Group Title: Reproductive Biology and Endocrinology 2003, 1:125
Title: Gonadotropin releasing hormone analogue (GnRHa) alters the expression and activation of Smad in human endometrial epithelial and stromal cells
CITATION PDF VIEWER THUMBNAILS PAGE IMAGE ZOOMABLE
Full Citation
STANDARD VIEW MARC VIEW
Permanent Link: http://ufdc.ufl.edu/UF00100219/00001
 Material Information
Title: Gonadotropin releasing hormone analogue (GnRHa) alters the expression and activation of Smad in human endometrial epithelial and stromal cells
Series Title: Reproductive Biology and Endocrinology 2003, 1:125
Physical Description: Archival
Creator: Luo X
Xu J
Chegini N
Publication Date: 37971
 Record Information
Bibliographic ID: UF00100219
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: Open Access: http://www.biomedcentral.com/info/about/openaccess/

Downloads

This item has the following downloads:

gonadotropin ( PDF )


Full Text


Reproductive Biology and 0

Endocrinology BioMedc



Research

Gonadotropin releasing hormone analogue (GnRHa) alters the
expression and activation of Smad in human endometrial epithelial
and stromal cells
Xiaoping Luo1, Jingxia Xu1,2 and Nasser Chegini*1


Address: 'Department of Obstetrics and Gynecology, University of Florida, Gainesville, Florida, USA and 2Present address: The Jackson Laboratory,
Bar Harbor, Main, USA
Email: Xiaoping Luo xiaoping@obgyn.ufl.edu; Jingxia Xu jingxia@obgyn.ufl.edu; Nasser Chegini* cheginin@obgyn.ufl.edu
* Corresponding author


Published: 16 December 2003
Reproductive Biology and Endocrinology 2003, 1:125


Received: 21 August 2003
Accepted: 16 December 2003


This article is available from: http://www.rbej.com/content/ 1/1/125
2003 Luo et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media
for any purpose, provided this notice is preserved along with the article's original URL.



Abstract
Gonadotropin releasing hormone analogues (GnRHa) are often used to regress endometriosis
implants and prevent premature luteinizing hormone surges in women undergoing controlled
ovarian stimulation. In addition to GnRH central action, the expression of GnRH and receptors in
the endometrium implies an autocrine/paracrine role for GnRH and an additional site of action for
GnRHa. To further examine the direct action of GnRH (Leuprolide acetate) in the endometrium,
we determined the effect of GnRH on endometrial stromal (ESC) and endometrial surface
epithelial (HES) cells expression and activation of Smads (Smad3, -4 and -7), intracellular signals
activated by transforming growth factor beta (TGF-beta), a key cytokine expressed in the
endometrium. The results show that GnRH (0.1 microM) increased the expression of inhibitory
Smad7 mRNA in HES with a limited effect on ESC, while moderately increasing the common Smad4
and Smad7 protein levels in these cells (P < 0.05). GnRH in a dose- (0.01 to 10 microM) and time-
(5 to 30 min) dependent manner decreased the rate of Smad3 activation (phospho-Smad3,
pSmad3), and altered Smad3 cellular distribution in both cell types. Pretreatment with Antide
(GnRH antagonist) resulted in further suppression of Smad3 induced by GnRH, with Antide
inhibition of pSmad3 in ESC. Furthermore, co-treatment of the cells with GnRH + TGF-beta, or
pretreatment with TGF-beta type II receptor antisense to block TGF-beta autocrine/paracrine
action, in part inhibited TGF-beta activated Smad3. In conclusion, the results indicate that GnRH
acts directly on the endometrial cells altering the expression and activation of Smads, a mechanism
that could lead to interruption of TGF-beta receptor signaling mediated through this pathway in
the endometrium.


Introduction
Excess production of ovarian steroids, as well as overex-
pression of their receptors, is believed to serve as an
underlying molecular mechanism that promotes uterine
abnormalities such as endometriosis, leiomyoma and
endometrial cancer. Gonadotropin releasing hormone
analogues (GnRHa) are often sought for medical manage-


ment of these disorders, due to hypoestrogenic condition
created by GnRHa therapy [1-3]. Short-term administra-
tion of GnRHa is also used to prevent premature luteiniz-
ing hormone (LH) surges in women undergoing
controlled ovarian stimulation [4-8]. GnRHa therapy acts
primary at the level of hypothalamus/pituitary/ovarian
axis. However, accumulating evidence for the expression


Page 1 of 13
(page number not for citation purposes)


central


^^3







Reproductive Biology and Endocrinology 2003, 1



of GnRH and GnRH receptors in several peripheral tis-
sues, including the uterus, implies an autocrine/paracrine
action for GnRH, and additional sites of action for GnRHa
therapy [9-13]. GnRH treatment is reported to alter the
rate of cell growth and apoptosis, and the expression of
cell cycle proteins, growth factors, cytokines, proteases,
and protease inhibitors in various cell types derived from
peripheral tissues, including the uterus [9-21]. In addi-
tion, administration of GnRHa in women undergoing
controlled ovarian stimulation is reported to induce an
imbalance in endometrial expression of ovarian steroid
receptors with a profound antimitotic effect, as compared
to endometrium of the natural cycle [8], a condition that
could result in an unfavorable environment for embryo
implantation [4-7].

Transforming growth factor beta (TGF-P) is a key regulator
of cell growth and differentiation, and the expression of
extracellular matrix, adhesion molecules, proteases, and
protease inhibitors [14,21-26]. TGF-P and TGF-P recep-
tors are expressed in the endometrium, where their
expression is regulated in part by ovarian steroids. Altered
expression of TGF-P has also been correlated with several
disorders [27], and in the uterus this includes endometri-
osis, leiomyoma and endometrial cancer [28-32]. The
uterine expression of TGF-P and TGF-P receptors is tar-
geted by GnRH treatment, and GnRH is reported to
inhibit ovarian steroid-induced TGF-P expression in leio-
myoma and myometrial smooth muscle cells, as well as
matrix metalloproteinases and their inhibitors in
endometrial stromal cells [9,12,14,17,21].

Binding of TGF-P to TGF-P receptors results in the activa-
tion of multiple intracellular signaling pathways, includ-
ing the Smad pathway [33]. Smad pathway, which
specifically mediates TGF-P receptors signaling from the
cell surface to the nucleus, is comprised of pathway-spe-
cific regulatory Smad (RSmad 1, 2, 3, 5 and 8), the com-
mon-Smad (Smad4), and the inhibitory Smad (Smad6
and -7) [33]. Smad2 and Smad3 are phosphorylated by
the activated TGF-P type I receptor, associate with Smad4
and their complex translocates into the nucleus, where
they direct specific transcriptional responses to TGF-P
actions. In contrast, the interaction of inhibitory Smads
with TGF-P type I receptors prevents phosphorylation of
RSmads, resulting in interruption of TGF-P receptor sign-
aling [33]. We have recently reported the expression of
Smad3, -4 and -7 in human endometrium, and demon-
strated that their expression and Smad3 activation are reg-
ulated by TGF-P in endometrial epithelial and stromal
cells [34]. Recent studies have also demonstrated a func-
tional interaction between GnRH and TGF-P as well as
activin, a member of TGF-P family, involving Smad and
MAPK pathways in the pituitary gonadotropes, resulting
in regulation of GnRH and GnRH receptor expression [35-


http://www.rbej.com/content/1/1/125


38]. In the present study we sought to extend our previous
work by examining the direct action of GnRH on Smad
expression and activation, as well as GnRH functional
interaction with TGF-P action in isolated endometrial
stromal cells and endometrial surface epithelial cell
(HES).

Materials and Methods
All the materials for isolation and culturing of endome-
trial cells, RT-PCR, Western blotting and immunocyto-
chemistry were purchased from commercial sources as
described [34,39]. Portions of endometrial tissue were
collected from premenopausal women (N = 3) who were
undergoing hysterectomy for symptomatic uterine leio-
myomas. These patients were not taking any hormonal
medications at least during the 3 months prior to surgery.
The tissues were collected at the University of Florida affil-
iated Shands Hospital with the approval of the Institu-
tional Review Board.

The endometrial tissues were used for isolation of
endometrial stromal cells (ESC) as previously described
[34]. Human endometrial surface epithelial cell line
(HES) was kindly provided by Dr. Douglas Kniss (Ohio
State University, Columbus Ohio). The isolated ESC and
HES were cultured as previously described [34]. To deter-
mine the direct action of GnRH (Leuprolide acetate; LA)
on Smads mRNA and protein expression, ESC and HES
were cultured at 1 x 106 cell/well in 6 well dishes in media
containing 10% FBS for 48 hrs. The cells were washed and
incubated in serum free/phenol red-free media for an
additional 24 hrs and then treated with GnRH (0.1 iM)
for 2, 4, 6 and 12 hrs to determine Smad mRNA expres-
sion, and 18, 24 and 36 hrs to determine Smads protein
production. Total RNA was isolated using Trizol (Invitro-
gen, Carlsbad, CA), and an equal amount of RNA (2 Gg)
was used to co-amplify Smads and glyceraldeyde-3 phos-
phate dehydrogenase (G3PDH) mRNA by RT-PCR as pre-
viously described [39]. Optimal amplification condition
within the logarithmic phase was established over a range
of 25-35 cycles with primers for Smads and G3PDH used
at equal concentrations with identical PCR buffer contain-
ing 1.5 mM MgCl2. The PCR temperature profile consist of
cycle denaturation (95 C for 1 min), annealing (55-
61 C for 30s), and extension (72C for 1 min) followed
by an additional 5 min extension at 72 C in the presence
of Taq polymerase added at the first annealing incuba-
tion. Controls included omission of the reverse-transcrip-
tion step before PCR amplification, co-amplification of
G3PDH mRNA, and inclusion of water blanks. After a 30-
35 cycle of amplification the co-amplified Smad:G3PDH
PCR products were separated on 1% agarose gels. The rel-
ative level of Smads mRNA expression was determined
from the band intensities and reported as fold changes in
Smad:G3PDH mRNA ratio. For protein analysis, the cells


Page 2 of 13
(page number not for citation purposes)







Reproductive Biology and Endocrinology 2003, 1



were directly lysed in a buffer containing 50 mM Hepes
(pH 7.4), 1% Nonidet P-40, 0.5% deoxycholate, 5 mM
EDTA, 1 mM sodium Ortho-vanadate, 5 mM NaF and
phosphatase and protease inhibitor cocktails (Sigma
Chemical, St Louis, MO). The cell lysates were centrifuged
at 14,000 x g for 15 min at 4 C the supematants were col-
lected and following determination of their total protein
content (Pierce, Rockford, IL), an equal amount of sample
proteins were subjected to immunoblot analysis [34,39].
The level of Smad protein production was determined
from the band intensity visualized using enhanced chemi-
luminescence reagents [34,39].

To determine the dose- and time-dependent effect of
GnRH on Smad activation (phosphorylated Smad3,
pSmad3), HES and ESC were cultured under serum-free/
phenol red-free conditions and then treated with 0.01 to
10 iM of GnRH for 15 min, or with 0.1 gM of GnRH for
5, 15 and 30 min. Total protein was isolated and subjected
to immunoblotting using anti pSmad2/3 and Smad3 anti-
bodies, determining the level of Smad3 activation in these
cells. To determine the effect of GnRH on translocation of
activated Smad3 into the nucleus, HES and ESC were cul-
tured in eight-well slides (Nalge Nunc, Naperville, IL) for
24 hrs in a medium containing 10% serum, and 24 hrs
under a serum-free/phenol red-free condition; washed
and treated with GnRH (0.1 iM) for 5, 15 and 30 min. The
cells were washed in phosphate-buffered saline (PBS),
fixed in methanol and immunostained with Smad3 anti-
bodies by fluorescein isothiocyanate (FITC)-labeled-indi-
rect method [34,40]. After incubation, the cells were
washed with PBS and Vectashield with DAPI (Vector Lab-
oratories) as the mounting medium. Images were cap-
tured using an Olympus IX70 microscope configured with
DAPI and FITC fluorescent excitation filters, and
equipped with digital camera and software image analysis
package (Olympus Inc., Melville, NY).

To determine the effect of GnRH on TGF-p-induced
Smad3 activation, HES and ESC were cultured under
serum-free/phenol red-free condition and the treated with
GnRH (0.1 iM), TGF-P1 (2.5 ng/ml) or GnRH + TGF-P1
for 15 min. The specificity of GnRH action on Smad3 acti-
vation was determined in serum-starved HES and ESC
pretreated with GnRH antagonist, Antide (10 iM) for 2
hrs prior to exposure to GnRH (0.1 iM) for 15 min. To
determine if the autocrine/paracrine action of TGF-P is in
part responsible for activation of Smad3, HES and ESC
were treated with TGF-P receptor type II antisense in order
to block the autocrine/paracrine action ofTGF-P. HES and
ESC were treated with 1 gtM of TGF-P type II receptor anti-
sense or sense 20 mer oligonucleotides for 24 hrs [21,39],
washed with PBS and then treated with GnRH (0.1 iM)
for 5, 15 and 30 min. Following treatments total protein
was isolated and subjected to immunoblotting to deter-


http://www.rbej.com/content/1/1/125


mine the level of Smad3 activation in parallel with total
Smad3 and P actin (control) as described [34,39,40].

The data is presented as mean + SEM of three independent
experiments. Experiments involving ESC the cells were
prepared from three different tissues. The data was ana-
lyzed using unpaired Student t-test and repeated measure
analysis of variance (ANOVA). A probability level of P <
0.05 was considered significant.

Results
To evaluate the effect of GnRH on Smad expression,
endometrial surface epithelial (HES) and endometrial
stromal cells (ESC) were maintained under serum-free
conditions and then treated with GnRH (0.1 iM) for 2, 4,
6 and 12 hrs to determine Smad mRNA expression, or for
18, 24 and 36 hrs to determine Smad protein production.
As shown in Figure 1, GnRH had little or no significant
effect on the expression of Smad3 and Smad4 mRNA in
HES and ESC (Bar graphs are not shown), while it signifi-
cantly increased the expression of Smad7 in HES (P <
0.05) with a limited effect on ESC. At the protein level,
GnRH inhibited Smad3 production in ESC and HES after
18 and 36 hrs respectively, which increased in ESC after
36 hrs (P < 0.05, Fig. 2). However, GnRH treatment
increased the production of Smad4 and Smad7 protein in
ESC after 18 to 24 hrs; while in HES GnRH action
occurred after 36 hrs of treatment with an inhibitory effect
on Smad7 at 18 hrs (P < 0.05; Fig. 2). The results indicate
that GnRH, in a time- and cell specific-manner, differen-
tially regulates the expression of Smads in endometrial
epithelial and stromal cells.

We then determine whether GnRH alters the activation of
Smad3, either alone, or Smad3-induced by TGF-P. Treat-
ment of serum-starved HES and ESC with GnRH resulted
in suppression of Smad3 activation (pSmad3) a time (Fig.
3A) and dose (Fig. 3B) dependent manner in both cells (P
< 0.05). Smad3 was primarily immunolocalized in cyto-
plasmic compartment of HES and ESC, and GnRH (0.1
iM) treatment for 5, 15 and 30 min resulted in accumula-
tion of Smad3 around the nuclear periphery, with limited
translocation into the nucleus (Fig. 4; show representative
after 15 min of treatment). To determine the specificity of
GnRH action on Smad3 activation, the cells were pre-
treated with GnRH antagonist, Antide (10 iM), for 2 hrs
prior to treatment with GnRH (0.1 iM). Treatment with
Antide inhibited Smad3 activation in ESC, with further
inhibition following co-treatment with Antide + GnRH
compared to GnRH in both cell types (P < 0.05; Fig. 5).
The results suggest that both GnRHa (GnRH and Antide)
alter Smad activation in endometrial epithelial and stro-
mal cells, however, whether GnRHa directly utilize Smad
pathway or their actions are mediated through crosstalk
with components of other pathways activated by GnRH


Page 3 of 13
(page number not for citation purposes)






Reproductive Biology and Endocrinology 2003, 1


HES


ESC


F 12


Smad3-
G3PDH
Smad4-*


Smad7-*


SHES
*ESC

b c







a a'


C'


,r1


Ctrl 2 4 6 12


Time (hrs)



Figure I
Time dependent action of GnRH (leuprolide acetate) on Smad3, Smad4 and Smad7 (arrows) mRNA expression in human
endometrial surface epithelial cells (HES) and isolated endometrial stromal cells (ESC). Serum-starved cells were treated with
GnRH (0.1 M) for 2 to 12 hrs and total RNA was isolated from treated and untreated control (Ctrl) cells and subjected to
semi-quantitative RT-PCR co-amplifying Smads and G3PDH (lower bands) mRNA shown from a representative experiment.
The bar graph show the mean SEM of fold change of ratio of Smad7:G3PDH mRNA expression from three independent
experiments. b, c, d, and e and c' and are significantly different from a and a', respectively (p < 0.05). M = DNA marker.



Page 4 of 13
(page number not for citation purposes)


http://www.rbej.com/content/l/1/125







Reproductive Biology and Endocrinology 2003, 1


HES

Ctrl 18 24 36


ESC

Ctrl 18 24 36


Smad3 -I


Smad4


Smad7 [ m I


p-actin 1


2


m 1.5

U
.r 1
4-
g 0.5
E
(n


Ctrl 18 24 36
Time (hrs)


Ctrl 18 24 36
Time (hrs)


Ctrl 18 24 36
Time (hrs)


Figure 2
Time dependent action of GnRH (leuprolide acetate) on Smad3, Smad4 and Smad7 protein expression in HES and ESC. Serum-
starved cells were treated with GnRH (0. I IM) for 18, 24 and 36 hrs and cell lysates were prepared from treated and
untreated control (Ctrl) and analyzed by immunoblotting for Smads and P-actin as loading control, all shown from a represent-
ative experiment. Bar graphs show the mean SEM of fold change in Smads expression from three independent experiments.
The denote d, b' and d' (Smad3), d, b', and c' (Smad4) and b, d, b' and c' (Smad7) are significantly different from a and a', respec-
tively (p < 0.05).


Page 5 of 13
(page number not for citation purposes)


http://www.rbej.com/content/l/1/125







Reproductive Biology and Endocrinology 2003, 1


HES
Ctrl 5 15 30

pSmad2/3

1-actin


ESC
Ctrl 5 15 30

IF- -

I _


2
O HES
ESC
c 1.5
5 a a'
1 --
-o b' c

E 0.5 d
L0. 1

[I"d0


Ctrl 5


15 30


Time (min)


B

pSmad2/3
p-actin


0
c 1.





E o.
Cn


HES


ESC


Ctrl 0.01 0.1 1 10 Ctrl 0.01 0.1 1 10

[ ~ ~ .............. .... ..r


Ctrl 0.01 0.1 1 10
GnRH (1M)


Figure 3
Time- (A) and dose- (B) dependent effects of GnRH (Leuprolide acetate) on the rate of Smad3 activation (phospho-Smad3;
pSmad3). HES and ESC were incubated under serum-free condition and treated with GnRH (0. I IM) for 5, 15 and 30 min, or
with GnRH at 0.01 to 10 IM for 15 min. The cell lysates were prepared and analyzed by immunoblotting for pSmad2/3 and 3-
actin as loading control, shown from a representative experiment. Bar graphs show the mean SEM of fold change in the rate
of Smad3 activation from three independent experiments. In Fig. A: denotes b, c, d, b', c', and d' and in Fig. B, c, d, e, b', c', d,'
and e'are significantly different from a and a', respectively (p < 0.05).



Page 6 of 13
(page number not for citation purposes)


http://www.rbej.com/content/1/1/125







Reproductive Biology and Endocrinology 2003, 1


Anit-Smad3
Conro









Conro


DAPI


Anit-Smad3


Figure 4
Immunofluorescence localization of Smad3 in HES and ESC. The cells were incubated under serum-free condition for 24 hrs
then treated with GnRH (0.1 gIM) for 5, 15 and 30 min. Note subcellular localization of Smad3 in untreated control with mostly
cytoplasmic and limited nuclear localization, while GnRH-treatment resulted in more cytoplasmic accumulation of Smad3. The
figures are shown after 15 min of GnRH treatment with FITC staining used to localize Smad3 and DAPI staining for the nuclei.


receptors remains to be investigated. Co-treatment of
serum-starved HES and ESC with GnRH + TGF-P also
resulted in inhibition of Smad3 activation compared to
TGF-P treated cells (P < 0.05; Fig. 6). The autocrine/para-
crine action of TGF-P in part account for Smad3 activation
since pretreatment of HES and ESC with TGF-P type II
receptor antisense prior to exposure to GnRH resulted in
further suppression of Smad3 activation compared to
sense-treated cells (Fig. 7).

Discussion
In the present study we demonstrated that GnRH, in a
dose, time, and cell specific manner, alters the endome-
trial epithelial and stromal cells expression and produc-
tion of Smad3, -4 and -7, intracellular proteins that
mediate TGF-P receptor signaling from the cell surface to
the nucleus. These actions of GnRH on Smads mRNA
expression were limited to Smad7, while it induced both
stimulatory and inhibitory effects on their protein pro-
duction. More specifically, GnRH inhibited Smad3 pro-
duction in ESC and HES, with an increased production in
ESC following longer exposure. GnRH increased the pro-
duction of Smad4 and Smad7 in ESC, however in HES


GnRH action occurred after 36 hrs, with an inhibition of
Smad7 after 18 hrs of treatment. Although the molecular
mechanism how GnRH alters the expression of Smads is
not known and requires detailed investigation, their dif-
ferential regulation at protein levels suggests possible
posttranscriptional regulation. The action of GnRH on
Smad expression could occur indirectly through the inhi-
bition of TGF-P and TGF-P receptors expression, since
TGF-P regulates its own expression and the expression of
Smads in several cell types [44-47]. In this respect we have
previously reported that GnRH alters the expression of
TGF-P, TGF-P receptors and Smads in leiomyoma and
myometrial smooth muscle cells, and TGF-P self-regula-
tion in endometrial stromal cells [9,21,39]. Alternatively,
GnRH-induced hypoestrogenic condition could result in
alteration of Smads expression, because ovarian steroids
regulate the endometrial expression of TGF-P and TGF-P
receptors [48-50]. A recent report indicated that direct
binding ofAP-1 (fos/jun) proteins to a Smad binding ele-
ment facilitates GnRH- and activin-mediated transcrip-
tional activation of the GnRH receptor gene [37].




Page 7 of 13
(page number not for citation purposes)


DAPI


HES







ESC


hftp://www. rbej. co m/co nte nt/l/l/l125






Reproductive Biology and Endocrinology 2003, 1


HES


ESC
|^K ^^ sl ^


pSmad2/3


f3-actin I m IA -


GnRH
Antide
1.5


1





0.5


0 L


+


- +


+


+ +


O HES


*ESC
a
I _- -_


b
bT


C
_


GnRH
Antide


Figure 5
The effect of GnRH (0. I IM) and GnRH antagonist (Antide, 10 rIM) on Smad3 activation in HES and ESC. Serum-starved cells
were treated with Antide for 2 hrs prior to treatment with GnRH for 15 min. The cells lysates were prepared from treated
and untreated control (-) and analyzed by immunoblotting for pSmad2/3 and P-actin as loading control, shown from a repre-
sentative experiment. Bar graph shows the mean SEM of fold change in the rate of Smad3 activation from three independent
experiments with denotes b, c, d, b', c' and d'are significantly different from a and a,' respectively (p < 0.05).

Page 8 of 13
(page number not for citation purposes)


- +


+ +


http://www.rbej.com/content/l/1/125





Reproductive Biology and Endocrinology 2003, 1


HES


ESC


pSmad2/3 -- m


p-actin

TGF-P
GnRH


Ai --E Ir w I l
WMjr- "t ^^^ 01'^1 mM


- +


- +


+ +


+


+





1.5



1



0.5


O HES
SESC


a
___L


b

=1i


-------T-----


TGF-0
GnRH


Figure 6
The effect of GnRH on TGF-P I-induced Smad3 activation in HES and ESC. Serum-starved HES and ESC were co-treated with
TGF-PI (2.5 ng/ml) and GnRH (0.1 rIM) for 15 min and cell lysates from treated (+) and untreated (-) groups were analyzed by
immunoblotting for pSmad2/3 and P-actin as loading control, shown from a representative experiment. Bar graph shows the
mean SEM of fold change in the rate of Smad3 activation from three independent experiments with denotes b, c, b' and c' are
significantly different from a and a', respectively (p < 0.05).


Page 9 of 13
(page number not for citation purposes)


- -- --- --- .


~--------I


http://www.rbej.com/content/1/1/125







Reproductive Biology and Endocrinology 2003, 1


HI FS
Ctrl 5 15 30 5


ESC
15 30 Ctrl 5 15 30 5


I, .- J -F W1


3-actin -"W-'-'-

GnRH + + + + + +
sense + + + -
antisense - + + +


+ + + + + +
- + + + -
- + + +


GnRH
sense
antisense


**








Ctrl 5 15 30 5 15 30

Time (min)


- + + + + + +
+ + + -


+ + +


Figure 7
The effect of GnRH (0.1 rIM) on Smad3 activation in HES and ESC pretreated with TGF-P type II receptor antisense (I rIM) or
sense (I rIM) oligonucleotides for 24 hrs. The cell lysates were prepared from GnRH-treated (+) and untreated (-) cells after 5,
15 and 30 min and analyzed by immunoblotting for pSmad2/3 and P-actin as loading control, shown from a representative
experiment. Bar graph shows the mean SEM of fold change in the rate of Smad3 activation from three independent experi-
ments with denotes ** are significantly different from untreated (*) controls (p < 0.05).


Because endometrial epithelial and stromal cells express
GnRH and TGF-P receptors, GnRH-induced alteration of
Smad expression could influence the outcome of TGF-P
autocrine/paracrine actions in the endometrium. Smad3
is phosphorylated by activated TGF-P receptor type I and
following complex formation with Smad4, translocate
into the nucleus where they regulate transcriptional acti-
vation of target genes in response to TGF-P. The inhibitory


Smad7 also interacts with TGF-P type I receptor, but pre-
vents RSmads activation [33]. In the presents study we
also found that GnRH altered the activation of Smad3 in
ESC and HES. These observations provide the first exam-
ple of interactions between GnRH and TGF-P signaling in
the endometrium, however, recent reports have also dem-
onstrated a functional interaction between GnRH, TGF-P
and activin, involving Smad and MAPK activation in the


Page 10 of 13
(page number not for citation purposes)


15 30


O HES
SESC

*


-1


q~. ii I lr lr


L


hftp://www. rbej. co m/co nte nt/l/l/l125


p~3rnadlo 11W


- C-l -- -1 ^ If


-







Reproductive Biology and Endocrinology 2003, 1



pituitary gonadotropes, resulting in regulation of GnRH
and GnRH receptor expression [35-38]. Collectively, these
results support a potential crosstalk between GnRH and
TGF-P receptors signaling, involving Smad pathway in the
endometrium and possibly the pituitary-gonadal axis.
However, translocation of activated Smad3 into the
nucleus is required for transcriptional activation of target
genes. Unlike TGF-P which induced nuclear accumulation
on Smad3 in HES and ESC [34], GnRH treatment resulted
in Smad3 accumulation around their nuclear peripheries.
The biological significance of GnRH action on Smad3 cel-
lular distribution around the nuclear periphery is
unknown, however, activated Smad3 complexes with
Smad4 prior to translocation into the nucleus. Therefore,
alteration in Smad3 cellular distribution may represent an
additional mechanism by which GnRH influences cellular
response to TGF-P autocrine/paracrine actions.

Our results also indicated that Antide (GnRH antagonist)
had an inhibitory effect on Smad3 activation in ESC and
HES, and found that TGF-P autocrine/paracrine action
may, in part, account for Smad3 activation in these cells.
Until recently it was considered that the biological action
of GnRH is mediated only through GnRH-I receptor.
Identification of a second form of GnRH receptor, which
is structurally and functionally distinct from GnRH-I
receptor, suggests that GnRH action could be mediated
through either or both receptors, with GnRH-I antagonists
having agonistic effects on GnRH-II receptor [41-43].
Human endometrium expresses both forms of GnRH
receptors, however it remains to be determined whether
GnRH and Antide interact with one or both GnRH recep-
tors, or Antide could convert an antagonist action into an
agonist as seen in other cell types [51,52]. Both GnRH
receptors are reported to mediate antiproliferative and
apoptotic effects of GnRH [13,53].

In addition to modulating Smad3 activation, GnRH also
altered the expression of Smad7 in HES and ESC. Smad7
is a key regulator of TGF-P receptor mediated signaling
whose expression is highly regulated by TGF-P [44-46].
TGF-P also regulates the expression of TGF-P receptors,
ECM, adhesion molecules, proteases, and proteases inhib-
itors [44-46], whose expression is documented in human
endometrium and influenced by GnRHa therapy
[14,15,17,25]. These molecules are key components of tis-
sue remodeling process which is critical to endometrial
tissue integrity during the menstrual cycle. Therefore,
GnRH either directly, or through alteration of TGF-P
receptor mediated signaling, could target endometrial tis-
sue remodeling in patients undergoing GnRHa therapy
for medical management of endometriosis and leiomy-
oma, or controlled ovarian stimulation [4-8]. A recent
study reported that during controlled ovarian stimulation,
GnRHa induced an imbalance in endometrial expression


http://www.rbej.com/content/1/1/125


of ovarian steroid receptors with a profound antimitotic
effect as compared to endometrium of the natural cycle
[8], resulting in an unfavorable environment for embryo
implantation [4-8].

How GnRH receptor-mediated actions lead to alteration
of Smads expression and activation of Smad3 remains to
be elucidated. However, interaction and crosstalk with
components of other signaling pathways such as PKC,
MAPK and calcium/calmodulin (Ca2+/CaM), that are acti-
vated by GnRH and TGF-P receptors can influence Smad
pathway [33,47,54-60]. For instance, Smad3 has been
shown to serve as a substrate for ERK2 [57,61], while
Ca2+/CaM alter receptor activated Smad functions [59].
Since co-treatment of HES and ESC with GnRH antago-
nized TGF-P action on Smad3 activation, it is quite possi-
ble that a crosstalk between GnRH and TGF-P receptor
system is operational in HES and ESC. Such interaction
between GnRH receptors signaling and crosstalk with
Smad was demonstrated in the experiment using TGF-P
receptor antisense which blocked/reduced TGF-P auto-
crine/paracrine action.

In conclusion, the results provide further evidence for the
direct action of GnRH in human endometrium. Specifi-
cally, we demonstrated that GnRH alters the expression
and activation of Smads in endometrial epithelial and
stromal cells, suggesting a functional interaction between
GnRH and TGF-P receptor signaling pathways, a mecha-
nism that could alter the endometrial response to TGF-P.
TGF-P is known to promote cell growth and differentia-
tion, migration, invasion, angiogenesis, and extracellular
matrix turnover, processes that influence the outcome of
embryo implantation, endometriosis implants, endome-
triosis-associated adhesions and endometrial cancer.

Acknowledgment
Presented in part at the 49th Annual Meeting of the Society for Gynecolog-
ical Investigation, Los Angeles CA, March 2002. Supported in part by a grant
HD37432 from the National Institute of Health

References
I. Takeuchi H, Kobori H, Kikuchi I, Sato Y, Mitsuhashi N: A prospec-
tive randomized study comparing endocrinological and clin-
ical effects of two types of GnRH agonists in cases of uterine
leiomyomas or endometriosis. J Obstet Gynoecol Res 2000,
26:325-331.
2. Grimbizis G, Tsalikis T, Tzioufa V, Kasapis M, Mantalenakis S: Regres-
sion of endometrial hyperplasia after treatment with the
gonadotrophin-releasing hormone analogue triptorelin: a
prospective study. Hum Reprod 1999, 14:479-484.
3. Burton JL, Wells M: Recent advances in the histopathology and
molecular pathology of carcinoma of the endometrium. His-
topathology 1998, 33:297-303.
4. Devroey P, Pados G: Preparation of endometrium for egg
donation. Hum Reprod Update 1998, 4:856-861.
5. Hernandez ER: Embryo implantation and GnRH antagonists:
embryo implantation: the Rubicon for GnRH antagonists.
Hum Reprod 2000, 15:121 I -1216.




Page 11 of 13
(page number not for citation purposes)








Reproductive Biology and Endocrinology 2003, 1


6. Kol S: Embryo implantation and GnRH antagonists: GnRH
antagonists in ART: lower embryo implantation? Hum Reprod
2000, 15:1881-1882.
7. Olivennes F, Cunha-FilhoJS, Fanchin R, Bouchard P, Frydman R: The
use of GnRH antagonists in ovarian stimulation. Hum Reprod
Update 2000, 8:279-290.
8. Bourgain C, Ubaldi F, Tavaniotou A, Smitz J, Van Steirteghem AC,
Devroey P: Endometrial hormone receptors and proliferation
index in the periovulatory phase of stimulated embryo trans-
fer cycles in comparison with natural cycles and relation to
clinical pregnancy outcome. Fertil Steril 2002, 78:237-244.
9. Chegini N, Rong H, Dou Q, Kipersztok S, Williams RS: Gonadotro-
pin releasing hormone (GnRH) and GnRH receptor gene
expression in human myometrial and leiomyomta and the
direct action of GnRH analogs on myometrial smooth mus-
cle cells interaction with ovarian steroids in vitro. Clin Endo-
crinolMetab 1996, 81:3215-3221.
10. Raga F, Casan EM, Kruessel JS, Wen Y, Huang HY, Nezhat C, Polan
ML: Quantitative gonadotropin-releasing hormone gene
expression and immunohistochemical localization in human
endometrium throughout the menstrual cycle. Biol Reprod
1998, 59:661-669.
I I. Dong KW, Marcelin K, Hsu MI, Chiang CM, Hoffman G, Roberts JL:
Expression of gonadotropin-releasing hormone (GnRH)
gene in human uterine endometrial tissue. Mol Hum Reprod
1998, 4:893-898.
12. Cheon KW, Lee HS, Parhar IS, Kang IS: Expression of the second
isoform of gonadotrophin-releasing hormone (GnRH-II) in
human endometrium throughout the menstrual cycle. Mol
Hum Reprod 2001, 7:447-452.
13. Grundker C, Gunthert AR, Millar RP, Emons G: Expression of
gonadotropin-releasing hormone II (GnRH-II) receptor in
human endometrial and ovarian cancer cells and effects of
GnRH-II on tumor cell proliferation.j Clin Endocrinol Metab 2002,
87:1427-1430.
14. Dou Q, Zhao Y, Tarnuzzer RW, Rong H, Williams RS, Schultz GS,
Chegini N: Suppression of TGF-ps and TGF-p receptors
mRNA and protein expression in leiomyomata in women
receiving gonadotropin releasing hormone agonist therapy.
J Clin Endocrinol Metab 1996, 81:3222-3230.
15. Dou Q, Tarnuzzer RW, Williams RS, Schultz GS, Chegini N: Differ-
ential expression of matrix metalloproteinases and their tis-
sue inhibitors in leiomyomata: a mechanism for
gonadotrophin releasing hormone agonist-induced tumour
regression. Mol Hum Reprod 1997, 3:1005-1014.
16. Vignali M: Molecular action of GnRH analogues on ectopic
endometrial cells. Gynecol Obstet Invest 1998, 45(Suppl 1):2-5.
17. Raga F, Casan EM, Wen Y, Huang HY, Bonilla-Musoles F, Polan ML:
Independent regulation of matrix metalloproteinase-9, tis-
sue inhibitor of metalloproteinase-l (TIMP-1), and TIMP-3
in human endometrial stromal cells by gonadotropin-releas-
ing hormone: implications in early human implantation.J Clin
EndocrinolMetab 1999, 84:636-642.
18. Imai A, Takagi A, Tamaya T: Gonadotropin-releasing hormone
analog repairs reduced endometrial cell apoptosis in
endometriosis in vitro. AmJ Obstet Gynecol 2000, 182:1142-1146.
19. Grundker C, Schlotawa L, Viereck V, Emons G: a Protein kinase C-
independent stimulation of activator protein-I and c-Jun N-
terminal kinase activity in human endometrial cancer cells
by the LHRH agonist triptorelin. Eur j Endocrinol 2001,
145:651-658.
20. Grundker C, Volker P, Emons G: Antiproliferative signaling of
luteinizing hormone-releasing hormone in human endome-
trial and ovarian cancer cells through G protein a (I)-medi-
ated activation of phosphotyrosine phosphatase. Endocrinology
2001, 142:2369-2380.
21. Chegini N, Tang XM, Ma C, Williams RS: The effects of gonado-
tropin releasing hormone analogues, add-back, antiestrogen
and antiprogestins on leiomyoma and myometrial smooth
muscle cells growth and transforming growth factor beta
expression. Mol Hum Reprod 2002, 12:1071 -1078.
22. Tang XM, Zhao Y, Rossi MJ, Abu-Rustum RS, Ksander GA, Chegini N:
Expression of transforming growth factor-beta (TGF p) iso-
forms and TGF p type II receptor messenger ribonucleic acid
and protein, and the effect of TGF ps on endometrial stro-


mal cell growth and protein degradation in vitro. Endocrinology
1994, 35:450-459.
23. Casslen B, Sandberg T, Gustavsson B, Willen R, Nilbert M: Trans-
forming growth factor betal in the human endometrium.
Cyclic variation, increased expression by estradiol and pro-
gesterone, and regulation of plasminogen activators and
plasminogen activator inhibitor-1. Biol Reprod 1998,
58:1343-1350.
24. Sandberg T, Casslen B, Gustavsson B, Benraad TJ: Human endothe-
lial cell migration is stimulated by urokinase plasminogen
activator:plasminogen activator inhibitor I complex
released from endometrial stromal cells stimulated with
transforming growth factor betal; possible mechanism for
paracrine stimulation of endometrial angiogenesis. Biol Reprod
1998, 59:759-767.
25. Ma C, Chegini N: Regulation of matrix metalloproteinases
(MMPs) and their tissue inhibitors in human myometrial
smooth muscle cells by TGF-p 1. Mol Hum Reprod 1999,
10:950-954.
26. Chegini N, Williams RS: Implication of growth factor and
cytokine networks in endometrium. In Cytokines in human
reproduction Edited by: Hill J. Wiley & Sons Publisher. New York;
2000:92-132.
27. Blobe GC, Schiemann WP, Lodish HF: Role of transforming
growth factor beta in human disease. N Engl ] Med 2000,
342:1350-1358.
28. Chegini N, Gold LI, Williams RS: Localization of transforming
growth factor beta isoforms TGF-p I, TGF-p2, and TGF-p3 in
surgically induced endometriosis in the rat. Obstet Gynecol
1994, 83:455-461.
29. Oosterlynck DJ, Meuleman C, Waer M, Koninckx PR: Transform-
ing growth factor-beta activity is increased in peritoneal fluid
from women with endometriosis. Obstet Gynecol 1994,
83:287-292.
30. Gold LI, Saxena B, Mittal KR, Marmor M, Goswami S, Nactigal L, Korc
M, Demopoulos RI: Increased expression of transforming
growth factor beta isoforms and basic fibroblast growth fac-
tor in complex hyperplasia and adenocarcinoma of the
endometrium: evidence for paracrine and autocrine action.
Cancer Res 1994, 54:2347-2358.
31. Bruner KL, Rodgers WH, Gold LI, Korc M, Hargrove JT, Matrisian
LM, Osteen KG: Transforming growth factor beta mediates
the progesterone suppression of an epithelial metalloprotei-
nase by adjacent stroma in the human endometrium. Proc
NatlAcad Sci USA 1995, 92:7362-7366.
32. Lea RG, Underwood J, Flanders KC, Hirte H, Banwatt D, Finotto S,
Ohno I, Daya S, Harley C, Michel M: A subset of patients with
recurrent spontaneous abortion is deficient in transforming
growth factor beta-2-producing "suppressor cells" in uterine
tissue near the placental attachment site. Am Reprod Immunol
1995, 34:52-64.
33. Dennler S, Goumans MJ, ten Dijke P: Transforming growth factor
beta signal transduction. J Leukoc Biol 2002, 71:731-740.
34. Luo X, Xu J, Chegini N: The expression of Smads in human
endometrium and regulation and induction in endometrial
epithelial and stromal cells by transforming growth factor-
beta. J Clin Endocrinol Metab 2003, 88:4967-4976.
35. Yamada Y, Yamamoto H, Yonehara T, Kanasaki H, Nakanishi H, Miya-
moto E, Miyazaki K: Differential activation of the luteinizing
hormone {beta}-subunit promoter by activin and gonadotro-
pin-releasing hormone: A role for the mitogen-activated
protein kinase signaling pathway in L{beta}T2 gonadotrophs.
Biol Reprod 2003. [Epub ahead of print]
36. Ellsworth BS, Burns AT, Escudero KW, Duval DL, Nelson SE, Clay
CM: The gonadotropin releasing hormone (GnRH) receptor
activating sequence (GRAS) is a composite regulatory ele-
ment that interacts with multiple classes of transcription
factors including Smads, AP-I and a forkhead DNA binding
protein. Mol Cell Endocrinol 2003, 206:93- III.
37. Norwitz ER, Xu S, Xu J, Spiryda LB, Park JS, Jeong KH, McGee EA,
Kaiser UB: Direct binding of AP-I (Fos/Jun) proteins to a
SMAD binding element facilitates both gonadotropin-releas-
ing hormone (GnRH)- and activin-mediated transcriptional
activation of the mouse GnRH receptor gene. Biol Chem 2002,
277:37469-37478.




Page 12 of 13
(page number not for citation purposes)


hftp://www. rbej. co m/co nte nt/l/l/l125








Reproductive Biology and Endocrinology 2003, 1




38. Norwitz ER, Xu S, Jeong KH, Bedecarrats GY, Winebrenner LD, Chin
WW, Kaiser UB: Activin A augments GnRH-mediated tran-
scriptional activation of the mouse GnRH receptor gene.
Endocrinology 2002, 143:985-997.
39. Xu J, Luo X, Chegini N: Differential expression, regulation, and
induction of Smads, transforming growth factor-beta signal
transduction pathway in leiomyoma, and myometrial
smooth muscle cells and alteration by gonadotropin-releas-
ing hormone analog. j Clin Endocrinol Metab 2003, 88:1350-1361.
40. Chegini N, Rossi Mj, Masterson BJ: Platelet-derived growth fac-
tor (PDGF), epidermal growth factor (EGF), and EGF and
PDGF beta-receptors in human endometrial tissue: localiza-
tion and in vitro action. Endocrinology 1992, 130:2373-2385.
41. Kraus S, Naor Z, Seger R: Intracellular signaling pathways medi-
ated by the gonadotropin-releasing hormone (GnRH)
receptor. Arch Med Res 2001, 32:499-509.
42. Leung PC, Cheng CK, Zhu XM: Multi-factorial role of GnRH-I
and GnRH-II in the human ovary. Mol Cell Endocrinol 2003,
202:145-153.
43. Millar RP: GnRH II and type II GnRH receptors. Trends Endocrinol
Metab 2002, 14:35-43.
44. Afrakhte M, Moren A, Jossan S, Itoh S, Sampath K, Westermark B,
Heldin CH, Heldin NE, ten Dijke P: Induction of inhibitory Smad6
and Smad7 mRNA by TGF-beta family members. Biochem Bio-
phys Res Commun 1998, 249:505-51 I.
45. Massague J, Wotton D: Transcriptional control by the TGF-p/
Smad signaling system. EMBOJ 2000, 19:1745-1754.
46. Stopa M, Anhuf D, Terstegen L, Gatsios P, Gressner AM, Dooley S:
Participation of Smad2, Smad3, and Smad4 in transforming
growth factor beta (TGF-p)-induced activation of Smad7.
The TGF-p response element of the promoter requires func-
tional Smad binding element and E-box sequences for tran-
scriptional regulation. Biol Chem 2000, 275:29308-29317.
47. Garcia-Montero AC, Vasseur S, Giono LE, Canepa E, Moreno S,
Dagorn JC, lovanna JL: Transforming growth factor beta-I
enhances Smad transcriptional activity through activation of
p38 gene expression. Biochem 2001, 357:249-253.
48. Pouliot F, Labrie C: Expression profile of agonistic Smads in
human breast cancer cells: absence of regulation by
estrogens. Intj Cancer 1999, 81:98-103.
49. Brodin G, ten Dijke P, Funa K, Heldin CH, Landstrom M: Increased
smad expression and activation are associated with apopto-
sis in normal and malignant prostate after castration. Cancer
Res 1999, 59:2731-2738.
50. Matsuda T, Yamamoto T, Muraguchi A, Saatcioglu F: Cross-talk
between transforming growth factor-beta and estrogen
receptor signaling through Smad3. j Biol Chem 2001,
276:42908-42914.
51. Ott TR, Troskie BE, Roeske RW, Illing N, Flanagan CA, Millar RP:
Two mutations in extracellular loop 2 of the human GnRH
receptor convert an antagonist to an agonist. Mol Endocrinol
2002, 16:1079-1088.
52. Sun YM, Flanagan CA, Illing N, Ott TR, Sellar R, Fromme B, Hapgood
J, Sharp P, Sealfon SC, Millar RP: A chicken gonadotropin-releas-
ing hormone receptor that confers agonist activity to mam-
malian antagonists. Identification of D-Lys(6) in the ligand
and extracellular loop two of the receptor as determinants.
j Biol Chem 2001, 276:7754-7761.
53. Everest HM, HislopJN, Harding T, UneyJB, Flynn A, Millar RP, McAr-
die CA: Signaling and antiproliferative effects mediated by
GnRH receptors after expression in breast cancer cells using
recombinant adenovirus. Endocrinology 2001, 142:4663-4672.
54. Schiffer M, Bitzer M, Roberts IS, Kopp JB, ten Dijke P, Mundel P, Bot-
tinger EP: Apoptosis in podocytes induced by TGF-p and
Smad7. j Clin Invest 200 108:807-816.
55. Landstrom M, Heldin NE, Bu S, Hermansson A, Itoh S, ten Dijke P,
Heldin CH: Smad7 mediates apoptosis induced by
transforming growth factor beta in prostatic carcinoma
cells. Curr Biol 2000, 10:535-538.
56. Chegini N, Kornberg L: Gonadotropin releasing hormone ana-
logue (GnRHa) therapy alters signal transduction pathways
involving MAP and focal adhesion kinases in leiomyoma.j Soc
Gynecol Investig 2003, 10:21-26.
57. Yue J, Mulder KM: Requirement of Ras/MAPK pathway activa-
tion by transforming growth factor beta for transforming


http://www.rbej.com/content/1/1/125


growth factor beta I production in a Smad-dependent
pathway. j Biol Chem 2000, 275:30765-30773.
58. Zimmerman CM, Padgett RW: Transforming growth factor beta
signaling mediators and modulators. Gene 2000, 249:17-30.
59. Scherer A, Graff JM: Calmodulin differentially modulates
Smad I and Smad2 signaling. Biol Chem 2000, 275:41430-41438.
60. Yakymovych I, Ten Dijke P, Heldin CH, Souchelnytskyi S: Regulation
of Smad signaling by protein kinase C. FASEB j 2001,
I5:553-555.
61. Kretzschmar M, Doody Timokhina I, Massague j: A mechanism of
repression of TGFb/Smad signaling by oncogenic Ras. Genes
Dev 1999, 13:804-816.


Page 13 of 13
(page number not for citation purposes)


Publish with BioMed Central and every
scientist can read your work free of charge
"BioMed Central will be the most significant development for
disseminating the results of biomedical research in our lifetime."
Sir Paul Nurse, Cancer Research UK
Your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
cited in PubMed and archived on PubMed Central
yours you keep the copyright

Submit your manuscript here: BioMedcentral
http://www.biomedcentral.com/info/publishingadv.asp




University of Florida Home Page
© 2004 - 2010 University of Florida George A. Smathers Libraries.
All rights reserved.

Acceptable Use, Copyright, and Disclaimer Statement
Last updated October 10, 2010 - - mvs