ARTICLE
Auteur(s) :, Namiko Setsu Hironori Matsuura, Satoshi
Hirakawa, Jirô Arata, Keiji Iwatsuki
Department of dermatology, Okayama University Graduate School of
Medicine, Dentistry and Pharmaceutical Sciences, Shikata-cho 2-5-1,
Okayama 700-8558, Japan
accepté le 5 Octobre 2005
Arachidonic acid (AA) is metabolized into eicosanoids including
prostaglandins (PGs), thromboxanes, leukotrienes (LTs) and
hydroxyeicosatetraenoic acids (HETEs). These eicosanoids exert
proinflammatory and immunoregulatory actions through their effects
on blood vessels and inflammatory cells [1]. AA is metabolized via
three major biochemical pathways: the cyclooxygenase (COX) pathway
leading to PGs, prostacyclin and thromboxanes; the lipoxygenase
(LOX) pathway giving rise to various hydroperoxy and hydroxyl fatty
acids as well as LTs; and the P450-dependent epoxygenase pathway
generating epoxyeicosatrienoic acids.There are six LOX molecules in
human beings: 5-LOX, 12S-LOX, 12R-LOX, 15-LOX-1, 15-LOX-2 and
epidermal LOX type 3 [1-4]. These LOXs have been proposed to play
different contributory roles in inflammatory skin diseases, such as
psoriasis [1, 5], and disorders of keratinization, such as
non-bullous congenital ichthyosiform erythroderma [4, 6]. 5-LOX
generates LTB4 from AA to cause erythema, edema,
vasoconstriction and hyperplasia of the epidermis in the skin.
12-LOX generates 12-HETE, of which functions are similar to those
of LTB4. Several cell types were found to contain
15-LOX, which generates 15-HETE, a predominant LOX product in the
skin, lung and prostate. The function of 15-HETE remains unclear.
For a long time it was believed that there was only a single
isozyme of 15-LOX. However, in 1997 Brash et al. [3] reported the
existence of a second isozyme of 15-LOX, which is designated as
15-LOX-2 in contrast to the prototypic isozyme termed 15-LOX-1.
15-LOX-2 was cloned from human hair roots [3], and 15-LOX-2 mRNA
was found to be expressed in epithelial tissues including skin,
cornea, lung, and prostate [7, 8].The level of 15-HETE, a 15-LOX
product, is increased in the sputum of asthma patients [9], the
skin of psoriasis patients [5], and interferon (IFN)-γ–treated
normal human epidermal keratinocytes (NHEKs) [10]. Furthermore, the
level of 15-LOX-2 is substantially or completely reduced in the
majority of prostate adenocarcinomas [11] and sebaceous neoplasms
of the skin [12]. These findings indicate that 15-LOX and 15-HETE
play an important role in the pathophysiology of inflammation and
tumorigenesis in the skin. However, it is unclear whether
pro-inflammatory cytokines, such as IFN-γ, induce the expression of
either of the 15-LOX isozymes in NHEKs. The present study examines
the effect of pro-inflammatory cytokines, such as IFN-γ, on 15-LOX
isozyme expression in cultured NHEKs. In addition, the pattern of
15-LOX-2 expression in psoriatic skin, actinic keratosis and
squamous cell carcinoma is also examined to determine the role of
15-LOX in these skin diseases.
Materials and methods
Cell culture and stimulation with IFN-γ
NHEKs from human foreskins were purchased from Toyobo (Tokyo,
Japan) and maintained in keratinocyte growth medium (KGM,
Clonetics, San Diego, CA, USA) supplemented with human recombinant
epidermal growth factor (0.1 ng/ml), bovine pituitary extract,
insulin (5 μg/ml), hydrocortisone (0.5 μg/ml), and
gentamycin/amphotericin B (50 μg/ml and 50 ng/ml, respectively) at
37° C in 5% CO2. An immortalized, non-tumorigenic
human epidermal keratinocyte cell line, HaCaT, was kindly given by
Dr. Fusenig (German Cancer Center, Heiderberg, Germany). A squamous
carcinoma cell line, A431, was purchased from American Type Culture
Collection. These cell lines were also maintained in KGM. After the
cells reached approximately 50% confluence in 150 mm culture
dishes, they were washed twice in phosphate buffer saline (PBS) and
treated with or without IFN-γ (200 units/ml) (R&D, Minneapolis,
MN, USA).
Preparation of cDNA probes and Northern blot analysis
Total RNA was isolated from cultured NHEK cells using Trizol
reagent (Invitrogen) according to the manufacturer’s instructions.
The human cDNA fragments for 15-LOX-2 were amplified by reverse
transcription polymerase chain reaction (RT-PCR). The PCR products
were ligated into the pCR-Script Amp SK (+) cloning vector
(Stratagene, CA, USA), and both DNA strands were sequenced with the
ABI Big Dye terminator cycle sequencing kit (Perkin Elmer, CA,
USA). The cloned cDNA fragments corresponded to nucleotides (nt)
78-529 of human 15-LOX-2 (GenBank accession No.U78294). All probes
were gel-purified and labeled with [α-32P]dCTP (3,000 Ci/mmol,
Amersham Pharmacia Biotech) using random primers. For Northern blot
analyses, RNA (10 μg) was electrophoretically separated through a
1.0% denaturing agarose-formaldehyde gel, transferred to Hybond-N+
membrane (Amersham Pharmacia, Uppsala, Sweden), UV cross-linked,
and hybridized in QuikHyb hybridization solution (Stratagene, CA,
USA) for 1 hour at 68 ˚C. Blots were washed in 0.1 × SSC/0.1%
SDS at 60 ˚C and exposed to an X-ray film at
– 80 ˚C.
In situ hybridization
Plasmids encoding human 15-LOX-2 (as prepared above) were used to
generate cRNA probes. The labeling reaction was performed according
to the manufacturer’s instructions (Roche). Briefly, the plasmids
were linearized with the appropriate restriction enzymes, treated
with proteinase K (Sigma), extracted with phenol/chloroform,
precipitated, and labeled by in vitro transcription with a
digoxigenin (DIG) RNA labeling kit (Roche) using SP6, T3, or T7 RNA
polymerases to generate cRNA anti-sense probes. In control
experiments, an excess amount of non-labeled anti-sense RNA
corresponding to the respective cDNA was added to the mixture.
Lesional and nonlesional psoriatic skin specimens were derived from
biopsies obtained for diagnostic or therapeutic purposes from three
untreated patients with plaque-type psoriasis. All patients gave
informed consent. Tissue samples were removed and frozen quickly.
Fresh frozen 5 μm sections were prepared, treated with
1 μg/ml of proteinase K (Sigma), acetylated in acetic
anhydride solution, and then dehydrated. Hybridization with freshly
denatured RNA probes in 50% formamide was performed in humidified
chambers for 15 h at 50 ˚C. The sections were washed
after hybridization at 50 ˚C under highly stringent
conditions. Prior to immunodetection of the in situ hybridization
signal, the sections were incubated in blocking solution (DIG
Nucleic Acid Detection Kit; Roche). Incubation with polyclonal
sheep anti-DIG Fab fragments conjugated to alkaline phosphatase
(Roche) was performed for 15 h in humidified chambers at
4 ˚C. The sections were stained by incubation in nitroblue
tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate solution
(Roche) containing 0.1% levamisole in darkness at room temperature.
Reagents
Sheep polyclonal anti 15-LOX-1 antibodies and cDNA for 15-LOX-1
were obtained from Cayman Chemicals (Ann Arbor, MI, USA). Rabbit
polyclonal anti-15-LOX-2 antibody and 12-LOX protein, which was
used as a positive control for anti-15-LOX-1 antibody, were
purchased from Oxford Biomedical Research (Oxford, MI, USA).
Western blot analyses
Cells were washed in PBS and then collected in extraction buffer
(1.5% SDS, 0.05 M Tris HCl pH6.8, 2 mM PMSF, 1 mM EDTA, 10 μg/ml
each pepstatin A, antipain, leupeptin, chymostatin). Protein
concentrations were determined by the DC protein assay (Bio-Rad).
Proteins (25 μg) were separated by SDS-polyacrylamide gel (Daiichi
Kagaku, Tokyo, Japan) and then transferred electrophoretically to
Hybond-P membranes (Amersham Corp.). For detection of 15-LOX-1 and
15-LOX-2, anti-15-LOX-1 antibody (Cayman chemicals) and
anti-15-LOX-2 antibody (Oxford Biomedical Research) were used as
the primary antibodies. Peroxidase-conjugated anti-mouse or
anti-rabbit IgG (1:2,000 dilution, Amersham Corp.) was used as the
secondary antibody. Antibodies were diluted in PBS containing 1%
milk powder and 0.05% Tween 20 Tris buffer. Detection was performed
by chemiluminescence using the ECL-Plus system (Amersham Bio).
Immunohistochemistry
Biopsies of lesional and nonlesional psoriatic skin were obtained
(as described above) and immediately embedded in Tissue-Tec OCT
compound (Sakura, Japan) and then sectioned (5 μm thickness)
on a cryostat (Microm HM505E). Sections were fixed with acetone for
10 min at 4 ˚C. For blockage of endogenous peroxidase
activity, the sections were dipped in methanol that contained 3%
H2O2 for 20 min, immersed with 10% sheep
or rabbit serum for 30 min and then incubated overnight with
anti-15-LOX-2 antibody diluted in PBS. The sections were washed and
stained with the DAKO LSAB2 kit, according to the manufacturer’s
instructions.
Formalin-fixed, paraffin-embedded samples of skin tumors,
including actinic keratosis (n = 3), and noninvasive and invasive
squamous cell carcinoma (n = 5), were sectioned to 4-μm thickness
and deparaffinized. After antigen retrieval was performed by
treating sections with instant retrieval solution (Mitsubishi
Kagaku Iatron, Japan) according to the manufacturer’s protocol,
immunostaining was performed as described above.
Results
15-LOX isozyme expression in NHEK cells treated with IFN-γ
In a previous study, we demonstrated that IFN-γ induces 15-HETE
production in cultured NHEKs [10]. To examine whether IFN-γ induces
the expression of 15-LOX isozymes in NHEKs, cells were treated with
IFN-γ, and the levels of 15-LOX-1 and 15-LOX-2 protein and mRNA
were analyzed by Western blot and Northern blot analysis,
respectively. Exponentially growing NHEK cells expressed low levels
of 15-LOX-2 mRNA, while the expression of 15-LOX-1 mRNA was not
observed (( figure
1 )). The 15-LOX-2 mRNA transcripts reached a higher level
48 h after treatment with IFN-γ. The induction of 15-LOX-2
mRNA expression by IFN-γ was accompanied by an increase in 15-LOX-2
protein (( figure
2 )). 15-LOX-2 protein was also detected at a low level
until 12 h and then increased between 12 h and 48 h
of IFN-γ treatment in a time-dependent manner (( figure 2 )). 15-LOX-1
protein was not detected in NHEKs treated with IFN-γ. Our results
suggest that the increased production of 15-HETE in IFN-γ–treated
NHEK cells is related to the increased level of 15-LOX-2. In a
control study, 15-LOX-2 protein at 48 h in untreated NHEK
cells was slightly increased. These results indicate that 15-LOX-2
expression in NHEK cells is low and may be affected by the
conditions of cell culture, such as contact inhibition at high
density or the induction of squamous differentiation in NHEK cells.
Expression of 15-LOX-2 in HaCaT and A431 cell lines
Squamous cell carcinoma cells have been reported to show many
changes in the control of growth and differentiation; some of these
changes involve alterations in growth factors and cytokine
signaling pathways. To determine whether such changes exist in
immortalized cells or squamous cell carcinoma cell lines, we
studied the effect of IFN-γ on 15-LOX-2 expression in HaCaT and
A431 cell lines. IFN-γ did not induce 15-LOX-2 protein expression
in A431 cells; however, there was an slight increase in HaCaT cells
(( figure 3 )).
These cells were found to be resistant to the growth-inhibitory and
differentiation-inducing effects of IFN-γ [10]. These observations
demonstrate that A431 cells are refractory to the 15-LOX-2–inducing
effects of IFN-γ and that HaCaT cells have changes in IFN-γ
signaling pathways as compared with NHEK cells.
The distribution of 15-LOX-2 in non-lesional and lesional
psoriatic skin, revealed by in situ hybridization and
immunohistochemistry
To determine the localization of 15-LOX-2 in vivo, we next examined
the expression pattern of 15-LOX-2 mRNA in non-lesional and
lesional psoriatic skin by using in situ hybridization and
immunohistochemistry. In non-lesional psoriatic skin, uniform weak
15-LOX-2 in situ hybridization was observed in the basal layer and
the lower part of the spinous layer, while strong 15-LOX-2 in situ
hybridization was observed in the basal, spinous, and granular
layers of lesional psoriatic skin (( figure 4 )). We also
observed strong 15-LOX-2 in situ hybridization in the sebaceous
glands of skin (data not shown), which is consistent with a
previous report [12]. Immunohistochemistry was used to identify the
localization of 15-LOX-2 protein and confirm the results of in situ
hybridization. Positive 15-LOX-2 immunostaining was shown in
non-lesional and lesional psoriatic skin (( figure 5A-C )). Its pattern
was similar to that of in situ hybridization.
The distribution of 15-LOX-2 in actinic keratosis and squamous
cell carcinoma, as revealed by immunohistochemistry
It has been reported that immunostaining and in situ hybridization
of 15-LOX-2 were reduced according to tumor progression in
sebaceous glands and Meibomian glands [12]. Thus, we examined
whether similar findings are observed in actinic keratosis and
squamous cell carcinomas. In actinic keratosis, positive 15-LOX-2
immunostaining was noted in the basal cell layer and the spinous
layer (( figure
6A )). In contrast to the expression of 15-LOX-2 in the
basal cell layer, the expression in the spinous layer was weaker.
Weak to intermediate immunostaining was generally detected in
noninvasive and invasive squamous cell carcinomas (( figure 6B and C )).
However, we could not find a strong relationship between 15-LOX-2
immunostaining and the progression of squamous cell carcinomas;
this is unlike the previously reported association between 15-LOX-2
immunostaining and tumorigenesis in sebaceous glands and Meibomian
glands.
Discussion
IFN-γ is a pro-inflammatory cytokine that affects growth and
differentiation in cultured epidermal keratinocytes and has been
implicated in several inflammatory skin diseases, such as allergic
contact dermatitis [13] and psoriasis [14-17]. We have previously
demonstrated that IFN-γ regulates the expression of COX-2 and
causes the increased synthesis of PGE2 and 15-HETE in
NHEK cells [10], which suggests that eicosanoid pathways also play
important roles in inflammatory skin diseases including psoriasis
[1, 5] and that IFN-γ also regulates 15-LOXs as well as COX-2 in
epidermal keratinocytes. In the present study, we showed that IFN-γ
treatment induces the expression of 15-LOX-2 in NHEK cells, while
there was no induction of 15-LOX-1 expression. These findings
indicate that the increased level of 15-LOX-2, but not 15-LOX-1, is
probably responsible for the synthesis of 15-HETE in IFN-γ-treated
NHEK cells and that 15-HETE may be associated with the inflammation
induced by IFN-γ in the skin. A similar regulation of 15-LOXs by
other cytokines, interleukin (IL)-4 and IL-13, has been reported.
15-LOX-1, but not 15-LOX-2, was induced by IL-4 and IL-13 in
cultured normal human bronchial epithelial cells [9]. Taken
together, these results indicate that both 15-LOX-1 and 15-LOX-2
may be regulated by several cytokines in a different manner in each
cell type or tissue. In addition, both the kinetics and product
profiles of 15-LOX-2 differ from those of 15-LOX-1. 15-LOX-2
produces only 15-HETE, while 15-LOX-1 can generate 12-HETE as well
as 15-HETE. 15-LOX-1 is rapidly auto-inactivated. In contrast,
15-LOX-2 continues to catalyze the reaction until substrate becomes
limiting [18]. Therefore, 15-LOX-2 may play an important role in
chronic inflammation of the skin. We showed that the level of
15-LOX-2 mRNA transcripts in NHEK cells remained low until
24 h after treatment with IFN-γ when the transcripts reached a
higher level. This delay in the induction of 15-LOX-2 suggests that
these genes are regulated by IFN-γ through an indirect mechanism.
The biological role and function of 15-HETE, a metabolite of
15-LOX, have remained unclear; however, several studies demonstrate
that 15-HETE may be an inhibitor of pro-inflammatory
LTB4 generated by 5-LOX [1, 19] and an endogenous ligand
for the nuclear receptor peroxisome proliferator activated
receptor-γ that regulates lipid metabolism. Recently, 15-LOX-2 has
been considered to be a negative cell cycle regulator in normal
prostate epithelial cells and a suppressor of prostate cancer
development [20, 21]. The expression of 15-LOX-2 was observed in
colorectal carcinoma cells during apoptosis [22]. Our findings
showed the defective regulation of IFN-γin A431 and HaCaT cells, as
compared with NHEK cells. These results support the concept that
15-LOX-2 may be associated with the progression of skin cancer,
however, we could not find defective immunostaining of 15-LOX-2 in
actinic keratosis and squamous carcinomas, nor did we find
decreased 15-LOX-2 expression associated with progression of
squamous carcinomas. Finally, given the strong expression of
15-LOX-2 in lesional psoriatic skin, the roles of this enzyme and
IFN-γ in psoriasis should be considered. Previous reports have
demonstrated that IFN-γ is an important cytokine in the development
of psoriasis [14-17]. The symptoms of human psoriasis vulgaris are
improved after intralesional injection of 15-HETE [19], and
anti-hyperproliferative effects of 15-HETE in pig skin have been
observed [23]. It has been speculated that these anti-inflammatory
and anti-hyperproliferative effects on the skin are mediated by the
protein kinase C/mitogen-activated protein kinase pathways [23]. We
observed strong immunostaining of 15-LOX-2 in psoriatic skin. This
discrepancy might be explained by a loss of sensitivity to 15-HETE
or the overproduction of inactive 15-HETE in psoriatic skin.
Further studies will be needed to clarify the effect of 15-HETE in
vivo. However, the results of the present study support the idea
that there is an anti-inflammatory and anti-hyperproliferative
mechanism via 15-LOX-2 in the skin and that the mechanism may be
triggered by IFN-γ.
In summary, we demonstrated the expression of 15-LOX-2 mRNA and
protein in IFN-γ–treated NHEK cells and psoriatic skin; this
expression may be associated with the pathogenesis of psoriasis
vulgaris.
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