Báo cáo khoa học: Characterization of the serotoninergic system in the C57BL/6 mouse skin potx

Characterization of the serotoninergic system in the C57BL/6 mouse skin Andrzej Slominski 1 , Alexander Pisarchik 1 , Igor Semak 2 , Trevor Sweatman 3 and Jacobo Wortsman 4 1 Department of Pathology, University of Tennessee Health Science Center, Memphis, TN, USA; 2 Department of Biochemistry, Belarus State University, Minsk, Belarus; 3 Pharmacology, University of Tennessee Health Science Center, Memphis, TN, USA; 4 Department of Internal Medicine, Southern Illinois University, Springfield, IL, USA We showed expression of the tryptophan hydroxylase gene and of tryptophan hydroxylase protein immunoreactivity in mouse skin and skin cells. Extracts from skin and melano- cyte samples acetylated serotonin to N-acetylserotonin and tryptamine to N-acetyltryptamine. A different enzyme from arylalkylamine N-acetyltransferase mediated this reaction, as this gene was defective in the C57BL6 mouse, coding predominantly for a protein without enzymatic activity. Serotonin (but not tryptamine) acetylation varied according to hair cycle phase and anatomic location. Serotonin was also metabolized to 5-hydroxytryptophol and 5-hydroxy- indole acetic acid, probably through stepwise transform- ation catalyzed by monoamine oxidase, aldehyde dehydrogenase and aldehyde reductase. Activity of the melatonin-forming enzyme hydroxyindole-O-methyltrans- ferase was notably below detectable levels in all samples of mouse corporal skin, although it was detectable at low levels in the ears and in Cloudman melanoma (derived from the DBA/2 J mouse strain). In conclusion, mouse skin has the molecular and biochemical apparatus necessary to produce and metabolize serotonin and N-acetylserotonin, and its activity is determined by topography, physiological status of the skin, cell type and mouse strain. Keywords: mouse skin; serotonin acetylation; arylalkylamine N-acetyltransferase; tryptophan hydroxylase; hair cycle. The skin is the largest body organ and functions as a metabolically active biological barrier regulating internal homeostasis and separating the internal milieu from noxious environmental factors [1]. These functions are mediated by the skin immune, pigmentary, neuroendo- crine, adnexal and vascular systems [1–7]. Most recently we have uncovered local serotoninergic and melatoniner- gic systems as novel elements of the cutaneous neuro- endocrine components of human and hamster skin [8–12]. Serotonin is the product of a multistep metabolic pathway that starts with the hydroxylation of the aromatic aminoacid L -tryptophan by tryptophan hydroxylase (TPH) [13,14]. Serotonin can be acetylated by arylalkyl- amine N-acetyltransferase (AANAT) to N-acetylserotonin (NAS),whichisfurthertransformedtomelatoninby hydroxyindole-O-methyltransferase (HIOMT) [13,15]. Serotonin can act as a neurohormone, regulator of vascular tone, immunomodulator and growth factor, while melatonin can act as a hormone, neurotransmitter, cytokine or biological modifier [2,15–17]. Some of these functions may be pertinent to skin physiology, which exhibits basic differences among the mammalian species. In rodents (mostly nocturnal animals) the skin is shielded from the damaging effect of solar radiation by fur [18], and the morphology of the entire mouse skin changes in close coordination with the cyclic activity of the hair follicle [19]. Mouse hair follicle cycling is characterized by a precisely regulated, time frame-restricted and differential pattern in the expression and activity of melanogenesis related proteins, PH, pterins and thioredoxin reductase [20]. Hair cycle-dependent changes also involve adrenergic innervation and specific patterns of b2-adrenergic receptor expression [21]. Our previous studies raised the possibility that the level of activity of an endogenous serotoninergic pathway would specifically determine whether its products are for internal use (intracrine regulation), or for external secretion (para- or autocrine regulation). Because mouse skin differs from human skin, we anticipated interspecies heterogeneity in the cutaneous expression of elements of the serotoninergic pathway. Therefore, we have tested the expression of dif- ferent elements of the serotoninergic system in the C57BL/ 6 mouse and related their activity in the skin to the phase of the hair cycle and to the cutaneous cellular compartments. Correspondence to A. T. Slominski, Department of Pathology, University of Tennessee Health Science Center, 930 Madison Ave Rm 519, Memphis, TN, USA. Fax: + 1 901 448 6979, Tel.: + 1 901 448 3741, E-mail: aslominski@utmem.edu Abbreviations: TPH, tryptophan hydroxylase; AANAT, arylalkyl- amine N-acetyltransferase; NAS, N-acetylserotonin; HIOMT, hydroxyindole-O-methyltransferase; PH, phenylalanine hydroxylase; CDL, curved desolvation line; TBST, Tris buffered saline with Tween 20; NAT, arylamine N-acetyltransferase; Bis, bisubstrate analog coenzyme A-S-acetyltryptamine; MAO, monoamine oxidase; 5HIAA, 5-hydroxyindolacetic acid; 5HTPOL, 5-hydroxytryptophol. Note: a website is available at http://www.utmem.edu/pathology (Received 30 April 2003, revised 4 June 2003, accepted 9 June 2003) Eur. J. Biochem. 270, 3335–3344 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03708.x Experimental procedures Tissue Murine samples consisted of skin isolated at telogen and anagen stages of the hair cycle as described previously, and of brain, pituitary and spleen tissues [12,19]. Experiments performedintheUSAusedC57BL/6strainfemalemice (8 weeks old) purchased from Taconic (NY) and housed in community cages at the animal facilities of the Albany MedicalCollege,Albany,NY.LC/MSassays,performedin Belarus, also used C57BL/6 mice (males 18 weeks old) obtained from the Vivarium of the Department of Bio- testings of the Institute of Bioorganic Chemistry (Belarus State University, Minsk, Belarus). The animals were housed in a temperature-controlled room on a 12-h light : 12-h dark schedule (lights turned on at 06.00 h) with food and water available ad libitum. The animals were killed under pento- barbital anesthesia and selected organs as well as back skin were collected following protocols routinely used in our laboratory, and then stored at )80 °C until use [12,19]. The Institutional Animal Care and Use Committee at Albany Medical College approved the original experimental proto- col, and a similar protocol for mice was approved at University of Tennessee Health Science Center. Approval for the experiments performed in Minsk, Belarus was granted by the Belarus University Animal Care and Use Committee. Cell culture Tested cell lines comprised mouse Cloudman S91 (sublines 6 and M3) and hamster AbC-1 melanoma cells, and mouse immortalized normal melanocytes (MelA). Melanoma cells were grown in Ham’s F10 medium as described previously, and the media were supplemented with 10% (v/v) fetal bovine serum and 1% antibiotic/antimycotic mixture (Gibco) [22]. MelA (the gift of D. Bennett, St George’s Hospital, London, UK), was cultured in RPMI 1640 media, supplemented with 10% (v/v) 200 n M bovine serum (phorbol-12-myristate-13-acetate), in the presence of 10% (v/v) CO 2 . After washing with NaCl/P i , melanoma cells were detached using Ca- and Mg-free Tyrode’s solution, containing 1 m M EDTA, while normal immortalized mouse melanocytes were trypsinized. The cells were centrifuged then frozen at )80 o C, for use in further analyses. Enzymatic assays Arylakylamine/arylamine N-acetyl transferase acti- vity. N-acetyl transferase activity was measured either by the method of Thomas et al. [23], using a modified RP-HPLC separation with fluorimetric detection of the reaction products [24] or by direct LC/MS detection of metabolic intermediates [8]. For both methods, tissue or cell samples were homogenized in an ice-cold 0.25 M potassium phosphate buffer (pH 6.8) containing 1 m M dithiothreitol, 1m M EGTA and protease inhibitor cocktail (2 lLÆmL )1 homogenization mixture, Sigma). Homogenates were cen- trifuged at 15 000 g for 10 min at 4 °C. Supernatants were used to measure serotonin N-acetyl transferase in the presence of 1 m M serotonin or tryptamine and 0.5 m M of acetyl coenzyme A in 0.25 M potassium phosphate buffer (pH 6.8) for 1 h or 1.5 h (when indicated) at 37 °C. The enzymatic reaction was stopped by the addition of HClO 4 . After centrifugation, the supernatant was subjected to HPLC in a system equipped with a Novapak C 18 reverse- phase column (100 · 5 mm, 4 lm particle size; 60 A ˚ pore size) and a fluorometric detector with excitation and emission wavelengths set at 285 and 360 nm, respectively. The elution was carried out isocratically at ambient temperature with a flow rate of 1.5 mLÆmin )1 for the mobile phases chosen according to the amine substrate to be used. The mobile phase contained 4 m M sodium 1-octane- sulfonate as an ion-pairing agent, 50 m M ammonium formate (pH 4.0) vs. methanol (80 : 20, v/v) for serotonin and (75 : 25, v/v) for tryptamine. Elution peaks of N-acetylserotonin and N-acetyltryptamine were verified by coelution with the authentic standards. The peak areas were quantified in relation to known concentrations of N-acetylserotonin and N-acetyltryptamine standards. Back- ground controls consisted of the reaction mixture incubated without substrate or enzyme source. For LC/MS analysis, the final concentrations of acetyl CoA and serotonin in reaction mixtures were 0.5 m M and 5m M , respectively. Aliquots of the final reaction super- natants (see above) were separated on an LCMS-QP8000a (Shimadzu, Japan) through Restec Allure C18 reverse-phase column (150 · 4.6 mm; 5 lm particle size; 60 A ˚ pore size). The elution was carried out isocratically at a flow rate of 0.3 mLÆmin )1 at 30 °C by mobile phases consisting of 20% (v/v) methanol and 0.1% (v/v) acetic acid. The effluent from the HPLC system was routed to the MS electrospray interface used in the positive mode. Nitrogen was used as nebulizing gas. MS parameters were as follows: nebulizer gas flow rate was 4.5 LÆmin )1 ; the electrospray voltage was 4.5 kV; CDL heater temperature was 250 °C. Selected ion monitoring mode was applied to detect ions with m/z ¼ 219. The LC/MS workstation CLASS -8000 software was used for system control and data acquisition (Shimadzu, Japan). Quantitative determination of N-acetylserotonin was made by comparing the observed peak areas with the peak areas of known concentrations of the NAS standard. Hydroxyindole-O-methyl-transferase activity. Hydroxy- indole-O-methyl-transferase activity was assayed as des- cribed previously [24]. Briefly, tissue homogenates were centrifuged at 15 000 g for 10 min at 4 °C. Supernatants were used to measure enzymatic activity in the presence of 0.5 m M of S-adenosyl- L -methionine and 1 m M of N-acetyl- serotonin in 0.05 M sodium phosphate buffer (pH 7.9). After incubation for 1 h at 37 °C, the enzymatic reaction was stopped by the addition of HClO 4 and, after centri- fugation, the supernatants were subjected to HPLC in the system described above for measurement of acetyl trans- ferase activity, with tryptamine as the substrate. Compar- ison with the retention times of the authentic standards identified elution peaks for N-acetylserotonin and melato- nin. Protein concentration was determined with a dye- binding method using BSA as the standard [24]. Western blot analysis Cultured cells were detached in Tyrode’s solution plus 1 m M EDTA, centrifuged at 200 g for 10 min at 4 °C and the cell 3336 A. Slominski et al.(Eur. J. Biochem. 270) Ó FEBS 2003 pellets were then washed with NaCl/P i and frozen at )70 °C. For protein isolation, frozen cell pellets or skin samples were homogenized with a glass homogenizer in ice-cold buffer A containing 20 m M Tris, pH 7.5, 5 m M EDTA, 120 lgÆmL )1 leupeptin, 3 l M pepstatin and 3 m M amino-ethylbenzene sulfonyl fluoride. The homogenates were centrifuged at 16 000 g for 10 min at 4 °Ctoremove cell debris and centrifuged at 100 000 g for 1 h at 4 °C. The supernatants representing the cytosol fraction were removed and stored at )80 °C for further analysis. Separate aliquots of 5 lL were used for protein determination using the Micro Protein Kit (Sigma). Fifty micrograms of protein were separated on a 12% (w/v) SDS polyacrylamide gel, transferred to immobilion-P poly(vinylidene difluoride) membrane (Millipore Corp, Bedford, MA, USA) and nonspecific binding sites were blocked by incubation in 5% (w/v) nonfat powdered milk in TBST (50 m M Tris, pH 7.5, 150 m M NaCl, 0.01% Tween 20) for 4 h at room temperature. Immunodetection of the TPH or AANAT proteins was performed after overnight incubation with polyclonal rabbit anti-human TPH (dilution 1 : 1000, at 4 °C) as the primary antibody (Chemicon, Temecula, CA) or with rabbit anti-(rat AANAT 25-200 ) serum (dilution 1 : 10 000, room temperature; gift of D. Klein, NIH, Bethesda, MD, USA). In parallel incubation we used preimmune rabbit serum at the same dilution as the corresponding antiserum (gift of D. Klein, NIH). The following day membranes were washed twice in TBST for 10 min. Goat anti-rabbit IgG coupled to horseradish peroxidase was used as a secondary antibody (dilution 1 : 4000, 1 h) (Santa Cruz Biotechnology). Membranes were washed twice in TBST and once in TBS. Bands were visualized using ECL reagent (Amersham Pharmacia Bio- tech) according to the manufacturer’s instructions. RNA extraction and cDNA preparation Tissues were pulverized in liquid nitrogen using a mortar and then suspended in Trizol (Invitrogen) and the isolation of RNA followed the manufacture’s protocol. The synthesis of first-strand cDNA was performed using the Superscript preamplification system (Invitrogen). Total RNA (5 lgper reaction) was reverse transcribed according to the manu- facturer’s protocol, using oligo(dT) as the primer. All samples were standardized for analysis by the amplification of housekeeping gene GAPDH as described previously [25]. Mouse TPH was amplified by a single PCR, while serotonin AANAT was amplified by nested PCR. The localization of the primers in corresponding genes is presented in Figs 1A and 3A. The reaction mixture (25 lL) contained 2.5 m M MgCl 2 ,0.25 M of each dNTP, 0.4 l M of each primer, 75 m M Tris/HCl (pH 8.8), 20 m M (NH 4 ) 2 SO 4 , 0.01% Tween 20 and 1.25 U of Taq DNA polymerase (Promega). The mixture was heated to 94 °Cfor 2.5minandthenamplifiedfor35or30cyclesasspecified: 94 °C for 30 s (denaturation), 60 °C for 45 s (annealing) and 72 °C for 1 min (extension). TPH was amplified by a single PCR using primers P108 (5¢-CTTTCGAGTCTTTCACTGCACTC-3¢) and P109 (5¢-CATTCATGGCACTAGTTATGCTC-3¢). Exons 1–2 of mouse AANAT were amplified by primers P242 (5¢-CCAGCGAGTTCCGTTGCCTTAC-3¢) and P243 (5¢-GCCTGTGCAGTGTCAGTGACTC-3¢) in the first round and primers P244 (5¢-CGTGTTTGAGATTGAGC GTGAAG-3¢) and P245 (5¢-CTTGTCCCAAAGTGAGC CGATG-3¢) in the second round of PCR. Primers for the first PCR of exons 3–4 of mouse AANAT were P145 (5¢-ACTTGGATGAGATCCGGCACTTCC-3¢) and P148 (5¢-GGCTGACTGCCCAGGTGGTGAAG-3¢). Primers for the second round were P146 (5¢-GTCCAGAGCTGT CACTGGGC-3¢)andP147(5¢-AGGACAGAGCCCT TGCCCTGCTG-3¢). Annealing temperature for the ampli- fication of exons 3 and 4 was 67 °C. Amplification products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining according to the standard protocol used in our laboratory [8,24,25]. The identified PCR products were excised from the gel and purified by GFX PCR DNA using the gel band purification kit (Amersham-Pharmacia-Biotech). PCR frag- ments were cloned in pGEM-T easy vector system (Pro- mega) and purified by plasmid purification kit (Qiagen). Sequencing was performed in the Molecular Resource Center at the University of Tennessee HSC (Memphis) using an Applied Biosystems 3100 Genetic Analyzer and the BigDye TM Terminator Kit. Results Tryptophan hydroxylase expression Using mouse-specific primers for mouse tryptophan hydroxylase (Fig. 1A) we subjected RNA from different tissues and cell lines to RT-PCR. The amplified fragments of 530 bp were sequenced and shown to have complete (100%) homology with the corresponding gene fragment. Thus, the tryptophan hydroxylase gene was expressed in the brain, pituitary, spleen, Cloudman S91 melanoma cells, MelA immortalized normal melanocytes, and anagen and catagen skin (growing and involutional phases of the hair cycle, respectively). The TPH gene was either absent (two experiments) or present (one experiment) in telogen (resting phase of the hair cycle) (Fig. 1B, Table 1). Western blot analysis using two different antibodies was performed in cytoplasmic extracts from mouse skin, MelA Fig. 1. TPH mRNA expression in murine tissues and skin cell lines. (A) Localization of the primers to the TPH coding exons. The numbers correspond to protein coding exons revealed after comparison of mRNA (NM-009414) and genomic DNA. (B) Expression of a 530 bp TPH transcript in brain (2), anagen IV (3), anagen V (4), middle anagen VI (5), late anagen VI (6), and telogen skin (7), spleen (8), subline 6 of S91 melanoma (9) and subline M3 of S91 melanoma (10). DNA markers are shown in lanes 1 and 11. Ó FEBS 2003 Serotoninergic system in mouse skin (Eur. J. Biochem. 270) 3337 immortalized normal melanocytes, S91 (clone 6) mouse melanoma cells and pig pineal gland control. These tests identified a specific protein of 53–55 kDa precipitated by anti-TPH Igs (Fig. 2; arrow). Additional proteins of both higher (83–85 kDa) and lower molecular mass were also detected by the same antibodies (Fig. 2). AANAT gene expression Using different pairs of specific primers located at exons 1 and 2, and 3 and 4 of the AANAT gene we subjected RNA from mouse tissue and normal and malignant melanocytes to RT-PCR amplification (Fig. 3A). RT-PCR with primers located at exons 1 and 2 demonstrated the presence of a 163 bp fragment in all tissues and cells tested; this fragment showed 100% homology with the corresponding fragment of mouse AANAT cDNA (Fig. 3B). In addition, an aberrantly spliced isoform of 252 bp was detected in the brain, pituitary, spleen and M3 subline of S91 melanoma, but not in the C57BL/6 mouse skin or the MelA melano- cytes (Fig. 3B, Table 1). This isoform had the insertion of 89 bp from an intron leading to a frame shift after the first exon. Translation of this transcript would produce a protein of 59 amino acids with a molecular mass of 6.5 kDa, devoid of enzymatic activity. Tests performed with primers located at exons 3 and 4 of the AANAT gene yielded bands of 187 bp, 289 bp and 118 bp (Fig. 2A,C, Table 1). The 187 bp band corresponding to the normal AANAT cDNA was detected in the brain, pituitary, parental subline M3 of Cloudman S91 melanoma and anagen IV mouse skin. It was not detected in skin at telogen, anagen V, early and late anagen VI and catagen phases of the hair cycle (Fig. 2C, Table 1). The 289 bp band represented the aberrantly spliced isoform described previously by Roseboom et al. [26] with the insertion of a 102 bp fragment that produced a frame shift; translation of this mRNA should generate an inactive enzyme [26]. It was detected as the predominant AANAT species in brain, pituitary and anagen IV skin of the C57BL/6 mouse, but only as a minor component in Cloudman S91 melanoma (Fig. 2C, Table 1). The 118 bp band was detected only in the spleen, and had a deletion of 69bp(24bpfromexon3and45bpfromexon4)buta preserved reading frame. Thus, this transcript would produce a protein with a deletion of 23 amino acids and an apparent molecular mass of 20.4 kDa. It is, however, unclear whether this protein posses enzymatic activity. As the deleted fragment does not include any of the residues critical for substrate binding or enzymatic activity, it is highly probable that it may be enzymatically active. Western blot analysis showed a protein with the expec- ted size for AANAT (24 kDa) immunoprecipitated by Table 1. Tissue and cell line expression pattern of TPH and AANAT genes from mouse source. Numbers 118 (GenBank Accession Number AY131261), 163 (GenBank Accession Number AF004108), 252 (GenBank Accession Number AY131262), 187 (GenBank Accession Number AF004108) and 289 (GenBank Accession Number AF004111) represent the size of corresponding transcripts (bp) detec- ted by RT-PCR. Specimens TPH AANAT Exons 1 and 2 AANAT Exons 3 and 4 Brain (+) 163, 252 187, 289 Pituitary (+) 163, 252 187, 289 Skin (anagen IV) (+) 163 (–)187, 289 Skin (anagen V) (+) 163 (–) Skin (middle anagen VI) (+) 163 (–) Skin (late anagen VI) (+) 163 (–) Skin (telogen) (–)(–)(+) 163 (–) Spleen (+) 163 118 Melanoma S91 (subline M3) (+) 163, 252 187, 289 MelA melanocytes (+) 163 (–) Fig. 2. Detection of TPH immunoreactive proteins in mouse skin and cultured melanocytes and melanoma cells. (A) Immunoprecipitation using rabbit anti-TPH Igs: skin at catagen (1), anagen III (2) and anagen V (3) phases of hair growth, MelA melanocytes (4), Cloudman S91 melanoma (5), pig epiphysis (6). The arrow indicates a TPH-like immunoreactivity of 53 kDa. (B) Immunoprecipitation using sheep anti-TPH Igs. Molecular masses in kDa are indicated on the left; skin at telogen (1), anagen III (2), catagen (3) phases of hair growth, MelA melanocytes (4). The arrow indicates a TPH-like immunoreactivity of 53 kDa. (C) Immunoprecipitation control for B. The panel presents the blot incubated with secondary antibody only. Explanation of numbersandarrowisasabove(B). 3338 A. Slominski et al.(Eur. J. Biochem. 270) Ó FEBS 2003 anti-(rat AANAT) serum (anti-rAANAT25-200) in control rat brain, Cloudman S91 and hamster AbC-1 melanomas but not in the mouse skin (Fig. 4). Detection of this AANAT-like immunoreactivity appeared to represent true expression, as it was not seen in control membranes incubated with preimmune serum. Acetylation of serotonin and tryptamine by skin extracts Using the RP-HPLC system with fluorimetric detection or LC/MSwewereabletofirmlyestablishthatextractsof C57BL/6 mouse skin and of cultured normal and malignant melanocytes after addition of acetyl-CoA transformed serotonin to N-acetylserotonin (Figs 5 and 6, Tables 2 and 3). In contrast, the acetylation of tryptamine by skin extracts was less efficient (Table 2). The experiments with LC/MS confirm NAS identity by showing the appearance of an adduct ion (M + H) + at m/z ¼ 219 with the same retention time as the correspond- ing NAS standard, e.g. m/z ¼ 219 (calculated mass ¼ 218 Da) at a retention time of 27 min (Fig. 6B). Kinetic analysis of N-acetylase activity showed K m and V max values of 0.56 m M and 174 pmolÆh )1 for serotonin substrate, respectively (Fig. 7). We also tested the effect of bisubstrate analog coenzyme A-S-acetyltryptamine (Bis; a specific inhibitor of arylalkylamine activity) on the enzymatic Fig. 4. AANAT-like immunoreactivity is absent in C57BL/6 mouse skin and present in hamster AbC-1 and mouse S91 Cloudman melanomas. Immunoprecipitation using rabbit anti-AANAT Igs (upper panel). Lower panel presents blots incubated with secondary antibody only. Markers in kDa are shown on the left; rat brain (1), hamster AbC-1 melanoma (2), mouse S91 Cloudman melanomas (3), C57BL/6 mouse skin at anagen III (4), anagen V (5) and catagen (6) phases of hair growth. Arrow indicates AANAT-like immunoreactivity of 24 kDa. Fig. 5. HPLC chromatogram obtained from reaction mixture in which S-91 melanoma cells were used as the enzyme source. Experimental incubation with acetyl CoA and serotonin (A) or tryptamine (B) and corresponding control extracts without amine substrate (C and D). N-acetylserotonin or N-acetyltryptamine indicate the elution position of corresponding standards. Fig. 3. AANAT mRNA expression in murine tissues and cell lines. Structure of the murine AANAT gene. Open boxes represent exons. Shadowed and black boxes are fragments of coding sequence located after the frame shift and cryptic exons, respectively. Primers are shown by arrows. (B) Detection of 163 and 252 bp AANAT PCR bands amplified by primers located at exons 1 and 2. DNA ladder (1, 14), pituitary (2), brain (3), MelA melanocytes (4), M3 subline of S91 mel- anoma (5), anagen IV (6), anagen V (7), early anagen VI (8), middle anagen VI (9), late anagen VI (10), and telogen (11) skin; #6 subline of S91 melanoma (12); telogen stain (13). (C) Expression of 187 and 289 bp AANAT transcripts amplified by primers located at exons 3 and 4. DNA ladder (1), brain (2), pituitary (3), M3 subline of S91 mel- anoma (4), anagen IV (5); late anagen VI (6), telogen skin (7), spleen (8). Ó FEBS 2003 Serotoninergic system in mouse skin (Eur. J. Biochem. 270) 3339 activity in skin extracts at a concentration of < 1 l M ,and found that it inhibited serotonin N-acetyltransferase activity by approximately 65%, with minimal additional effects at concentrations > 1 l M (Fig. 6C,D). Serotonin N-acetylase activity was dependent on the phase of the hair cycle being low in telogen skin, increasing during anagen to a peak at late anagen VI and decreasing during catagen (Table 2). The enzymatic activity towards tryptamine did not show clear hair cycle dependence. Thus, the ratio between enzymatic activity toward tryptamine and serotonin changed during hair cycling from approximately 4intelogenandanagenIIIto16and17inmiddleandlate anagen VI, being 14 in catagen. The same ratio was 1.5 in the ear and 1 in S91 melanoma cells (Table 3). While testing cultured cells, we noted significantly lower enzymatic activity in preparations of frozen cells as compared to fresh cells (data not shown). Serotonin and NAS metabolism in mouse skin LC/MS analysis of the reaction products of arylalkylamine/ arylamine activity in mouse corporal skin showed two metabolites with retention times of 19 min and 23.5 min, corresponding to 5-hydroxytryptophol (5HTPOL; m/z of 178 [M + H] + , calculated mass ¼ 177 Da) and 5-hydroxy- indolacetic acid (5HIAA; m/z of 198 [M + H] + ,calcula- ted mass ¼ 197 Da) (Fig. 8). Accumulation of both compounds was inhibited by the monoamine oxidase inhibitor pargyline (Fig. 8). HIOMT activity was below the level of detectability in corporal back skin at telogen, anagen VI and catagen phases of the hair cycle (not shown). However, the chromatograms of products from the HIOMT assay did show nine additional fluorescent products, apart from the NAS substrate. The pattern of expression of these products changed during progression of the hair cycle (Fig. 9). RP-HPLC separation of the reaction products of the HIOMT assay from mouse ear and Cloudman S91 mouse melanoma cells showed weak but detectable transformation of NAS to a species with a retention time identical to melatonin (Fig. 10). These results suggest that both Cloud- man melanoma cells (derived from the DBA 12J mouse) and the ears of the C57BL/6 mouse express HIOMT activity, albeit at low levels. The same activity, however, is undetectable in the corporal skin of the C57BL/6 mouse. Discussion The current study demonstrates that mouse skin and skin cells have the molecular and biochemical apparatus neces- sary to produce and metabolize serotonin and N-acetyl- serotonin. Activity of this serotoninergic system varied depending on anatomical location, phase of hair cycle and skin cell type. In this study, we show the specific expression of the tryptophan hydroxylase gene in normal and malignant mouse melanocytes, in anagen and catagen skin, and in pituitary and spleen. In telogen skin gene expression was low. Expression of the TPH gene in skin and in skin-derived normal and malignant melanocytes was accompanied by detection of TPH immunoreactive protein with its expected molecular mass of 53–55 kDa. The molecular mass of newly translated TPH is 51 kDa and it increases to 53–60 kDa after post-translational modification [14,27]. Variants of higher and lower molecular mass have also been described and are believed to represent products of enzyme turnover Fig. 6. LC/MS analysis of serotonin transformation to NAS by mouse skin. Control reaction mixture (A) contains only substrates and cofactors without addition of the extract. Experimental incubation enzyme extracts, acetyl CoA and serotonin without (B) or with (C) 1 l M of Bis. The arrow identifies m/z ¼ 219 at a retention time of 26 min, corresponding to the NAS product. The dose-dependent effect of Bis on serotonin N-acetyltransferase activity is shown in (D). 3340 A. Slominski et al.(Eur. J. Biochem. 270) Ó FEBS 2003 [28,29]. In mastocytoma cells, ubiquitination of TPH can generate species of higher molecular mass (80–93 kDa) as intermediates in a very fast turnover process driven by proteasomes, that leads to the final degradation of native TPH to species of lower molecular mass [28,29]. Such turnover process would be consistent with our detection of TPH immunoreactive species with the expected relative molecular mass (53–55 kDa) as well as species of both higher and lower relative molecular mass. Thus, the high molecular mass TPH-like species may represent ubiquiti- nated TPH, whereas the low molecular mass TPH-like species could represent degradation products. However, as alternative splicing has already been reported for the TPH gene [30,31], the possibility that part of the diversity in TPH-like immunoreactivity molecular mass may be due to translation of alternatively spliced TPH mRNAs cannot be totally excluded. The observed expression of the TPH gene in spleen and pituitary deserves further study to assess the possible production of serotonin by these organs. Our extensive molecular analyses of AANAT transcripts in the C57BL/6 mouse demonstrate genetic defects that result in the predominant transcription of aberrant isoforms encoding protein(s) without enzymatic activities (Table 1). Thus, our data support the conclusion of Roseboom et al. [26] that this species is a natural ÔknockdownÕ for the AANAT. However, because we document separately the capability of C57BL/6 mouse skin to acetylate serotonin and tryptamine, the above reactions are likely to be catalyzed by arylamine N-acetyltransferase (NAT). NAT is a cytosolic enzyme that acetylates nitrogen or oxygen atoms of aromatic amines, hydrazines and N-hydroxyl- amines, thus playing an important detoxifying role [32–35]. There are two isozymic forms of the enzyme, NAT-1 and NAT-2, encoded by different genetic loci. Each isoform has at least 15 different allelic forms. Expression of NAT-1 cDNA in mammalian or bacterial cells has demonstrated that the enzyme is capable of acetylating both endogenously derived arylalkylamines and exogenous arylamines. NAT-1 is ubiquitously expressed in different tissues including the skin, and expression of the NAT-1 gene has been demon- strated by in situ hybridization in rat skin [36–38]. Therefore, we suggest that serotonin and tryptamine acetylation by skin extracts of the C57BL/6 mouse is probably mediated by one of the allelic forms of NAT-1. Testing for the effect of Bis uncovered a dual action in the cutaneous acetylation process, e.g. significant inhibition at concentrations equal to or below 1 l M , indicative of selectivity towards arylalkylamines [39], and insensitivity to Table 2. Hair cycle dependent changes in skin serotonin acetyl trans- ferase activity. Values represent means ± SEM of two to three assays. Mouse skin Enzyme Activity pmolÆmin )1 Æmg protein )1 (Mean ± SEM) Activity Ratio (serotonin/ tryptamine) Serotonin Tryptamine Telogen 3.4 ± 0.33 0.91 ± 0.001 4 Anagen III 5.64 ± 0.55 1.57 ± 0.2 4 Anagen IV 10.52 ± 0.27 1.02 ± 0.05 10 Early anagen VI 12.48 ± 1.3 0.78 ± 0.09 16 Late anagen VI 16.75 ± 2.46 1.0 ± 0.03 17 Catagen 12.68 ± 0.16 0.91 ± 0.001 14 Ears 9.65 ± 0.51 6.1 ± 1.02 1.5 Table 3. Enzymatic activity in normal and malignant melanocytes. Values represent means ± SEM of two to three assays. bd, below detectability; na, not applicable. Cell line Enzyme Activity pmolÆmin )1 Æmg protein )1 (Mean ± SEM) Activity Ratio (serotonin/ tryptamine)Serotonin Tryptamine S91 melanoma (6) 1.3 ± 0.01 1.02 ± 0.09 1 MelA melanocytes 2.2 ± 0.48 bd na S91 melanoma (M3) 15.32 ± 0.001 18.6 ± 1.09 1 Fig. 7. Michaelis–Menten and Lineweaver–Burk (insert) plots of N-acetyltransferase activity for serotonin in mouse skin. Fig. 8. LC/MS of products of reaction mixture in which skin extract was incubated with acetyl CoA and serotonin. 5-Hydroxytryptophol (arrow pointing to m/z ¼ 178 with a retention time of 20.2 min) was identified in the reaction mixture (A) and its accumulation was inhibited by pargyline (B). HIAA (arrow pointing at m/z ¼ 192 with a retention time of 25 min) was identified in the reaction mixture (C) and its accumulation was inhibited by pargyline (D). Ó FEBS 2003 Serotoninergic system in mouse skin (Eur. J. Biochem. 270) 3341 Bis > 1 l M (less than 25% decrease in activity), suggestive of preferential activity towards arylamines [40]. These results indicate that transformation of serotonin to NAS in mouse skin extract is mediated by enzymatic activities different from AANAT. Nevertheless, in at least the DBA/ 2 J mouse strain (S91 melanoma), the reaction could still be mediated by AANAT. Thus, that cell type expressed both the correct AANAT transcript and the protein with the expected molecular mass. Therefore, we suggest that serotonin acetylation is an intrinsic property of rodent skin; moreover depending on species [41] or specific strains the reaction can either be mediated by NAT-1 or AANAT, or by both NAT-1 and AANAT. The C57BL/6 mouse strain has undetectable production of melatonin in the pineal gland, and very low-to-undetect- able concentrations in plasma [42]. This is in agreement with the genetic defect in AANAT that led Roseboom et al.[26] to postulate this mouse species as a natural melatonin ÔknockdownÕ. It must nevertheless be noted that significant production of melatonin has been reported in peripheral organs of the same species, most notably in bone marrow- derived cells [43,44]. Our own studies that document the skin capability to produce NAS raise the possibility of alternative AANAT-independent pathways to produce this obligatory precursor to melatonin in peripheral organs. While our enzymatic studies excluded corporal skin of the C57BL/6 mouse as a site of melatonin production, we did detect low HIOMT activity in mouse ears and in S91 melanoma cells. Thus, we tentatively agree with the notion that mice may produce melatonin at selected extracranial sites [43,44]. Serotonin is a potent biological agent, and as such needs tight regulation at the tissue level [16,17], provided by monoamine oxidase (MAO) pathways. MAO deaminates serotonin to 5-hydroxyindoleacetaldehyde, which is further oxidized to 5HIAA by aldehyde dehydrogenase or reduced to 5-HTPOL by aldehyde reductase [13]. Indeed, when serotonin was incubated with skin extracts, 5HIAA and 5-HTPOL were readily detected by LCMS, whereas addi- tion of the MAO inhibitor, pargyline, blocked production of these compounds. This indicates that serotonin degrada- tion in the skin includes its oxidative deamination. The H 2 O 2 produced during this reaction may also be used for the oxidation of serotonin and other indoleamines, similar to the intestinal metabolism of tyramine [45]. NAS metabolism was extensive and hair cycle-dependent in mouse back skin, producing several as yet unidentified Fig. 9. HPLC chromatograms of products of HIOMT assays in telogen (A), anagen VI (B) and catagen (C) skin. Numbers over peaks with retention times different from NAS represent unknown products of NAS changing metabolism through the different phases of the hair cycle, e.g. telogen (A), anagen VI (B) and catagen (C). Fig. 10. HPLC chromatogram shows transformation of N-acetylsero- tonin to melatonin in ear (A and B) and S91melanoma (C and D) extracts. Experimental incubation with N-acetylserotonin (A and C) and corresponding control incubation without N-acetylserotonin (B and D). The numbers represent the elution position of standards: 1, melatonin; 2, N-acetylserotonin. 3342 A. Slominski et al.(Eur. J. Biochem. 270) Ó FEBS 2003 metabolites of the indoleamine. As NAS has been shown to be a substrate for horseradish peroxidase [46] it is possible that these metabolites could be the products of NAS oxidation by skin hemoproteins. Although NAS oxidative mechanisms have not been fully elucidated, it is possible that its metabolites may include kynuramines (N 1 -acetyl- N 2 -formyl-5-methoxykynuramine and N-acetyl-5-methoxy- kynuramine), similar to the oxidation of melatonin [47,48]. To summarize, mouse skin has both the capability of producing serotonin and the machinery for its extensive metabolism. Data currently available suggest the model for the serotoninergic pathway in the C57BL/6 mouse skin that is presented in Fig. 11. It would involve stepwise transfor- mation of tryptophan to serotonin including action by tryptophan hydroxylase, serotonin metabolism by NAT-1 to NAS and further processing to unidentified products (presumably kynuramine derivatives). An alternative degra- dation pathway would include MAO with the production of 5HTPOL and 5HIAA as intermediate products. This interpretation is consistent with the work of Schallreuter et al. [49–51] showing cutaneous synthesis of tetrahydro- biopterin (a necessary cofactor for TPH) and expression of MAO-A activity, and of Debiec-Rychter et al.[36]demon- strating NAT-1 gene expression in rodent epidermis. In mice, hair growth is a complex, highly synchronized process regulating physiology and morphology of the entire skin [19]. We now add serotonin acetylation to the hair cycle phase-dependent skin functions. The constancy of tryptamine acetylation throughout the hair cycle emphasizes the selectivity of cutaneous NAT activity for serotonin. Such selectivity could have physiological and pathological significance because serotonin transformation to NAS would limit serotonin effects in the skin (pro- edema, vasodilatory, pruritogenic and proinflammatory activities). The further metabolism of NAS in hair cycle- dependent fashion implies an additional regulatory func- tion of NAS in skin physiology. In summary, we present the molecular and biochemical characterization of the apparatus producing and metabo- lizing serotonin and N-acetylserotonin in the skin of C57BL/6 mouse. We define further some of the factors determining the activity of this apparatus that include anatomical location, phase of hair cycle and skin cell type. Acknowledgements We thank Dr D. Klein from NIH for antibodies against AANAT, Bis inhibitor and constructive criticism, and Dr D. Bennett (St George’s Hospital, London, UK) and Dr V. Hearing (NIH) for immortalized mouse melanocytes (MelA). The work was supported in part by grants from the Center of Excellence for Diseases of Connective Tissue, UTHSC, and from the Center of Genomics and Bioinformatics, UTHSC, to AS. References 1. Slominski, A. & Wortsman, J. (2000) Neuroendocrinology of the skin. Endocrine Rev. 21, 457–487. 2. Bos, J.D., Ed. (1997) Skin Immune System (Sis). Cutaneous Immunology and Clinical Immunodermatology.CRCPress,Boca Raton, FL. 3. 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Schallreuter, K.U., Wood, J.M., Pittelkow, M.R., Gu ˆ tlich, M., Lemke,R.,Rodl,W.,Swanson,N.N.,Hitzemann,K.&Ziegler,I. (1994) Regulation of melanin biosynthesis in the human epidermis by tetrahydrobiopterin. Science 263, 1444–1446. 3344 A. Slominski et al.(Eur. J. Biochem. 270) Ó FEBS 2003 . heterogeneity in the cutaneous expression of elements of the serotoninergic pathway. Therefore, we have tested the expression of dif- ferent elements of the serotoninergic system in the C57BL/ 6 mouse. in skin physiology. In summary, we present the molecular and biochemical characterization of the apparatus producing and metabo- lizing serotonin and N-acetylserotonin in the skin of C57BL/6 mouse. . shown). Serotonin and NAS metabolism in mouse skin LC/MS analysis of the reaction products of arylalkylamine/ arylamine activity in mouse corporal skin showed two metabolites with retention times of 19 min