ARTICLE
Auteur(s) : Yan Zheng1, Suju Luo1,
Guorong Wang2, Zhenhui Peng1,
Weihui Zeng1, Shengshun Tan1, Yanping
Xi1, Jinhua Fan1
1Department of Dermatology, Second Hospital of Xi’an
Jiaotong University, 157 Xi Wu Road, Xi’an, Shaanxi, China,
710004
2Department of Gastroenterology, People’s Hospital of
Shaanxi Province, Xi’an, Shaanxi, China
accepté le 22 Juin 2008
It is known that retinoids exercise their effects through their
nuclear receptors, and there are two classes of nuclear retinoid
receptors that have been identified: the retinoic acid receptor
(RAR) and retinoid X receptor (RXR), and they are each composed of
three different subtypes: α, β, and γ [1]. Tazarotene, as an
RARβ/γ-selective synthetic retinoid, was found to induce three
genes, namely tazarotene-induced genes (TIG) 1, 2 and 3 [2, 3].
TIG2 was first identified as a retinoid-responsive gene which was
abundantly expressed in non-lesional psoriatic skin, but at lower
levels in psoriatic lesions [3]. The expression of TIG2 was
up-regulated by RAR-specific retinoids, but is not affected by
RXR-specific retinoids or by 1,25-dihydroxyvitamin D3. TIG2 is
unique in that it is retinoid-inducible only when keratinocytes and
fibroblasts form a 3-dimensional tissue [3]. In contrast to TIG1
and TIG3, TIG2 exhibits no anti-proliferation effects, but is
implicated in the physiology of skin [3].
The TIG2-encoded protein, which has been renamed recently as
chemerin, is a ligand of Chem23, a member of a G-protein coupled
receptor family [4]. Chemerin is secreted as a precursor with a low
biological activity, called prochemerin. Prochemerin is then
cleaved by a protease, resulting in the mature protein chemerin [4,
5]. Chemerin can be found in human inflammatory fluids and
hemofiltrate. TIG2 mRNA is not only detected in skin, but also in
pancreas, liver, spleen, prostate, ovary, small intestine, and
colon [3]. Chemerin was detected in bone stromal cells after
steroid and 1,25-dihydroxy-vitamin D3 treatments [6], and in dermal
endothelial vessels of lupus erythematosus skin lesions [7]. In
addition, the activation of chemerin receptors in chemoattracting
dendritic cells, macrophages and T cells [8], results in the
release of intracellular calcium and the promotion of the
phosphorylation of the mitogen-activated protein kinases ERK.
Studies have indicated that bioactive chemerin is synthesized
during the early stage of inflammation, and serves as a bridge
between innate and adaptive immunity [9].
The retinoid tazarotene is a drug that can treat non-malignant
(psoriasis, acne), and malignant (SCC, basal cell carcinoma,
actinic keratoses) skin diseases, even though the mechanism is
poorly understood. Since TIG2 is considered to be an anti-oncogene,
we detected the expression of TIG2 in normal tissues and tissues
from patients with SCC.
Materials and methods
Reagents
A rabbit monoclonal antibody against the human chemerin was
purchased from Phoenix pharmaceuticals (Catalog No. G-002-52). The
antibody can detect both prochemerin and chemerin. The working
dilution of the antibody is 1:100. The secondary antibody is a goat
anti-rabbit IgG-HRP. An oligonucleotide probe was synthesized by
Takara. The probe was 5’ -labeled with digoxin. The probe sequence
is as following: 5’-TTCACAAGCACCCGCCCGTGCAGTGGGCCTTCCAG. The probe
sequence shares no apparent sequence similarity with other human
genes.
Tissue samples
The study comprised 22 SCC paraffins diagnosed by histology and
clinical features which include ten 1-2 grade SCC and twelve 3-4
grade SCC, as well as uninvolved skin samples adjacent to the
lesions. Among them, 13 cases were males and 9 were females; and 2
cases were 35-50 years old and 20 cases were 51-72 years old.
Normal skin samples were obtained from 19 volunteers, with 11 males
and 8 females; and one from 35-50 years old and 18 from 51-73 years
old. There was no significant difference between the two groups.
All patients and volunteers had given their consent to the study.
The Grades of SCC 1-4 are defined by increasing percentages of
cells that are undifferentiated: grade 1, < 25% of the cells are
undifferentiated; grade 2, < 50%; grade 3, < 75%; grade 4,
> 75%.
Immunohistochemistry
Tissues were fixed in 10% formalin solution and embedded by
paraffin, following a procedure described previously [10]. A
positive reaction was visualized with DAB and then counterstained
with hematoxylin. A concentration-matched goat IgG was used as the
negative control for anti-chemerin antibody.
In situ hybridization
Paraffin sections fixed in 10% formalin were investigated as
described previously using a slightly modified non-radioactive in
situ hybridization technique with the digoxigenin-labeled
oligonucleotide as a probe [11].
Statistical comparisons between the groups were performed by
using the x2 test and exact probabilities.
Results
We found that chemerin was present in all layers of epidermis in
normal skin tissues (figure 1A). In uninvolved
epidermis, there was also brown staining in all layers, while there
was no staining in some part of basal layer or in the lower layers
of stratum spinosum (figure 1B). Furthermore,
in the marginal part of SCC, chemerin was concentrated in the
granular layers to the upper or middle layers of stratum spinosum
(figure 1C). In
grades 1-2 SCC, low levels of chemerin were present around the
keratin pearls that were highly differentiated (figure 1D), whereas no
chemerin was detected in grades 3-4 SCC (figure 1E). The
distribution of mRNA is almost the same as the protein distribution
observed above (figures
2A-E).
TIG2 expression was less in SCC compared with the normal skin
and uninvolved tissues from SCC patients. The positive rates of the
TIG2 protein and mRNA in normal and uninvolved tissues were 18/19
(94.7%) and 20/22 (90.9%) respectively, whereas the positive rates
in keratin pearls in SCC were 6/22 (27.3%). The differences between
the former two and the latter were statistically significant, with
x2 values of 19.1 and 18.4 (P < 0.01),
respectively.
TIG2 expression was absent in the lower layers of the stratum
spinosum and the basal layer of the marginal part of SCC compared
with normal skin and uninvolved skin from SCC patients: the
positive rates in the basal layer of normal epidermis and
uninvolved tissues were 18/19 (94.7%) and 17/22 (77.3%), while the
positive rates in the marginal part of SCC was 2/22 (9.09%). The
differences were statistically significant with x2
values of 29.9 and 18.6 (P < 0.01). TIG2 expression
was also less in SCC 3-4 than in the marginal part of SCC. TIG2
positive rates were 18/22 (81.8%) in suprabasal layers of the
marginal part of SCC and 1/12 (8.3%) in SCC 3-4. Again, the
differences between these two types of tissues were statistically
significant (x2 = 17.0, P < 0.01).
Discussion
High levels of TIG2 transcription and translation in normal
epidermis imply that TIG2 may be involved in normal keratinocyte
differentiation [3]. In our studies, there was no protein-staining
in some parts of the basal layer in uninvolved tissues adjacent to
SCC and nor in marginal parts of SCC. There is no TIG2 expression
in the lower layers of the stratum spinosum either. These
observations support that the expression of TIG2 is associated with
terminal keratinocyte differentiation [12], and the more
differentiated the keratinocytes, the higher the levels of TIG2
expression observed, indicating that the upregulation of TIG2
expression is part of the normalization process, whereas
suppression of TIG2 expression might contribute to the abnormal
keratinocyte differentiation. Expression of TIG2 in suprabasal
layers in the marginal part of SCC and in the keratin pearls may
help to regulate normal terminal differentiation [12].
Studies indicated that if skin is affected by inflammation or
other harmful stresses, such as allergy or tumor, some kinds of
cells will be chemoattracted to the target sites, and neutrophils
in these sites have the ability to convert inactive prochemerin
into active chemerin [5]. Chemerin, a member of the
cathelicidin/cystatin family, is able to chemoattract immature
dendritic cells and macrophages [8], and the infiltration of tumors
by these cells contributed to their aggressive phenotype by
supplying different classes of factors, such as growth and
angiogenic factors and proteolytic enzymes for tumor cell
proliferation and metastasis [9].
As shown in the data above, we hypothesize that in the initial
phases of carcinoma, prochemerin converts into bioactive chemerin
through proteolytic processing under the conditions mentioned
above, following antigen-presenting cell recruitment, such as
dendritic cells and macrophages [8, 13], and then chemerin binds to
its receptors, leading to the release of intracellular calcium and
an increase of ERK phosphorylation [9]. With the release of
calcium, the keratinocyte differentiation changes, and with
phosphorylation of ERK [14], cytokines and chemokines could be
produced, such as interleukin-8 and tumor necrosis factor α, which
are produced by monocytes and also overexpressed in SCC [15]. This
cascade of events eventually leads to the development of carcinomas
[16].
Conclusion
Prochemerin in normal epidermis has an effect on normal
keratinocyte differentiation and on normal epidermal configuration.
In the early stages of SCC, various stresses lead to activation of
chemerin through proteolytic processing. The bioactive chemerin
chemoattracts antigen-presenting cells to the target sites,
resulting in immune responses and the release of cytokines and
chemokines, which promote invasive tumors. Later, the transcription
of TIG2 is inhibited, resulting in the loss of chemerin in SCC.
Acknowledgements
This work was supported by a grant from National Natural Science
Foundation of China (No. 30400387). Conflict of interest: none.
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