimen and cultured HaCaT keratinocytes, SKMEL188 melanoma and C1–4 squamous cell carcinoma. An additional HIOMT PCR fragment of 396 bp representing mRNA with all the exons was present in pituitary, adrenal gland, myometrium, epidermal and follicular melanocytes and keratinocytes, dermal and dermal papilla fibroblasts, and selected skin samples.
We tested the effect of ultraviolet radiation (UVR) on expression of the above genes within the following cell lines: human HaCaT keratinocytes, squamous cell carcinoma C1–4, and SKMEL188 melanoma cells. Though UVR had no effect on TPH gene expression in HaCaT keratinocytes, it did inhibit gene expression in squamous cell carcinoma C1–4 cells and human melanoma cells. UVR had no effect on AANAT expression in any of the three cell lines tested, nor did it change significantly HIOMT gene expression in SKMELL188 cell line. UVR (5 mJ/cm2) inhibited HIOMT gene expression in squamous cell carcinoma line, whereas in HaCaT keratinocytes the same irradiation shifted the splicing pattern: treated cells produced increased amount of only one HIOMT mRNA transcript, a 510 bp fragment (GeneBank accession no. AY037933) as opposed to the two 171 and 510 bp PCR bands observed in control cells. The 510 bp PCR band represents an aberrantly spliced isoform with the exons 5–7 spliced out and the introns D and F inserted in the final mRNA transcript. Introns D and F have multiple terminator codons that would stop gene translation to produce truncated proteins. These results imply that UVR may actively contribute to regulation of the molecular apparatus responsible for the generation of melatonin from tryptophan. Thus, the selective functional consequences of solar irradiation on the activity of the skin serotoninergic and melatoninergic systems may represent a new research frontier in cutaneous biology.
2. Detection of serotonin and NAS in the skin
Extracts of human skin subjected to RP-HPLC showed fluorescence at the retention times of serotonin and NAS standards, indicating endogenous stores of the amines. Detailed analysis was performed in immortalized HaCaT keratinocytes by RP-HPLC with electrochemical detection, which showed the presence of serotonin and its metabolite 5-hydroxyindole-acetic acid (5HIAA). Further investigations with tandem LC/MS showed an adduct ion (M+H)+ with mass spectrum and retention time consistent with NAS (m/z at 219 with RT of 14 min; calculated mass was 218 Da). We made a special search for melatonin in the cell extracts; although we detected an adduct ion with the same mass spectrum at m/z 233 (calculated mass of 232 Da) and RT (23 min) as melatonin standard, the level was only slightly above background, thus only suggestive of the presence of the indole.
3. Biochemical detection of AANAT and HIOMT activities
Detection of products of AANAT activity was accomplished in human skin and in all melanomas and immortalized normal melanocytes and keratinocytes using RP-HPLC system with fluorimetric detection. When human skin extracts were incubated with acetyl-CoA, serotonin converted to NAS and tryptamine to N-acetyltryptamine, thus confirming the presence of the enzyme in the skin and consistent with expression of the corresponding gene. These biochemical assays showed there were linear relationships between enzyme activity and protein concentration and between enzyme activity and time of incubation (over a 75 min period). Kinetic analysis of the acetylation reaction using serotonin as substrate showed Km and Vmax for human skin of 0.69 ± 0.08 mM and 36.64 pmol/h, respectively. The calculated Km for the same reaction in immortalized melanocytes and HaCaT keratinocytes did not differ significantly, albeit they were fourfold lower than in the skin (e.g., 3.96 ± 0.6 and 2.75 ± 0.57 mM with Vmax of 40.64 and 44.3 pmol/h, respectively). This suggests that AANAT may have varying degrees of affinity for serotonin across the different cellular skin compartments. Comparative analysis of Km values reported in the literature showed that keratinocyte and melanocyte AANAT is similar to human AANAT expressed in COS-7 cells (2.6 mM) but higher than in bacterially expressed AANAT of human (1.3 mM) or ovine (0.31 mM) origin. In the ovary, the apparent AANAT Km was calculated at 0.15 mM, being one-fourth of that in the skin. Since RT-PCR in the immortalized normal melanocytes failed to detect AANAT gene expression and AANAT activity toward tryptamine was below detection, we conclude that this cell line expresses an enzyme that differs from other skin cells (Table 1
). Thus, serotonin metabolism was highly specific for the existing conditions and dependent on cellular environment in vitro or histological location in vivo. Indeed, the calculated AANAT activity ratios for tryptamine vs. serotonin as substrates were close to 1 for all melanoma lines and HaCaT keratinocytes, whereas they ranged from 2.5 to 6 for whole skin from three white patients; the activity ratios were zero in immortalized normal melanocytes and in whole skin of a black patient, because the activity toward tryptamine was below detection. Since AANAT activity for serotonin was higher in melanoma cells than in either whole skin or immortalized keratinocytes and melanocytes, racial pigmentation and cutaneous pathology (melanoma) may be important determinants of serotonin acetylation rate and specificity.
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The products of HIOMT activity were separated by RP-HPLC, which showed NAS transformation to melatonin in samples of skin of either Caucasian or African-American origin. HIOMT activity was detected in HaCaT keratinocytes and melanoma cells. The identity of newly formed melatonin was verified by its coelution with authentic melatonin standard. HIOMT activity was higher in melanoma cells than in whole skin or keratinocytes. Thus, human skin can be defined as another extrapineal and extraretinal organ with the intrinsic capability to synthesize melatonin. Expression of the HIOMT gene in keratinocytes and melanocytes of epidermal and follicular origin, as well as in dermal and follicular papilla fibroblasts, implies that melatoninergic pathways may operate independently in the different epidermal, adnexal, and dermal compartments.
The RP-HLC chromatogram of human skin showed, besides melatonin, species absent from control extracts and with retention time different from melatonin ( 7.6, 12, 17.6, 18.8, 19.8, 24, and 28.5 min). Since species with retention times of 7.6, 17.6, 18.8, 19.8, and 24 min were detected in samples containing NAS without the cofactor S-adenosyl-L-methionine, they are likely products of non-HIOMT-mediated NAS metabolism. The species with retention time 12 and 28.5 min, absent under the above conditions, represent probable products of melatonin metabolism. Although the nature of these compounds remains to be clarified, these findings indicate that NAS may enter either of two alternative metabolic pathways: direct metabolism independent of NAS methylation or progression to melatonin production and its rapid, subsequent metabolism. Frog skin shows extensive metabolism of melatonin with a degradation rate higher than in the brain or retina. Further characterization of these pathways will be the subject of future research.
CONCLUSIONS
We provide definitive evidence that human skin and cultured skin-derived cells express the intrinsic capability to transform L-tryptophan to serotonin and to metabolize serotonin to NAS and melatonin. This conclusion is based on the accumulated results of our experiments demonstrating expression of the TPH, AANAT, and HIOMT genes, metabolism of serotonin to NAS and melatonin, and identification of the intermediate reaction products.
We found the TPH gene expressed in all samples tested (except for HaCaT keratinocytes), suggesting that different cellular populations in epidermis and dermis share the potential capacity to hydroxylate L-tryptophan. Identification of serotonin and 5HIAA in immortalized HaCaT keratinocytes implies that these cells must express serotonin transporters to allow for serotonin cellular uptake and serotonin cellular degradation, the latter probably mediated by monoamine oxidase and aldehyde dehydrogenase. Indeed, a serotonin transport system has already been described in melanoma cells, and we recently identified 5HTP transporter immunoreactivity in cultured HaCaT cells. Hence, paracrine and autocrine mechanisms may coexist within the epidermal compartment to regulate serotonin availability and its conversion to biologically active compounds. Thus, serotonin produced by melanocytes may be either transformed in situ to NAS and possibly melatonin or released into the extracellular medium to be taken up by keratinocytes for metabolism to NAS and melatonin. This model is supported by our detection of AANAT and HIOMT activities in skin and cultured skin cells, of NAS in skin samples, and our identification of NAS and melatonin in immortalized keratinocytes.
Serotonin is a recognized neurotransmitter, vasodilator, immunomodulator, and growth factor; in the skin, serotonin may exert pro-edema, vasodilatory, proinflammatory, and/or pruritogenic actions. Melatonin is known to act as a hormone, neurotransmitter, cytokine, biological modifier, and immunomodulator; in the skin itself, melatonin may inhibit melanogenesis, regulate hair growth, and act as a free radical scavenger. It has been reported to exhibit tumorostatic properties in some rodent melanomas. The present demonstrations of local serotonin and melatonin synthesis provide mechanistic support for the expression of those actions. The compounds may act through para- or autocrine mechanisms, acting in concert to regulate skin functions at a highly compartmentalized level. Thus, the present findings uncover a new layer of skin function with potential for regulation and deregulation and implications for cutaneous physiology and pathology. Though the full significance of this intrinsic production of serotonin, melatonin, and their metabolites is yet to be determined, we have already detected in cutaneous cells the expression of receptors for melatonin and serotonin.
In conclusion, we demonstrate expression of fully developed, local serotonin and melatonin biosynthetic pathways in the human skin. Pathway activities appeared to vary according to skin cellular compartment, human racial pigmentation, and cutaneous pathology.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0952fje; to cite this article, use FASEB J. (April 23, 2002) 10.1096/fj.01-0952fje. 
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