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
SuperscriptTM 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
dNTPmix 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,
RNaseOUTTM 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 SuperScriptTM
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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