Original Article

Subject Category: Photobiology

Journal of Investigative Dermatology (2003) 121, 390–398; doi:10.1046/j.1523-1747.2003.12354.x

Quantitative Real-Time Reverse Transcription–Polymerase Chain Reaction Analysis of Drug Metabolizing and Cytoprotective Genes in Psoriasis and Regulation by Ultraviolet Radiation

Gillian Smith, Robert S Dawe*, Colin Clark*, Alan T Evans†, Muriel M Comrie, C Roland Wolf, James Ferguson* and Sally H Ibbotson*

  1. *Biomedical Research Center, Photobiology Unit, University of Dundee, Ninewells Hospital and Medical School, Dundee, UK
  2. †Biomedical Research Center, Department of Molecular and Cellular Pathology, University of Dundee, Ninewells Hospital and Medical School, Dundee, UK

Correspondence: Dr Sally H. Ibbotson, Photobiology Unit, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK. Email: s.h.ibbotson@dundee.ac.uk

Received 9 September 2002; Revised 17 December 2002; Accepted 5 March 2003.

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Abstract

There are unpredictable inter-individual differences in response to ultraviolet radiation, used in the treatment of psoriasis and other common skin diseases. It is therefore essential that we attempt to identify phenotypic markers that correlate with individual treatment outcomes. Exposure of human skin to ultraviolet radiation results in the generation of reactive intermediates and oxidative stress. Hepatic drug metabolizing and cytoprotective genes are induced as an adaptive response to xenobiotics and reactive intermediates; as several of these genes are present in skin, we hypothesized that their cutaneous expression and regulation may be implicated in responses to ultraviolet radiation. We used quantitative real-time reverse transcription–polymerase chain reaction to investigate interindividual differences in the cutaneous expression of a variety of drug metabolizing and cytoprotective genes, including cytochrome P450s, glutathione S-transferases and drug transporters, and investigated the regulation of gene expression by ultraviolet radiation and in lesional psoriatic skin. We confirmed significant induction of cyclooxygenase 2 (mean 3.63-fold, range 0.14–22.6, p<0.0001) by ultraviolet radiation and showed more modest (approximately2-fold) inductions of glutathione peroxidase, and novel inductions of glutathione S-transferase P1 and the drug transporter multidrug resistance associated protein-1. Glutathione S-transferase P1 (3.74-fold, 1.3–33.1, p< 0.0001) and multidrug resistance associated protein-1 (4.06-fold, 1.3–24.8, p<0.0001) were also significantly increased in psoriatic plaque, as were P450 CYP2E1 (3.64-fold, 1–28.9 p<0.0001) and heme oxygenase-1 (10.19-fold, 2.9–49.7, p<0.0001), implying a differential adaptive response to oxidant exposure in lesional psoriatic skin. We found considerable interindividual variation in constitutive gene expression and inducibility, indicating that these genes may be associated with individuality in response to ultraviolet radiation.

Keywords:

adenosine triphosphate-binding cassette transporters, cytochrome P450, gene expression profiling, reverse transcription–polymerase chain reaction, skin

Abbreviations:

COX-2, cyclooxygenase-2; CPR, NADPH cytochrome P450 reductase; GPx-1, glutathione peroxidase-1; GST, glutathione S-transferase; HO-1, heme oxygenase-1; MED, minimal erythema dose; MDR1, multidrug resistance protein-1; MPD, minimal phototoxic dose; MRP1, multidrug resistance associated protein-1; P450, cytochrome P450; ROS, reactive oxygen species

There are unpredictable interindividual differences in sensitivity and response to photo(chemo)therapy in patients with psoriasis (Green et al, 1992;Ibbotson and Farr, 1999). It is therefore essential that we attempt to identify phenotypic markers that may be associated with the response of individual patients to ultraviolet radiation (UVR), as ultimately this may enable prediction of treatment outcomes.

Hepatic drug metabolizing enzymes and cytoprotective genes are known to be important determinants of systemic drug handling and defense against reactive oxygen species (ROS), and have been extensively studied in human liver and in rodent and cell culture models. Individuality in the hepatic expression of drug metabolizing enzymes has previously been shown to contribute to interindividual differences in response to drugs, environmental toxins, and ROS (Wolf and Smith, 1999;Wolf et al, 2000). In addition to their role in classical "drug metabolism", many of these genes are regulated as an adaptive response to environmental challenge (Nebert, 2000). For example, ultraviolet radiation (UVR) and psoralen UVA (PUVA) are both known to induce oxidative stress and generate ROS (Mukhtar and Elmets, 1996) and inheritance of specific loss of function alleles of, for example, the glutathione S-transferases (GST) has been associated with UV sensitivity and increased susceptibility to skin cancer (Kerb et al, 1997,2002;Lear et al, 2000). Human skin is the largest organ of the body and the first line of defense against environmental insult. It therefore seems logical that it would be a rich source of drug metabolizing and cytoprotective genes; however, relatively little is known about the expression and regulation of these genes in human skin.

Cytochrome P450s (P450) are a multigene family of monooxygenase enzymes, which, together with their redox partner NADPH P450 reductase (CPR), catalyze the oxidative metabolism of the vast majority of drugs and xenobiotics to which we are exposed (Gibson and Skett, 2001). P450 in subfamilies CYP1 to CYP4 are responsible for the majority of foreign compound metabolism, have unique but overlapping substrate specificities and, in general, are regulated by substrate-induced activation of gene transcription (Gonzalez et al, 1993). P450 expression is highest in the liver, but there is increasing evidence that many P450s are also expressed at significant levels in extrahepatic tissues (Tanaka, 1998). There is marked interindividual variation in the expression and activity of many hepatic P450s, which has been correlated with treatment outcomes (Wolf and Smith, 1999;Wolf et al, 2000).

Cutaneous P450 expression, measured as aryl hydrocarbon hydroxylase activity, has been shown to be inducible following the topical application of crude coal tar, a rich source of polycyclic aromatic hydrocarbons, known to be potent inducers of P450 CYP1A1 (formerly aryl hydrocarbon hydroxylase) gene transcription (Bickers and Kappas, 1978;Lawrence et al, 1984). Constitutive expression of CYP1A1, and the closely related isozyme CYP1B1, in human keratinocytes was recently confirmed in a series of reverse transcription–polymerase chain reaction (reverse transcription–PCR) experiments, which also identified mRNAs encoding CYP2B6, CYP2E1, and CYP3A5, and demonstrated CYP1A1 induction following benzanthracene treatment (Baron et al, 2001). In a complementary study, mRNAs encoding P450 CYP2A6, CYP2B6, and CYP3A4 were identified in primary human keratinocytes using RNase protection analysis (Janmohamed et al, 2001). Interestingly, recent data suggest that UVB irradiation can induce the cutaneous expression of CYP1A1, CYP1B1 (Katiyar et al, 2000), and CYP4A11 (Gonzalez et al, 2001), and we have recently shown that a novel P450, CYP2S1, is expressed in human skin, and is inducible by both UVR, PUVA and topical chemicals used to treat psoriasis (Smith et al, 2003).

Solar and artificial UVR irradiation generate cytotoxic ROS (Mukhtar and Elmets, 1996). As an adaptive response to repeated UVR exposure, the cutaneous expression of antioxidant enzymes, including superoxide dismutase and catalase, is induced in order to neutralize ROS and protect the cell from oxidative stress (Mewes et al, 2001). One of the most important cellular antioxidants is glutathione, which is responsible for a number of free radical quenching reactions (Tyrrell et al, 1991) and is an essential cofactor, for example, for the antioxidant glutathione peroxidase (GPx) enzymes. Glutathione can also be conjugated to reactive electrophilic species, frequently produced in P450-mediated reactions, by GSTs. The GSTs are also a multigene family of enzymes with diverse substrate specificities (Hayes and Strange, 2000). Specific GSTs, including GSTP1 (Del Boccio et al, 1987) and GSTA4 (Desmots et al, 2001) have been identified in human skin.Table I


An additional cellular detoxification mechanism involves the active efflux of toxins from the cell, catalyzed by a family of ABC (adenosine triphosphate binding cassette) transporter proteins. The expression of several ABC transporters, including the multidrug resistance-associated protein 1 (MRP1), has recently been reported in human keratinocytes (Baron et al, 2001). Human skin is known to express a number of cytoprotective genes, including cyclooxygenase-2 (COX-2,Leong et al, 1996) and heme oxygenase-1 (HO-1,Applegate et al, 1995). COX-2, the rate-limiting enzyme in the production of prostaglandins from arachidonic acid, is inducible by a number of inflammatory stimuli, including UVR (Buckman et al, 1998), whereas HO-1 catalyzes the rate-limiting step in heme catabolism and is a key determinant of cellular iron homeostasis (Tenhunen et al, 1968). HO-1 mRNA expression is known to be induced by cellular stresses, including free radical ROS and UVA (Applegate et al, 1991).

The expression of many drug metabolizing enzymes and cytoprotective genes is subject to genetic polymorphism, resulting in a diverse population distribution of alleles, often with significantly different levels of expression and catalytic activities (Wolf and Smith, 1999;Wolf et al, 2000). For example, the GSTM1 and GSTT1 genes are deleted in approximately 50% and 20% of the Caucasian population, respectively, with a corresponding loss of mRNA and protein expression (Seidegard et al, 1988;Pemble et al, 1994). Inheritance of these "null" GST alleles has been associated with increased susceptibility to skin cancers (Lear et al, 2000) and has been proposed as a determinant of cutaneous UVR erythemal sensitivity (Kerb et al, 1997,2002). Polymorphisms in NADPH-quinone oxidoreductase (NQO1), a flavoprotein that protects against quinone-induced oxidative stress, have also been associated with susceptibility to basal cell carcinoma (Clairmont et al, 1999), suggesting that interindividual differences in cutaneous cytoprotective gene expression may be important determinants of individuality in defense against environmental toxins.

Although there is increasing evidence that skin is metabolically active, the majority of previous reports of cutaneous drug metabolizing and cytoprotective gene expression have used cell culture or animal models and have not considered gene expression in human skin in vivo. In addition, other in vivo studies of gene expression have analyzed only a limited number of genes in small numbers of subjects, making it difficult to investigate interindividual differences in expression, and to assess the relative contribution of individual genes to constitutive cutaneous expression. Based on previous observations that specific genes are inducible by UVR, we hypothesized that other cutaneous drug metabolizing and cytoprotective genes may be expressed and regulated by UVR and may be implicated in individual responses to phototherapy. Recent advances in analytical methods have allowed us to perform a detailed characterization of drug metabolizing and cytoprotective gene expression in human skin, using quantitative "Taqman" reverse transcription–PCR. We have therefore examined the constitutive expression of a variety of these genes and investigated how gene expression is regulated by UVR, PUVA, and in lesional psoriatic skin.

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Materials and Methods

Study participants

Adults with stable untreated chronic plaque psoriasis (median age 39 y (range 20–71), 21 males, eight females, skin types I (n=2), II (n=15), III (n=11), and V (n=1)), about to commence narrow band UVB (TL-01) phototherapy (n=26) or oral PUVA (n=3) and a control group of healthy volunteers (n=6, median age 41 y (range 36–54), two males, four females, skin types II (n=2), III (n=2), and IV (n=2)) were invited to participate in the study. Patients were excluded if they had received UVB, PUVA, or systemic therapy for psoriasis within 6 mo prior to recruitment. No topical anti-psoriatic therapies (other than emollients), and in particular, no tar-based preparations had been used on buttock sites in the majority of patients (21 of 29) in the week prior to recruitment. Four study participants (patients 6, 11, 14, and 15) had used Eumovate (clobetasone butyrate) (GlaxoWellcome UK Ltd, Uxbridge, UK) and an additional four participants (patients 34, 35, 41, and 43) had used Dovonex (calcipotriol) (Leo Pharma, Buckinghamshire, UK) at other body sites, but confirmed on questioning that these topical preparations had not been applied to buttock sites. We are therefore confident that these treatments are extremely unlikely to have influenced gene expression. Informed consent was obtained from all participants and the study was approved by the Tayside Committee on Medical Research Ethics. according to the Helsinki principles.

Prior to commencement of photo(chemo)therapy, irradiations were performed on photoprotected buttock sites of patients with psoriasis using either a solar simulator UVR source (150 W xenon arc lamp, 290–400 nm, 770 mW per cm2, 3.9–48 J per cm2) (n=26), or a UVA dose series (Waldmann F15W/T8, 320–400 nm, 5.7 mW per cm2, 0.5–7.9 J per cm2) 2 h after ingestion of 8-methoxypsoralen (0.6 mg per kg; Meladinine, Galderma, Buckinghamshire, UK) (n=3). At 24 h after solar simulator irradiation or 72 h after PUVA exposure, the minimal dose of radiation required to cause a just perceptible erythema (the minimal erythema dose (MED) or minimal phototoxic dose for PUVA (MPD)) was determined and biopsies performed. Full thickness (4 mm diameter) punch biopsies were taken from: (1) an irradiated site (1–4timesMED or 3–4timesMPD); (2) adjacent untreated psoriatic plaque; and (3) adjacent control site. Similarly, full thickness punch biopsies were taken from photoprotected untreated buttock sites of a control group of healthy volunteers (n=6). All biopsy samples were immediately snap frozen in liquid nitrogen and stored at –70°C prior to analysis.

RNA preparation and cDNA synthesis

Total RNA was extracted from 4 mm diameter skin punch biopsies using Qiagen RNeasy (Qiagen, Ltd, West Sussex, UK) spin columns, according to the manufacturer's instructions, with the addition of Proteinase K and DNase digestion steps. RNA concentration and purity was determined spectrophotometrically by measuring fluorescence at 260 nM and 280 nM. Total RNA (200 ng) was reverse transcribed into cDNA in a total volume of 50 muL using PE Applied Biosystems Taqman Reverse (Biosystems, Cheshire, UK) transcription reagents according to the manufacturer's instructions. One microliter, corresponding to approximately 4 ng of input RNA was used in subsequent Taqman analysis.

Taqman quantitative real-time PCR analysis

Sequence-specific primers and probes for Taqman quantitative PCR analysis of mRNA expression were designed using PE Applied Biosystems Primer Express software, according to the manufacturer's protocol (please see table published with the web version of this paper at http://www.blackwellpublishing.com/products/journals/
suppmat/JID/jid12354/jid12354sm.htm). Each assay was designed such that the probe spanned an intron/exon boundary to minimize the possibility of coamplifying genomic DNA. PCR (1times(50°C, 2 min, 95°C, 10 min), 40times(92°C, 15 s, 60°C, 1 min) was performed in the presence of 0.6timesTaqman Universal PCR Master Mix (PE Applied Biosystems), forward and reverse primers and a sequence-specific fluorescent probe. To ensure specificity of amplification, all primer sequences were designed to include a minimum of two unique nucleotides at the 3' end. Optimal probe and primer concentrations were determined for each assay to ensure maximum specificity. A panel of cDNA clones, representative of each gene of interest, was used to confirm specificity of amplification. Real-time PCR was performed on an ABI Prism 7700 Sequence Detector ("Taqman"), where fluorescent output, measured as cycle threshold (Ct), was directly proportional to input cDNA concentration. A Ct value of 40 was interpreted as absence of gene expression, whereas Ct values in the range 35 to 40 were interpreted as being at the limit of detection of the Taqman and were therefore not quantitatively analyzed. Input cDNA concentrations were normalized to 18S ribosomal RNA, using PE Applied Biosystems Ribosomal RNA control reagents. Oligonucleotide primers were synthesized by MWG Biotech (MWG Biotech, Milton Keynes, UK) and fluorescent Taqman probes by PE Applied Biosystems.

Statistical analysis

Triplicate measurements of skin mRNA expression were made from each sample, and the arithmetic mean values taken. After logarithmic transformation, within-subject differences in mRNA expression between "lesional" (irradiated or psoriatic plaque) and control skin approximated a Gaussian distribution, and the paired t test was used to compare values in lesional versus control skin. The arithmetic mean differences between lesional versus control skin log mRNA expression and their confidence intervals (CI) become, when back-transformed, a ratio of geometric mean mRNA expression values that represent a measure of average fold induction. No statistical corrections were made for multiple comparisons, but statistical significance was taken as p<0.01, and corresponding 99% CI are given. Stata (Intercooled Stata for Windows version 7.0, Stata Corp., College Station, Texas, 2002) software was used. Differences in gene expression between healthy volunteers and psoriasis patients were determined using unpaired t tests, where statistical significance was taken as p<0.05.

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Results

Constitutive cytoprotective gene expression in human skin

Quantitative real-time "Taqman" PCR analysis was used to compare the expression of the major human glutathione-dependent enzymes, P450 genes, drug transporters, and cytoprotective genes in nonlesional skin taken from photo-protected buttock sites in a representative panel of patients with psoriasis (n=10; Figure 1). GSTP1 was the most abundantly expressed of the glutathione-dependent enzymes and was expressed in all individuals (Figure 1A). In contrast, and in agreement with our genotyping analysis (data not shown), GSTM1 expression was detectable in only five of the 10 individuals examined. Similarly, although GSTT1 mRNA expression was relatively low in human skin, there was complete concordance between GSTT1 genotype and GSTT1 mRNA expression (data not shown). GSTA4 mRNA expression was consistently detected, in contrast to the alpha class GST isozymes GSTA1 and GSTA2, which were not expressed. The catalytic or "heavy" subunit of gamma-glutamyl cysteine synthetase, the rate-limiting enzyme in glutathione biosynthesis, was expressed at low levels in all individuals, whereas GPx-1 was more abundantly expressed.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Constitutive cytoprotective gene expression in human skin. Quantitative real-time reverse transcription–PCR was used to investigate the constitutive expression of a variety of (A) glutathione-dependent enzymes, (B) P450, (C) drug transporters, and (D) stress response genes in nonlesional skin, taken from untreated photoprotected buttock sites of a representative panel of the patients with psoriasis (n=10), as described in Materials and Methods. Gene expression was normalized to 18S ribosomal RNA to ensure equality of loading. All samples were analyzed in triplicate. Data are presented as mean gene expression across the panel, with the exception of GSTM1 and GSTT1 where the data represent mean expression in GSTM1 (n=5) and GSTT1 (n=8) genotype-positive individuals. The standard deviation represents the extent of interindividual variation across the panel.

Full figure and legend (19K)

CYP1B1 was the most abundantly expressed P450 in human skin (Figure 1B). CYP1A1 mRNA expression was also consistently detected, but at much lower levels than CYP1B1, whereas the closely related enzyme CYP1A2 was present only at the limit of detection. In contrast, CYP2E1 was expressed at relatively high levels. The most abundant P450 isozyme in human liver, CYP3A4, was expressed only at very low levels in human skin and at consistently lower levels than the closely related isozyme CYP3A5, as was CYP2B6, whereas CYP2A6, CYP2C8, CYP2C9, CYP2C18, and CYP2C19 were not detected. The recently described extrahepatic P450, CYP2S1, was consistently expressed, as was the P450 redox partner CPR (Smith et al, 2003).

MRP1 was the most highly expressed of the drug transporter genes analyzed (Figure 1C). In contrast, MRP2 was expressed at relatively low levels, as was the breast cancer resistance protein, the expression of which has been described in various extrahepatic tissues, including lung, placenta, and the gastrointestinal tract (Maliepaard et al, 2001), but which has not previously been shown to be expressed in human skin. Expression of the organic anion transporter protein (OATP1) was not detected, whereas the multidrug resistance protein (MDR1) was expressed in all individuals.

Constitutive expression of the cytoprotective genes, COX-2, HO-1, and NADPH-quinone reductase (NQO1) was also detected in all individuals (Figure 1D). There were marked interindividual differences in the constitutive expression of many of the genes studied, e.g., GSTP1, CYP1B1, CYP2E1, and MRP1 (Figure 1).

We selected a subset of genes, representative of the cutaneously expressed glutathione-dependent enzymes, P450s, drug transporters, and cytoprotective genes, to study the effects of UVR exposure on gene expression and to compare gene expression in nonlesional and lesional psoriatic skin. Eleven genes (GPx-1, GSTP1, GSTM1, CYP1B1, CYP2E1, CYP2S1, CYP3A5, CPR, MRP1, COX-2, and HO-1) were selected either because they were expressed at relatively high levels in human skin or because they were known to be regulated by UVR (e.g., COX-2;Leong et al, 1996).

Comparison of constitutive cytoprotective gene expression in nonlesional skin from patients with psoriasis and healthy volunteers

We compared constitutive gene expression between clinically "normal" nonlesional skin from patients with psoriasis and skin obtained from healthy volunteers. There was a trend to lower gene expression consistently in nonlesional buttock skin from randomly selected psoriasis patients (n=6) than in photoprotected buttock skin from healthy volunteers (n=6, Table I), with the reduction in gene expression reaching statistical significance (p<0.05, unpaired t test) for GSTM1 (p=0.04), CYP1B1 (p=0.01), CYP2E1 (p=0.02), CYP3A5 (p=0.003), and HO-1 (p=0.02).

Regulation of cytoprotective gene expression by UVR/PUVA

We compared gene expression in skin exposed to a solar simulator at 1–4 MED UVR with untreated skin (n=26) (Figure 2). Consistent with previous reports (Buckman et al, 1998), COX-2 was markedly induced by UVR, with a ratio of geometric mean induction of 3.63 (99% CI 2.07–6.36, p<0.0001) and a maximum induction of 22.6-fold (Figure 2A). More modest inductions were seen for GSTP1 (1.90-fold, 99% CI 1.35–2.67, p<0.0001, maximum 16.25-fold, Figure 2B), CYP1B1 (1.48-fold, 99% CI 1.01–2.18, p=0.009, maximum 5.41-fold, Figure 2C), CYP2E1 (1.13-fold, 99% CI 0.76–1.66, p=0.40, maximum 5.13-fold, Figure 2D), GPx-1 (1.61-fold, 99% CI 1.23–2.11, p<0.0001, maximum 4.95-fold, Figure 2E), and MRP1 (1.42-fold, 99% CI 1.03–1.96, maximum 6.59-fold, p=0.0057, Figure 2F), whereas no consistent induction was seen for CYP3A5, CPR, GSTM1, or HO-1 (data not shown). CYP2S1 was also induced by UVR (Smith et al, 2003). Interestingly, we observed induction of gene expression for CYP1B1, CYP2E1, CYP2S1, CPR, GSTP1, GPx-1, MRP1, HO-1, and COX-2 by PUVA in at least two of the three PUVA patients studied (Figure 2).

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Induction of gene expression by UVR. Changes in gene expression for: (A) COX-2; (B) GSTP1; (C) CYP1B1; (D) CYP2E1; (E) GPx-1; and (F) MRP1 in UVR irradiated skin (< 2timesMED, dark blue bars; 2–3timesMED, light blue bars; >3timesMED, green bars) compared with control skin in the same patients are shown. Expression in PUVA-treated skin is also shown (red bars). All gene expression was normalized to 18S ribosomal RNA to ensure equality of loading and all samples were analyzed in triplicate. Each bar represents data derived from a single individual.

Full figure and legend (36K)

Considerable interindividual variation in UVR inducibility was seen for all the UVR-responsive genes examined. Doses of UVR ranged from 1timesMED to 4timesMED UVR (2–3timesMED in 20 of 26 patients; Figure 2), but there was no detectable correlation between UVR dose and the degree of induction of gene expression. In an attempt to identify a subset of gene "inducers" and "non-inducers" following UVR exposure, we identified the five individuals with the highest and lowest fold induction for each gene. Individuals 8, 19, 38, 40, and 41 were the most consistent UVR "inducers", whereas individuals 5, 12, 16, 17, and 18 consistently failed to show significant gene induction by UVR (Figure 2).

Altered drug metabolizing and cytoprotective gene expression in psoriatic plaque

In order to determine whether drug metabolizing or cytoprotective gene expression was altered in lesional psoriatic skin, gene expression was compared in biopsies taken from untreated lesional psoriatic plaque and from adjacent clinically normal-appearing control skin (n=29) (Figure 3). HO-1 was consistently markedly induced in psoriatic skin (10.19-fold induction, 99% CI 6.86–15.16, p<0.0001, maximum 49.68-fold; Figure 3A) as were GSTP1 (3.74-fold, 99% CI 2.56–5.46, p<0.0001, maximum 33.11-fold; Figure 3B), CYP2E1 (3.64-fold, 99% CI 2.40–5.51, p<0.0001, maximum 28.84-fold; Figure 3C), MRP1 (4.06-fold, 99% CI 2.98–5.53, p<0.0001, maximum 24.76-fold; Figure 3D), and CPR (3.42-fold, 99% CI 2.44–4.79, p<0.0001, maximum 16.60-fold; Figure 3E). GPx-1 (1.38-fold, 99% CI 1.06–1.79, p=0.002, maximum 3.77-fold), COX-2 (1.86-fold, 99% CI 1.29–2.70, p=0.0001, maximum 12.72-fold), and GSTM1 (1.64-fold, 99% CI 1.16–2.34, p=0.0008, maximum 2.66-fold) were also significantly induced in psoriatic plaque. CYP2S1 was also markedly induced in psoriatic skin (Smith et al, 2003). In contrast, significant decreases in gene expression in psoriatic skin were observed for CYP1B1 (0.64-fold, 99% CI 0.45–0.93, p=0.0025) and CYP3A5 (0.71-fold, 99%CI 0.55–0.90, p=0.0006). Again, we found significant interindividual differences in mRNA expression in psoriatic plaques (Figure 3).

Figure 3.
Figure 3 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Induction of gene expression in psoriatic plaque. Changes in gene expression for: (A) HO-1; (B) GSTP1; (C) CYP2E1; (D) MRP1; (E) CPR; and (F) GPx-1 in lesional psoriatic skin compared with control skin in the same patients are shown. All gene expression was normalized to 18S ribosomal RNA to ensure equality of loading and all samples were analyzed in triplicate. Each bar represents data derived from a single individual.

Full figure and legend (36K)

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Discussion

In addition to its function as a physical barrier, human skin is increasingly recognized as a metabolically active organ. Skin is constantly exposed to chemical and environmental stresses, such as UVR, and it is therefore not surprising that it expresses a wide variety of drug metabolizing enzymes and cytoprotective genes, the expression of which is thought to have evolved as an adaptive response to xenobiotic challenge (Nebert, 2000). Many previous studies have shown that inherited interindividual differences in the hepatic expression of specific drug metabolizing enzymes, e.g., the P450 enzymes CYP2C9 and CYP2D6, are critical determinants of systemic drug handling (Wolf and Smith, 1999;Wolf et al, 2000) and recent evidence suggests that similar variation in the cytoprotective GST genes GSTM1 and GSTTI influences sensitivity to UVR (Kerb et al, 1997,2002).

We found marked interindividual differences in the constitutive expression of the majority of the drug metabolizing and cytoprotective genes analyzed, and identified significant differences in the expression of a number of genes in skin from healthy volunteers compared with nonlesional skin from patients with psoriasis. We additionally found marked differences in gene expression comparing nonlesional and lesional psoriatic skin in patients and interindividual differences in the cutaneous expression of a number of genes in response to UVR, supporting our hypothesis that cutaneous drug metabolizing and cytoprotective gene expression can be regulated in response to UVR and PUVA.

There have now been several reports in which P450, GST, and other cytoprotective genes have been identified in human skin (e.g.,Katiyar et al, 2000;Baron et al, 2001); however, the majority of studies considered only a limited number of genes or were performed in vitro and are therefore not necessarily representative of gene expression in vivo. A major advantage of our quantitative reverse transcription–PCR approach is that it has allowed us to compare and contrast the expression of a wide variety of genes both within and between individuals. In previous studies using northern or Western blotting techniques or immunohistochemical analysis, for example, it has not always been possible to uniquely identify individual genes, particularly when investigating the expression of specific P450s or GSTs, which are members of extensive, highly homologous gene families. The use of reverse transcription–PCR, in the presence of a fluorescent gene-specific probe, not only provides us with an extremely sensitive approach, but allows us to be confident that our analysis is isozyme specific.

Consistent with their role in cellular defense against ROS, the glutathione-dependent enzymes were the most abundantly expressed of the genes examined (Figure 1), with the pi-class GST isozyme GSTP1 expressed at the highest level. The use of gene-specific Taqman probes allowed us to identify the alpha-class enzyme expressed in human skin as GSTA4, in agreement with immunohistochemical analysis of GSTA4 expression in several extrahepatic tissues (Desmots et al, 2001). GSTA1 and GSTA2 were not constitutively expressed in human skin or inducible by UVR. As expected, we found complete concordance between lack of cutaneous expression of GSTM1 and GSTT1 and inheritance of the null GST alleles (Seidegard et al, 1988; Pemble et al, 1994).

In contrast to human liver, where the CYP3A and CYP2C enzymes are the most abundantly expressed P450 isozymes (Shimada et al, 1994), and in agreement with previous studies in other extrahepatic tissues (Spink et al, 1994;Murray et al, 1997), we found CYP1B1 to be the most abundantly expressed P450 in human skin. Contrary to the findings ofKatiyar et al (2000) andBaron et al (2001) who reported similar levels of CYP1A1 and CYP1B1 expression in human epidermal keratinocytes, we found CYP1A1 to be constitutively expressed at very low levels in human skin in vivo. Cutaneous CYP1A1 or "aryl hydrocarbon hydroxylase" has been shown in human skin and in animal models to be inducible by coal tar, a rich source of polycyclic aromatic hydrocarbons (Bickers and Kappas, 1978;Li et al, 1995). We have independently shown that topical coal tar induces CYP1A1 expression in human skin and have shown very similar effects for CYP1B1 (unpublished data). Our CYP1A1 and CYP1B1 RNA expression data suggest that previous reports of constitutive aryl hydrocarbon hydroxylase activity may in fact be more attributable to constitutive CYP1B1 rather than CYP1A1 expression. The cutaneous expression of CYP2E1 was particularly interesting, as CYP2E1 has previously been implicated in defense against ROS (Sun and Sun, 2001). CYP2E1 mRNA expression was also detected in human skin byBaron et al (2001) using reverse transcription–PCR. Consistent with our data, these authors also reported the constitutive expression of CYP2B6 and CYP3A5. Whereas we found CYP3A5 to be present in all individuals tested, CYP2B6 expression was variable and present only at the limit of detection in the majority of individuals. We recently identified the cutaneous expression of CYP2S1, a novel P450, which is predominantly expressed in extrahepatic tissues (Rylander et al, 2001) and the presence of CPR, the redox partner, and electron donor to all P450s, suggesting that human skin has the ability to support functional P450 expression (Smith et al, 2003).

Drug transporter proteins function as membrane-associated adenosine triphosphate-dependent efflux pumps, which protect the cell from xenobiotic challenge (Litman et al, 2001). Increased expression of MDR1, the gene encoding P-glycoprotein, has been associated with the development of many drug-resistant cancers through active extrusion of drug from the cell (Gottesman et al, 2002). MDR1 or P-glycoprotein expression has previously been demonstrated in human skin (Baron et al, 2001), although its function is unclear. The multidrug resistance associated protein MRP1 is involved in the active transport of glutathione conjugates (Keppler et al, 1997). Consistent with our finding that the glutathione-dependent enzymes are expressed at high levels in human skin, we also found relatively high levels of MRP1 expression.

A number of cytoprotective genes are thought to have evolved to protect the cell from oxidative challenge, following exposure to ROS (Afaq and Mukhtar, 2001). We found the constitutive expression of the cytoprotective genes COX-2, HO-1, and quinone reductase (NQO1) to be relatively low in comparison with the glutathione-dependent enzymes, P450s, and drug transporters (Figure 1).

Comparison of constitutive gene expression in photoprotected skin from healthy volunteers and patients with psoriasis produced interesting results, with a trend to reduced expression of all the genes examined in clinically unaffected skin of psoriasis patients and significant reduction in expression of a subset of genes, notably CYP1B1, CYP2E1, and CYP3A5, reduced expression of which has previously been reported in response to inflammatory mediators (Morgan, 1997,2001). Our data suggest that there may be fundamental differences in metabolizing and detoxifying capacity between patients with psoriasis and healthy volunteers, although clearly these observations will need to be substantiated in a larger study.

The effects of UVR on drug metabolizing and cytoprotective gene expression were, in general, less pronounced than the changes seen in psoriatic plaque expression (Figure 2 and Figure 3). Consistent with previous reports (Buckmann et al, 1998), we saw significant induction of COX-2 gene expression in response to UVR and PUVA, although there were marked interindividual differences in response (Figure 2). In contrast to the results ofKatiyar et al (2000) who reported UVB-mediated induction of both CYP1A1 and CYP1B1 gene expression, we found significant CYP1B1 induction in only a subset of individuals and saw no consistent induction of CYP1A1, the constitutive expression of which was seen only at the limit of detection in all individuals tested. UVR exposure induced the expression of a number of other drug metabolizing and cytoprotective genes in some individuals, including CYP2E1, GSTP1, GPx-1, and MRP1, suggesting that there may be common mechanisms through which these genes respond to oxidative stress. A number of transcription factors including activator protein-1 and nuclear factor-kappaB have been shown to mediate the effects of both inflammatory mediators and ROS on gene transcription (Bäuerle and Henkel, 1994;Karin et al, 1997). Activator protein-1 binds c-fos/c-jun heterodimers, both of which have been shown to be expressed in the skin (Welter and Eckert, 1995) and, through increased transcription of c-fos and c-jun, is known to mediate induction of the cutaneous expression of genes in response to a variety of extracellular stimuli, including UVR (Fisher et al, 1997).

The expression of many of the genes examined was markedly increased in lesional psoriatic skin (Figure 2). The greatest increase in gene expression was seen for HO-1, increased transcription of which occurs as an adaptive response to provide cellular defense against oxidative stress (Applegate et al, 1991), and GSTP1, the expression of which has previously been shown to be induced in response to cellular hyperproliferation (Satoh et al, 1985). Consistent with our observations, increased HO-1 expression has previously been reported in lesional psoriatic skin (Hanselmann et al, 2001) and GSTP1 expression has been shown to be increased in psoriatic scale (Aceto et al, 1992). Significant and previously unreported increases in gene expression in psoriatic plaques were observed for CYP2E1, CPR, and MRP1, suggesting that there is a global upregulation of drug metabolizing potential in lesional psoriatic skin. In light of these findings, it is of interest that perilesional or nonlesional skin of patients with psoriasis is more susceptible than lesional skin to irritancy following treatment with coal tar or dithranol and is more likely to develop phototherapy-induced erythema. There were clear interindividual differences in the level of constitutive expression and inducibility in psoriatic plaques of each of the enzymes analyzed. Further studies are required to determine whether these differences are also reflected in induction of protein expression and to investigate whether individuality in gene expression is of clinical relevance.

These data demonstrate, for the first time, that there are marked interindividual differences in the constitutive expression of a variety of drug metabolizing and cytoprotective enzymes in human skin, in psoriatic plaques and in response to UVR and PUVA. There is unequivocal evidence that individuality in hepatic drug metabolizing enzyme and cytoprotective gene expression is a determinant of the adaptive response to systemic drugs and environmental toxins. At present, however, we have no clinical predictors of response to phototherapy and further studies are therefore needed to investigate the potential role of these genes as phenotypic markers of treatment response.

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Acknowledgments

We would like to thank Drs Irene Man, Paula Beattie and Alyson Bryden and Staff Nurse Susan Yule for help with the clinical part of the study, Murray Wilkie for technical assistance and the Chief Scientist Office for Financial Support (Ref K/MRS/50/C2769). GS acknowledges additional financial support from the Medical Research Council (Grant reference G0000281).

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