Expression and Function of Fatty Acid-binding Protein 4 in Epithelial
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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
Biron-Shental T, Schaiff WT, Ratajczak CK, Bildirici I, Nelson DM, Sadovsky Y.
Hypoxia regulates the expression of fatty acid-binding proteins in primary term human
trophoblasts. Am J Obstet Gynecol. 2007;197:516 e1-6.
Elmasri H, Karaaslan C, Teper Y, Ghelfi E, Weng M, Ince TA, et al. Fatty acid binding
protein 4 is a target of VEGF and a regulator of cell proliferation in endothelial cells.
FASEB J. 2009;23:3865-73.
Fischer H, Gustafsson T, Sundberg CJ, Norrbom J, Ekman M, Johansson O, et al. Fatty
acid binding protein 4 in human skeletal muscle. Biochem Biophys Res Commun.
2006;346:125-30.
Girona J, Rosales R, Plana N, Saavedra P, Masana L, Vallve JC. FABP4 induces vascular
smooth muscle cell proliferation and migration through a MAPK-dependent pathway.
PLoS One. 2013;8:e81914.
Kralisch S, Fasshauer M. Adipocyte fatty acid binding protein: a novel adipokine
involved in the pathogenesis of metabolic and vascular disease? Diabetologia.
2013;56:10-21.
Kralisch S, Stepan H, Kratzsch J, Verlohren M, Verlohren HJ, Drynda K, et al. Serum
levels of adipocyte fatty acid binding protein are increased in gestational diabetes
mellitus. Eur J Endocrinol. 2009;160:33-8.
Lamounier-Zepter V, Look C, Alvarez J, Christ T, Ravens U, Schunck WH, et al.
Adipocyte fatty acid-binding protein suppresses cardiomyocyte contraction: a new link
between obesity and heart disease. Circ Res. 2009;105:326-34.
Layne MD, Patel A, Chen YH, Rebel VI, Carvajal IM, Pellacani A, et al. Role of
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macrophage-expressed adipocyte fatty acid binding protein in the development of
accelerated atherosclerosis in hypercholesterolemic mice. FASEB J. 2001;15:2733-5.
Makowski L, Boord JB, Maeda K, Babaev VR, Uysal KT, Morgan MA, et al. Lack of
macrophage fatty-acid-binding protein aP2 protects mice deficient in apolipoprotein E
against atherosclerosis. Nat Med. 2001;7:699-705.
Makowski L, Brittingham KC, Reynolds JM, Suttles J, Hotamisligil GS. The fatty acid-
binding protein, aP2, coordinates macrophage cholesterol trafficking and inflammatory
activity. Macrophage expression of aP2 impacts peroxisome proliferator-activated
receptor gamma and IkappaB kinase activities. J Biol Chem. 2005;280:12888-95.
Shangguan X, Liu F, Wang H, He J, Dong M. Alterations in serum adipocyte fatty acid
binding protein and retinol binding protein-4 in normal pregnancy and preeclampsia. Clin
Chim Acta. 2009;407:58-61.
Shum BO, Mackay CR, Gorgun CZ, Frost MJ, Kumar RK, Hotamisligil GS, et al. The
adipocyte fatty acid-binding protein aP2 is required in allergic airway inflammation. J
Clin Invest. 2006;116:2183-92.
Tian Z, Zhao ZA, Liang XH, Zhang XH, Sha AG, Zhang ZR, et al. Expression and
function of fatty acid-binding protein 4 during mouse decidualization. Fertil Steril.
2011;95:2749-52 e1-5.
Wang H, Dey SK. Roadmap to embryo implantation: clues from mouse models. Nat Rev
Genet. 2006;7:185-99.
Zimmerman AW, Veerkamp JH. New insights into the structure and function of fatty
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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