help button home button Endocrine Society Molecular Endocrinology ENDO 08 Sessions Library
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Molecular Endocrinology, doi:10.1210/me.2006-0125
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
20/12/3336    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pon, Y. L.
Right arrow Articles by Wong, A. S. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pon, Y. L.
Right arrow Articles by Wong, A. S. T.
Molecular Endocrinology 20 (12): 3336-3350
Copyright © 2006 by The Endocrine Society

Gonadotropin-Induced Apoptosis in Human Ovarian Surface Epithelial Cells Is Associated with Cyclooxygenase-2 Up-Regulation via the ß-Catenin/T-Cell Factor Signaling Pathway

Yuen Lam Pon and Alice S. T. Wong

Department of Zoology, University of Hong Kong, Hong Kong

Address all correspondence and requests for reprints to: Alice S. T. Wong, 4S-14 Kadoorie Biological Sciences Building, Department of Zoology, University of Hong Kong, Pokfulam Road, Hong Kong. E-mail: awong1{at}hku.hk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Gonadotropins play a prominent role in ovarian function and pathology. We have shown that treatment with gonadotropins (FSH and LH/human chorionic gonadotropin) reduces the amount of N-cadherin with a concomitant induction of apoptosis in human ovarian surface epithelial (OSE) cells, but precise molecular mechanisms remain to be elucidated. Here, we demonstrated activation of ß-catenin/T-cell factor (TCF) signaling by gonadotropins. We further showed that ectopic expression of N-cadherin was sufficient to recruit ß-catenin to the plasma membrane, thereby blocking ß-catenin/TCF-mediated transactivation in gonadotropin-treated cells. Transfection with ß-catenin small interfering RNA or expression of dominant negative TCF inhibited apoptosis, whereas expression of dominant stable ß-catenin (S37A) caused significant apoptosis, thus supporting a proapoptotic role for ß-catenin/TCF in human OSE. In addition, we showed that gonadotropins enhanced ß-catenin/TCF transcriptional activity through inactivation of glycogen synthase kinase-3ß in a phosphatidylinositol 3-kinase/Akt-dependent manner, indicating cross talk between the phosphatidylinositol 3-kinase/Akt and ß-catenin signaling pathways through glycogen synthase kinase-3ß. Furthermore, gonadotropins increased cyclooxygenase-2 (COX-2) expression via the ß-catenin/TCF pathway. COX-2 also played a role in gonadotropin-induced apoptosis, as treatment with the COX-2-specific inhibitor NS-398 or COX-2 small interfering RNA blocked gonadotropin-dependent apoptotic activity. These findings suggest that the participation of ß-catenin in adhesion and signaling may represent a novel mechanism through which gonadotropins may regulate the cellular fate of human OSE.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
EPITHELIAL OVARIAN CARCINOMAS are thought to arise in the ovarian surface epithelium (OSE), which is a simple squamous-to-cuboidal cell layer covering the ovary. In vivo, this epithelium undergoes programmed cell death before ovulation and rapidly proliferates to repair the surface of the ovulatory follicle after ovulation (1, 2). This process, which involves the disruption and reestablishment of epithelial cell-cell contacts, changes with the reproductive cycle; thus, it is likely to be hormone dependent. In the human ovary, N-cadherin is the major adhesion protein. Our recent study shows that disruption of N-cadherin-mediated cell-cell adhesion is an important molecular event in the apoptosis of human OSE, and gonadotropins [FSH and LH/human chorionic gonadotropin (hCG)] are crucial regulators in this process (3). In the cytoplasm, cadherin is linked to the actin cytoskeleton via ß-catenin (4). In addition to a structural role in mediating cell-cell adhesion, ß-catenin is part of the Wnt signaling pathway. Whether there is biological significance and relevance of the dual activity of ß-catenin in human OSE is an issue that we attempted to address.

Because cadherins bind ß-catenin tightly and stabilize it at the membrane, changes in cell-cell adhesion in model systems have been found to alter ß-catenin signaling, suggesting that cadherin may be a negative regulator of ß-catenin signaling. Disruption of adherens junctions releases ß-catenin from the adherens complex, thus making it available for nuclear import and subsequent increased transactivation of ß-catenin responsive genes (5), whereas forced expression of cadherins can antagonize ß-catenin signaling by binding and sequestering it from the nuclear signaling pool (6, 7, 8, 9, 10). However, most of these studies were done based on experimental manipulations of cadherin levels. Whether changes in endogenous cadherin expression under physiological conditions can similarly alter ß-catenin signaling is still largely unknown. Alternatively, there is evidence that loss of cadherin function is not associated with enhanced ß-catenin signaling (11, 12). For example, in the SW480 colorectal tumor cell line, the regulation of ß-catenin transcriptional activity can occur in an adhesion-independent manner (12). It is also possible that cadherins control survival via interactions with other receptor systems, including EGF receptor (13).

Stability of ß-catenin is a critical point in Wnt signaling that is regulated by many cytoplasmic proteins including glycogen synthase kinase-3ß (GSK3ß), Axin, and adenomatous polyposis coli. Stabilized ß-catenin translocates to the nucleus where it binds to members of the T cell factor (TCF)/lymphoid enhancer binding factor 1 (LEF1) transcription factor family and stimulates transcription of the target genes that direct cell fate, polarity, and proliferation (4). Deregulation of ß-catenin signaling has been detected in a number of malignancies, such as colon cancer, melanoma, hepatocellular carcinoma, ovarian cancer, breast cancer, and prostate cancer (4).

ß-Catenin has been shown to affect cell proliferation either positively or negatively, depending on the cell type. A direct implication of ß-catenin in apoptosis and cell cycle arrest has been demonstrated in retinal and wing development of Drosophila (14, 15). Forced expression of ß-catenin induces apoptosis in normal fibroblasts, epidermal keratinocytes, and tumor cells (16, 17, 18). Conversely, there are reports showing that ß-catenin functions as an oncogene by promoting cell proliferation and/or protecting cells from apoptosis (19, 20, 21, 22).

In this report, we extend our studies on the mechanisms of gonadotropin regulation of apoptosis in human OSE and examine the effect of gonadotropins on interactions between N-cadherin and components of Wnt signaling pathway. Our results suggest a novel role for gonadotropins to modulate the participation of ß-catenin in adhesion and signaling. In addition, the effect of ß-catenin/TCF on the apoptotic cell death of OSE may involve cyclooxygenase-2 (COX-2). These findings represent a novel and potentially important mechanism underlying the effects of gonadotropins on OSE.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Gonadotropin Treatment Induces Redistribution of ß-Catenin
ß-Catenin interacts directly with N-cadherin in adhesion complexes (4). Therefore, down-regulation of N-cadherin in response to gonadotropins might result in the redistribution of membrane-localized ß-catenin. To test this hypothesis, OSE cells were treated with or without recombinant human FSH (0.4 IU/ml) and hCG (1 IU/ml), and the subcellular localization of ß-catenin was determined by cell fractionation analyses using a detergent-free/hypotonic lysis method as described in Materials and Methods and analyzed by Western blot. As previously reported (3), FSH and hCG treatment led to a dramatic decrease in the levels of N-cadherin protein (Fig. 1AGo). Similar results were obtained with purified LH (data not shown). In contrast, however, the total levels of ß-catenin were largely unaltered. Interestingly, although the expression level of ß-catenin was not affected, the amount of ß-catenin in the membrane fraction was reduced, correlating with the removal of N-cadherin from the plasma membrane (Fig. 1BGo). This was accompanied by a corresponding cytosolic and nuclear accumulation of ß-catenin (Fig. 1Go, C and D). The nuclear fraction contained little or no membrane contaminants, as indicated by the absence of N-cadherin (data not shown). Together, these results show the redistribution of ß-catenin from the plasma membrane to the cytoplasm and nucleus.


Figure 1
View larger version (47K):
[in this window]
[in a new window]
  		Fig. 1. Gonadotropins Induce Redistribution of ß-Catenin

Cell fractionation analysis was carried out in OSE cells either untreated or treated with recombinant FSH (0.4 IU/ml) or hCG (1 IU/ml) for 24 h. The total cell lysate (A), membrane pellet (B), cytosol (C), and nuclear fraction (D) of control and treated cells were subjected to standard SDS-PAGE and immunoblot analysis with specific anti-N-cadherin or anti-ß-catenin antibodies. Immunoblots were reprobed with ß-actin antibodies to confirm equal loading of total, membrane, and cytosolic proteins, and with histone H1 antibodies for nuclear proteins. Right panel, Immunoblots were quantified by densitometry and expressed as the ratio of ß-catenin relative to ß-actin or histone H1 for each sample. Data points shown represent the mean, and the error bars represent SD (n =3). The asterisk indicates significant difference from control cells with P < 0.05.

 
Gonadotropins Activate Downstream ß-Catenin/TCF-Dependent Transcription
Cytosolic/nuclear ß-catenin binds and activates transcription factors of the TCF/LEF family, which in turn regulate target genes by binding to TCF/LEF elements within the promoter of the gene (4). To determine whether gonadotropins were able to stimulate TCF/LEF transcription, OSE cells were transfected with a TCF reporter plasmid encoding multimerized wild-type (TOPflash) or mutant (FOPflash) TCF binding sites followed by a luciferase reporter gene (23). As a control for transfection efficiency, a simian virus 40-driven ß-galactosidase cDNA was cotransfected. Relative luciferase activities were calculated as described in Materials and Methods. As shown in Fig. 2AGo, exposure to FSH or hCG increased the transcriptional activity of TOPflash by approximately 1.5-fold as compared with untreated control cells (P < 0.05).


Figure 2
View larger version (40K):
[in this window]
[in a new window]
  		Fig. 2. Activation of ß-Catenin/TCF Transcription in OSE

A, OSE cells were transfected with 0.5 µg of either the TOPflash or FOPflash luciferase reporter constructs containing wild-type and mutated TCF binding sites, respectively, together with 5 ng of ß-galactosidase for normalization of transfection efficiency. Four hours after transfection, cells were treated for 24 h with recombinant FSH or hCG. B, TOPflash or FOPflash reporter constructs were transiently transfected with empty vector (pcDNA3) or vector expressing wild-type N-cadherin (N-cad) into OSE cells. Luciferase and ß-galactosidase activities were measured. Values are normalized luciferase activity (TOPflash activity minus the activity devoted to FOPflash and normalized to the ß-galactosidase activity) and are shown as mean ± SD of three independent experiments performed in triplicate. Inset, The expression of ectopic protein is shown by immunoblotting of total cell lysates of transfected OSE cells using antibodies to N-cadherin. ß-Actin served as a loading control. C, Left panel, OSE cells were transfected with 0.5 µg of N-cadherin constructs or empty vector (pcDNA3) as a control and were posttreated with vehicle, FSH (0.4 IU/ml), or hCG (1 IU/ml) for 24 h. The total cell lysate, membrane, cytosolic, and nuclear proteins were separated by standard SDS-PAGE and immunoblotted with anti-ß-catenin antibody. Immunoblots were reprobed with ß-actin antibodies to confirm equal loading of total, membrane, and cytosolic proteins, and with histone H1 antibodies for nuclear proteins. Right panel, Immunoblots were quantified by densitometry and expressed as the ratio of ß-catenin relative to ß-actin or histone H1 for each sample. *, P < 0.05 vs. control; a, P < 0.05 vs. FSH treatment in the absence of N-cadherin; b, P < 0.05 vs. hCG treatment in the absence of N-cadherin.

 
Because accumulation of ß-catenin in a cadherin-free pool correlates with TCF-induced activation of transcription, we postulated that the gonadotropin effects on N-cadherin could modulate ß-catenin. We therefore transiently transfected OSE cells with the TOPflash reporter and N-cadherin expression vector. Figure 2BGo shows that the expression of ectopic N-cadherin dramatically inhibited both FSH- and hCG-mediated activation of the TCF reporter. The empty vector used had no inhibitory effect. Overexpression of N-cadherin was confirmed by Western blot analysis (Fig. 2BGo, inset). Next, we assessed the effects of N-cadherin expression on the localization of ß-catenin. As shown in Fig. 2CGo, the expression of ectopic N-cadherin, which resulted in increased membrane-localized N-cadherin, was able to sequester ß-catenin in the cell membrane and inhibited gonadotropin-induced increase in cytosolic/nuclear ß-catenin without significant changes in total levels of ß-catenin. These results suggest that alterations in N-cadherin expression can modulate ß-catenin/TCF signaling by regulating the intracellular localization of ß-catenin in OSE cells.

To provide direct evidence that ß-catenin/TCF signaling was a key component of the gonadotropin-induced apoptosis, we asked whether expression of ß-catenin small interfering RNA (siRNA) or a dominant negative form of TCF known to inhibit ß-catenin/TCF nuclear signaling (24) could inhibit apoptosis in OSE cells. Endogenous ß-catenin protein levels were efficiently and specifically reduced in the presence of ß-catenin siRNA but not with the control nonspecific siRNA (Fig. 3AGo). ß-Catenin siRNA, as well as dominant negative TCF, resulted in a significant decrease in gonadotropin-induced ß-catenin/TCF transcriptional activity (Fig. 3BGo) and apoptosis (Fig. 3CGo). No inhibition was observed for nonspecific siRNA (Fig. 3Go). The ability of ß-catenin siRNA to inhibit apoptosis was examined by transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) (Fig. 3CGo, upper panel) as well as by counting 4',6-diamidino-2-phenylindole (DAPI)- (Fig. 3CGo, lower panel) or Hoechst- (data not shown) stained cells with condensed nuclei, and a decrease in gonadotropin-induced apoptosis was observed. Conversely, we asked whether expression of a stable mutant of ß-catenin (S37A), which cannot be phosphorylated and degraded by the proteosome complex, could promote apoptosis in OSE cells. Because of the presence of a binding epitope, expression of green fluorescent protein (GFP) by anti-GFP antibody and the exogenous GFP-tagged protein (119 kDa) by anti-ß-catenin was clearly demonstrable in transfected clones, whereas expression was not evident in the empty vector control cells (Fig. 4AGo). The expression of S37A ß-catenin was further confirmed by significant increases in TCF transcriptional activity as compared with controls (Fig. 4BGo). Expression of S37A increased the fraction of apoptotic cells to 19% by counting GFP-positive cells that were also positive for the TUNEL test after transient transfection (Fig. 4CGo). Further flow cytometry analysis showed that expression of S37A resulted in the accumulation of cells at G2/M (from 24.7–32.5%) and a significant increase in the percentage of apoptotic cells (from 7–21.8%) (P < 0.05) (Fig. 4DGo). This magnitude of change was comparable with the fold induction by gonadotropin treatment (3). These observations clearly demonstrate a critical role of ß-catenin/TCF signaling in regulating OSE cell survival and apoptosis.


Figure 3
View larger version (41K):
[in this window]
[in a new window]
  		Fig. 3. Inhibition of ß-Catenin Nuclear Signaling Prevents OSE from Undergoing Apoptosis

A, OSE expressing nonspecific (NS) siRNA or siRNA against ß-catenin were subjected to immunoblotting with specific anti-ß-catenin antibodies. Immunoblots were reprobed with ß-actin antibodies to confirm equal loading of proteins. B, Cells were transfected with either the TOPflash or FOPflash luciferase reporter, along with NS siRNA, specific ß-catenin siRNA, or dominant negative TCF. ß-Galactosidase vector was cotransfected for normalization of transfection efficiency. FSH (0.4 IU/ml) or hCG (1 IU/ml) was added for 24 h before harvest. Luciferase and ß-galactosidase activities were measured. Values are normalized luciferase activity (TOPflash activity minus the activity devoted to FOPflash and normalized to the ß-galactosidase activity) and are shown as mean ± SD of three independent experiments performed in triplicate. C, Left panel, Apoptosis was measured by TUNEL (upper panel) and DAPI (lower panel) staining and visualized by fluorescence microscopy. DAPI-stained cells exhibiting condensed, pyknotic, or fragmented nuclei were representative of apoptotic cells. Right panel, Data points shown represent the mean percentage of apoptotic cells counted in four fields in replicate wells from three independent experiments. Bars represent SD. *, P < 0.05 vs. control; a, P < 0.05 vs. FSH treatment in untransfected cells; b, P < 0.05 vs. hCG treatment in untransfected cells.

 

Figure 4
View larger version (38K):
[in this window]
[in a new window]
  		Fig. 4. Constitutively Active ß-Catenin Sensitizes OSE Cells to Apoptosis

A, OSE cells were transfected with empty vector (pEGFP) as a control or a transcriptionally active mutant of ß-catenin (S37A). Cellular protein was extracted and the presence of exogenous ß-catenin was analyzed by immunoblotting using anti-GFP antibodies (upper panel) or anti-ß-catenin antibodies (lower panel). Immunoblots were reprobed with ß-actin antibodies to confirm equal loading of proteins. B, Cells were transfected with either the TOPflash or FOPflash luciferase reporter, along with empty vector or S37A. ß-Galactosidase vector was cotransfected for normalization of transfection efficiency. Twenty-four hours after transfection, luciferase and ß-galactosidase activities were measured. Values are normalized luciferase activity (TOPflash activity minus the activity devoted to FOPflash and normalized to the ß-galactosidase activity) and are shown as mean ± SD of three independent experiments performed in triplicate. C, Apoptosis was measured by TUNEL (left panel) and DAPI staining, and visualized by fluorescence microscopy. Right panel, Data points shown represent the mean percentage of apoptotic cells counted in four fields in replicate wells from three independent experiments. Bars represent SD. The asterisk indicates significant differences from controls with P < 0.05. D, Cells expressing empty vector (pEGFP) or S37A were stained with propidium iodide, and representative example of cell cycle analyses by flow cytometry was shown. Sub-G1 peak corresponds to the apoptotic cells. Sizes of cell subpopulations are given as percentage of total populations.

 
The Effect of GSK3ß Phosphorylation on ß-Catenin Transcriptional Activity
Inactivation of GSK3ß as a result of phosphorylation at an N-terminal Ser9 residue has been shown to promote ß-catenin protein stability and transactivation activity (4). We therefore investigated whether gonadotropin-mediated change in the subcellular localization of ß-catenin would involve GSK3ß phosphorylation. As shown in Fig. 5AGo, gonadotropins promoted a significant increase in the Ser9 phosphorylated GSK3ß. Treatment with lithium chloride (LiCl), a pharmacological agent well known for its inhibitory effect on GSK3ß activity (25, 26) (Fig. 5AGo), enhanced the cytosolic expression of ß-catenin (2.5-fold) (Fig. 5BGo) and ß-catenin/TCF transcriptional activity (2.4-fold) (Fig. 5CGo) and resulted in a high rate of apoptosis (26%) (Fig. 5DGo). Similar results were obtained with another specific inhibitor of GSK3ß (SB415286) (data not shown). To further demonstrate a functional role of GSK3ß in mediating the effects of gonadotropins on OSE, we used a constitutively active form of GSK3ß (S9A-GSK3ß) in which Ser9 was replaced with alanine, thus preventing phosphorylation and inactivation of the kinase (27, 28). Inhibition of GSK3ß phosphorylation by S9A-GSK3ß transfection was confirmed by immunoblot analysis (Fig. 6AGo). Expression of S9A-GSK3ß reverted the effects of gonadotropins on ß-catenin expression (Fig. 6AGo) and TOPflash activity (Fig. 6BGo), indicating that GSK3ß inhibition was required for gonadotropin stimulation of ß-catenin/TCF-dependent transcription. We also observed that overexpression of S9A-GSK3ß abolished the effect of gonadotropins on apoptosis, as manifested by TUNEL and DAPI staining (Fig. 6CGo). These findings indicate a role for GSK3ß in the regulation of the ß-catenin signaling pathway in response to gonadotropins.


Figure 5
View larger version (30K):
[in this window]
[in a new window]
  		Fig. 5. GSK3ß Plays a Role in ß-Catenin-Dependent Apoptosis Induced by Gonadotropins

A, OSE cells were treated with recombinant FSH (0.4 IU/ml) or hCG (1 IU/ml) or incubated with 20 mM of LiCl. Levels of GSK3ß phosphorylated at Ser9 were analyzed by immunoblotting. Immunoblots were reprobed with nonphosphorylated GSK3ß antibodies to confirm equal loading of proteins. Right panel, Immunoblots were quantified by densitometry and expressed as the ratio of phosphorylated GSK3ß relative to total GSK3ß for each sample. B, Cells were incubated with 20 mM of LiCl or NaCl, which served as controls for 8 h. Cellular protein was extracted and cytosolic levels of ß-catenin were analyzed by immunoblotting. C, Cells were transiently transfected with either the TOPflash or FOPflash luciferase reporter and then treated with 20 mM LiCl or NaCl as control for 8 h. ß-Galactosidase vector was cotransfected for normalization of transfection efficiency. Luciferase and ß-galactosidase activities were measured. Values are normalized luciferase activity (TOPflash activity minus the activity devoted to FOPflash and normalized to the ß-galactosidase activity) and are shown as mean ± SD of three independent experiments performed in triplicate. D, Apoptosis was measured by TUNEL (left panel) and DAPI staining and visualized by fluorescence microscopy. Right panel, Data points shown represent the mean percentage of apoptotic cells counted in four fields in replicate wells from three independent experiments. Bars represent SD. The asterisk indicates significant differences from controls with P < 0.05.

 

Figure 6
View larger version (35K):
[in this window]
[in a new window]
  		Fig. 6. GSK3ß Inhibition Was Required for Gonadotropin Stimulation of ß-Catenin/TCF-Dependent Transcription

A, OSE cells were transfected with empty vector (pcDNA3) or constitutively active GSK3ß (S9A-GSK3ß). Four hours after transfection, cells were treated for 24 h with recombinant FSH or hCG. Levels of GSK3ß phosphorylated at Ser9 and cytosolic levels of ß-catenin were analyzed by immunoblotting. B, Cells were transiently cotransfected with either the TOPflash or FOPflash luciferase reporter. Values are normalized luciferase activity and are shown as mean ± SD of three independent experiments performed in triplicate. C, Apoptosis was measured by TUNEL (left panel) and DAPI staining and was visualized by fluorescence microscopy. Right panel, Data points shown represent the mean percentage of apoptotic cells counted in four fields in replicate wells from three independent experiments. Bars represent SD. The asterisk indicates significant differences from controls with P < 0.05.

 
Phosphorylation of GSK3ß by Gonadotropins Occurs via a Phosphatidylinositol 3-Kinase (PI3K)/Akt-Dependent Pathway
Akt inhibited GSK3ß activation by inducing its phosphorylation (29); therefore, we tested the involvement of the PI3K/Akt signaling pathway. Figure 7AGo shows that gonadotropins strongly stimulated the level of phosphorylation of Akt and phosphorylation of its downstream target GSK3ß. To establish that GSK3ß is a downstream substrate of Akt in the signaling activated by gonadotropins, we blocked Akt activity with the small molecule inhibitor of PI3K/Akt activity, LY294002 (Fig. 7AGo). Under these conditions, gonadotropins were unable to phosphorylate GSK3ß (Fig. 7AGo), thus showing a role for PI3K/Akt in GSK3ß inactivation induced by gonadotropins. The total level of Akt and GSK3ß were not affected by this incubation. No inhibitory effect was observed using the MAPK kinase 1 inhibitor PD98059 (data not shown).


Figure 7
View larger version (31K):
[in this window]
[in a new window]
  		Fig. 7. Inhibition of PI3K Blocks the Effects of Gonadotropins on Apoptosis of OSE Cells

A, Cells were preincubated for 30 min with or without 50 µM LY294002 in the presence or in the absence of FSH. Levels of Akt and GSK3ß phosphorylated at Ser473 and Ser9, respectively, were analyzed by immunoblotting. Immunoblots were reprobed with nonphosphorylated antibodies to confirm equal loading of proteins. B, Cells were transfected with either the TOPflash or FOPflash luciferase reporter, along with dominant negative Akt. In some experiments, cells were treated with 50 µM LY294002 or vehicle (dimethyl sulfoxide). ß-Galactosidase vector was cotransfected for normalization of transfection efficiency. FSH or hCG was added for 24 h before harvest. Luciferase and ß-galactosidase activities were measured. Values are normalized luciferase activity (TOPflash activity minus the activity devoted to FOPflash and normalized to the ß-galactosidase activity) and are shown as mean ± SD of three independent experiments performed in triplicate. C, Apoptosis was measured by TUNEL (left panel) and DAPI staining, and visualized by fluorescence microscopy. Right panel, Data points shown represent the mean percentage of apoptotic cells counted in four fields in replicate wells from three independent experiments. Bars represent SD. *, P < 0.05 vs. control; a, P < 0.05 vs. FSH treatment in the absence of PI3K/Akt inhibition; b, P < 0.05 vs. hCG treatment in the absence of PI3K/Akt inhibition.

 
Next, we investigated whether PI3K/Akt would play a role in the regulation of ß-catenin/TCF activity in a reporter gene assay. We observed a concomitant reduction in the transcriptional activity of the ß-catenin/TCF complex by the PI3K inhibitor (LY294002), as well as dominant negative Akt, correlating with the decrease in the percentage of apoptotic cells from about 20% to 5% (Fig. 7Go, B and C). Together, these results strongly implicate inactivation of GSK3ß by the PI3K/Akt pathway in the enhancement of ß-catenin/TCF-dependent apoptotic signaling in response to gonadotropins.

Induction of Apoptosis by ß-Catenin is Dependent on COX-2
Regulation of apoptosis by the ß-catenin/TCF signaling pathway raises the possibility that the effects of gonadotropins on apoptosis are mediated through the transcription of Wnt target genes. To explore the molecular mechanism for gonadotropin-induced apoptosis, we examined whether c-myc, a downstream target gene for ß-catenin and a known inducer of apoptosis (19, 30, 31), was up-regulated by FSH. However, no significant increase in c-myc levels was detected in these cells compared with untreated cells (data not shown). The level of the c-myc protein was also not affected by expression of activated ß-catenin (S37A) (data not shown). Therefore, we ruled out the possibility that the apoptotic effects of ß-catenin are caused by increases in the levels of c-myc.

Because COX-2, another potential target of ß-catenin and TCF (32), is a regulator of apoptosis (33) and a known local mediator of gonadotropin-stimulated ovulation (34), we next examined the possible involvement of COX-2 in the apoptotic effects of ß-catenin in human OSE. To study the role of COX-2 in the gonadotropin-induced cell death, we pretreated cells with NS-398, a specific inhibitor of COX-2. As shown in Fig. 8AGo, pretreatment of cells with NS-398 (10 µM) at concentration known to selectively inhibit COX-2 activity (35) blocked gonadotropin-induced apoptosis of OSE. The inhibitor alone exhibited no apparent cytotoxicity at this concentration. To further define the role of COX-2 induction by gonadotropins in the apoptosis of OSE cells, we chose to deplete COX-2 using siRNA. The efficacy of silencing COX-2 was determined by immunoblotting (Fig. 8BGo). Most importantly, reduced COX-2 expression with siRNA also markedly attenuated gonadotropin-induced apoptosis compared with cells transfected with nonspecific siRNA (Fig. 8CGo). These findings strongly suggest that COX-2 is a mediator of gonadotropin-induced apoptosis in human OSE.


Figure 8
View larger version (52K):
[in this window]
[in a new window]
  		Fig. 8. Gonadotropin-Induced Apoptosis Is COX-2 Dependent

A, OSE cells were pretreated with either vehicle or 10 µM NS-398 for 15 min before exposure to 0.4 IU/ml FSH or 1 IU/ml hCG for 24 h. Apoptosis was measured by TUNEL (left panel) and DAPI staining. Right panel, Data points shown represent the mean percentage of apoptotic cells counted in four fields in replicate wells from three independent experiments. B, Cells were transfected with 100 nM nonspecific (NS) and COX-2-specific siRNA before gonadotropin treatment. Effects on gonadotropin-induced COX-2 expression were measured by Western blot analysis. C, Apoptosis was measured by TUNEL (left panel) and DAPI staining and visualized by fluorescence microscopy. Right panel, Data points shown represent the mean percentage of apoptotic cells counted in four fields in replicate wells from three independent experiments. Bars represent SD. *, P < 0.05 vs. control; a, P < 0.05 vs. FSH treatment in the absence of COX-2 inhibition; b, P < 0.05 vs. hCG treatment in the absence of COX-2 inhibition.

 
Because COX-2 is a Wnt target gene (32), we investigated whether ß-catenin would play a role in the regulation of COX-2 in OSE cells. To test this hypothesis, the cells were treated with recombinant FSH or hCG in the absence or presence of the expression of ß-catenin siRNA. Figure 9AGo shows a significant increase in COX-2 levels when OSE cells were incubated with gonadotropins, and this induction of COX-2 expression was effectively suppressed by expression of ß-catenin siRNA but not nonspecific siRNA. Consistent with this finding, OSE cells that ectopically express transcriptionally active ß-catenin (S37A) and as well as treatment with LiCl had elevated levels of COX-2 (Fig. 9BGo), suggesting that COX-2 induction in OSE cells is mediated via the activation of ß-catenin/TCF signaling pathway.


Figure 9
View larger version (35K):
[in this window]
[in a new window]
  		Fig. 9. Gonadotropins Regulate the Levels of COX-2 in a ß-Catenin/TCF Dependent Manner

A, OSE cells transiently transfected with control nonspecific (NS) siRNA or specific ß-catenin siRNA were treated with or without FSH or hCG for 24 h and were investigated for the amount of COX-2. Immunoblots were reprobed with ß-actin antibodies to confirm equal loading of proteins. B, Lysates from OSE cells transiently transfected with either vector control or constitutively active ß-catenin (S37A) or cell treated with LiCl were investigated for the amount of COX-2. Immunoblots were reprobed with ß-actin antibodies to confirm equal loading of proteins. Right panel, Immunoblots were quantified by densitometry and expressed as the ratio of COX-2 relative to ß-actin for each sample. Data points shown represent the mean, and the error bars represent SD (n = 3). *, P < 0.05 vs. control; a, P < 0.05 vs. FSH treatment in untransfected cells; b, P < 0.05 vs. hCG treatment in untransfected cells.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Based on our findings from this and previous reports (3), we propose that gonadotropins can regulate OSE cell survival as a result of alterations in the expression and/or the distribution of ß-catenin. This can be achieved in two ways (Fig. 10Go): 1) from the release of ß-catenin from N-cadherin after a down-regulation of N-cadherin at the cell-cell contact; and 2) from the increase of free ß-catenin stability in the cytoplasm, which is mediated through the inhibition of GSK3ß after gonadotropin-mediated PI3K/Akt activation. To our knowledge, this is the first demonstration that gonadotropins can regulate ß-catenin signaling, thereby providing new insights into the role of these factors in the biology of the OSE. Regulation of ß-catenin by pituitary hormones has not been reported, although retinoids were shown to stabilize ß-catenin in breast cancer cells (36).


Figure 10
View larger version (11K):
[in this window]
[in a new window]
  		Fig. 10. Model for Signal Transduction Pathways Mediating Gonadotropin-Stimulated Apoptosis of OSE Cells

As shown in the model, down-regulation of N-cadherin induced by gonadotropins results in dissociation of ß-catenin from the adherens complex. Accumulation of ß-catenin in the nucleus leads to ß-catenin/TCF transactivation and induction of target genes, such as COX-2, through a GSK3ß-dependent manner. These events contribute to the activation of apoptosis in OSE.

 
The potential for gonadotropins to regulate OSE cell proliferation is suggested by the demonstration of receptors for these hormones in the OSE. Numerous studies have shown the expression of FSH and LH receptors in various normal (37, 38, 39, 40) and immortalized (40, 41, 42) OSE models. Moreover, levels of gonadotropin receptors seem to be retained in immortalized OSE cells, which exhibit similar responsiveness to gonadotropin stimulation as compared with normal OSE cells (40). However, the findings regarding the effect of FSH and LH/hCG on OSE are inconsistent. Whereas gonadotropins stimulated OSE proliferation in some studies (40, 41, 42, 43), they was noneffective or inhibitory in other cases (37, 44). The variations in hormone concentration used in different studies may account for the contradictory reports of FSH effects on OSE (3). Our findings are in line with a report showing that the growth-inhibitory effect of gonadotropins upon OSE is limited to high levels of FSH (44). It should also be pointed out that we tested the effects of FSH or LH/hCG on confluent OSE cultures (~1 x 105 cells per well of a 24-well plate), compared with the sparsely plated cells (1–2 x 104 cells) in other studies (40, 41, 42, 43). When we tested the effects of FSH and LH/hCG on N-cadherin-mediated apoptosis of sparsely plated OSE, we found no significant effect, even at very high hormone concentrations (data not shown). Conceivably, the high-density OSE cell culture might resemble the microenvironment of preovulatory OSE cells and, in response to FSH and LH/hCG, the OSE undergoes apoptosis, whereas the sparse or subconfluent OSE cells might mimic the ruptured OSE cells and gonadotropins may have different effects, including stimulation of the OSE proliferation. Such cell density-dependent effects have been observed in many other systems (45, 46). In particular, recent data indicate that cadherin-mediated signaling is essential for maintaining viability in dense cultures, and its disruption induces apoptosis (46). Changing cell densities did not alter the expression of gonadotropin receptors (data not shown), indicating that receptor changes were not responsible for these different responses. Similar observations are reported for aggregate and dispersed granulosa cells (47).

Free cytosolic ß-catenin, upon translocation into the nucleus, interacts with TCF family members. We found that ß-catenin consistently induced a 1.5-fold (P < 0.05) increase in TOPflash activity. This small increase in the transcriptional activation of the TOPflash reporter construct is in contrast to reports in other cell systems in which experimental or genetic manipulation of critical components of the Wnt pathway led to appreciable activity. However, the low level of ß-catenin signaling is not unprecedented and has been described in more physiological conditions of ß-catenin stabilization, such as glucocorticoid-mediated osteoblast differentiation (48) and during hair follicle lineage commitment and differentiation (49). Our data showing that blocking ß-catenin signaling by expressing a ß-catenin-specific siRNA resulted in significant inhibition of apoptosis, whereas the expression of a constitutive active ß-catenin promoted apoptosis, confirm an essential role of ß-catenin signaling in the regulation of OSE on apoptosis.

The positive involvement of ß-catenin in cellular apoptosis has also been supported by studies in different cell systems and some developmental context (14, 15, 16, 17, 18). Consistent with these findings, we observed that expression of stable ß-catenin is sufficient to induce apoptosis. Our results also support a role for ß-catenin in the regulation of the cell cycle (14, 15, 16, 18). However, whereas the apoptotic effect of ß-catenin may occur independent of its transactivation function with TCF of a few cell lines (17), our findings suggest that ß-catenin/TCF transcriptional activation contributes directly to OSE apoptosis because reduction in the activity of ß-catenin/TCF signaling using dominant negative TCF repressor prevented apoptosis in human OSE cells. In rat OSE, ß-catenin can promote apoptosis; however, constitutive activation of ß-catenin was reported not to be sufficient for mediating TCF-dependent transcription and inducing apoptosis (50). This variation, together with those from our previous work (3), suggests that growth regulatory signaling differ between the two species. Such differences might provide clues for their different susceptibility to developing epithelial ovarian cancer. In contrast to the humans, OSE-derived carcinomas are essentially nonexistent in rodents (51). A role for ß-catenin in apoptosis may also be functionally significant in the neoplastic transformation of OSE cells. Unlike most other tumors, E-cadherin is frequently up-regulated in ovarian tumors (52). The elevated expression of E-cadherin, which would reduce the free pool of cytoplasmic ß-catenin, in ovarian cancer could make the cells less susceptible to apoptosis and thereby favor tumor growth.

GSK3ß is a key component of the Wnt signaling pathway controlling ß-catenin levels and transcriptional responses. Here, we show that inhibition of GSK3ß (LiCl) led to an elevation of ß-catenin levels and subsequent activation of transcriptional activity. Of particular interest is our finding that the gonadotropin action on ß-catenin was negated not only by expression of constitutively active GSK3ß (S9A-GSK3ß), but also by inhibition of the PI3K/Akt pathway. The involvement of PI3K/Akt in the regulation of the ß-catenin signaling pathway has been described in other cell systems (53, 54), even though PI3K and Akt are not direct downstream effectors of the Wnt signal. This suggests that gonadotropins, like the Wnt signal, can activate the ß-catenin pathway by phosphorylating and inhibiting GSK3ß. Although GSK3ß might have been implied in tumor suppressor function because its inhibition leads to activation of the Wnt signaling pathway, GSK3ß can promote cancer cell proliferation and survival. Corroborating our current data, active GSK3ß has been shown to stimulate ovarian cancer cell growth and cyclin D1 expression may be involved in the regulation; however, the precise mechanisms still remain unclear (55).

Given a critical role of ß-catenin in cellular apoptosis, however, only a minority of the ß-catenin/TCF target genes described to date have been implicated as important factors in this process. Our data implicating COX-2 as a ß-catenin/TCF target gene in OSE are of particular interest. COX-2 has been shown to play an essential role in mediating the ovulatory response. In the ovary, gonadotropin surge before ovulation stimulates COX-2 expression (32, 56, 57, 58), and blockage of COX-2 activity using either gene knockout or enzymatic inhibitors resulted in the failure of ovulation (59, 60, 61). COX-2 induction is thought to be necessary for the rupture of the preovulatory follicle and subsequent release of oocyte during ovulation. As such, COX-2 has been shown to increase proteolytic activity and decrease synthesis of basement membrane components in the granulosa of growing follicles and OSE, permitting ovulation (62, 63). Our data show another mechanism by which COX-2 may facilitate ovulation is through the induction of apoptotic cell death of OSE. This seems paradoxical at first glance, because COX-2 expression is often thought to contribute to tumor cell proliferation and survival (64, 65). The discrepancy with previous reports may be explained by their differences in COX-2 expression. In contrast to other malignancies, ovarian tumor cells are found negative for COX-2 expression (66). It is not yet known why ovarian cancer cells inactivate their COX-2, but results emerging from this study suggest that it is possible that the loss of COX-2 expression may enable the ovarian tumor cells to escape apoptosis, which favors neoplastic transformation. In ovaries, studies on granulosa cells also demonstrate a role of COX-2 in promoting apoptosis (67). c-myc, another candidate ß-catenin/TCF-regulated gene that we have studied, has not shown similar activity in OSE cells, although c-myc functions as a potent inducer of apoptosis in certain circumstances (30, 31).

Although the precise underlying mechanisms responsible for the development of epithelial ovarian cancer remain unclear, various hypotheses have been proposed and, among them, incessant ovulation appears best related to this study. It is argued that incessant ovulation causes repeated minor trauma to the epithelial surface of the ovary and that the inflammatory reaction and mediators associated with the ovulatory process provide recurring oxidative stress that may be mutagenic (68). A recent report has demonstrated direct genotoxic effects of COX-2 (69). Similarly, in vivo, damaged DNAs are often found in the OSE surrounding the sites of ovulatory rupture, but they are not detected in extrinsic sites, greatly favoring the development of ovarian tumors.

In this study, we have demonstrated for the first time that gonadotropins have the capacity to regulate the dual role of ß-catenin, namely as an intercellular component of the cadherin adhesion complex and as a transcription factor, in determining the fate of OSE. Additionally, we identified COX-2 as a potentially important mediator of the apoptotic effect of ß-catenin in human OSE. These findings shed new light on the molecular mechanism underlying OSE survival and death, a process that is anticipated to have important implications in ovulation and ovarian cancer risk.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Reagents
Compounds added to culture experiments, including recombinant FSH (6650 IU/mg) and hCG (~6000 IU/mg) and purified LH (6100 IU/mg), were obtained from the National Hormone and Peptide Program (Harbor-UCLA Medical Center, Torrance, CA). LiCl was purchased from Sigma (St. Louis, MO), NS-398 was from Caymen (Ann Arbor, MI), and LY294002 and SB415286 were obtained from Calbiochem (San Diego, CA). An antibody against N-cadherin was from Zymed Laboratories Inc. (San Francisco, CA). The polyclonal phospho-specific antibodies to Akt (Ser473) and GSK3ß (Ser9) as well as polyclonal anti-Akt and GSK3ß were purchased from Cell Signaling, Inc (Austin, TX). Anti-COX-2 was from Caymen, and anti-ß-catenin was from Transduction Laboratories (Lexington, KY). Anti-histone H1 (clone AE-4) was from Santa Cruz Biotechnology. (Santa Cruz, CA). The monoclonal anti-c-myc antibody (clone 9E10) and polyclonal anti-ß-actin were from Sigma, and peroxidase-conjugated secondary antibodies were purchased from Bio-Rad (Hercules, CA).

Constructs
Human ß-catenin was first amplified from a cDNA library by PCR, and site-directed mutagenesis was then used to generate the activated mutant form of ß-catenin (S37A). The PCR product was confirmed by DNA sequencing and was subcloned into the KpnI and BamHI sites of the pEGFP expression vector (Clontech, Palo Alto, CA). siRNA oligonucleotide targeting ß-catenin mRNA (24), the SMART-pool siRNA for silencing COX-2 (catalog no. M-004557), and nonspecific RNA control were obtained from Dharmacon Research (Lafayette, CO). Dominant negative Akt, the TCF-binding site reporter plasmid (TOPflash), and a mutated control reporter (FOPflash) were purchased from Upstate Biotechnology (Lake Placid, NY). N-Cadherin expression vector and dominant negative TCF repressor were provided by Dr. Barry Gumbiner (University of Virginia, Charlottesville, VA). Constitutively active GSK3ß (S9A-GSK3ß) was a kind gift of Dr. James Woodgett (Ontario Cancer Institute, Toronto, Ontario, Canada).

Cell Culture and Transfection
The two immortalized nontumorigenic human OSE cell lines, IOSE-397 and IOSE-398, used in this study were kindly provided by Dr. Nelly Auersperg (University of British Columbia, Vancouver, British Columbia, Canada) (3, 70). It has been reported previously that FSH receptor is expressed in IOSE-398 cells (39), and the expression of FSH receptor and LH receptor in both cell lines used in this study were confirmed by RT-PCR (data not shown). Cells were grown in 1:1 media 199: MCDB105 supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 5% fetal bovine serum. Cultures were maintained at 37 C in a humidified incubator containing 95% room air and 5% CO2 atmosphere. Cells were seeded into a 24-well dish at 1 x 105 cells/well and were serum-starved 24 h before being treated with FSH (0.4 IU/ml; activity equivalent to concentration used in our previous report, Ref. 3) or LH/hCG (1 IU/ml). Transfections were performed using Lipofectamine reagent (Life Technologies, Inc., Gaithersburg, MD). Cells transfected with siRNA duplex oligos were incubated for 24 h before protein levels determination, TCF reporter gene assay, or apoptosis assay. To express cDNA constructs, 0.5 µg of plasmid DNA was used per 24-well plate.

TOPflash Luciferase Reporter Assays
In 24-well plates, cells were transiently transfected with 0.5 µg of the TOPflash or FOPflash reporter plasmid using Lipofectamine (Life Technologies). As a control for transfection efficiency, 5 ng of the ß-galactosidase construct was included in each transfection. Cells were harvested 24 h after transfection, and extracts were prepared in 200 µl of reporter lysis buffer (Promega, Madison, WI). Luciferase and ß-galactosidase activity were assayed according to the manufacturer’s protocol using the luciferase assay kit from Promega. Luciferase activity in each well was normalized to the ß-galactosidase activity. To allow easier comparison of the transcriptional activities, the background transcriptional activity represented by the FOPflash value was subtracted from the TOPflash value. All experiments were assayed in triplicate, and the assay was performed in three independent experiments.

Subcellular Fractionation
The purification of membrane, cytosolic, and nuclear fractions of ß-catenin was performed as described by Reinacher-Schick et al. (71). In brief, cells were washed once with PBS and scraped into a buffer containing 30 mM HEPES (pH 7.3) supplemented with protease inhibitors mixture. Cells were lysed by Dounce homogenization and then centrifuged at 500 x g for 5 min at 4 C to pellet unlysed cells and nuclei. The supernatant was centrifuged at 50,000 x g (Beckman TLA 120.2; Beckman Coulter, Fullerton, CA) for 60 min at 4 C to separate a soluble cytosolic fraction from a pellet containing membrane proteins. The pellet containing unlysed cells and nuclei were further fractionated by sucrose gradient centrifugation (72). After centrifugation at 10,000 x g for 60 min at 4 C, the resulting supernatant was considered the nuclear fraction. The total cell lysates, membrane pellet, cytosol, and nuclear fraction of control and treated cells were subjected to standard SDS-PAGE and immunoblot analysis.

Western Blot Analysis
Twenty micrograms of total proteins were separated by SDS-PAGE and transferred onto nitrocellulose membrane. The membrane was then incubated with antibodies specific for N-cadherin (1:1000), ß-catenin (1:5000), c-myc (1:1000), COX-2 (1:1000), phospho-Akt (1:1000), Akt (1:1000), phospho-GSK3ß (1:1000), GSK3ß (1:1000), ß-actin (1:1000), or histone H1 (1:100) overnight at 4 C and developed using enhanced chemiluminescence (Amersham Biosciences Inc., Piscataway, NJ). The density of the bands was quantified by densitometric analysis using an Image Tool (version 3.0) System.

Detection of Apoptosis
Cells were analyzed for apoptosis by TUNEL using a commercially available In Situ Cell Death Detection kit (Boehringer Mannheim, Indianapolis, IN) to find DNA strand breaks according to the manufacturer’s instructions, as described in previous reports (3). The number of TUNEL-positive cells was counted in four different fields, and representative fields were photographed. Each set of experiments was performed in replicate and was repeated three times, with at least 300 cells counted in each instance. To confirm the findings of TUNEL assay, apoptotic cells were detected by Hoechst 33258 (Molecular Probes, Eugene, OR) and DAPI (Vector Laboratories, Inc., Burlingame, CA) staining as reported previously (3). Apoptotic cells were identified by chromatin condensation and nuclei fragmentation. The percentage of apoptotic cells was calculated from the ratio of apoptotic cells to total cells counted.

Flow Cytometry
Cells were harvested, washed in PBS, fixed with 70% ethanol, and followed by DNA staining with propidium iodide (1 µg/ml) (Sigma). GFP-positive cells were gated and analyzed for cellular DNA content. Cell cycle analysis was performed on a Coulter flow cytometer (Beckman-Coulter, Inc.), and data were analyzed with WinMDI software.

Statistical Analysis
Treatment differences were analyzed by one-way ANOVA followed by Tukey’s least significant difference t test for post hoc analysis (GraphPad Software, San Diego, CA). Significance was accepted at P < 0.05.


   ACKNOWLEDGMENTS
 
We thank Dr. Nelly Auersperg (University of British Columbia, Vancouver, British Columbia, Canada) for the human OSE cell lines, Dr. Barry Gumbiner (University of Virginia, Charlottesville, VA) and Dr. James Woodgett (Ontario Cancer Institute, Toronto, Ontario, Canada) for the constructs, and Dr. Cara Gottardi (University of Northwestern, Chicago, IL) for stimulating discussions.


   FOOTNOTES
 
This work was supported by Hong Kong Research Grants Council Grants (to A.S.T.W.).

First Published Online August 31, 2006

Abbreviations: COX-2, Cyclooxgenase-2; DAPI, 4',6-diamidino-2-phenylindole; hCG, human chorionic gonadotropin; GFP, green fluorescent protein; GSK3ß, glycogen synthase kinase-3ß; LEF1, lymphoid enhancer binding factor 1; LiCl, lithium chloride; OSE, ovarian surface epithelium; PI3K, phosphatidylinositol 3-kinase; siRNA, small interfering RNA; TCF, T cell factor; TUNEL, transferase-mediated deoxyuridine triphosphate nick end labeling.

Received for publication March 20, 2006. Accepted for publication August 24, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Osterholzer HO, Johnson JH, Nicosia SV 1985 An autoradiographic study of rabbit ovarian surface epithelium before and after ovulation. Biol Reprod 33:729–738[Abstract]
  2. Murdoch WJ 1995 Programmed cell death in preovulatory ovine follicles. Biol Reprod 53:8–12[Abstract]
  3. Pon YL, Auersperg N, Wong AS 2005 Gonadotropins regulate N-cadherin-mediated human ovarian surface epithelial cell survival at both post-translational and transcriptional levels through a cyclic AMP/protein kinase A pathway. J Biol Chem 280:15438–15448[Abstract/Free Full Text]
  4. Polakis P 1999 The oncogenic activation of ß-catenin. Curr Opin Genet Dev 9:15–21[CrossRef][Medline]
  5. Cox RT, Kirkpatrick C, Peifer M 1996 Armadillo is required for adherens junction assembly, cell polarity, and morphogenesis during Drosophila embryogenesis. J Cell Biol 134:133–148[Abstract/Free Full Text]
  6. Heasman J, Crawford A, Goldstone K, Garner-Hamrick P, Gumbiner B, McCrea P, Kintner C, Noro CY, Wylie C 1994 Overexpression of cadherins and underexpression of ß-catenin inhibit dorsal mesoderm induction in early Xenopus embryos. Cell 79:791–803[CrossRef][Medline]
  7. Fagotto F, Funayama N, Gluck U, Gumbiner BM 1996 Binding to cadherins antagonizes the signaling activity of ß-catenin during axis formation in Xenopus. J Cell Biol 132:1105–1114[Abstract/Free Full Text]
  8. Sanson B, White P, Vincent JP 1996 Uncoupling cadherin-based adhesion from wingless signalling in Drosophila. Nature 383:627–630[CrossRef][Medline]
  9. Orsulic S, Huber O, Aberle H, Arnold S, Kemler R 1999 E-cadherin binding prevents ß-catenin nuclear localization and ß-catenin/LEF-1-mediated transactivation. J Cell Sci 112:1237–1245[Abstract]
  10. Eger A, Stockinger A, Schaffhauser B, Beug H, Foisner R 2000 Epithelial mesenchymal transition by c-Fos estrogen receptor activation involves nuclear translocation of ß-catenin and upregulation of ß-catenin/lymphoid enhancer binding factor-1 transcriptional activity. J Cell Biol 148:173–188[Abstract/Free Full Text]
  11. Gottardi CJ, Gumbiner BM 2004 Distinct molecular forms of ß-catenin are targeted to adhesive or transcriptional complexes. J Cell Biol 167:339–349[Abstract/Free Full Text]
  12. Gottardi CJ, Wong E, Gumbiner BM 2001 E-cadherin suppresses cellular transformation by inhibiting ß-catenin signaling in an adhesion-independent manner. J Cell Biol 153:1049–1060[Abstract/Free Full Text]
  13. Pece S, Gutkind JS 2000 Signaling from E-cadherins to the MAPK pathway by the recruitment and activation of epidermal growth factor receptors upon cell-cell contact formation. J Biol Chem 275:41227–41233[Abstract/Free Full Text]
  14. Ahmed Y, Hayashi S, Levine A, Wieschaus E 1998 Regulation of armadillo by a Drosophila APC inhibits neuronal apoptosis during retinal development. Cell 93:1171–1182[CrossRef][Medline]
  15. Johnston LA, Edgar BA 1998 Wingless and Notch regulate cell-cycle arrest in the developing Drosophila wing. Nature 394:82–84[CrossRef][Medline]
  16. Damalas A, Ben-Ze’ev A, Simcha I, Shtutman M, Leal JF, Zhurinsky J, Geiger B, Oren M 1999 Excess ß-catenin promotes accumulation of transcriptionally active p53. EMBO J 18:3054–3063[CrossRef][Medline]
  17. Kim K, Pang KM, Evans M, Hay ED 2000 Overexpression of ß-catenin induces apoptosis independent of its transactivation function with LEF-1 or the involvement of major G1 cell cycle regulators. Mol Biol Cell 11:3509–3523[Abstract/Free Full Text]
  18. Olmeda D, Castel S, Vilaro S, Cano A 2003 ß-catenin regulation during the cell cycle: implications in G2/M and apoptosis. Mol Biol Cell 14:2844–2860[Abstract/Free Full Text]
  19. He TC, Sparks AB, Rago C, Hermeking H, Zawel L, da Costa LT, Morin PJ, Vogelstein B, Kinzler KW 1998 Identification of c-MYC as a target of the APC pathway. Science 281:1509–1512[Abstract/Free Full Text]
  20. Orford K, Orford CC, Byers SW 1999 Exogenous expression of ß-catenin regulates contact inhibition, anchorage-independent growth, anoikis, and radiation-induced cell cycle arrest. J Cell Biol 146:855–868[Abstract/Free Full Text]
  21. Tetsu O, McCormick F 1999 ß-Catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature 398:422–426[CrossRef][Medline]
  22. Zhu AJ, Watt FM 1999 ß-catenin signalling modulates proliferative potential of human epidermal keratinocytes independently of intercellular adhesion. Development 126:2285–2298[Abstract]
  23. Korinek V, Barker N, Morin PJ, Barker N, Clevers H, Vogelstein B, Kinzler KW 1999 Constitutive transcriptional activation by a ß-catenin-TCF complex in APC–/– colon carcinoma. Science 275:1784–1787
  24. Wong AS, Gumbiner BM 2003 Adhesion-independent mechanism for suppression of tumor cell invasion by E-cadherin. J Cell Biol 161:1191–1203[Abstract/Free Full Text]
  25. Klein PS, Melton DA 1996 A molecular mechanism for the effect of lithium on development. Proc Natl Acad Sci USA 93:8455–8459[Abstract/Free Full Text]
  26. Stambolic V, Ruel L, Woodgett JR 1996 Lithium inhibits glycogen synthase kinase-3 activity and mimics wingless signaling in intact cells. Curr Biol 6:1664–1668[CrossRef][Medline]
  27. Stambolic V, Woodgett JR 1994 Mitogen inactivation of glycogen synthase kinase-3ß in intact cells via serine 9 phosphorylation. Biochem J 303:701–704[Medline]
  28. Eldar-Finkelman H, Argast GM, Foord O, Fischer EH, Krebs EG 1996 Expression and characterization of glycogen synthase kinase-3 mutants and their effect on glycogen synthase activity in intact cells. Proc Natl Acad Sci USA 93:10228–10233[Abstract/Free Full Text]
  29. Cross DAE, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA 1995 Inhibition of glycogen-synthase kinase-3 by insulin-mediated protein-kinase-B. Nature 378:785–789[CrossRef][Medline]
  30. Askew D, Ashmun R, Simmons B, Cleveland J 1991 Constitutive c-myc expression in IL-3-dependent myeloid cell line suppresses cycle arrest and accelerates apoptosis. Oncogene 6:1915–1922[Medline]
  31. Evan GI, Wyllie AH, Gilbert CS, Littlewood TD, Land H, Brooks M, Waters CM, Penn LZ, Hancock DC 1992 Induction of apoptosis in fibroblasts by c-myc protein. Cell 69:119–128[CrossRef][Medline]
  32. Araki Y, Okamura S, Hussain SP, Nagashima M, He P, Shiseki M, Miura K, Harris CC 2003 Regulation of cyclooxygenase-2 expression by the Wnt and ras pathways. Cancer Res 63:728–734[Abstract/Free Full Text]
  33. Li MH, Jang JH, Surh YJ 2005 Nitric oxide induces apoptosis via AP-1-driven upregulation of COX-2 in rat pheochromocytoma cells. Free Radic Biol Med 39:890–899[CrossRef][Medline]
  34. Duffy DM, Stouffer RL 2001 The ovulatory gonadotrophin surge stimulates cyclooxygenase expression and prostaglandin production by the monkey follicle. Mol Hum Reprod 7:731–739[Abstract/Free Full Text]
  35. Futaki N, Takahashi S, Yokoyama M, Arai I, Higuchi S, Otomo S 1994 NS-398, a new anti-inflammatory agent, selectively inhibits prostaglandin G/H synthase/cyclooxygenase (COX-2) activity in vitro. Prostaglandins 47:55–59[CrossRef][Medline]
  36. Byers S, Pishvaian M, Crockett C, Peer C, Tozeren A, Sporn M, Anzano M, Lechleider R 1996 Retinoids increase cell-cell adhesion strength, ß-catenin protein stability, and localization to the cell membrane in a breast cancer cell line: a role for serine kinase activity. Endocrinology 137:3265–3273[Abstract]
  37. Wright JW, Toth-Fejel S, Stouffer RL, Rodland KD 2002 Proliferation of rhesus ovarian surface epithelial cells in culture: lack of mitogenic response to steroid or gonadotropic hormones. Endocrinology 143:2198–2207[Abstract/Free Full Text]
  38. Zheng W, Lu JJ, Luo F, Zheng Y, Feng Y, Felix JC, Lauchlan SC, Pike MC 2000 Ovarian epithelial tumor growth promotion by follicle-stimulating hormone and inhibition of the effect by luteinizing hormone. Gynecol Oncol 76:80–88[CrossRef][Medline]
  39. Parrott JA, Doraiswamy V, Kim G, Mosher R, Skinner MK 2001 Expression and actions of both the follicle stimulating hormone receptor and the luteinizing hormone receptor in normal ovarian surface epithelium and ovarian cancer. Mol Cell Endocrinol 172:213–222[CrossRef][Medline]
  40. Syed V, Ulinski G, Mok SC, Yiu GK, Ho SM 2001 Expression of gonadotropin receptor and growth responses to key reproductive hormones in normal and malignant human ovarian surface epithelial cells. Cancer Res 61:6768–6776[Abstract/Free Full Text]
  41. Choi JH, Choi KC, Auersperg N, Leung PC 2004 Overexpression of follicle-stimulating hormone receptor activates oncogenic pathways in preneoplastic ovarian surface epithelial cells. J Clin Endocrinol Metab 89:5508–5516[Abstract/Free Full Text]
  42. Choi JH, Choi KC, Auersperg N, Leung PC 2005 Gonadotropins upregulate the epidermal growth factor receptor through activation of mitogen-activated protein kinases and phosphatidyl-inositol-3-kinase in human ovarian surface epithelial cells. Endocr Relat Cancer 12:407–421[Abstract/Free Full Text]
  43. Davies BR, Finnigan DS, Smith SK, Ponder BA 1999 Administration of gonadotropins stimulates proliferation of normal mouse ovarian surface epithelium. Gynecol Endocrinol 13:75–81[Medline]
  44. Ivarsson K, Sundfeldt K, Brannstrom M, Hellberg P, Janson PO 2001 Diverse effects of FSH and LH on proliferation of human ovarian surface epithelial cells. Hum Reprod 16:18–23[Abstract/Free Full Text]
  45. Bar J, Cohen-Noyman E, Geiger B, Oren M 2004 Attenuation of the p53 response to DNA damage by high cell density. Oncogene 23:2128–2137[CrossRef][Medline]
  46. Erez N, Zamir E, Gour BJ, Blaschuk OW, Geiger B 2004 Induction of apoptosis in cultured endothelial cells by a cadherin antagonist peptide: involvement of fibroblast growth factor receptor-mediated signaling. Exp Cell Res 294:366–378[CrossRef][Medline]
  47. Harandian F, Farookhi R 1998 Contact-dependent cell interactions determine hormone responsiveness and desensitization in rat granulosa cells. Endocrinology 139:1700–1707[Abstract/Free Full Text]
  48. Smith E, Frenkel B 2005 Glucocorticoids inhibit the transcriptional activity of LEF/TCF in differentiating osteoblasts in a glycogen synthase kinase-3ß-dependent and -independent manner. J Biol Chem 280:2388–2394[Abstract/Free Full Text]
  49. Lowry WE, Blanpain C, Nowak JA, Guasch G, Lewis L, Fuchs E 2005 Defining the impact of ß-catenin/Tcf transactivation on epithelial stem cells. Genes Dev 19:1596–1611[Abstract/Free Full Text]
  50. Peluso JJ, Pappalardo A, Hess SA 2000 Effect of disrupting cell contact on the nuclear accumulation of ß-catenin and subsequent apoptosis of rat ovarian surface epithelial cells in vitro. Endocrine 12:295–302[CrossRef][Medline]
  51. MacLachlan NJ 1987 Ovarian disorders in domestic animals. Environ Health Perspect 73:27–33[Medline]
  52. Auersperg N, Wong AS, Choi KC, Kang SK, Leung PC 2001 Ovarian surface epithelium: biology, endocrinology, and pathology. Endocr Rev 22:255–288[Abstract/Free Full Text]
  53. Delcommenne M, Tan C, Gray V, Rue L, Woodgett J, Dedhar S 1998 Phosphoinositide-3-OH kinase-dependent regulation of glycogen synthase kinase 3 and protein kinase B/AKT by the integrin-linked kinase. Proc Natl Acad Sci USA 95:11211–11216[Abstract/Free Full Text]
  54. Novak A, Hsu SC, Leung-Hagesteijn C, Radeva G, Papkoff J, Montesano R, Roskelley C, Grosschedl R, Dedhar S 1998 Cell adhesion and the integrin-linked kinase regulate the LEF-1 and ß-catenin signaling pathways. Proc Natl Acad Sci USA 95:4374–4379[Abstract/Free Full Text]
  55. Cao Q, Lu X, Feng YJ 2006 Glycogen synthase kinase-3ß positively regulates the proliferation of human ovarian cancer cells. Cell Res 1–7
  56. Tsafriri A, Reich R 1999 Molecular aspects of mammalian ovulation. Exp Clin Endocrinol Diabetes 107:1–11[Medline]
  57. Tokuyama O, Nakamura Y, Muso A, Honda K, Ishiko O, Ogita S 2001 Expression and distribution of cyclooxygenase-2 in human periovulatory ovary. Int J Mol Med 8:603–606[Medline]
  58. Richards JS, Russell DL, Ochsner S, Hsieh M, Doyle KH, Falender AE, Lo YK, Sharma SC 2002 Novel signaling pathways that control ovarian follicular development, ovulation, and luteinization. Recent Prog Horm Res 57:195–220[Abstract/Free Full Text]
  59. Lim H, Paria BC, Das SK, Dinchuk JE, Langenbach R, Trzaskos JM, Dey SK 1997 Multiple female reproductive failures in cyclooxygenase 2-deficient mice. Cell 91:197–208[CrossRef][Medline]
  60. Davis BJ, Lennard DE, Lee CA, Tiano HF, Morham SG, Wetsel WC, Langenbach R 1999 Anovulation in cyclooxygenase-2-deficient mice is restored by prostaglandin E2 and interleukin-1ß. Endocrinology 140:2685–2695[Abstract/Free Full Text]
  61. Pall M, Friden BE, Brannstrom M 2001 Induction of delayed follicular rupture in the human by the selective COX-2 inhibitor rofecoxib: a randomized double-blind study. Hum Reprod 16:1323–1328[Abstract/Free Full Text]
  62. Tsafriri A 1995 Ovulation as a tissue remodelling process. Adv Exp Med Biol 377:121–140[Medline]
  63. Butler TA, Zhu C, Mueller RA, Fuller GC, Lemaire WJ, Woessner Jr JF 1991 Inhibition of ovulation in the perfused rat ovary by the synthetic collagenase inhibitor SC 44463. Biol Reprod 44:1183–1188[Abstract]
  64. Prescott SM, Fitzpatrick FA 2000 Cyclooxygenase-2 and carcinogene