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Study on the expression of RXRα in patients with psoriasis vulgaris

European Journal of Dermatology. Volume 16, Number 1, 33-8, January-February 2006, Investigative report

Summary

Author(s) : Suying Feng, Lin Lin, Qinxue Wu, Wuqin Zhou, Changgeng Shao , Institute of Dermatology, Chinese Academy of Medical Sciences, Peking Union Medical College, Nanjing 210042, China.

Summary : Retinoic acid regulates keratinocyte proliferation and differentiation – processes that are disturbed in psoriatic skin – via binding to nuclear receptors, including retinoic acid receptor (RAR-α,β,γ) and the common heterodimer partners (RXR-α,β,γ). By RT-PCR and immunohistochemistry methods, the expression of RXRα was studied in psoriatic skin and controls. The expression of RXRα was down-regulated in patients with psoriasis\; moreover, its level was related to the stage of the disease\; in the progressive stage of the disease, the level of the RXRα was lower than in the stable stage. The results suggest that retinoid signaling is abnormal in lesional psoriatic skin, RXRα expression is mainly confined to differentiated keratinocytes.

Keywords : psoriasis, RXRα

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ARTICLE

Auteur(s) : Suying Feng, Lin Lin, Qinxue Wu, Wuqin Zhou, Changgeng Shao

Institute of Dermatology, Chinese Academy of Medical Sciences, Peking Union Medical College, Nanjing 210042, China

accepté le 30 Juillet 2005

It is commonly accepted that psoriasis is a disease with abnormal cellular immunity on a background of polygenic inheritance. The disease is characterized by epidermal hyperproliferation, abnormal differentiation of keratinocytes and skin inflammation [1]. Retinoids have been shown to be highly effective in the treatment of psoriasis and mediate their biologic effects through binding to nuclear receptors, known as retinoic acid receptors (RARs) or retinoid X receptors (RXRs) [2]. In the normal epidermis, the expression of RARγ/RXRα is the most abundant [3]. Although the ligands of the retinoid receptor superfamily and its derivatives are the first-line reagent to treat psoriasis, the specific function of retinoids on psoriasis is still obscure, which holds back the development and utilization of the new drugs relating to retinoids [2].Although most of the retinoic acid receptors known so far have been recognized in normal skin, their role in the pathogenesis and treatment of skin disorders remains unclear [4]. In particular, data on the expression level and distribution of retinoic acid receptors in psoriasis are still lacking. S. Noji et al. 1989 [5], J. Reichrath et al. 1995 [6] and H. Torma et al. 2000 [7] tried to do some work in psoriasis, but the results were not entirely consistent. All of biopsies in the above studies were obtained from patients with chronic stable psoriasis.RXR can combine with retinoid receptors or other nuclear receptors (VDR, TR, ER) to form heterodimers, then bind to their respective receptors. These ligand receptor complexes recognize specific regulatory sequences in the promoter region of certain genes and control their transcription [8], so that the RXR is the most important mediator in the nuclear receptor family, and RXRα is most abundant in the epidermis. So the contents of the research we study include: 1. to explore the level of expression of RXRαmRNA in lesional epidermis from 20 cases of progressive psoriasis vulgaris (15 guttate, 5 plaque) and compare with 10 normal controls, at the same time, the level of RXRαmRNA in 15 guttate patients being compared with that in 5 plaque patients; 2. to detect the level of expression of RXRα protein in lesional epidermis from 34 cases of psoriasis vulgaris (progressive stage: 13 guttate, 5 plaque, stable stage: 6 guttate, 10 plaque) and compare with 10 normal controls, also the level of RXRα in guttate patients being compared with that in plaque patients.

Material and methods

Source of tissue

Thirty-six patients with psoriasis (22 male and 14 female, age range 15-60 years) were included in the study. Among them, there were 16 cases of stable chronic plaque psoriasis (6 guttate and 10 plaque) and 20 cases of psoriasis in the progressive stage (15 guttate and 5 plaque). Progressive stages of psoriasis should meet one of the following criteria: 1. The new psoriatic lesions still present continuously. 2. The area of lesion becomes larger and the color more bright. 3. Koebner’s phenomenon is positive. On the other hand, there are no new lesions be found in the stable stage of psoriasis. No systemic or topical psoriasis therapy (excluding emollients) was allowed for at least 3 weeks prior to the study. The patients were healthy except for the skin disease. All patients gave their written consent in a protocol approved by Ethics Committee of Peking Union Medical College. The tissue from 34 (minus 2 guttate cases in the progressive stage) was used to do immunohistochemistry assays, otherwise, 20 cases in the progressive stage were subjected to RT-PCR assay. 8mm punch biopsies were taken from the central areas of lesional skin of the patients. Normal samples were obtained from the trunks of healthy people who were undergoing cosmetic surgery.

Preparation of the epidermis

The biopsies were washed in phosphate buffered saline (PBS) and digested in 0.5% dispase solution (Roche Diagnostic, Mannheim, Germany) for 16 h ~ 18 h at 0 ~ 4 °C. Then the epidermis was mechanically separated, frozen in liquid nitrogen, and stored at – 70 °C [9, 10].

Isolation of RNA

Total RNA was extracted from the epidermal biopsies by Trizol reagent (GiBCO BRL). In short, tissue samples were homogenized in 1 ml of Trizol Reagent per 50-100 mg of tissue using a power homogenizer (polytron) and total RNA was extracted according to the rule of the Trizol. The RNA was dissolved in 30 μl RNAse-free water. The purity of RNA was evaluated by an ultraviolet spectrophotometer. The quality of RNA was proved by agarose gel electrophoresis.

RT-PCR and statistical analysis

Superscript^TM II First-Strand Synthesis System for RT-PCR (GiBCO BRL) was used. (First-strand cDNA was synthesized from 4 μg of total RNA in a 20 reaction mixture using oligo-d(D)_12-18 as primer and RnaseH⁺M-MLV reverse transcriptase). The steps included were as follows : first, prepare RNA/primer mixtures, RNA template 8 μl (4 μg, 10 mmol/l dNTP_mix 1 μl, Oligo(DT)_12-18 1 μl, incubate each sample at 65 °C for 5 min, then place on ice for at least 1 min. then prepare the reaction mixture, adding each component following the indicated order, 10 × RT buffer 2 μl, 25 mmol/l MgCl₂ 4 μl, 0.1 mol/L DTT 2 μl, RNaseOUT^TM Recombinant Rnase inhibitor 1 μl, add 9 μl of reaction mixture to each RNA/primer mixture, mix gently, incubate at 42 °C for 2 min, add 1 μL of SuperScript^TM II RT to each tube, mix, and incubate at 42 °C for 50 min, terminate the reaction at 70 °C for 15 min, chill on ice. To nick the RNA strand, the sample was incubated with 1 μl RNAse H (Gibco BRL) for 20 min at 37 °C, then conserved below – 20 °C or the PCR was carried out directly.

The PCR mixture included 10 × PCR buffer 5 μl, 25 mmol/l Mgcl₂ 3 μl, 10 mmol/l dNTP mix 1 μl, 1 μl of each primer (50 μM), cDNA 2 μl, distilled water 36.5 μl, Taq DNA-Polymerase (Perkin-Elmer, Sundbyberg, Sweden) 0.5 μl (2.5 U): Total PCR volume was 50 μl. PCR was performed on an Perkln Elmer 9600 with preheating at 95 °C for 4 min, followed by 35 cycles of 30 s at 95 °C, 30 s at 60 °C and 45 s at 72 °C followed by 7 min at 72 °C. The primer of RXRα: 5′-TGGCAAGGACCGGAACGAGAATG-3′ and 5′-GCGGCGCCTCCAGCATCTCCATA-3′, the product was 800 bp. The housekeeping gene β-actin was taken as a control. Its primer was 5’-ATCATGTTTGAGACCTTCAACA-3’ and 5-’CATCTCTTGCTCGAAGTCCA-3’and the product was 318 bp.

Quantification of RT-PCR products and statistics: PCR reactions were separated by gel electrophoresis on 1.5% agarose gels stained with 0.4 mg per ml ethidium bromide. The gels were visualized over an on-line ultraviolet light source transilluminator (Gel Doc 1000 Video documentation System, Bio-Rad, Hercules, CA). The PCR products of the expected size (800 bp for RXRα, 318 bp for β-actin) were then manually defined and the band intensity was quantified using Quantity one quantitation software (Bio-Rad). The intensities of the PCR bands of RXRα were expressed normalized to β-actin.

An independent sample t-test was performed by SPSS 11.5. A p-value below 0.05 was considered to be significant.

Immunohistochemistry and statistical analysis

The RXRα was visualized on sections. Endogenous peroxidase activity was blocked by incubation in 0.3% H₂O₂ in phosphate buffered saline (PBS) for 15 min. The section was then allowed to react with 10% normal rabbit serum for 10 min to reduce non-specific staining. Thereafter they were incubated with a rabbit polyclinic antibody (Santa Crus-55C) against the RXRα (dilution 1/500) overnight at 4 °C. Biotinylated horse antirabbit IgG (dilution 1/200; vector) was used as a secondary antibody. Finally, in a third step the sections were incubated with an avidin-biotin complex (dilution 1/50). The peroxidase reaction was developed with 3-amino-9-ethylcarbazole. The slides were then counterstained with hematoxylin, dehydrated through graded ethanols and xylene, mounted, and coverslipped. Negative controls included use of buffer alone.

All evaluations were made by the same observer (ME) on coded sections from one biopsy from each patient. According to the staining intensity and the number of the immunoreactive cells, the results were evaluated and classified as four grades. (–) absence of staining, (+) weakness of staining or the number of the cells stained was ≤ 20%, (++) medium or between 20% and 50%, (+++) strong or ≥ 50%. Three whole section of each specimen were analyzed and the mean number was calculated.

Statistical differences were evaluated by the Mann-Whitney test and performed by SPSS 11.5.

Results

Evaluation of the quality and purity of the RNA

The ratio of A260/A280 of the RNA was between 1.7 and 1.9, which indicated that the RNA was not contaminated. The result of the electrophoresis showed two bands of 18 s, 28 s (( figure 1 )).

Confirmation of the product of RCR

The product of the PCR reactions was separated by gel electrophoresis on 1.5% agarose gels stained with ethidium bromide: DNA Marker (DL2, 000, TaKaRa) as the molecular weight standard. The picture showed that the product of RXRαmRNA was 800 bp (( figure 2 )).

The mRNA expression of RXRα in lesional skin of psoriasis vulgaris

Our study using RT-PCR and normalization to β-actin expression revealed that the RXRα expression was decreased in psoriasis patients as compared to normal controls. Moreover, there was no significant difference in the RXRα expression between guttate (n = 15) and plaque (n = 5). Table 1( Table 1 ) indicates the expression of RXRα in progressive stage psoriatic skin and controls normalized to the expression of β-actin. Table 2( Table 2 ) shows the mean ± SEM in progressive stage psoriasis and controls, and the level of RXRα expressions were significantly lower in the progressive stage of psoriatic skin (p < 0.01). Table 3( Table 3 ) shows that the mean ± SEM in guttate and plaque patients, and the level of RXRα in guttate and plaque of progressive psoriasis was not different (p > 0.05)
Table 1 The expression of RXRα/βactin in 20 patients with progressive stage psoriasis and 10 controls

Guttate (n = 15)	0.112	0.123	0.211	0.033	0.203.	0.321	0.332	0.114	0.134	0.322	0.145	0.217	0.341	0.111	0.340
Plaque (n – 5)	0.112	0.210	0.233	0.127	0.211
Control (n = 10)	0.876	0.456	0.455	0.544	0.644	0.567	0.534	0.654	0.458	0.679

Table 2 The comparison of the expression of RXRα/β Actin in 20 progressive stage psoriasis with 10 controls (mean ± SEM)

Group	N	RXRα/β-Actin (mean ± SEM)
Patients	20	0.19760 ± 0.02086
Controls	10	0.58670 ± 0.04163
T-value		9.377
P-value		< 0.01

Table 3 The comparison of the expression of RXRα/β Actin of 10 guttate patients in progressive stage psoriasis with that of plague patients (mean ± SEM)

Group	N	RXRα/β-Actin (mean ± SEM)
Guttate	10	0.20393 ± 0.02680
Plaque	5	0.17860 ± 0.02459
T-value		0.516
P-value		0.612 (> 0.05)

The level of protein expression

The most prominent staining reaction for rabbit anti- RXRα antisera in all skin sections was seen in the cells of the epidermis and the epidermal appendages. RXRα immunoreactivity was demonstrable in the nuclei of all viable epidermal cell layers, with less cytoplasmic immunoreactivity. The result of RXRα expression was qualitatively consistent in all control skin biopsies (figures 3 and 4). RXRα was detected in keratinocytes of normal and psoriatic human skin, moreover, it was expressed more strongly in keratinocytes of the up per layers of the viable epidermis than in the basal layer. The expression of RXRα was down-regulated in psoriatic keratinocytes. In psoriatic patients, the level of expression of RXRα was related to the stage of the disease. In the progressive psoriasis patients, the expression of the RXRα was lower than in RXRα at a stable stage. The level of the RXRα was not related to the form of the rash: comparing guttate with plaque patients, the RXRα expression was not different (figures 5, 6, 7, 8, table 4( Table 4 )).

In the hair follicle, RXR α immunoreactivity was consistently most markedly expressed in the nuclei of the keratinocytes of the root sheath (( figure 9 )). Similarly, the nuclei of the cells of the sebaceous glands and accrine sweat gland also stained intensely (figures 10 and 11).
Table 4 The expression of RXRα in the lesional skin of psoriasis and controls. Analysis with SPSS 11.5 software (Mann-Whitney Test). The level of RXR α in lesional skin of psoriasis (n = 34) was lower than that of controls (N = 10) (p < 0.01). The level of the RXR α in the lesional skin from the progressive patients (n = 18) was lower compared with that from the stable patients (n = 16) (P < 0.05). The level of the RXRα of guttate patients is no different from that of the plaque patients (P > 0.05) in the progressive stage as well as in the stable stage (13 versus 5 in the progressiv stage, 6 versus 10 in the static stage)

Groups	n	–	+	++	+++
Progressive stage psoriasis (guttate)	13	3	9	1	0
Progressive stage psoriasis (plaque)	5	1	3	1	0
Static stage psoriasis (guttate)	6	0	2	4	0
Static stage psoriasis (plaque)	10	0	4	6	0
Controls	10	0	0	0	10

Discussion

Retinoids are thought to exert their effects on the target cells by activating the retinoid receptor. Retinoid receptors are members of the steroid-thyroid hormone superfamily [2]. Two receptor families (RARs and RXRs) have been suggested to mediate retinoid activity at the molecular level. They act as ligand-dependent transcriptional factors, and they are bound by receptor heterodimers (RXR/RAR) with a higher affinity than for individual receptors. The retinoid molecule/retinoid receptor dimmer complex activates genes that possess specific short DNA sequences in their promoter regions (RAREs and RXREs) [11]. RARs can bind both all-trans and 9-cis-retinoic acid with high affinity, while RXRs can selectively interact with 9-cis-retinoic acid. In contrast, 13-retinoic acid can show low affinity for RARs [2].

RXRα is one of the subtypes of retinoid receptors, the level of its expression in the epidermis is five times more than that of RARγ, which always forms heterodimers with RXRα (RAR γ/ RXRα) to produce effects [2]. Not only can RXR take the form of heterodimers with RAR, but also it can form heterodimers with receptors of other hormones (e.g., with the thyroid hormone receptor and vitamin D3 receptor) and take over the gene regulation [11]. So the function of RXRα in regulating the behavior of the epidermis is very important.

There are several excellent experiments to show the important function of RXRα in the development of the epidermis. For example, Mei Li et al. (2001) [12] and Mei Li et al. (2000) [13] have used pre-mediated recombination to selectively disrupt the mouse gene for RXRα in epidermal and hair follicle keratinocytes. They demonstrated that RXRα ablation results in epidermal interfollicular hyperplasia with keratinocyte hyperproliferation and aberrant terminal differentiation. Segaert et al. 2000 [14] found that proliferating keratinocytes expressed RXRα at the lowest but gradually increasing expression during squamous differentiation, whereas VDR was regulated in the opposite way. RXRα seemed to be an early differentiation marker.

We found that the level of RXRα was lower in psoriasis patients than that of controls, which was in line with the above study and consistent with the result detected by Torma et al. in 2000 [7]. It is known that psoriasis is a kind of disease that is characterized by epidermal hyperproliferation and abnormal differentiation of keratinocytes, so the lower RXRα expression often occurs in the hyperproliferative state of psoriasis, i.e. RXRα expression is mainly confined to differentiated keratinocytes.

We also found in the study that the level of the RXRα protein was different between the distinct stages of the psoriasis: the level of RXRα protein in progressive stages was lower than in stable stages. It seemed that there was a compensatory mechanism in psoriasis; in stable patients, the expression of the RXRα protein was recovered to some extent for an unknown reason. Because we did not study other special types of psoriasis and had no research on the change of the RXRα protein after the patients were exposed to the retinoic acid reagent, we have not drawn a conclusion as to whether the level of the retinoic acid receptor was related to the degree of the severity of the disease or not.

With regard to RXR gene regulation, it remains even more difficult to elucidate the mechanism because its promoter has not been cloned yet. However, it is likely that transcriptional as well as post-transcriptional mechanisms are involved.

The reduced expression of RXR in lesional skin implied a several-fold increase in the VDR: RXR ratio or RAR: RXR ratio in psoriasis, which suggests that RAR-selective agonists (Tazarotene) [15] or VDR-selective agonist (Calcipotriol) [16] may have therapeutic advantages over other retinoids used in psoriasis treatment; moreover, the level of RXR is lower in psoriasis, so the RXR ligands (Bexarotene) [17] have low activity on psoriasis, and if they were used in psoriasis, a higher dose would be indicated.

Vissers et al. (2005) [18] carried out an inventory of T-cell subsets, cells expressing NK receptors and markers for epidermal growth and differentiation in flexural and chronic plaque psoriasis, and proposed that the decreased quantity of lesional CD161+ cells in the dermis of flexural psoriatic lesions might result from chronic microbial challenge in flexural psoriasis. These authors’ experiences give us a clue. We know guttate and plaque psoriasis are two different patterns of psoriasis and maybe the expression of RXR differs. However, the results of the study revealed no difference between guttate psoriasis and plaque psoriasis. We should be cautious about the results, because the patient groups were not balanced between guttate and plaque, which could introduce bias to the conclusion.

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