Molecular Human Reproduction, Vol.19, No.3 pp.

160– 168, 2013

Advanced Access publication on November 25, 2012 doi:10.1093/molehr/gas055

ORIGINAL RESEARCH

Leptin receptor is induced in

endometriosis and leptin stimulates
the growth of endometriotic epithelial
cells through the JAK2/STAT3 and ERK
pathways
Hoon Kyu Oh 1, Youn Seok Choi 2, Yeong-In Yang 3,4, Ji-Hyun Kim3,4,
Peter C.K. Leung 5, and Jung-Hye Choi 3,4,*
1
Departments of Pathology, School of Medicine, Catholic University of Daegu, Daegu, Republic of Korea 2Department of Obstetrics and
Gynecology, School of Medicine, Catholic University of Daegu, Daegu, Republic of Korea 3Department of Molecular Biology, College of
Pharmacy, Kyung Hee University, Hoegi-Dong 130-701, Seoul, Republic of Korea 4Department of Life & Nanopharamceutical Science, Kyung
Hee University, Seoul, Republic of Korea 5Department of Obstetrics and Gynecology, Child and Family Research Institute, University of
British Columbia, Vancouver, British Columbia, Canada

*Correspondence address. Fax: +82-2-962-0860, E-mail: [email protected]

Submitted on August 28, 2012; resubmitted on November 8, 2012; accepted on November 17, 2012

abstract: Leptin acts as a potential growth stimulator in several normal and neoplastic cells. Recent studies have shown the presence of
increased levels of leptin in the peritoneal fluid of patients with endometriosis, implicating leptin in the pathogenesis of endometriosis.
However, the specific function of leptin in the induction of mitogenesis in endometriosis is not known. This study investigated the expression
of the leptin receptor (ObR) in endometrioma tissues and immortalized endometriotic cells, and the effect of leptin on cell growth. ObR
expression was higher in endometriomas than in the normal endometrium, and it was detected in 74% of epithelial and 30% of stromal
endometrioma tissues. In addition, human endometriotic epithelial cells (11Z and 12Z) showed a high level of ObR when compared
with endometrial cells and endometriotic stromal cells (22B). Furthermore, leptin treatment stimulated the growth of 11Z and 12Z cells,
but not that of 22B cells. Knockdown of the ObR in 11Z and 12Z cells impaired the ability of leptin to induce cell growth. Leptin
induced the activation of Janus Kinases 2 (JAK2), signal transducers and activators of transcription 3 (STAT3) and extracellular signal-regu-
lated kinase (ERK) in endometriotic epithelial cells. Moreover, pretreatment with the JAK2/STAT3 inhibitor AG490 and the ERK inhibitor
PD98059 significantly inhibited leptin-induced cell growth. The present results show that the ObR is induced in endometriosis, and that leptin
stimulates the growth of endometriotic epithelial cells through the JAK2/STAT3 and ERK pathways.
Key words: endometriosis / leptin receptor / leptin / JAK2/STAT3 / ERK

Although discrepancies exist in the measured leptin serum levels in

Introduction endometriosis patients (Matarese et al., 2000; Somigliana et al., 2006),
Endometriosis is a common chronic disease characterized by the multiple lines of evidence have suggested that leptin levels are signifi-
ectopic implantation and growth of endometrial tissue. The disorder cantly elevated in the peritoneal fluid of patients with endometriosis
is a common cause of pelvic pain, and accounts for over 20% of all (Matarese et al., 2000; Mahutte et al., 2003; Bedaiwy et al., 2006;
cases of infertility in women. Its defining feature is the presence of Barcz et al., 2008; Alviggi et al., 2009). Leptin, a product of the
endometrial implants outside the uterus, primarily on the pelvic peri- obese (ob) gene, is predominantly secreted by adipocytes and
toneum and ovaries. Accumulating evidence suggests that the concen- shows a strong positive correlation with total body fat and body
trations of various substances such as cytokines, chemokines, mass index (BMI). The effects of leptin are mediated by the transmem-
prostaglandins and growth factors in the peritoneal fluid are aberrant brane leptin receptor (ObR), which belongs to the cytokine receptor
in women with endometriosis and these molecules are associated with superfamily. In human tissues, at least four isoforms of ObR with dif-
the pathogenesis of the disease (Kyama et al., 2003). ferent COOH-terminal cytoplasmic domains exist, including a long

& The Author 2012. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
For Permissions, please email: [email protected]

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 Effect of leptin on endometriotic cell growth 161

form and a short form. The long form of ObR (ObRL) activates clas- Cell culture
sical cytokine Janus Kinases 2 (JAK2)/signal transducers and activators Immortalized human endometriotic epithelial cells (11Z and 12Z) and
of transcription 3 (STAT3) pathways (Yamashita et al., 1998) as well as stromal cells (22B) (Zeitvogel et al., 2001) and immortalized human endo-
the ras/extracellular signal-regulated kinase (ERK) (Yamashita et al., metrial surface epithelial cells (HES) and stromal cells (HESC) (Desai et al.,
1998; Catalano et al., 2004) and phosphatidyl-inositol 3′ -kinase 1994; Krikun et al., 2004) were maintained in Dulbecco’s modified Eagle’s
(PI3K)/Akt (O’Rourke et al., 2001) pathways. medium (DMEM)/F12 medium supplemented with 5% fetal bovine serum,
Although the primary function of leptin is the regulation of food 100 U/ml penicillin G and 100 mg/ml streptomycin (Life Technologies,
intake and energy consumption through its effects on the brain, Grand Island, NY, USA) in a humidified atmosphere of 5% CO2 –95%
leptin is known to play multiple roles in various cell systems, including air at 378C. The endometriotic cells were generously provided by Dr
reproduction, angiogenesis and immune homeostasis (La Cava et al., Starzinski-Powitz (Johann-Wolfgang-Goethe-Universitaet, Germany). The
endometrial cells HES, recently established by Dr Krikun (Yale University,
2004). Interestingly, recent studies have demonstrated that this
New Haven, CT, USA), and HESC were kindly provided by Dr Asgi
hormone stimulates growth, migration, invasion and angiogenesis in
Fazleabas of University of Illinois at Chicago. MCF7 cells (Korean Cell
tumor cell models, suggesting that leptin is capable of promoting an Line Bank, Seoul, Korea) were maintained in DMEM.
aggressive cancer phenotype (Lang and Ratke, 2009). The similarity
between these phenotypes and the pathogenesis of endometriosis MTT and clonogenic assay
led to the question of whether leptin could play a role in endometri-
Cell growth was estimated using the MTT (3-[4,5-dimethylthiazol-2-yl]-
osis. To address this, the expression of the ObR was examined in
2,5-dipheyl tetrazolium bromide; Sigma-Aldrich) assay. Cells were
endometrioma tissues and the potential effect of leptin on cell seeded in 96-well plates at a density of 5 × 103 cells per well and incu-
growth and its underlying molecular mechanism in immortalized endo- bated for 24 h. To examine the growth stimulatory effect of leptin, cells
metriotic cells were investigated. were treated with leptin for 48 h. On the day of collection, 50 ml of
MTT solution was added to the medium and the cells were incubated
at 378C for 4 h. The MTT-containing medium was removed and the
cells were solubilized in DMSO (100 ml) for 30 min. The absorbance at
Materials and Methods 490 nm was determined using a microplate spectrophotometer (Fisher
Scientific Ltd., Ottawa, Ontario, Canada). To determine long-term
Patient subjects effects, clonogenic assay was performed. Cells were seeded in 6-well
Ovarian endometriosis tissue samples were obtained from 44 women of plates at a cell density of 5000 cells per well and treated with leptin at
reproductive age (24– 47 years old) who underwent laparoscopic various concentrations. Cells were allowed to form colonies for 14 days,
surgery for ovarian endometriomas at the Department of Obstetrics which were counted after staining with crystal violet (Sigma). The
and Gynecology, Daegu Catholic University Medical Center in Daegu, density of colony in the well was quantified in pixels using Scion Image Soft-
Korea. The study was approved by the Institutional Review Board of ware (Scion Corp, Frederick, MD, USA).
Daegu Catholic University Medical Center. Endometriosis was classified
according to American Society for Reproductive Medicine classification. Immunoblot assay
The patients have not received any hormonal therapy before the The cells were washed twice with ice-cold phosphate buffered saline and
surgery. The mean BMI was 20.7 + 3.0 (kg/m2). Nineteen women were lysed in ice-cold RIPA buffer [150 mM NaCl, 1% Nonidet P-40, 0.5% deox-
in the proliferative phase and 25 were in the secretory phase. Normal ycholate, 0.1% SDS, 50 mM Tris [pH, 7.5], 1 mM phenylmethylsulphonyl
endometrial tissues from disease-free patients of reproductive age under- fluoride, 10 mg/ml leupeptin and 100 mg/ml aprotinin). The extracts
going hysterectomy for leiomyoma or ovarian pathology (n ¼ 40) were were placed on ice for 15 min and centrifuged to remove cellular debris.
also collected at the time of laparoscopy. BMI was 23.3 + 3.4 (kg/m2). The protein concentration of supernatants was determined using the Brad-
Twenty-four out of 40 women were in the proliferative phase. The histo- ford assay (Bio-Rad Laboratories, Hercules, CA, USA). Thirty micrograms of
logical slides of the excised ovarian endometriomas were evaluated by total protein was run on 10% SDS – polyacrylamide gels and electrotrans-
one pathologist (H.K.O.), who was blind to the clinical variables of the ferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Oak-
patient. Routine hematoxylin and eosin staining was used to diagnose ville, Ontario, Canada). The membranes were blocked in 5% non-fat dry
endometriosis. milk for 1 h, rinsed and incubated with specific antibodies against ObR,
p-JAK2, p-STAT3, p-ERK and b-actin (1:1000 –2000) in Tris-buffered
saline (TBS) containing Tween-20 (0.1%) overnight at 48C. Primary antibody
was removed by washing the membranes three times in TBS-T, and incu-
Materials
bated for 1 h with horseradish peroxidase (HRP)-conjugated secondary
Recombinant leptin was purchased from Sigma-Aldrich (Oakville, Ontario, antibody (1:2000). Following three times of washing in TBS-T, immuno-
Canada). PD98059 [2-(2-amino-3-methoxyphenyl)-4H-1-benzopy-ran-4- positive bands were visualized by enhanced chemiluminescence and
one], a MAPK/ERK kinase (MEK) inhibitor, and AG490 [a-cyano-(3,4- exposed to X-ray film (Amersham Pharmacia Biotech). Quantification of
dihydroxy)-N-benzyl-cinnamide], a JAK2/STAT3 inhibitor, and wortman- the immunoblots was performed on Scion image software (Scion Corp.,
nin, a PI3K inhibitor, were purchased from New England Biolabs Frederick, MD, USA). Intensities of interested protein bands were
(Beverly, MA, USA) and Calbiochem (La Jolla, CA, USA), respectively. scanned and quantified by density plot.
Antibodies against ObR (rabbit polyclonal) and b-actin (goat polyclonal)
were purchased from Abcam (Cambridge, MA, USA) and Santa Cruz Bio-
Reverse transcription – polymerase chain
technology (Santa Cruz, CA, USA), respectively. Phospho-STAT3 (mouse
monoclonal), phospho-JAK2 (mouse monoclonal) and phospho-ERK reaction
(mouse monoclonal) were obtained from Cell Signaling Technology Total RNA was extracted from immortalized endometrial and endometrio-
(Beverly, MA, USA). tic cells using the TRIzol reagent (Invitrogen Canada, Burlington, Ontario,

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 162 Oh et al.

Canada), according to the manufacturer’s instructions. Total RNA (2.5 mg) Transfection of siRNA
was reverse transcribed into first-strand cDNA (Amersham Pharmacia
ObR and control small interfering RNAs (siRNAs) were synthesized by
Biotech) following the manufacturer’s procedure. The synthesized cDNA
Bioneer technology (Daejon, South Korea). Cells were transfected with
was used as a template for polymerase chain reaction (PCR) amplification.
siRNA at a final concentration of 20 nmol/l using lipofectamine (Invitro-
Real-time PCR was performed using Thermal Cycler Dice Real-Time PCR
gen, Carlsbad, CA, USA) according to the manufacturer’s suggested
System (Takara, Japan). The primers used for SYBR Green real-time RT–
protocol. Briefly, cells were plated in 6-well culture dishes and allowed
PCR were as follows: for ObRL, sense primer, 5′ -AGG ACG AAA GCC
to attach and grow 24 h before transfection. Each transfection mixture
AGA GAC AAC C-3′ and antisense primer, 5′ -GCC TGG GCC TCT
was prepared by mixing up the siRNA and lipofectamine in serum-free
ATC TCC CA-3′ ; for GAPDH sense primer, 5′ -GAG TCA ACG
Opti-MEM (Invitrogen) and incubating for 15 min at room temperature.
GAT TTG GTC GT-3′ and antisense primer, 5′ -TTG ATT TTG GAG
The transfection mixture was slowly added to the cells, which were
GGA TCT CG-3′ . A dissociation curve analysis of ObRL and GAPDH
allowed to recover for an additional 24 h before experimental treatments.
showed a single peak. PCRs were carried out for 40 cycles using the follow-
ing conditions: denaturation at 958C for 5 s, annealing at 578C for 10 s and
elongation at 728C for 10 s. The results for ObR mRNA are normalized to a Data analysis
control gene GAPDH (Klein et al., 2011; Gebeh et al., 2012) and expressed Statistical analysis for cell growth data were performed by one-way analysis
as the fold increase over HES cells. of variance (ANOVA). Relationships between staining intensity of ObR in
endometrioma and clinical parameters (size of endometrioma, BMI, stage
of disease, menstrual phase and age) and correlation between epithelial
and stromal expression of ObR were evaluated by calculation of Spear-
Immunohistochemistry man’s correlation coefficient. x2- test was used for correlation between
Immunohistochemistry was carried out on 40 normal endometrium and ObR expression and endometriosis. In all statistical analysis used in this
44 endometrioma tissue sections and 2 well-differentiated hepatocellular study, P , 0.05 was considered statistically significant.
carcinoma tissue sections for positive control using the Bond Polymer
Intense Detection System (Leica microsystems, VIC, Australia) according
to the manufacturer’s instruction with minor modifications. In brief, the Results
5 mm sections of formalin-fixed and paraffin-embedded tissues were
deparaffinized by Bond Dewax Solution (Leica microsystems), and an Expression of the leptin receptor
antigen retrieval procedure was done using Bond ER Solution (Leica
in endometrioma tissues
microsystems) for 30 min at 1008C. The endogenous peroxidase was
quenched by incubation with hydrogen peroxide for 5 min. Sections In this study, the levels of ObR were evaluated in 40 normal endome-
were incubated for 15 min at ambient temperate with a rabbit poly- tria from endometriosis-free patients and 44 endometrioma tissues
clonal Leptin receptor antibody (ab60042, 1:150; Abcam, Cambridge, from patients with endometriosis by immunohistochemical analysis.
UK) using biotin-free polymeric HRP-linker antibody conjugate system In endometrioma tissues, ObR immunoreactivity was found in 32 of
in a Bond-Max automatic slide stainer (Leica microsystems). Heptocellu- 44 (72.8%) epithelial and 14 of 44 (31.8%) stromal tissues, while
lar carcinoma (ObR) tissues, obtained from patient who had undergone only 18 of 40 (45%) epithelial and 5 of 40 (12.5%) stromal tissues
surgical resection at Daegu Catholic University Medical Center, were of normal endometrium were ObR positive (Table I). Notably, ObR
used as a positive control (Wang et al., 2006). A negative control
expression in endometrioma epithelium was significantly more exten-
was included by using non-specific bovine serum instead of primary anti-
sive and stained more intensely than in the epithelial compartment of
body. The expression of ObR in samples was scored separately in epi-
thelium and stromal cells by using a four-point scale: 0, ,10% positive the normal endometrium (P , 0.01). In endometrioma tissues, ObR
cells; 1+, 10 – 50% positive cells with weak staining; 2+, .50% positive expression in the epithelium was significantly correlated with the ex-
cells with weak staining; 3+, .50% positive cells with strong staining. pression of the receptor in the stroma (Supplemental data, Table
The expression of ObR in endometriotic tissues was classified as nega- SI). Figure 1A is the representative staining of endometrioma tissue
tive (score 0) or positive (score 1 – 3). to show the strong epithelial and relatively mild stromal staining. In

Table I The epithelial and stromal expression of ObR in normal endometrium and endometrioma.

Tissues Staining intensity Endometrium (n 5 40) Endometrioma (n 5 44) P-value

....................................................
Endometrium versus endometrioma
.............................................................................................................................................................................................
Epithelial cells 0 22 12 0.009*
+1 18 24
+2 0 7
+3 0 1
Stromal cells 0 35 30 0.065**
+1 5 11
+2 0 3
+3 0 0

*x2 test to compare the difference in staining intensity of ObR between epithelial cells of normal endometrium and that of ovarian endometriomas.
**x2 test to compare the difference in staining intensity of ObR between stromal cells of normal endometrium and stromal cells of ovarian endometriomas.

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 Effect of leptin on endometriotic cell growth 163

the representative staining of normal endometrium (Fig. 1B), very The expression of ObR was also assessed in immortalized human
weak ObR expression was observed in the epithelial cells. Endome- endometriotic epithelial and stromal cells. As seen in Fig. 2A, human
trioma tissue with no ObR expression is shown as a negative endometriotic epithelial cells 11Z and 12Z showed high levels of
control (Fig. 1C) and hepatocellular carcinoma tissue is shown as a ObR protein (130 kDa) when compared with endometrial cells
positive control (Fig. 1D; Wang et al., 2006). The expression of (HES and HESC) and endometriotic stromal cells (22B). In parallel
ObR did not correlate with disease stage, size of the endometrioma, with ObR protein levels, the expression of ObRL mRNA was signifi-
menstrual phase (proliferative versus secretory phase), age or BMI cantly enhanced in 11Z and 12Z cells (Fig. 2B). MCF7 cells have
(Supplementary data, Tables SII and SIII). been used as a positive control for ObR expression (Yin et al.,
2004; Garofalo et al., 2006). This observation is consistent with our
findings in primary tissues showing that ObR expression is increased
in endometrioma tissues compared with normal endometrium
(Table I).

Effect of leptin on cell growth

in endometriotic cells
Although the primary function of leptin is the regulation of food intake
and energy consumption, leptin also promotes cell growth in various
cell systems. To evaluate the effect of leptin on cell growth, endome-
triotic epithelial and stromal cells were treated with leptin and cell
growth was assessed by MTT assay. Leptin treatment (0.05, 0.2,
1 mg/ml) stimulated the growth of endometriotic epithelial cells 11Z
and 12Z but did not induce significant changes in endometriotic
stromal cells 22B (Fig. 3A). Knockdown of the ObR in 11Z cells
impaired the ability of leptin to induce cell growth (Fig. 3B), suggesting
that leptin-induced cell growth is mediated through the ObR. In add-
ition, a clonogenic assay was performed to assess the long-term effect
of leptin treatment. As shown in Fig. 4, clonogenic survival of both 11Z
and 12Z cells was increased in a dose-dependent manner after expos-
ure to leptin (0.2 and 1 mg/ml).
Figure 1 Immunohistochemical staining for ObR in human normal
endometrium and endometrioma tissues. (A) Endometrioma tissues Involvement of the JAK2/STAT3 and ERK
show strong cytoplasmic expression in epithelial cell (EP, arrowhead) pathways in leptin-induced cell growth in
and a few stromal cells (ST, double arrows). (B) Normal proliferative endometriotic cells
endometrium with weak expression. (C) Endometrioma tissue with
no ObR expression. (D) Hepatocellular carcinoma tissue section Leptin signaling in normal endometrial and endometriotic cells has not
show strong cytoplasmic expression as positive control. The insert been studied to date. In other types of cells including adipocytes,
in (D) shows a representative microphotograph of a section stained leptin stimulates multiple pathways, including the JAK2/STAT3, ERK
with non-specific bovine serum instead of primary antibody. Original and PI3K/Akt pathways. To test the effect of leptin on the activation
magnification ×400. of JAK2/STAT3, ERK and PI3K/Akt signaling cascades, phosphory-
lated forms of JAK2, ERK, Akt were detected by immunoblot analysis.

Figure 2 Expression of ObR in endometrial and endometriotic cell lines. The protein (A) and mRNA expression (B) of ObR was investigated by
western blot and real-time RT– PCR, respectively. HES, human immortalized endometrial epithelial cells; HESC, human immortalized endometrial
stromal cells; 11Z and 12Z, human endometriotic epithelial cells; 22B, human endometriotic stromal cells; MCF7, human breast cancer cells.
Values are the mean + SD for three individual experiments (n ¼ 3).

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 164 Oh et al.

Figure 3 Effect of leptin on cell growth in endometriotic cells. (A) Human endometriotic epithelial cells (11Z and 12Z) and stromal cells (22B) were
treated with different concentrations of leptin (0.05, 0.2, 1.0 mg/ml) for 2 days. Cell growth was measured using MTT assay. (B) Cells were transiently
transfected with ObR siRNA (20 nM) or control (con) siRNA, and treated with leptin (1.0 mg/ml) for 2 days. Cell growth was measured using MTT
assay. Values are the mean + SD for three individual experiments (n ¼ 5). *P , 0.05 versus untreated control; #P , 0.05 versus 1.0 mg/ml leptin-
treated group; significant differences between groups were determined using one-way ANOVA.

Figure 4 Effect of leptin on clonogenic survival of endometriotic epithelial cells 11Z and 12Z. Cells were seeded in 6-well plates at a cell density of
5000 cells per well and treated with leptin (0.2 and 1.0 mg/ml) for 2 weeks. The colony formation was analyzed as described in Material and Method
section. Values are the mean + SD for three individual experiments (n ¼ 5). *P , 0.05 versus untreated control; significant differences between
groups were determined using one-way ANOVA.

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 Effect of leptin on endometriotic cell growth 165

Figure 6 Effect of JAK2/STAT3, ERK and PI3K/Akt inhibitors on

leptin-induced cell growth in 12Z cells. The cells were treated for 2
days with leptin (0.2 and 1 mg/ml) following pretreatment (30 min)
with JAK2 inhibitor AG490 (10 mM), ERK inhibitor PD98059
(10 mM) and PI3K/Akt inhibitor wortmannin (100 nM). Cell growth
was measured using MTT assay. Values are the mean + SD for
three individual experiments (n ¼ 5). *P , 0.05 versus untreated
control; #P , 0.05 versus leptin-treated group (0.2 or 1 mg/ml); sig-
nificant differences between groups were determined using one-way
ANOVA.
Figure 5 Effect of leptin on JAK2, ERK and Akt activation in 12Z
cells. Cells were treated with leptin (0.2 mg/ml) for the indicated
times (15, 30, 60 min). Western blot analysis was performed using endometriosis. These authors also showed that expression of leptin
specific antibodies for phospho- or total JAK2, STAT3, ERK and Akt. is induced by hypoxia in endometriosis (Wu et al., 2007). Whether
the exact origin of peritoneal leptin is fat, endometriotic tissues or
other cell types remains to be elucidated.
As shown in Fig. 5, leptin significantly increased the activation of JAK2,
Accumulating evidence indicates that leptin has specific biological
STAT3 and ERK in 12Z, but not that of Akt. To further investigate the
functions in the human normal endometrium and plays a role in
specific signaling pathways involved in the stimulation of growth by
embryo implantation and remodeling of the endometrium (Cervero
leptin in endometriotic epithelial cells, the effect of specific signal
et al., 2004; Tanaka and Umesaki, 2008). Leptin signaling appears to
transduction inhibitors on leptin-induced cell growth was examined.
be essential for endometrial cell adhesion, proliferation, survival and
The results showed that AG490, a JAK2/STAT3 inhibitor and
migration (Carino et al., 2008; Tanaka and Umesaki, 2008).
PD98059, an ERK inhibitor, markedly blocked leptin-stimulated cell
However, few studies on the functional role of leptin in endometriosis
growth of endometriotic cells, whereas the PI3K/Akt inhibitor wort-
exist to date. A study using a murine endometriosis model showed
mannin did not cause a substantial inhibition (Fig. 6). In 11Z cells,
that disruption of leptin signaling by i.p. injection of a pegylated
leptin also induced the activation of JAK2/STAT3 and ERK and pre-
leptin peptide receptor antagonist impairs the establishment of
treatment with the AG490 and PD98059 significantly inhibited
endometriosis-like lesions and results in a reduction of viable orga-
leptin-induced cell growth (Supplemental data, Fig. S1). These
nized glandular epithelium, vascular endothelial growth factor-A ex-
results indicate that leptin may increase 11Z and 12Z cell growth by
pression and mitotic activity (Styer et al., 2008). Despite these
activating the JAK2/STAT3 and ERK signaling pathway.
observations, the role of leptin in endometriotic cell biology and in
the pathogenesis of endometriosis remains to be fully elucidated and
the exact mechanism of the response to leptin is not clearly
Discussion understood.
Over the past few years, several lines of evidence have demonstrated ObR is expressed in various tissues including the peripheral repro-
that the peritoneal fluid concentration of leptin is increased in women ductive system, such as placenta and ovary (Cioffi et al., 1997). It is
with endometriosis (Matarese et al., 2000; Mahutte et al., 2003; also expressed in the human normal endometrium and in endometrial
Bedaiwy et al., 2006; Barcz et al., 2008; Alviggi et al., 2009). cancer (Kitawaki et al., 1999; Gonzalez-Robayna et al., 2000). One
However, whether this aberrant high level of leptin is due to obesity study showed that ObR is expressed in epithelial and stromal cells
is still unclear, as the association between obesity and endometriosis in the endometrium and stromal cells in endometriotic tissues (Ness
is controversial (Ferrero et al., 2005; Yi et al., 2009). In addition to et al., 2002). However, among 25 endometriotic tissues tested in
body fat as a cellular source of peritoneal leptin, other mechanisms re- this study, the number of samples showing ObR immunoreactivity
sponsible for aberrant expression of leptin have been studied. For and the tissue immunolocalization of ObR were not clearly described.
example, Wu et al. (2002) suggested that ectopic endometriotic Significant differences in ObR expression in the eutopic endometrium
lesions may be important sites of leptin production, thereby elevating between women with endometriosis and controls were not detected
leptin concentration in the peritoneal fluid of women with (Lima-Couy et al., 2004). In the present study, ObR expression and

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 166 Oh et al.

immunolocalization were assessed in 44 endometrioma tissue samples present study, activation of both the JAK2/STAT3 and ERK pathways
and compared with ObR expression in 40 normal endometrium correlated with leptin-induced proliferation in epithelial endometriotic
tissues. Although ObR expression was not significantly correlated cells. The molecular mechanisms underlying the interaction between
with the stage of the disease, size of the endometrioma, menstrual these two pathways leading to the induction of cell growth remain
phase, age or BMI, endometrioma tissues expressed higher levels of to be elucidated.
ObR than normal endometrium. Interestingly, ObR immunoreactivitiy The previously reported elevated levels of leptin found in the periton-
was more frequently observed in epithelial cells than in stromal cells of eal fluid of women with endometriosis (Matarese et al., 2000; Mahutte
both the normal endometrium and that of endometriosis patients. The et al., 2003; Bedaiwy et al., 2006; Barcz et al., 2008; Alviggi et al., 2009),
present finding of strong epithelial ObR staining is consistent with a together with the presence of ObRs in endometriotic tissues revealed in
previous study done in endometrial tissues. Lima-Couy et al. (2004) this study suggest that leptin may play a role in the development and/or
demonstrated that a clear positive signal is observed in the luminal progression of endometriosis. We found that leptin stimulates the
and glandular epithelium of the endometrium compared with growth of endometriotic epithelial cells through the JAK2/STAT3 and
stromal cells. Other studies showed ObR protein expression in the ERK pathways in this study. Despite these findings, the role of leptin
glandular and luminal epithelium of the human endometrium and in in endometriosis-related pelvic pain and infertility remains to be eluci-
cultured endometrial epithelial cells (Kitawaki et al., 1999; Gonzalez- dated. In fact, recent studies suggested that leptin in the peritoneal
Robayna et al., 2000). In this study, the increased expression of fluid is associated with chronic pelvic pain and inflammation, but not in-
ObR in endometriotic tissues, which are likely exposed to a high fertility, in endometriosis patients (Wertel et al., 2005; Bedaiwy et al.,
leptin environment from peritoneal fluid, suggested that the potential 2006; Milewski et al., 2008). Leptin-induced cell growth revealed in
role of leptin in the development and progression of endometriosis this study may accelerate the peritoneal inflammation and chronic
required further investigation. pelvic pain. In addition, leptin-induced activation of the JAK/STAT and
In addition to its known role in metabolism, and cardiovascular and ERK pathway may be involved in the pelvic pain by regulating the expres-
renal function, there is convincing evidence that leptin functions as a sion of pro-inflammatory cytokines. For example, the STAT3 signaling
mitogen in various cancers, including breast, prostate and gastrointes- was shown to regulate the expression of tumor necrosis factor-a and
tinal cancer (Lang and Ratke, 2009). Despite this, it is unclear whether IL-1b (Pang et al., 2010), which are well documented to play a role in
leptin plays a role in promoting the growth of endometriotic cells, and chronic pain (Sommer and Kress, 2004; Marchand et al., 2005). More
the exact molecular mechanism of the response to leptin has yet to studies are needed to evaluate the exact role of leptin in pelvic pain
be investigated. To investigate the effect of leptin on the cell growth and inflammation.
of endometriotic cells, immortalized endometriotic cells were used as This study demonstrated for the first time that epithelial expression
the experimental model in the present study. The source and availability of ObR is significantly greater in the endometrioma than in the endo-
of primary tissues and the ethical concerns in obtaining endometriotic metrium. This study also revealed that leptin is a potent mitogenic
tissues are among the major obstacles for study on endometriosis. In factor in endometriotic epithelial cells, but not in endometriotic
addition, there have been no appropriate in vivo and in vitro models avail- stromal cells. These data suggest that elevated leptin levels in the peri-
able for studying the characteristics of the active phase of endometriosis toneal environment, which have been reported before, can be a
in humans. Immortalized endometriotic cells 11Z, 12Z and 22B used in stimulating factor of cell growth in ObR positive endometriosis, thus
this study were established from active endometriotic lesions from emphasizing the need to assess ObR and leptin status in endometri-
women with endometriosis, and these lines retained the phenotypic osis patients. Furthermore, targeting the ObR and its signaling
characteristics and several in vivo properties of the active phase of endo- pathway could result in potent anti-proliferative effects in endometrio-
metriosis (Banu et al., 2008; Grund et al., 2008). The results show that tic epithelial cells and become a new strategy for the treatment of
leptin significantly increased cell growth and clonogenic survival of the endometriosis.
endometriotic epithelial cell lines 11Z and 12Z, while no significant
effect was observed in stromal 22B cells. As shown in Fig. 2, endome-
triotic epithelium and immortalized endometriotic epithelial cells (11Z
Supplementary data
and 12Z) expressed significantly higher levels of ObR than their Supplementary data are available at http://molehr.oxfordjournals.
normal and stromal counterparts. These data support the potential org/.
role of leptin in the pathogenesis of endometriosis via the regulation
of endometriotic epithelial cell growth.
The activity of leptin as a signaling molecule is known to be
Acknowledgements
mediated by multiple pathways, including the STAT3 (Yamashita We sincerely thank Dr Asgerally T. Fazleabas (University of Illinois at
et al., 1998), ERK (Yamashita et al. 1998; Catalano et al., 2004) and Chicago, Chicago, IL, USA) and Dr Graciela Krikun (Yale University,
PI3K/Akt (O’Rourke et al., 2001) pathways. The concomitant activa- New Haven, CT, USA) for providing HESC and HES cells as generous
tion of multiple signaling pathways by leptin was observed in certain gifts.
types of cells (Bedaiwy et al., 2006; Gao et al., 2009). For example,
in hepatocellular carcinoma cells, the concomitant activation of
JAK2/STAT3, ERK and PI3K/AKT signaling is associated with leptin-
Authors’ roles
mediated promotion of invasion and migration (Zeleznik et al., H.K.O. performed the immunohistochemical analysis and participated
2003). However, there are no reports in the current literature de- in study design, manuscript writing and critical discussion. Y.S.C. par-
scribing leptin signaling pathways in endometriotic cells. In the ticipated in study design, analysis, manuscript writing and critical

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 Effect of leptin on endometriotic cell growth 167

discussion. Y.-I.Y. and J.-H.K. performed immunoblot assay. P.C.K.L. endometrium from healthy and ectopic pregnant women. Int J Mol Sci
participated in critical discussion. J.H.C. participated in study design, 2012;13:2810 – 2826.
execution, analysis, manuscript writing and critical discussion. Gonzalez-Robayna IJ, Falender AE, Ochsner S, Firestone GL, Richards JS.
Follicle-Stimulating hormone (FSH) stimulates phosphorylation and
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Funding signaling by FSH in granulosa cells. Mol Endocrinol 2000;14:1283– 1300.
This study was supported by a grant of the Korean Health Technology Grund EM, Kagan D, Tran CA, Zeitvogel A, Starzinski-Powitz A, Nataraja S,
R&D Project, Ministry of Health & Welfare, Republic of Korea (No. Palmer SS. Tumor necrosis factor-alpha regulates inflammatory and
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Mol Pharmacol 2008;73:1394 – 1404.
Kitawaki J, Kusuki I, Koshiba H, Tsukamoto K, Honjo H. Leptin directly
Conflict of interest stimulates aromatase activity in human luteinized granulosa cells. Mol
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Klein C, Rutllant J, Troedsson MH. Expression stability of putative
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