Histochem Cell Biol (2008) 129:321–329 DOI 10.1007/s00418-007-0367-6

ORIGINAL PAPER

Characterization of cationic amino acid transporters (hCATs) 1 and 2 in human skin Kristin Jaeger · Friedrich Paulsen · Johannes Wohlrab

Accepted: 2 December 2007 / Published online: 3 January 2008 © Springer-Verlag 2007

Abstract In the present study, we characterized the distribution of human cationic amino acid transporters 1 (hCAT1) and 2 (hCAT2) in healthy skin and compared it to psoriatic skin lesions by means of immunohistochemistry. Moreover, we tested the hypothesis that L-arginine and L-ornithine inXuence the expression and synthesis of hCAT1 and hCAT2 in cell culture experiments by means of realtime-PCR and Western blot. Immunohistochemical comparison between healthy and psoriatic skin revealed a decreased amount of hCAT1, especially in the stratum granulosum of psoriatic skin; the distribution pattern of hCAT2 was not signiWcantly aVected in psoriatic skin. Cell culture experiments showed that supraphysiological concentrations of 15 mM L-arginine (72 h) lead to a signiWcant increase of the hCAT1-mRNA and protein expression, whereas other concentrations had no signiWcant inXuence. In contrast, L-arginine concentrations of 2 mM led to a signiWcant increase of the hCAT2B mRNA-expression after 24 h. However, 48 and 72 h revealed no signiWcant changes and high concentrations (15 mM L-arginine) led to a signiWcant downregula-

K. Jaeger · F. Paulsen Department of Anatomy and Cell Biology, Martin-Luther-University Halle-Wittenberg, Grosse Steinstrasse 52, 06097 Halle, Germany K. Jaeger Department of Anatomy and Cell Biology, Martin-Luther-University Halle-Wittenberg, Halle, Germany J. Wohlrab (&) Department of Dermatology and Venereology, Martin-Luther-University Halle-Wittenberg, Ernst-Grube-Strasse 40, 06120 Halle, Germany e-mail: [email protected]

tion of the hCAT2B transporter over all time points analyzed. L-ornithine had no eVect on the hCAT1 expression of mRNA and protein level. On the other hand the expression of hCAT2B was signiWcantly up regulated at a 5-mM concentration of L-ornithine at all analyzed time points. Other concentrations had no eVect. For the Wrst time, the Wndings yield data about hCAT1 and hCAT2 on proteinlevel and suggest that L-arginine is a worthwhile object of studies, which investigated L-arginine as a possible therapeutic agent to reduce psoriatic symptoms. Keywords Human · Cationic amino acid transporter · Psoriasis · Native human epidermal keratinocytes

Introduction The skin is continuously exposed to an environment with potentially noxious stimuli and represents a barrier that maintains internal homeostasis (Slominski and Wortsman 2000; Slominski 2000). The disturbance of this barrier function is one pathogenetic factor, which may cause the psoriatic lesion (Lebwohl and Herrmann 2005). Psoriasis is a systemic chronic-inXammatory disease with the involvement of skin, bone and joint that aVects 1–3% of the European population. Several studies have demonstrated the pathogenetic role of genetic underpinnings, which cause an excessive growth and aberrant diVerentiation of keratinocytes as well as a complex metabolic dysfunction, triggered by the activation of cellular immune system, with T cells, dendritic cells and various immunerelated cytokines and chemokines (GriYths et al. 2006; Lowes et al. 2007). One of the connectors between inXammation and aberrant diVerentiation is L-arginine, which can be metabolized

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by two pathways. First by nitric oxide synthase (NOS) to L-citrulline and nitric oxide (NO), and second by arginase forming L-ornithine and urea (Heys et al. 1997; Jenkinson and Grigor 1994). InXammatory responses like those present in psoriasis may inXuence the balance between these pathways. In skin, urea plays a decisive role as a moisturizer in maintaining the epidermal barrier function. L-ornithine is a promoter of cell proliferation and matrix synthesis and NO is a regulator of cutaneous microcirculation (Williams-Asham and Pegg 1981; Brittenden et al. 1997). It has been demonstrated that low NO level increases the proliferation of keratinocytes, whereas a high NO level arrests cell proliferation and initiates cell diVerentiation (Krischel et al. 1998). Psoriatic keratinocytes express inducible NOS (iNOS) mRNA and protein (BruchGerharz et al. 1996; Kolb-Bachofen and Bruch-Gerharz 1999; Kolb-Bachofen et al. 1994; Sirsjö et al. 1996), potentially leading to high-output NO synthesis, which normally should stop epidermal hyperproliferation. However, this does not occur in psoriasis. A reason for this might be that in psoriasis the NOS induction is paralleled by an abnormal overexpression of arginase 1 that limits the availability of L-arginine (Schnorr et al. 2003). This would suggest an increased synthesis of urea and L-ornithine. Indeed it has been shown that L-ornithine is increased in blood plasma of patients with psoriasis (Schnorr et al. 2005). However, in contrast to expectations, urea is reduced in the stratum corneum of psoriatic patients by as much as 40% (Wellner and Wohlrab 1993). One limiting component for the NOS and arginase pathways is transport of the semi-essential amino acid Larginine into the keratinocytes. This process is mainly enabled by cationic amino acid transporters (CATs), y+ transporters. There are at least four diVerent human genes (hCAT1–4) (Deves and Boyd 1998) exhibiting diVerential expression patterns and tissue localization (Vekony et al. 2001). Mammalian cells ubiquitously and constitutively express CAT-1 (Verrey et al. 2004). By contrast, de novo expression of CAT-2B is observed in many diVerent cell types under inXammatory conditions only, providing increased L-arginine availability, in particular for iNOS (Kawahara et al. 2001). The notable exceptions to this, known so far, are hepatocytes and smooth muscle cells with constitutive expression of the splice-variant CAT2A, the amino acid from sequence of which diVers in a stretch of 42 amino acids from that of CAT-2B, resulting in ten times less substrate aYnity (Habermeier et al. 2003). The apparent impact of hCAT1 and hCAT2 on the integrity of human skin led us to a detailed analysis of their synthesis and regulation in human skin keratinocytes in the healthy state, and to an investigation of potential diVerences in the presence of psoriasis.

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Materials and methods Healthy and inXamed paraYn-embedded samples Psoriasis samples (n = 50) were collected from 50 diVerent male and female Caucasians, aged between 20 and 50 years. Patients were included if aZicted by clinically manifest psoriasis, measured by the Psoriasis Area and Severity Index (PASI). Age- and sex-matched healthy controls (n = 50) were collected from 50 persons of the general population and were included in this study if without clinical or diagnostic evidence of psoriasis or other inXamed skin disease. The study was approved by the Ethics Board of the Medical Faculty of Martin Luther University Halle-Wittenberg. Antibody generation and testing To produce antibodies to the individual hCAT proteins 1 and 2, appropriate peptides were Wrst designed and used to immunize rabbits (anti-human CAT1 and CAT2-IgG; DPC Biermann GmbH, Bad Nauheim, Germany; Biocarta, Hamburg, Germany). The generated antibodies were tested before treatment for their speciWcity. For this purpose, the respective peptides [hCAT1-peptide [C] QMLRRKVVDCSREETRSLR-30 (NP003036.1, XP 007137.1, P30825); hCAT2-peptide RDENNEDAYPDNVHAAA EEK-611 (CAT-2 BAA06271.1, D29990, 52569, CTR_HUMAN)] were diluted into diVerent concentrations in the incubation solution and tested with the hCAT antibodies. It was shown that a speciWc 70 kDa band reduction was equivalent to the increase in peptide concentration (data not shown) for hCAT1. The speciWcity of the antiserum against hCAT2 was evaluated in a same manner in human adult low-calcium hightemperature keratinocytes (data not shown). Additionally, in immunohistochemistry, the speciWc staining of hCAT1 and hCAT2 antibodies did not appear after preincubation with the respective blocking peptides (data not shown). Immunohistochemistry The immunohistochemical procedures followed the methods described in the manufacturer’s instructions (DAKO Diagnostika GmbH, Hamburg, Germany) and were performed on 7-
m paraYn-embedded tissue sections [18 (hCAT1) and 32 (hCAT2) healthy skin biopsies as well as 33 (hCAT1) and 17 (hCAT2) psoriatic skin biopsies]. In order to make the individual reactions comparable, all investigations were performed with an antibody dilution of 1:100. Two negative control sections were used in each case: one was incubated with the secondary antibody (antirabbit IgG, biotinylated, Calbiochem, La Jolla, CA, USA) only, the other with the primary antibody only. Liver tissue was included with each investigation batch as positive control for hCAT2 and as negative control for hCAT1 (Fig. 1).

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323

Morphologic analysis We tried to evaluate the slides by the use of SigmaScanPro version 4.01 software. However, the system was not able to diVerentiate between high-, medium- and low-staining intensity. So the immunohistochemical slides were evaluated regarding the reaction intensity of the antibodies in the diVerent epidermal strata and were correlated with microvascular endothelial cells (MEC), which were present in each section. A total of 50 sections from healthy skin (n = 18 for hCAT1, healthy samples 1–18, Table 1 and n = 32 for hCAT2, healthy samples 19–50, Table 1) and 50 sections from psoriatic skin (n = 33 for hCAT1, psoriatic samples 1–33, Table 1 and 17 for hCAT2, psoriatic samples 34–50, Table 1) were evaluated for each antibody. The staining intensity was scored as high (+++), medium (++), low (+) or none (¡). MEC were always scored as highstaining intensity (+++), these cells were designated as the internal standard. To semiquantitate the data, each “+” was assigned a value of 50 points. Thus, the highest score that could be obtained was 150 points for +++. All points for 50 sections in healthy subjects and all points of 50 sections in psoriatic subjects were added together, then divided by the

Table 1 Summary of the immunohistochemical staining pattern of hCAT1 in healthy skin and in psoriatic skin, and of hCAT2 in healthy skin and in psoriatic skin Layer

Weak

Medium

High

Intensity of hCAT1 in healthy skin; n = 18 Str. basale

1

9

8

lStr. spinosum

2

14

2

uStr. spinosum

2

15

1

Str. granulosum

0

0

18

Intensity of hCAT1 in psoriatic skin; n = 33 Str. basale Fig. 1 Immunohistochemical proof of hCAT1 and hCAT2. a-d Proof of hCAT1 and hCAT2-protein in healthy epidermis and corium. a Reactivity against hCAT1 is visible in all epidermal layers except stratum corneum. b Microvascular endothelial cells in the dermis react strongly positive with the antibody to hCAT1. c Reactivity against hCAT2 is somewhat weaker in comparison to hCAT1, but here as well all epidermal layers except stratum corneum (not visible) react positive with the antibody. d Microvascular endothelial cells in the dermis react positive with the antibody to hCAT2. In the left upper corner stratum basale and stratum spinosum of the epidermis are visible. e-h Comparisons of healthy (e and g) with psoriatic skin biopsies (f and h). e Reactivity of the hCAT1 antibody is as described in Fig. 2b. Dermal microvascular endothelial cells react strongly positive with the antibody to hCAT1. f The reactivity of the hCAT1 antibody is markedly reduced in psoriatic skin, especially in the stratum granulosum (arrows). g Reactivity of the hCAT2 antibody is as described in Fig. 2c. h No change is visible in the staining intensity of hCAT2 in cases of psoriasis compared with healthy skin. i Negative control for hCAT1, no reactivity of hCAT1 in human liver, j Positive control of hCAT2, positive reactivity of hCAT2 in human liver. Counterstains: hemalum (a-h). Scale bars a, c, d, e, i, j 38
m, b 21
m, f 320
m g, h 165
m

1

29

3

lStr. spinosum

21

12

0

uStr. spinosum

30

3

0

Str. granulosum

27

6

0 31

Intensity of hCAT2 in healthy skin; n = 32 Str. basale

0

1

lStr. spinosum

0

0

32

uStr. spinosum

1

1

30

Str. granulosum

0

0

32

Intensity of hCAT2 in psoriatic skin; n = 17 Str. basale

9

5

3

lStr. spinosum

13

4

0

uStr. spinosum

13

4

0

Str. granulosum

5

9

3

The numbers in the Welds are related to sections analyzed in total. For example 14 means that 14 of the 18 sections analyzed in total revealed medium staining reactivity in the lower stratum spinosum

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number of specimens analyzed. This was performed for diVerent epidermal strata that included stratum basale (StB), lower stratum spinosum (lStS), upper stratum spinosum (uStS) and stratum granulosum (StG). All slides were scored by two independent investigators. Cell culture Native human epidermal keratinocytes (NHEK) were isolated and cultivated from neonatal prepuce tissue of various donors. Culturing followed methods described by Wohlrab et al. (2002). RNA-isolation Isolation of total RNA was performed with a High Pure RNA Isolation Kit (Roche, Mannheim, Germany) following the manufacturer’s instructions. The RNA was pretreated with 10 U RNase-free DNase I to prevent DNAcontaminations. Construction of RNA standards For the construction of standard RNA, a composite primer was synthesized (Table 2, the 5⬘-SP6 RNA polymerase binding site is underlined). The forward primer contained a 5⬘-SP6 RNA polymerase binding site followed by a speciWc sequence of the appropriate gene to be ampliWed. The PCR product obtained with forward and reverse primer was gelpuriWed (QIA quick Gel Extraction Kit, Qiagen, Hilden, Germany) followed by in vitro transcription from a SP6 promoter (Roche, Mannheim, Germany). The copy-RNA was quantiWed at 260 nm and used as the standard in the quantitative RT-PCR reaction. cDNA synthesis The cDNA synthesis was performed using the Superscript II RT Kit (Invitrogen, Karlsruhe, Germany) following the manufacturer’s instructions.

Quantitative RT-PCR Assignment to the genes hCAT1 and hCAT2 was made by comparing the sequences determined with the database of the National Center of Biotechnology Information (Table 2). AmpliWcation products obtained were then eluted from an agarose gel and sequenced. To quantify the obtained PCR products using real-time PCR, NHEK were incubated with various concentrations of L-arginine and L-ornithine hydrochloride (Merck KGaA, Darmstadt, Germany) for a period of 24, 48 or 72 h, or untreated cells were used to determine the basic expression amplitude. RNA was isolated from the cells and transcribed into cDNA. Relative quantiWcation of hCAT1 and hCAT2B gene expression was performed with Rotor-Gene software, version 4.6, in the comparative quantiWcation mode. This mode allows the comparison of diVerently treated samples relative to a control sample. For this, a standard of each of the hCAT products was created via in vitro transcription, which was included in deWned concentrations in each PCR and used to calculate the products obtained for the individual samples. The second derivative of the raw data was taken to calculate the take-oV point. Based on the take-oV point and the reaction eYciency, the software calculated the relative concentration of each sample and performed a comparison with the corresponding control sample. Western blot The cells that were used for RNA analyses were also tested in Western blot analyses. The cells were cleaved by treatment for 3 min in an ultrasonic bath and boiled for 10 min at 95°C. Finally, proteins were released. Twenty micrograms each of the total protein were separated in a 10% SDS gel and transferred to nitrocellulose membranes, which were incubated for 3 h with anti-hCAT1 and hCAT2 antibodies (dilution 1:1,000) and for 1 h with a secondary antibody (anti-rabbit IgG, peroxidase-conjugated, Calbiochem, Canada). The intensity of the peroxidase-conjugated protein bands was assessed densitometrically using the IMAGE Master®VDS Software 2.0 (Pharmacia Biotech,

Table 2 GenBank accession no. and sequence of primers used for real-time PCR Product/GenBank accession no. Human CAT1/NM/003045 Human CAT2B/U76369

Strand

Sequence

Fragment size

Sense

5⬘-GATTTAGGTGACACTATAGAATACATCTGCTTCATCGCCTACTT-3⬘

228 bp

Anti-sense

5⬘-TAGCAGTCCATCCTCAGCCATG-3⬘

Sense

5⬘-GATTTAGGTGACACTATAGAATACCCCAATGCCTCGTGTAATCT-3⬘

Anti-sense

5⬘-TGCCACTGCACCCGATGATAAAGT-3⬘

120 bp

Cycle protocol: 95°C 5 min; cycle 1 (eight replicates) 95°C 15 s, 64°C 30 s, 72°C 30 s, 80°C 10 s; cycle 2 (30 replicates) 95°C 20 s, 55°C 30 s, 72°C 30 s, 80°C 15 s, 40°C 30 s. Measurement of the ampliWcates obtained was made via the intercalated substance SyberGreen every 7 s. The SP6 regions are underlined

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Freiburg, Germany). All experiments were performed independently at least three times.

325

Results hCAT1 and hCAT2 protein are produced in healthy skin

Statistical analysis The results of the experiments are reported as means and standard deviation. The statistical analysis was made by using SigmaStat software for Windows V3.00 (SPSS Inc.). Variance analysis was applied for determination of three and more samples (treatment groups) of normally-distributed populations with equal variance. Since only one factor was examined in the various mean groups, the simple variance analysis (one-way ANOVA) was used. Random samples with unknown common variance were examined. The Bonferroni t-test was performed to compare three or more groups with reference to a control. All data represent a minimum of three separate experiments performed in triplicate. A standardized t-test was used and P-values were generated to establish the signiWcance level of the data and comparisons between groups. Immunohistochemical data were analyzed using a
2 test. A P-value of <0.05 was considered statistically signiWcant. All analyses were performed using SPSS 11.0 software (SPSS, Chicago, IL, USA).

Immunoreactivity of healthy skin sections with antibodies against hCAT1 was visible as an intensive and signiWcantly raised red reaction product in the StG compared to stratum spinosum and StB in cross-sections through the epidermis (Fig. 1a). The stratum corneum revealed no reactivity with the antibody. Comparison with MEC of the subcutis (Fig. 1b) showed diVerences in the reaction intensity of the antibodies in the diVerent epidermal layers (Table 1) that were scored as described in the Sect. ”Materials and methods” with + (weak reactivity), ++ (medium reactivity) and +++ (high reactivity). DiVerences between the diVerent individuals were analyzed by this method, with the results shown in Fig. 2a for hCAT1. Reactivity of antibodies against hCAT2 (Fig. 1c, d) reactivity was evenly distributed throughout StB, lStS and uStS. Reactivity in the StG was somewhat more intensive, and was comparable to reactivity in MEC (Fig. 3a). The stratum corneum showed no reactivity. The staining of hCAT2 was very homogenous in all investigated layers (Fig. 1c).

Fig. 2 Subjective scoring of hCAT1 immunhistoreactivity in diVerent epidermal layers in comparison with microvascular endothelial cells (MEC). The scoring is based on the evaluation of diVerent healthy (n = 17; a) and psoriatic (n = 32; b) skin sections and compares diVerent epidermal strata that include stratum basale (StB), lower stratum spinosum (lStS), upper stratum spinosum (uStS) and stratum granulosum (StG)

Fig. 3 Subjective scoring of hCAT2 immunhistoreactivity in diVerent epidermal layers in comparison with microvascular endothelial cells (MEC). The scoring is based on the evaluation of diVerent healthy (n = 17; a) and psoriatic (n = 32; b) skin sections and compares diVer-

ent epidermal strata that include stratum basale (StB), lower stratum spinosum (lStS), upper stratum spinosum (uStS) and stratum granulosum (StG)

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Reduced reactivity of antibodies against hCAT1 and hCAT2 in psoriatic skin Comparison of the 18 (hCAT1) and 32 (hCAT2) healthy skin biopsies with skin biopsy specimens from 33 (hCAT1) and 17 (hCAT2) patients with psoriasis revealed synthesis of hCAT1 and hCAT2 in all epidermal layers except stratum corneum. In psoriatic lesions there was a reduction in the staining intensity visible in all layers for both hCAT1 and hCAT2 (Fig. 1f, h; Table 1) compared to healthy skin (Fig. 1e, g; Table 1). The staining of hCAT1 in healthy skin is signiWcantly higher in StG compared to the other three investigated layers, especially the StB (Fig. 2a). Conspicuously, this layer is signiWcantly less intensively colored than the StB in the psoriatic skin (Fig. 2b; Table 1). The staining intensity of hCAT2 was only slightly reduced in psoriatic skin (Fig. 3b).

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eVect after 72 h at 15 mM. The protein expression was increased about fourfold compared to untreated control of 72 h (Fig. 5a, b). The expression of hCAT2B increased signiWcantly after a 24-h incubation with 2 mM L-arginine to 150% compared to the controls (Fig. 6). However, longer incubation times and higher concentrations revealed no eVect, with the exception of 15 mM L-arginine. These concentrations revealed, for all time points analyzed, a signiWcant downregulation of the hCAT2B mRNA (Fig. 6). Application of diVerent L-ornithine concentrations showed no eVect on hCAT1 expression at mRNA level (data not shown), and protein level (data not shown). The expression of hCAT2B mRNA was upregulated at a 5-mM concentration of L-ornithine at all analyzed time points. Other concentrations had no eVect (Fig. 7).

L-Arginine

and L-ornithine regulate their own transport via feedback mechanisms

To investigate the regulation of the expression of the CAT, native keratinocytes were incubated with diVerent nontoxic concentrations of L-arginine and L-ornithine, which were speciWed using a proliferation assay kit. Real-time PCR of cultured keratinocytes revealed no signiWcant change in hCAT1 expression due to application of diVerent L-arginine concentrations within 24–48 h. However, the expression of hCAT1 increased signiWcantly by 70–120% after 72 h treatment with 15 mM L-arginine (Fig. 4). To examine the inXuence of L-arginine or L-ornithine on the protein level, a semiquantitative Western blot analysis was performed. No assessment was possible for hCAT2, since the expression of protein was very low and not detectable in the Western blot. With regard to hCAT1, L-arginine only produced an

Fig. 4 Expression and regulation of hCAT1 after L-arginine application. Real-time PCR. The expression of hCAT1 increases signiWcantly by 60% after 72 h treatment with 15 mM L-arginine. No eVect is visible after 24 and 48 h. The values are related to corresponding untreated controls (n = 3, *P < 0.05, compared to the corresponding control)

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Fig. 5 a Western blot. The Wgure shows a representative Western blot with a speciWc band for hCAT1 at 70 kDa after L-arginine application of diVerent concentrations and times. MWS (prestained protein ladder 10–180 kDa, Fermentas, Germany) b Semiquantitative assessment of hCAT1 protein expression after L-arginine stimulation (n = 3, *P < 0.5, compared to untreated controls = 0 mM). The Wndings are comparable to hCAT1 mRNA expression (Fig. 5a). Only 15 mM show eVect. The protein expression of hCAT1 increased signiWcantly by 120% after 72 h of treatment with 15 mM L-arginine. At other time points or L-arginine concentrations there is no signiWcant change observable in the hCAT1 expression. The protein expression of the hCAT1 is aligned with the housekeeping gene -actin and densitometrically evaluated

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Fig. 6 Real-time PCR of hCAT2B mRNA expression after L-arginine application. The values are related to corresponding untreated controls (n = 3, *P < 0.05, compared to the corresponding control). The expression of hCAT2B increases signiWcantly after 24 h incubation with 2 mM L-arginine to 150% compared to controls. 15 mM L-arginine application reduces hCAT2B mRNA expression signiWcantly at all time points investigated

Fig. 7 Real-time PCR of the hCAT2B mRNA expression after L-ornithine application. The values are related to corresponding untreated controls (n = 3, *P < 0.05, compared to the corresponding control). The expression of hCAT2B mRNA is signiWcantly upregulated at a 5mM concentration of L-ornithine at all analyzed time points

Discussion The two enzymes arginase 1 and iNOS are overexpressed in psoriasis in both the mRNA and protein levels (BruchGerharz et al. 2003). Arginase 1 and iNOS metabolize the cationic amino acid L-arginine. Since both enzymes use L-arginine as a substrate and are dependent on its availability, transport of L-arginine through the cell membrane is of great relevance. Arginase 1 has been shown to be upregulated by Th2-cytokines, whereas iNOS is increased by Th1 cytokines (Bruch-Gerharz et al. 2003). In the present study we investigated on protein level the involvement of the y+ transporters hCAT1 and hCAT2 in psoriasis pathogenesis. Immunohistochemical comparisons of the distribution pattern of the two hCAT proteins were carried out between healthy and psoriatic skin biopsies. We

327

were able to show a signiWcant decrease of immunoreactivity of hCAT1 protein expression in psoriatic skin, especially in the StG. Immunoreactivity with an antibody to hCAT2 had no signiWcant eVect in distribution of hCAT2 in psoriatic skin. With regard to hCAT2, a comparable Wnding was recently made at the mRNA level (Schnorr et al. 2005). However, hCAT1 is highly upregulated in lesional and nonlesional psoriatic skin at the mRNA level in comparison to healthy skin (Schnorr et al. 2005). Schnorr et al. (2005) assumed that the upregulation of hCAT1 mRNA in psoriatic skin occurs due to reduced L-arginine concentration based on increased L-arginine turnover by arginase 1 in the psoriatic skin areas (Schnorr et al. 2005). An explanation for the contrary Wndings between upregulation of hCAT1 at the mRNA-level (Schnorr et al. 2005) and downregulation at the protein level as detected in the present work may be that the turnover rate of keratinocytes is increased to such a degree in psoriasis that there is not enough time for protein maturation in adequate amounts. This would also explain the discrepancy between high arginase 1 and low urea concentrations in psoriatic skin. Moreover, Schnorr et al. (2005) found a direct connection between L-arginine and L-ornithine on the one hand and psoriasis on the other. They described blood plasma L-arginine concentrations that were signiWcantly decreased in psoriatic patients compared to healthy controls and found that, by contrast, L-ornithine was signiWcantly elevated in the plasma of these patients. To gain deeper insight into the regulation of hCAT1 and hCAT2, we analyzed the inXuence of various L-arginine and L-ornithine concentrations in cell culture experiments. The addition of L-arginine led to an increase in hCAT1 expression and protein expression and a decreased hCAT2 expression, at the same time points and at similar L-arginine concentrations. It should be stated that we were unable to realize an evaluable Western blot analysis for hCAT2 protein determination in native keratinocytes (NHEK), probably due to insuYcient protein synthesis. The increase in mRNA and protein expression of hCAT1 after stimulation with L-arginine was expected. Normally, hCAT1 is associated with an isoenzyme of NOS, the endothelial NOS (eNOS) (McDonald et al. 1997). eNOS expression can be strongly upregulated by the addition of L-arginine in vitro as well as in vivo (deNigris et al. 2003). These Wndings, as well as our own, show that the upregulation of hCAT1 by L-arginine seems to be a major mechanism providing the associated enzyme (eNOS) with substrate, thus ensuring a basic level of L-arginine supply to the cell. The observed time and concentration-dependent (15 mM) decrease of hCAT2B expression can be explained by an increase in the NO concentration, for example during inXammation, since hCAT2B is induced quickly and in a pronounced manner under inXammatory conditions (Nicholson

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et al. 2001). A proliferation assay evaluated on the basis of BrdU incorporation excluded a toxicity-dependent decrease of hCAT2B expression at 15 mM of L-arginine. L-arginine is toxic at 25 mM after 48 h incubation. Thus, a sustained NO synthesis requires hCAT2 for high arginine turnover, as shown recently for macrophages (Nicholson et al. 2001). Inactivated macrophages, hCAT1 mRNA levels decrease, whereas hCAT2 levels increase (Nicholson et al. 2001). This suggests that a hCAT2-mediated L-arginine transport might regulate the L-arginine-NO pathway (Kakuda et al. 1998). At concentrations of 2 mM L-arginine, we observed a signiWcant increase in hCAT2 mRNA expression after 24 h. Since hCAT2 is physiologically associated with a further isoenzyme of the NOS, iNOS (Nicholson et al. 2001; Manner et al. 2003), this Wnding suggests that the upregulation takes place to guarantee the highest possible availability of L-arginine as a substrate for the iNOS. At later time points the upregulation has already taken place and the cell corrects the upregulation so that no further signiWcant increase is visible and the values decline to control levels. Comparable to L-arginine, treatment of cultured keratinocytes with L-ornithine also revealed a diVerent regulation of hCAT1 and hCAT2 expression. hCAT1 is mostly unaVected by L-ornithine treatment at the mRNA level and protein level. HCAT2B increased at the mRNA level after the addition of 5 mM L-ornithine at all investigated time points, whereas other L-ornithine concentrations had no signiWcant eVect. The response of the amino acid transporter gene to L-ornithine is diVerent from that of hCAT1. Comparable results were obtained by Durante et al. (1997) who used lipopolysaccharide for stimulation. In contrast to hCAT1, which seems to be important for the basic transport of L-arginine into the cell, hCAT2 is rapidly and pronouncedly upregulated during potentially dangerous processes such as inXammation. However, it is not clear why higher supraphysiological concentrations such as 10 mM L-ornithine had no signiWcant eVect on hCAT2B mRNA expression. In conclusion, our Wndings show that hCAT1 on protein level is diVerentially regulated in keratinocytes, especially in the cases of inXammatory processes of psoriasis. Possibly this fact is a hint for the dysfunction between the metabolism and immunological regulation of keratinocytes. Observed changes in protein expression of hCATs lead to diVerences in the distribution of L-arginine in cellular subcompartments. Therefore, consumption of L-arginine by NOS increases and withdraws arginase its substrate. This steal eVect could be one reason for the deWcit of urea synthesis, which leads to dysfunctions of the epidermal barrier in combination with disturbances in keratinocyte diVerentiation. The preferred synthesis of NO by keratinocytes has an impact on the regulation of the immune system and causes proinXammatory conditions. In this context we recommend

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further studies, to Wnd out whether L-arginine should be adopted as a possible topical therapeutic treatment. Acknowledgments The authors would like to thank Dr Oliver Schnorr, Research Group Immunobiology, Heinrich Heine University of Düsseldorf and Dr Luminita Göbbel, Department of Anatomy and Cell Biology, Martin-Luther-University of Halle-Wittenberg for critical evaluation and comments on the manuscript, Michael Beall, Kiel, for editing the English and Dr Sandra Otto for her help in evaluating the staining intensity of immunostained sections.

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