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Research Article Cell Biology International
10.1002/cbin.10429
Expression and Function of Fatty Acid-binding Protein 4 in
Epithelial
Cell of Uterine Endometrium
Running Title: FABP4 is necessary for endometrial cell
Qiuyuan Zhu, Yan Jin, Peng Wang, Hanzhi Wang, Bingjian Lu
Zhengping Wang, Minyue Dong
Womens Hospital, School of Medicine, Zhejiang University
Key Laboratory of Reproductive Genetics, Ministry of
Education
Key Laboratory of Womens Reproductive Health of Zhejiang
Province
Key words: biology, endometrial epithelial cell, fatty
acid-binding protein 4, function, maintenance, proliferation
Corresponding Author Minyue Dong, MD/PhD Womens Hospital, School of
Medicine, Zhejiang University Add: 1 Xueshi Road, Hangzhou,
Zhejiang Province, 310006, China Tel: (+86) 571 8999 1011(work)
Fax: (+86) 571 8706 1878 Email:[email protected] This article
has been accepted for publication and undergone full peer review
but has not been through the copyediting, typesetting, pagination
and proofreading process, which may lead to differences between
this version and the Version of Record. Please cite this article as
doi: [10.1002/cbin.10429] This article is protected by copyright.
All rights reserved Received: 21 June 2014; Revised: 27 November
2014; Accepted: 19 December 2014
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Abstract
The aims of this study were to delineate the expression of
fatty-acid binding protein
(FABP) 4 in human uterine endometrium and its function in the
regulation of
proliferation, migration and invasion of epithelial cells.
Immunohistochenistry,
immunofluorence and Western blotting were used to determine the
expression and
cellular localization of FABP4 in endometrium and endometrial
epithelial cell lines.
Interference of small ribonuclear acid (siRNA) and specific
FABP4 inhibitor were used
to inhibit FABP4. The proliferation, migration and invasion of
epithelial cells were
evaluated with CCK-8 assay, wound-healing test and transwell
analysis respectivley. We
found that FABP4 was expressed by epithelial cells of
proliferative endometrium and
epithelial and stromal cells of secrectory endometrium.
Epithelial cell lines Ishikawa and
RL-952 expressed FABP4 and this expression was decreased by
FABP4 siRNA. FABP4
siRNA and specific FABP4 inhibition significantly decreased the
proliferation, migration
and invasion of epithelial cell lines. We concluded that FABP4
is functionally expressed
in endometrial epithelium and is necessary for maintaining the
cell function of epithelial
cells of endometrium.
Key words: biology, endometrial epithelial cell, fatty
acid-binding protein 4, function,
maintenance, proliferation
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1 Introduction
Fatty acid binding protein(FABP) 4 (FABP4, also named adipocyte
FABP or aP2) is a
member of FABP superfamily consisting of 9 highly conserved
cytosolic proteins that are
abundantly expressed in a tissue-specific manner with some
overlap(Zimmerman and
Veerkamp, 2002). FABPs are capable of binding a variety of
hydrophobic ligands, such
as long-chain fatty acid, encosanoids, leukotrienes, and
postglandins, and are critical in
several cellular processes including uptake and trafficking of
fatty acid, regulation of
gene expression, as well as cell proliferation and
differentiation (Zimmerman and
Veerkamp, 2002). FABP4 has been originally identified as an
adipocyte-specific protein
and have been considered a marker of adipogenesis and is
important in the maintenance
of glucose and lipid metabolism (Kralisch and Fasshauer, 2013).
It had been
demonstrated that FABP4 is constitutively expressed in human
macrophage (Layne et al.
, 2001, Makowski et al. , 2001, Makowski et al. , 2005),
bronchial epithelial cell (Shum et
al. , 2006), endothelial cell (Elmasri et al. , 2009), skeletal
muscle (Fischer et al. , 2006),
vascular smooth muscle cell (Girona et al. , 2013),
cardiomyocyte (Lamounier-Zepter et
al. , 2009), and trophoblast (Biron-Shental et al. , 2007).
These observations indicate that
FABP4 is more widely expressed than originally thought and may
have additional
biological role in other cell types.
Tian et al (Tian et al. , 2011) described that FABP4 messenger
ribonuclear acid (mRNA)
level in human uterine endometrium was significantly higher on
day LH+7 (day of
embryonic implantation) than that on day LH+2 (day of
fertilization) and FABP4 was
highly expressed in mouse deciduas and significantly induced by
in vitro decidualization.
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To further characterize the expression and function of FABP4 in
human endometium, we
performed immunohistochemical analysis of FABP4 in human
endometrium and found
that FABP4 is constitutively expressed in epithelial cells.
Thus, we hypothesized that
FABP4 is important in maintaining the function of epithelial
cells. To verify our
hypothesis, we observed the effects of FABP4 on the
proliferation, migration and
invasion of epithelial cells of human uterine endometrium.
2 Materials and Methods
2.1 Tissue collection and Immunohistochemistry
Uterine samples were routinely fixed in formalin solution after
hysterectomy, and
endometrial tissues were grossly examined and cut by a
pathologist for
immunohistochemistry from patients with benign ovarian cyst or
uterine myoma. The
tissue splices were processed with a standard protocol by the
Leica Tissue Processor
ASP300S (Leica Biosystems, Heidelberger, Germany), and embedded
in paraffin by the
Leica Embedding Workstation EG1160 (Leica Biosystems,
Heidelberger, Germany).
The paraffin blocks were cut at 4 m with the Microm rotary
microtome X-ActCut 4050
(Microm International GmbH, Walldorf, Germany). Heamotoxylin and
Eosin staining
was performed for pathological examination with the Sakura
Tissue-Tek DRS 2000 A-
D1 Autostainer (Sakura Finetek Co., Ltd, Tokyo, Japan).
Additional 4 m sections were
stained with anti-human FABP4 antibody (Cell Signaling
Technology, USA; catalog
number: 3544) using the 2-step En Vision method according to the
manufacturers
instructions and visualized with 3-diaminobenzidine
tetrachloride (Sigma Co., St Louis,
MO, USA). The second monoclonal antibodies and the detection kit
were from DAKO
(DAKO Inc., Glostrup, Denmark). The omission of primary bodies
was used as the
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negative control. Brown cytoplasmic staining was considered
positive for FABP4. The
protocol of the current investigation was approved by the Ethics
Committee of Womens
Hospital, School of Medicine, Zhejiang University and informed
consents were obtained
from sample providers.
2.2 Cell culture and Immunofluorescence
Ishikawa cell was cultured in RPMI-1640 medium (phenol red free)
and RL952 cell in
Dulbecco's Modified Eagle Media: Nutrient Mixture F-12 (F12/DMEM
1:1; phenol red
free) supplemented with 10% fetal bovine serum (FBS) and 1%
penicillin/ streptomycin
at 37C in a humidified atmosphere of 5% CO2. For immuno-
fluorescence, the cells
were fixed with 4% formaldehyde for 15 min, washed with PBS,
incubated with 5% BSA
for 30 min at room temperature and with anti-human FABP4
antibody (Cell Signaling
Technology Co.) overnight at 4oC. The cells were incubated with
goat FITC-conjugated
anti-mouse secondary antibody for 2 h at 4oC and then incubated
with 4', 6-diamidino-2-
phenylindole (DAPI) for 2 min at room temperature, washed twice
with phosphate-
buffered solution (PBS), and observed under an inverted
fluorescence microscope.
2.3 Transfection of siRNA and functional inhibition of FABP4
Cells were transfected with FABP4 siRNA (Santa Cruz Inc., CA,
USA; catalog number:
sc-43592; concentration: 10 mol/mL) or negative control siRNA
(Santa Cruz; sc37007)
using Lipofectamine 2000 (Life Technologies Co., Taipei, Taiwan)
according to the
manufacturers protocol. The transfection medium was replaced
with complete medium 6
h after transfection, and the cells were incubated for further
experiment. To inhibit the
function of FABP4, a selective inhibitor of FABP4 (EMD Millipore
Inc., Darmstadt,
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Germany; catalog number: 341310),
((2-(5-Ethyl-3,4-diphenyl-1H-pyrazol-1-yl)(1,1-
biphenyl)-3-yl)oxy)-acetic acid, which targets fatty acid
binding pocket, was added to the
medium at the indicated concentrations for 12 hours and then the
medium was replaced.
2.4 Western blotting
Cells were collected and lyzed in 50 l cell lysis buffer
containing protease inhibitors.
The protein concentration was quantified using the BCA Protein
Kit (Applygen Inc. Ltd,
Beijing, China). The cell lysates were separated on 10% SDS-PAGE
and the proteins
were transferred to polyvinylidene difluoride (PVDF) membranes
(Millipore,
Massachusetts, USA), blocked with Tris-buffered saline (TBS) and
0.1% Tween 20
(TBS/T) containing 5% bovine serum albumin, and then incubated
with anti-human
FABP4 antibody (Cell Signaling Technology, USA; diluted at
1:1000 in TBS/T) or anti-
human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody
(Cell Signaling
Technology Co.; diluted at 1:2000 in TBS/T) ) at 4C overnight.
The membranes were
washed three times with TBS/T and then incubated with the
appropriate HRP-conjugated
secondary antibodies for 1 hour at room temperature. The protein
bands were detected by
enhanced-chemiluminescence and visualized by autoradiography.
The relative expression
of FABP4 was normalized against internal control GAPDH.
2.5 Cell proliferation assay
Cells were seeded in 96-well plates at a density of 5000
cells/well. The medium was
replaced with the corresponding serum-free medium for 24 h to
synchronize the cells, and
then the culture media was replaced with complete medium
containing the drugsat at the
indicated concentrations for 48 h. Then, 10 L CCK8 solution
(Dojindo Inc., Kumamoto,
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Japan) was added, the plates were incubated for 3 h, and
absorbance was measured at 450
nm using a MRX II microplate reader (BioTek Inc., Vermont, USA).
Cell proliferation
was calculated as the percentage of control cells.
2.6 Wound-healing assay
Cell migration was analyzed with the in vitro scratch assay.
Cells were seeded in six-well
plates at a density of 3 105 cells/well after FABP4 was
quiescence or inhibited and
cultured with medium containing 10% FBS overnight, after which,
the medium was
changed to corresponding FBS-free medium and the cells were
cultured for a further 24 h
until >90% confluence. A single scratch wound was created in
the middle of cell
monolayer by the gentle removal of attached cells with a sterile
plastic pipette tip.
Micrographs were taken with an inverted phase contrast
microscope (Olympus, Tokyo,
Japan; magnification, 40) at indicated time intervals. The ratio
of the healed area
relative to the initial wound area was calculated and the wound
area was quantified using
software Image-Pro Plus v 6.0.
2.7 Transwell analysis
After transfection with FABP-4 siRNA (10 mol/mL) or treatment
with FABP-4
inhibitor (25 mole/L), the cells were seeded at a density of
2105 cells/well in the upper
chamber of a Transwell 24-insert plate with medium. The upper
chambers were coated
with Matrigel and the lower chamber contained 10% FBS medium.
After 24 h, the
bottom of the inserts were fixed in methanol for 10 min and
stained with hematoxylin and
eosin (H&E). The cells that had invaded to the lower surface
were photographed and
counted using an inverted phase contrast microscope (40).
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2.8 Statistical analysis
The Kolmogorov-Smirnov test was used to evaluate the
distribution of data. The data
were presented in means SD of at least 3 separate experiments.
Student t-test and one-
way analysis of variance (ANOVA) followed by a Dunnetts posthoc
test were used for
the comparison of normally distributed data. SPSS statistical
package (Statistical
Analysis System, Chicago, IL, USA) was used for the statistic
analysis. Values of P
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(Figure 4B; P
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Tian et al (Tian, Zhao, 2011) described that FABP4 is expressed
in human and mouse
uterine endometrium and is critical for the decidualization.
They thought FABP4 is
present in stromal cells of deciduas but did not determine its
cellular localization. In this
study, we reveled with immunohistochemistry that FABP4 is
constitutively expressed in
the epithelial cells of endometrium at proliferative and
secretory phases while FABP4 is
positive in stromal cells of secretory phase. Basically, our
findings were in accordance
with those of Tian. We also confirmed Tians finding that FABP4
mRNA expression is
higher on day LH+7 than that on day LH+2 (data not shown). We
also observed that
FABP4 expression was up-regulated by estradiol and further
up-regulated by the
combination of estradiol and progesterone (data not shown).
Taken together, our findings
indicate FABP4 may play a role in the biological function. To
clarify whether FABP4 is
involved in the regulation of cell function of endometrial
epithelium, we further observed
the effects of silence of FABP4 and selective inhibition of
FABP4 on the proliferation,
migration and invasiveness and found FABP4 interfering or
inhibition suppressed the
proliferation, migration and invasiveness, suggesting FABP4 is
necessary for the
maintenance of cell function of endometrial epithelium.
Proliferation and migration are important cell function of
endometrial epithelium and are
important for the periodic maturation of endometrium and
menstruation. Apposition and
adhesion are initial steps of embryonic implantation occurring
on the endometrium
(Wang and Dey, 2006), and then FABP4 may have a role in
embryonic implantation and
thus involved in pregnancy loss. When the roles of FABP4 on cell
function of
endometrial epithelium is considered, our findings imply FABP4
may also participate the
development of endometrial pathogenesis, such as dysfunctional
uterine bleeding,
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hyperplasia, carcinoma, endometriosis and adenomyosis.
In summary, FABP4 is functionally expressed in human endometrial
epithelium and
necessary for the maintenance of cell function of epithelial
cells. It would be interesting
to clarify the potential roles of FABP4 in the regulation of
cell function and biological
function of endometrial epithelium, and subsequently in the
development of pathogenesis
of endometrium.
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5 References
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Fischer H, Gustafsson T, Sundberg CJ, Norrbom J, Ekman M,
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Girona J, Rosales R, Plana N, Saavedra P, Masana L, Vallve JC.
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Kralisch S, Fasshauer M. Adipocyte fatty acid binding protein: a
novel adipokine
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Drynda K, et al. Serum
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Lamounier-Zepter V, Look C, Alvarez J, Christ T, Ravens U,
Schunck WH, et al.
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Layne MD, Patel A, Chen YH, Rebel VI, Carvajal IM, Pellacani A,
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macrophage-expressed adipocyte fatty acid binding protein in the
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Makowski L, Brittingham KC, Reynolds JM, Suttles J, Hotamisligil
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5 Funding
This work was supported by Natural Scientific Foundation of
China (81170572) and
National Basic Research Program of China (2012CB944903).
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Figure Legends
Figure 1: The expression and cellular localization of FABP4 in
human uterine
endometrium. The positive staining of FABP4 (brown) was
localized to epithelial cells of
proliferative (A) and secretary (B) endometrium. FABP4 was
negative in stromal cells of
proliferative endometrium (A) but positive in stromal cells of
secretary endometrium (B).
Figure 2: FABP4 expression by cell lines of human uterine
endometrial epithelium.
Immunefluorence showed positive cytosol signal of FABP4 in
Ishikawa and RL-952 cells
(A). Western blotting revealed specific bands of FABP4 in
Ishikawa (B) and RL-952
cells (C). FABP4 siRNA significantly reduced FABP4 expression of
Ishikawa (B) and
RL-952 cells (C).
Figure 3: The effect of FABP4 siRNA and inhibition on cell
proliferation. There were
significant differences in cell proliferation among control,
negative siRNA and FABP4
siRNA (P
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experiments of Ishikawa.
Figure 5: The effect of FABP4 siRNA and inhibition on cell
invasion. There were
significant differences in the cell numbers invaded to low
chamber among control,
negative siRNA and FABP4 RNA (P
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5