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Endocrinology, doi:10.1210/en.2003-0986
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Endocrinology Vol. 144, No. 12 5322-5328
Copyright © 2003 by The Endocrine Society

Cyclooxygenase-2 Overexpression Inhibits Cathepsin D-Mediated Cleavage of Plasminogen to the Potent Antiangiogenic Factor Angiostatin

Gabrielle B. Perchick and Henry N. Jabbour

Medical Research Council Human Reproductive Sciences Unit, Centre for Reproductive Biology, The University of Edinburgh Academic Centre, Chancellor’s Building, Edinburgh, Scotland EH16 4SB, United Kingdom

Address all correspondence and requests for reprints to: Dr. Henry N. Jabbour, Medical Research Council Human Reproductive Sciences Unit, The University of Edinburgh Academic Centre, Chancellor’s Building, 49 Little France Crescent, Edinburgh, Scotland EH16 4SB, United Kingdom. E-mail: h.jabbour{at}hrsu.mrc.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Overexpression of cyclooxygenase (COX)-2 and enhanced synthesis of prostaglandin E2 (PGE2) have been implicated in human endometrial pathologies. To investigate the molecular role of COX-2, the Ishikawa human endometrial epithelial cell line was stably transfected with the pIRES2 vector containing COX-2 cDNA in either the sense or antisense directions. PGE2 concentrations were significantly elevated in the cells transfected with the COX-2 sense compared with wild-type cells or cells transfected with the antisense cDNA (P < 0.01). Elevated PGE2 synthesis was associated with enhanced expression and signaling of PGE2 receptors (EP). cDNA array analysis revealed differential expression of cathepsin D between the COX-2 sense and antisense cells. Cathepsin D RNA and protein expression was 6.7- and 2.1-fold lower in the COX-2 sense compared with COX-2 antisense cells respectively. Cathepsin D is known to cleave plasminogen to the potent antiangiogenic factor angiostatin. To investigate differential angiostatin generation, conditioned media from COX-2 sense, COX-2 antisense and wild-type cells were incubated with plasminogen and subsequently subjected to Western blot analysis. In comparison to wild-type cells, the cleavage of plasminogen to angiostatin was abolished when incubated in COX-2 sense cells conditioned media and elevated when incubated in COX-2 antisense cells conditioned media. Coincubation of plasminogen with the cathepsin D inhibitor pepstatin A inhibited the cleavage of plasminogen to angiostatin in the COX-2 antisense conditioned media. These data demonstrate that COX-2 exerts a negative feedback on the expression of cathepsin D. This in turn reduces the generation of the antiangiogenic factor angiostatin, hence promoting a proangiogenic environment.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TWO PREDOMINANT ISOFORMS of the cyclooxygenase (COX) enzymes have been identified (COX-1 and COX-2). COX-1 is constitutively expressed in many cell types and has been shown recently to be inducible in certain cancers (1, 2, 3, 4). COX-2 is the readily inducible form of the enzyme and is commonly associated with several pathological conditions including tumorigenesis (5, 6). The COX enzymes catalyze the rate-limiting step in the biosynthetic pathway of prostanoids. There are five endogenous prostanoids PGD2, PGE2, PGF2{alpha}, prostacyclin (PGI2) and thromboxane A2 (7). Arachidonic acid, once released from the membrane phospholipids is converted to the prostanoid intermediate PGH2 by the COX enzymes. PGH2 acts as a substrate for synthases specific to each prostanoid such as PGE synthase (PGES) for PGE2 (8, 9). Once synthesized, PGE2 elicits its effects via its seven trans-membrane G protein-coupled receptors, of which four have been identified (EP1, EP2, EP3, and EP4). These receptors signal via alternate and in some cases opposing signaling pathways (7, 10). EP1 receptor activation leads to elevated inositol-3-phosphate and Ca2+ levels, activation of both EP2 and EP4 results in increased intracellular cAMP levels and depending on the splice variant, EP3 activation either decreases or increases cAMP levels (7).

Numerous studies have demonstrated that overexpression of COX-2 in epithelial cells is associated with enhanced production of angiogenic factors (11, 12). These factors act in a paracrine manner to promote endothelial cell migration and microvascular tube formation (12). In the female reproductive tract, a role for COX enzymes and PGE2 in normal and pathological angiogenesis has been proposed. In the human endometrium expression/synthesis and signaling of COX-2, PGE2, and EP receptors colocalize in glandular epithelial and endothelial cells of the normal and neoplastic endometrium (13, 14, 15, 16, 17, 18, 19). Moreover, overexpression of COX enzymes in epithelial cells of the reproductive tract has been shown to promote the expression of various angiogenic factors (4).

A role for COX enzymes has also been proposed in benign pathologies of the endometrium such as endometriosis, dysmenorrhea, and heavy menses (20, 21, 22, 23, 24, 25, 26). Furthermore, several studies have associated heavy menses with abnormalities in vasodilatatory prostanoid production such as PGE2 from the uterus (20, 21, 22). PGE2 synthesis and PGE2 binding sites in uterine tissues are greater in women diagnosed with heavy menses compared with women with normal blood loss (18, 21, 27, 28, 29). The elevated prostanoids detected in menstrual flow of patients with heavy menses has lead to the administration of COX enzyme inhibitors as a means of therapy (30). COX enzyme inhibitors such as ibuprofen have been shown to reduce menstrual blood loss (31). This suggests that the degree or duration of menstrual bleeding in women diagnosed with menorrhagia is augmented following elevation of vasodilatatory factors by COX enzyme products.

This study was designed to investigate the potential role of COX-2 in regulating endometrial epithelial cell function. The specific aims of the study were to examine the effect of COX-2 overexpression in an endometrial epithelial cell line (Ishikawa) on PGE2 secretion, EP receptor expression and signaling and to identify genes regulated by COX-2 that may be associated with endometrial function and angiogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Ishikawa wild-type, COX-2 sense, and COX-2 antisense cells were routinely maintained in DMEM nutrient mixture F-12 with glutamax-1 and pyridoxine, supplemented with 10% fetal bovine serum and 1% antibiotics (500 U/ml penicillin and 500 µg/ml streptomycin) at 37 C and 5% CO2 (vol/vol). In addition, COX-2 sense and COX-2 antisense cells were maintained in media containing 800 µg/ml G418 (Calbiochem, Nottingham, UK).

Transfection of cells
The pBS(SK-)PSHI containing the full-length COX-2 cDNA (kindly supplied by Dr. Stephen Prescott, University of Utah, Salt Lake City, UT) was used as the template plasmid. The COX-2 cDNA was excised from the template plasmid and ligated at the EcoR1 site of the pIRES2 vector (CLONTECH, Hampshire, UK). The orientation of the COX-2 cDNA insert was determined by dideoxy DNA sequencing using sequence specific primers for pIRES2. Wild-type Ishikawa cells were plated in a 12-well plate at a density of 1.2 x 105 cells per well and left to attach overnight. The following day, the pIRES2 vector containing the COX-2 cDNA in either the sense or antisense directions was transfected into the Ishikawa endometrial epithelial cell line using pfx-5 (Invitrogen, Paisley, Scotland, UK) diluted in Optimem (Life Technologies, Inc., Paisley, Scotland, UK). Following a 4-h incubation at 37C, 5% CO2 (vol/vol), the transfection mixture was replaced with fresh complete media. The transfected cells were allowed to grow for 24 h and then seeded with wild-type cells and selected using G418 (Calbiochem; at a concentration of 800 µg/ml). A total of 120 colonies with the COX-2 sense cDNA and 60 colonies with the COX-2 antisense cDNA were picked using cloning rings. The clones were screened for COX-2 protein expression using Western blot analysis. Initial experiments were performed on four COX-2 sense and two COX-2 antisense clones. All of the COX-2 sense clones generated significantly higher PGE2 into the culture media compared with COX-2 antisense and wild type. The data presented in the manuscript and all further investigations were performed using the COX-2 sense 72 clone and COX-2 antisense 15 clone as these generated the highest and lowest levels of PGE2, respectively.

Protein extraction and Western blot analysis
Ishikawa wild-type, COX-2 sense, and COX-2 antisense cells were seeded at a density of 7.5 x 105 cells in six-well plates, allowed to attach for 24 h in complete media and then cultured in serum free media overnight (n = 4 independent experiments). Subsequently, cells were lysed on the plates for 20 min with the lysis buffer [150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 10 mM EDTA, 0.6% Nonidet P-40, 1 mM Na3VO4, 10% glycerol, 10 µg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride]. The lysates were clarified by centrifugation and the supernatants collected. Protein concentration was determined by the modified Lowry method (Bio-Rad D2 Protein Assay kit, Bio-Rad Laboratories, Hemel Hempstead, UK). A total of 20-µg protein for cathepsin D expression and 40 µg protein for COX-2, COX-1, and ß-actin, expression were denatured and subjected to SDS-PAGE on 4–12% Tris-glycine gels (Invitrogen). The proteins were transferred onto polyvinylidene difluoride membrane and blocked for 1 h in TBS-Tween [50 mM Tris-HCl, 150 mM NaCl, and 0.05% (vol/vol) Tween 20 containing 5% skimmed milk powder]. The membranes were probed with one of the following antibodies: COX-2 (sc-1745; at dilution of 1:1000), COX-1 (sc-1752; at dilution of 1:500), ß-actin (sc-1616; at dilution of 1:1000), cathepsin D (sc-6486; at dilution of 1:2000) or plasminogen (sc-15034; at dilution of 1:500) overnight followed by rabbit antigoat conjugated to alkaline phosphatase secondary antibody at a dilution of 1:30,000 (Sigma, Poole, UK). The specificity of some of the antibodies has been confirmed previously in our laboratory by preadsorbtion of the antibodies to the respective blocking peptides (4). All the primary antibodies were purchased from Santa Cruz Biotechnology (Autogenbioiclear, Whiltshire, UK). The membranes were developed and revealed by PhosphorImager analysis using the ECF chemifluorescence system according to the manufacturer’s instructions (Amersham Biosciences UK Ltd., Little Chalfont, UK). The molecular weights of the proteins were determined by comparing mobility on the gel with a molecular weight standard (Invitrogen). Protein bands were semiquantified by densitometry using STORM 860 system (Molecular Dynamics, Amersham Biosciences, Buckinghamshire, UK). Relative expression of cathepsin D protein was calculated by normalizing with ß-actin and expressed as mean ± SEM.

PGE2 assay
Ishikawa wild-type, COX-2 sense, and COX-2 antisense cells were seeded in six-well plates at a cell density of 3.5 x 105 cells per well, allowed to attach for 24 h in complete media and then cultured in serum free media overnight (n = 4 independent experiments). The cells were incubated for a further 48 h in serum-free media containing 5 µg/ml arachidonic acid in the presence or absence of 10 µM NS398 (Calbiochem, Nottingham, UK). PGE2 secretion in the culture media was assayed using an ELISA as described by Denison et al. (32). The data are presented as mean ± SEM. The intra- and interassay coefficients of variation were 7.8% and 15.0%, respectively, with an assay detection limit of 10 pg/ml.

Taqman quantitative RT-PCR
To determine the effect of COX-2 on EP receptor expression, wild-type, COX-2 sense, and COX-2 antisense cells were seeded at a density of 5 x 105 in six-well plates, allowed to attach for 24 h in complete media and then cultured in serum-free media overnight (n = 3 independent experiments). These experiments were conducted in the absence of NS398 as recent data suggest that this inhibitor up-regulates the expression of EP receptors (33). Thereafter RNA was extracted using Tri Reagent (Sigma) following the manufacturer’s instructions. RNA samples were quantified and reverse transcribed using 5.5 mM MgCl2, 0.5 mM each deoxynucleotide triphosphates, 2.5 µM random hexamers, 0.4 U/ml ribonuclease inhibitor, and 1.25 U/ml Multiscribe reverse transcriptase (all from PE Applied Biosystems, Warrington, UK). A total of 400 ng template RNA was added to RT mix and incubated for 60 min at 25 C, 45 min at 48 C, and 5 min at 95 C. The PCR mix consisted of 1x Universal PCR Mastermix, forward and reverse primers for either EP1, EP2, EP3, or EP4 (300 nM) and EP1, EP2, EP3, or EP4 probe (200 nM) and ribosomal 18S forward primer, reverse primer and probe (50 nM; all from PE Applied Biosystems, Warrington, UK). For PCR, 48 µl of PCR mix was mixed with 2 µl cDNA and subsequently a volume of 24 µl of this mixture was placed in duplicate into wells on a PCR plate along with a no template control. The wells were sealed using optical lids and the PCR was carried out using an ABI Prism 7700 (PE Applied Biosystems). The primers for the EP receptors and 18S were designed using PRIMER express software (PE Applied Biosystems) and the sequences are presented in Table 1Go. 18S rRNA was used as an internal standard to normalize the samples for RNA loading. Results were expressed relative to a positive standard (cDNA obtained from a single sample of endometrial tissue) run in each PCR. Relative EP receptor expression was calculated by dividing EP receptor expression in the COX-2 sense and COX-2 antisense cells by expression detected in wild type. Data are presented as mean ± SEM.


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  		TABLE 1. Oligonucleotide sequences for the various EP receptor primers and probes

 
cAMP turnover
Ishiwawa wild-type, COX-2 sense, and COX-2 antisense cells were seeded in six-well plates at a cell density of 2 x 105 cells per well, allowed to attach for 24 h in complete media and then cultured in serum free media overnight (n = 4 independent experiments). The media was then replaced with fresh serum free media containing 1 mM 1-methyl-3-isobutylxanthine (Sigma) for 1.5 h. Cells were stimulated with 0 or 100 nM PGE2 for 10 min. The media was removed and the cells were lysed using 0.1 M HCl. cAMP generation was quantified using a cAMP ELISA kit (Biomol, Affiniti, Exeter, UK) according to the manufacturer’s instructions and normalized to protein concentrations of the homogenate. Protein concentrations were determined using protein assay kits (Bio-Rad Laboratories). The data are presented as the mean (±SEM), fold induction of cAMP in COX-2 sense and antisense cells after treatment with PGE2 where fold induction was calculated relative to the wild-type samples. The intra- and interassay coefficients of variation were 8.3% and 11.6%, respectively.

cDNA array analysis
Differential gene expression in the COX-2 sense and antisense cells was assessed using cDNA array analysis. COX-2 sense and COX-2 antisense cells were grown to approximately 70% confluency in complete media. The cells were cultured in serum free media overnight and then harvested by trypsinization for 5 min. The cells were resuspended in PBS, pelleted by centrifugation, snap frozen on dry ice, and stored at -70 C. The cell pellets were sent to CLONTECH Laboratories Inc. (Palo Alto, CA) for custom cDNA array analysis using the Atlas plastic human 8K gene Microarray service. This array includes a list of 8000 genes that are involved in diverse molecular and cellular functions (for further information visit www.clontech.com). The results were analyzed using AtlasImage software and expressed as a comparison between the COX-2 sense and COX-2 antisense cells.

Determination of angiostatin generation
Ishikawa wild-type, COX-2 sense, and COX-2 antisense cells were seeded in six-well plates at a density of 3.5 x 105, allowed to attach for 24 h in complete media and then cultured in serum-free media overnight (n = 3 independent experiments). Following culture, the media was collected, spun at 1000 x g for 5 min to pellet cell debris, and the supernatant was aspirated. To investigate the differential cleavage of plasminogen to angiostatin by the three cell lines, aliquots of cell media (100 µl) were incubated with 25 µg /ml plasminogen at 37 C for 0, 4, 8, or 24 h. To investigate whether cathepsin D mediates the cleavage of plasminogen to angiostatin, 100 µl of media from COX-2 antisense cells were incubated in the presence or absence of 1 µM pepstatin A (Sigma) for 8 h. Subsequently, a total of 20 µl of each reaction was denatured and subjected to SDS-PAGE on 4–12% Tris-glycine gels (Invitrogen). The generation of angiostatin from plasminogen was assessed by Western blot analysis and probed with a plasminogen antibody as described above

Statistical analyses
The data in this study were analyzed by ANOVA using StatView 5.0 software (Abacus Concepts, Berkeley, CA). Statistical significance was taken as P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Stable transfection of the Ishikawa human endometrial epithelial cells with the pIRES2 vector containing COX-2 cDNA in the sense direction resulted in the overexpression of COX-2 protein. Western blot analysis revealed strong COX-2 protein expression detected as 72-kDa band in the COX-2 sense cells compared with the COX-2 antisense and wild-type cells (Fig. 1AGo). Basal levels of COX-2 were detected in the wild-type Ishikawa cells that was abolished by transfection with COX-2 antisense cDNA. (Fig. 1AGo). Stable transfection of the COX-2 cDNA in either the sense or antisense direction had no effect on COX-1 protein expression (Fig. 1BGo); no differences were detected in COX-1 protein levels between the wild-type, COX-2 sense, or COX-2 antisense cell lines.



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  		FIG. 1. Western blot analysis of 40 µg protein from wild-type Ishikawa cells (WT) and Ishikawa cells stably transfected with COX-2 cDNA in either the sense (S) or antisense (AS) directions. A, Specific bands for COX-2 and ß-actin proteins were detected at approximately 72 and 46 kDa, respectively. B, COX-1 and ß-actin proteins were detected by specific bands at 71 and 46 kDa, respectively.

 
To confirm functionality of the transfected cDNA, PGE2 synthesis was measured by ELISA in COX-2 sense, COX-2 antisense and wild-type cells (Fig. 2Go). PGE2 secretion was significantly increased (P < 0.01) in the culture media of the COX-2 sense cells (2391.25 ± 311.09 pg/ml) compared with wild-type and COX-2 antisense cells (359 ± 71.54 and 78.96 ± 50.9 pg/ml, respectively). Coculture of the cells with the specific COX-2 enzyme inhibitor NS398 reduced the increased secretion of PGE2 in the wild-type, COX-2 sense, and COX-2 antisense cells to almost undetectable levels. To investigate the effect of COX-2 overexpression on EP receptors, real time quantitative RT PCR was performed to quantify the expression of EP1, EP2, EP3, and EP4 receptors (Fig. 3AGo). Relative expression of EP2 and EP3 mRNA (compared with expression levels detected in wild-type cells) was significantly higher (P < 0.05) in the COX-2 sense cells compared with COX-2 antisense cells (EP2 receptor: 3.73 ± 0.43 vs. 1.19 ± 0.39 for COX-2 sense and antisense cells, respectively; EP3 receptor: 2.35 ± 0.3 vs. 1.03 ± 0.105 for COX-2 sense and antisense cells, respectively). EP1 mRNA was not detectable in any of the cell lines and EP4 receptor expression showed no significant differences between the COX-2 sense and antisense cells. Functionality of the EP receptors was investigated by measuring the generation of cAMP in COX-2 sense, COX-2 antisense and wild-type cells in response to treatment with 100 nM PGE2 for 10 min (Fig. 3BGo). Fold induction of cAMP relative to that measured in wild-type cells was significantly higher (P < 0.05) in the COX-2 sense cells compared with the COX-2 antisense cells (2.57 ± 0.16 vs. 0.88 ± 0.33 for COX-2 sense and antisense cells, respectively).



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  		FIG. 2. The functionality of the transfected COX-2 was assessed by ELISA to measure PGE2 secretion by wild-type (WT), COX-2 antisense (AS), and COX-2 sense (S) cells into culture media following incubation with 5 µg arachidonic acid (AA) for 48 h in the presence or absence 10 µM of the COX-2 enzyme inhibitor NS398. The data are mean ± SEM of n = 4 experiments. Different letters denote statistical significance (P < 0.05).

 


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  		FIG. 3. A, Relative expression of EP2, EP3, and EP4 receptors in Ishikawa cells stably overexpressing COX-2 in either the sense (S) or antisense (AS) directions. Relative expression was determined by dividing expression detected in COX-2 sense and antisense cells by expression detected in wild-type cells. The data are mean ± SEM of n = 3 experiments. Different letters for each of the receptors denote statistical significance (P < 0.05). B, ELISA for cAMP generation. Fold induction of cAMP generation in COX-2 antisense (AS) and COX-2 sense (S) cells following treatment with 100 nM PGE2 for 10 min. Fold induction is calculated by dividing cAMP generation in COX-2 sense and antisense cells by cAMP generation in wild-type cells. The data are mean ± SEM of n = 4 experiments. Different letters denote statistical significance (P < 0.05).

 
Differential gene expression between COX-2 sense and COX-2 antisense cells was investigated using RNA extracted from the two cell lines and the CLONTECH Atlas human 8K microarrays. A number of genes demonstrated differential expression between the two cell lines. Of the genes differentially expressed, one of the greatest differences observed was cathepsin D. Cathepsin D mRNA expression demonstrated a 6.7-fold difference between the COX-2 sense and the COX-2 antisense cells; cathepsin D RNA expression was reduced in the COX-2 sense compared with the COX-2 antisense cells (Fig. 4AGo). Western blot analysis conducted using proteins extracted from the wild-type, COX-2 sense, and COX-2 antisense cell lines confirmed lower cathepsin D protein expression in the COX-2 sense and elevated expression in the COX-2 antisense cells compared with the wild-type cells. Cathepsin D protein expression in the COX-2 sense and COX-2 antisense cell lines was 0.8 ± 0.13 and 1.68 ± 0.39-fold relative to wild-type cells. The difference in cathepsin D expression was 2.16 ± 0.22-fold lower in the COX-2 sense compared with COX-2 antisense cells P < 0.05 (Fig. 4Go, B and C).



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  		FIG. 4. A, Atlas plastic 8K Human cDNA array image following hybridysation with cDNA from untreated COX-2 antisense and COX-2 sense cells. Arrows correspond to cathepsin D position. B, Western blot analysis of 20 µg protein isolated from untreated Ishikawa wild-type (WT), COX-2 antisense (AS), and COX-2 sense (S) cells. The blot was probed with cathepsin D antibody that detected procathepsin D (1 ), pseudocathepsin D (2 ) and cathepsin D (3 ) protein expression at 52 kDa, 51 kDa, and 32 kDa, respectively. C, Relative expression of cathepsin D in COX-2 antisense and COX-2 sense cells normalized for ß-actin and expressed relative to expression detected in wild-type cells. Bands were semiquantified as outlined in Materials and Methods and presented as mean ± SEM relative expression of n = 4 experiments. Different letters denote statistical significance (P < 0.05).

 
We investigated the potential effect of differential cathepsin D expression in the three cell lines, on cleavage of plasminogen to angiostatin. Plasminogen was cultured in the media collected from COX-2 sense, COX-2 antisense, and wild-type cells for 0, 4, 8, and 24 h. Using antiplasminogen antibody and Western blot analysis, angiostatin bands were detected at 36 and 32 kDa, which are within the reported range (34, 35). Angiostatin production was elevated following culture of plasminogen in media collected from COX-2 antisense cells compared with wild-type cells. However, angiostatin production was abolished when plasminogen was incubated in the culture media collected from COX-2 sense cells (Fig. 5Go). To confirm that the cleavage of plasminogen to angiostatin is mediated by cathepsin D, the incubation experiments were repeated by incubating plasminogen in COX-2 antisense conditioned media in the presence of the cathepsin D inhibitor pepstatin A. Coincubation of plasminogen and cathepsin D in the conditioned media from COX-2 antisense cells for 8 h resulted in inhibition of angiostatin production (Fig. 6Go).



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  		FIG. 5. Generation of angiostatin following incubation of 25 µg /ml plasminogen for 0, 4, 8, and 24 h in serum-free conditioned media collected from Ishikawa wild-type, COX-2 antisense, and COX-2 sense cells. Angiostatin production was detected by Western blot analysis using 20 µl of media. Plasminogen and angiostatin were detected at expected molecular masses of 97 and 36/32 kDa, respectively.

 


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  		FIG. 6. Inhibition of angiostatin generation by pepstatin A following incubation of 25 µg/ml plasminogen in media collected from COX-2 antisense cells in the presence or absence of 1 µM pepstatin A for 8 h. Angiostatin production was detected by Western blot analysis using 20 µl of media. Plasminogen and angiostatin were detected at expected molecular masses of 97 and 36/32 kDa, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we describe the establishment of an endometrial epithelial cell line stably overexpressing COX-2. COX-2 protein expression was higher in the sense clone compared with the antisense and wild type. Overexpression of COX-2 in the Ishikawa endometrial epithelial cells was associated with elevated secretion of PGE2 that was abolished by the COX-2 selective inhibitor NS398. This is in agreement with previous studies that have demonstrated increased prostanoid generation in response to the overexpression of COX-2 (12). Similar expression of COX-1 protein was detected in all three cell lines, suggesting that COX-1 expression is not regulated by COX-2. Treatment of the cells with NS398 reduced PGE2 secretion in wild-type, COX-2 sense, and COX-2 antisense cells to almost undetectable levels. This suggests that PGE2 secretion in the wild-type, COX-2 sense, and COX-2 antisense cell lines is predominantly a result of COX-2 activity. COX-2 overexpression was also associated with increased EP2/EP3 receptor expression and signaling with no effect on EP1 and EP4 receptor expression. The elevation in EP receptor expression in the COX-2 sense cells suggests that COX enzyme products act in an autocrine/paracrine manner to up-regulate EP receptor expression. The COX-2 sense cells displayed also significantly higher fold induction of cAMP relative to the COX-2 antisense cells. The EP2 receptor is associated with Gs coupled signaling and results in enhanced cAMP generation (7), whereas a number of splice variants have been described for the EP3 receptor and these have been shown to be linked to either the Gs- or Gi-coupled signaling (7). The net increase in cAMP in response to exogenous PGE2 in the COX-2 sense cells suggests an overall balance toward Gs coupled signaling in response to exogenous PGE2 is favored. Hence, these data confirm that prostanoids such as PGE2 secreted in response to COX-2 regulate the expression of their receptors and regulate gene expression in an autocrine/paracrine manner.

To identify differential gene expression between the COX-2 sense and the COX-2 antisense cells, we employed cDNA array analysis. We demonstrated reduced cathepsin D mRNA and protein expression in the COX-2 sense cells compared with wild-type and antisense cells. In the human endometrium, cathepsin D expression has been localized to glandular and stromal cells, and glandular expression has been shown to be highest during the secretory phase of the menstrual cycle (36). The elevated expression of cathepsin D is thought to be positively regulated by progesterone. However, cathepsin D expression remains elevated in the mid to late secretory phase despite a reduction in progesterone receptor activity. This has prompted the suggestion that cathepsin D expression is also under the control of other factors (37). Our data demonstrating that cathepsin D expression is inhibited via a COX-2 mediated action suggests COX-2 may be one of those factors. COX-2 expression in the human endometrium is highest during the late secretory and proliferative phases of the menstrual cycle (14) when cathepsin D levels have been reported to be at their lowest. Interestingly, elevated COX-2 and reduced cathepsin D have independently been associated with a poor prognosis in reproductive tract carcinoma (38, 39, 40). Moreover, COX-2 has been shown to be induced in endometrial adenocarcinomas (15, 16, 17). Hence it is plausible to suggest that increased COX-2 may be an indicator of low cathepsin D expression.

Angiostatin is a potent antiangiogenic factor that is proteolytically derived from plasminogen (41). Angiostatin inhibits vasodilation, proliferation, and migration of endothelial cells and endothelial tube formation possibly via the induction of apoptosis in endothelial cells (41, 42, 43, 44, 45). Conditioned medium of human prostate carcinoma cells has been demonstrated to cleave plasminogen to angiostatin by the action of procathepsin D. Furthermore, purified mature cathepsin D can cleave plasminogen to angiostatin (46). We initially investigated the generation of angiostatin from plasminogen in conditioned media collected from COX-2 sense, COX-2 antisense, and wild-type cells. Angiostatin accumulation was abolished in the media from COX-2 sense cells and elevated in the media from the COX-2 antisense cells compared with the wild-type cells supporting a role for cathepsin D in the generation of angiostatin in endometrial epithelial cells. The differential cleavage of plasminogen to angiostatin in the three cell lines is reflective of the varying degrees of COX-2 expression. Coincubation of plasminogen in conditioned media from antisense cells with the cathepsin D inhibitor, pepstatin A, reduced angiostatin formation. This suggests that the cleavage of plasminogen to angiostatin is mediated in part by cathepsin D. Other proteolytic enzymes are known to cleave plasminogen to angiostatin. These include matrix metalloproteinases, plasminogen activators, pepsin, and cathepsin E (35, 46, 47, 48, 49, 50). Hence, it is plausible to suggest that, in addition to cathepsin D, other enzymes may be involved in the regulation of angiostatin production that may be inhibited by pepstatin A.

Overexpression of COX-2 and prostanoid receptors such as EP2 play a role in angiogenesis by promoting the formation of proangiogenic factors (51). Once synthesized the angiogenic factors act in a paracrine manner on endothelial cells to promote enhanced cell migration and tubular formation (12). However, it is now well accepted that the promotion of an angiogenic environment is the result of a balance in the production of angiogenic and anti angiogenic factors (52). Recently, inhibition of COX-2 has been demonstrated to up-regulate the generation and expression of the antiangiogenic factors endostatin and thrombospondin-1 (53, 54). The data presented herein demonstrate an alternative pathway by which COX-2 can regulate angiogenesis in endometrial epithelial cells through inhibition of production of antiangiogenic factors such as angiostatin. Hence it is hypothesized that, in endometrial pathologies that are associated with elevated COX-2 enzyme expression, vascular function may be promoted through overexpression of angiogenic factors and reduced production of antiangiogenic factors such as angiostatin. However, the underlying cellular and molecular mechanisms by which COX-2 down-regulates cathepsin D expression resulting in reduced angiostatin generation remains to be elucidated.

In conclusion, the data outlined here demonstrate overexpression of COX-2 results in a concomitant induction of PGE2 secretion and EP2/EP3 receptor expression. COX-2 inhibits cathepsin D expression in endometrial epithelial cells via an unknown mechanism, which contributes to an inhibition of the formation of angiostatin. These data outline a novel function of COX-2 in promoting a proangiogenic environment through suppression of production of angiostatin.


   Acknowledgments
 
The authors acknowledge Dr. Kurt Sales for constructing the COX-2 sense and COX-2 antisense plasmids, Dr. Rodney Kelly for supplying the PGE2 assays, and Sheila Boddy for her general technical assistance.


   Footnotes
 
G.B.P. was a recipient of a Ph.D. scholarship from the Medical Research Council.

Abbreviations: COX, Cyclooxygenase; EP, PGE2 receptor; PGE, prostaglandin E; PGI2, prostacyclin; PGES, PGE synthesis.

Received August 1, 2003.

Accepted for publication August 29, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Bauer AK, Dwyer-Nield LD, Malkinson AM 2000 High cyclooxygenase 1 (COX-1) and cyclooxygenase 2 (COX-2) contents in mouse lung tumors. Carcinogenesis 21:543–550[Abstract/Free Full Text]
  2. Hwang D, Scollard D, Byrne J, Levine E 1998 Expression of cyclooxygenase-1 and cyclooxygenase-2 in human breast cancer. J Natl Cancer Inst 90:455–460[Abstract/Free Full Text]
  3. Kirschenbaum A, Klausner AP, Lee R, Unger P, Yao S, Liu XH, Levine AC 2000 Expression of cyclooxygenase-1 and cyclooxygenase-2 in the human prostate. Urology 56:671–676[CrossRef][Medline]
  4. Sales KJ, Katz AA, Howard B, Soeters RP, Millar RP, Jabbour HN 2002 Cyclooxygenase-1 is up-regulated in cervical carcinomas: autocrine/paracrine regulation of cyclooxygenase-2, prostaglandin E receptors, and angiogenic factors by cyclooxygenase-1. Cancer Res 62:424–432[Abstract/Free Full Text]
  5. Vane JR, Bakhle YS, Botting RM 1998 Cyclooxygenases 1 and 2. Annu Rev Pharmacol Toxicol 38:97–120[CrossRef][Medline]
  6. DuBois RN, Radhika A, Reddy BS, Entingh AJ 1996 Increased cyclooxygenase-2 levels in carcinogen-induced rat colonic tumors. Gastroenterology 110:1259–1262[CrossRef][Medline]
  7. Breyer RM, Bagdassarian CK, Myers SA, Breyer MD 2001 Prostanoid receptors: subtypes and signaling. Annu Rev Pharmacol Toxicol 41:661–690[CrossRef][Medline]
  8. Murakami M, Naraba H, Tanioka T, Semmyo N, Nakatani Y, Kojima F, Ikeda T, Fueki M, Ueno A, Oh S, Kudo I 2000 Regulation of prostaglandin E2 biosynthesis by inducible membrane-associated prostaglandin E2 synthase that acts in concert with cyclooxygenase-2. J Biol Chem 275:32783–32792[Abstract/Free Full Text]
  9. Forsberg L, Leeb L, Thoren S, Morgenstern R, Jakobsson P 2000 Human glutathione dependent prostaglandin E synthase: gene structure and regulation. FEBS Lett 471:78–82[CrossRef][Medline]
  10. Ashby B 1998 Co-expression of prostaglandin receptors with opposite effects: a model for homeostatic control of autocrine and paracrine signaling. Biochem Pharmacol 55:239–246[CrossRef][Medline]
  11. Masferrer JL, Leahy KM, Koki AT, Zweifel BS, Settle SL, Woerner BM, Edwards DA, Flickinger AG, Moore RJ, Seibert K 2000 Antiangiogenic and antitumor activities of cyclooxygenase-2 inhibitors. Cancer Res 60:1306–1311[Abstract/Free Full Text]
  12. Tsujii M, Kawano S, Tsuji S, Sawaoka H, Hori M, DuBois RN 1998 Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell 93:705–716[CrossRef][Medline]
  13. Milne SA, Perchick GB, Boddy SC, Jabbour HN 2001 Expression, localization, and signaling of PGE2 and EP2/EP4 receptors in human nonpregnant endometrium across the menstrual cycle. J Clin Endocrinol Metab 86:4453–4459[Abstract/Free Full Text]
  14. Jones RL, Kelly RW, Critchley HO 1997 Chemokine and cyclooxygenase-2 expression in human endometrium coincides with leukocyte accumulation. Hum Reprod 12:1300–1306
  15. Tong BJ, Tan J, Tajeda L, Das SK, Chapman JA, DuBois RN, Dey SK 2000 Heightened expression of cyclooxygenase-2 and peroxisome proliferator-activated receptor-{delta} in human endometrial adenocarcinoma. Neoplasia 2:483–490[CrossRef][Medline]
  16. Jabbour HN, Milne SA, Williams AR, Anderson RA, Boddy SC 2001 Expression of COX-2 and PGE synthase and synthesis of PGE2 in endometrial adenocarcinoma: a possible autocrine/paracrine regulation of neoplastic cell function via EP2/EP4 receptors. Br J Cancer 85:1023–1031[CrossRef][Medline]
  17. Ferrandina G, Legge F, Ranelletti FO, Zannoni GF, Maggiano N, Evangelisti A, Mancuso S, Scambia G, Lauriola L 2002 Cyclooxygenase-2 expression in endometrial carcinoma: correlation with clinicopathologic parameters and clinical outcome. Cancer 95:801–807[CrossRef][Medline]
  18. Rees MC, Anderson AB, Demers LM, Turnbull AC 1984 Endometrial and myometrial prostaglandin release during the menstrual cycle in relation to menstrual blood loss. J Clin Endocrinol Metab 58:813–818[Abstract]
  19. Rees MC, Parry DM, Anderson AB, Turnbull AC 1982 Immunohistochemical localisation of cyclooxygenase in the human uterus. Prostaglandins 23:207–214[CrossRef][Medline]
  20. Buttram Jr VC, Reiter RC 1981 Uterine leiomyomata: etiology, symptomatology, and management. Fertil Steril 36:433–445[Medline]
  21. Smith SK, Abel MH, Kelly RW, Baird DT 1981 Prostaglandin synthesis in the endometrium of women with ovular dysfunctional uterine bleeding. Br J Obstet Gynaecol 88:434–442[Medline]
  22. Smith SK, Abel MH, Kelly RW, Baird DT 1981 A role for prostacyclin (PGI2) in excessive menstrual bleeding. Lancet 1:522–524[Medline]
  23. Rees MC 1989 Heavy, painful periods. Baillieres Clin Obstet Gynaecol 3:341–356[CrossRef][Medline]
  24. Rees MC, Demers LM, Anderson AB, Turnbull AC 1984 A functional study of platelets in menstrual fluid. Br J Obstet Gynaecol 91:667–672[Medline]
  25. Ota H, Igarashi S, Sasaki M, Tanaka T 2001 Distribution of cyclooxygenase-2 in eutopic and ectopic endometrium in endometriosis and adenomyosis. Hum Reprod 16:561–566[Abstract/Free Full Text]
  26. Haney AF 1993 Endometriosis-associated infertility. Baillieres Clin Obstet Gynaecol 7:791–812[CrossRef][Medline]
  27. Hofmann GE, Rao CV, Barrows GH, Sanfilippo JS 1983 Topography of human uterine prostaglandin E and F2 {alpha} receptors and their profiles during pathological states. J Clin Endocrinol Metab 57:360–366[Abstract]
  28. Adelantado JM, Lopez Bernal A, Turnbull AC 1988 Topographical distribution of prostaglandin E receptors in human myometrium. Br J Obstet Gynaecol 95:348–353[Medline]
  29. Adelantado JM, Rees MC, Lopez Bernal A, Turnbull AC 1988 Increased uterine prostaglandin E receptors in menorrhagic women. Br J Obstet Gynaecol 95:162–165[Medline]
  30. Stirrat GM 1999 Choice of treatment for menorrhagia. Lancet 353:2175–2176[CrossRef][Medline]
  31. Makarainen L, Ylikorkala O 1986 Primary and myoma-associated menorrhagia: role of prostaglandins and effects of ibuprofen. Br J Obstet Gynaecol 93:974–978[Medline]
  32. Denison FC, Grant VE, Calder AA, Kelly RW 1999 Seminal plasma components stimulate interleukin-8 and interleukin-10 release. Mol Hum Reprod 5:220–226[Abstract/Free Full Text]
  33. Nasrallah R, Laneuville O, Ferguson S, Hebert RL 2001 Effect of COX-2 inhibitor NS-398 on expression of PGE2 receptor subtypes in M-1 mouse CCD cells. Am J Physiol Renal Physiol 281:F123–F132
  34. Scapini P, Nesi L, Morini M, Tanghetti E, Belleri M, Noonan D, Presta M, Albini A, Cassatella MA 2002 Generation of biologically active angiostatin kringle 1–3 by activated human neutrophils. J Immunol 168:5798–5804[Abstract/Free Full Text]
  35. Westphal JR, Van’t Hullenaar R, Geurts-Moespot A, Sweep FC, Verheijen JH, Bussemakers MM, Askaa J, Clemmensen I, Eggermont AA, Ruiter DJ, De Waal RM 2000 Angiostatin generation by human tumor cell lines: involvement of plasminogen activators. Int J Cancer 86:760–767[CrossRef][Medline]
  36. Maudelonde T, Martinez P, Brouillet JP, Laffargue F, Pages A, Rochefort H 1990 Cathepsin-D in human endometrium: induction by progesterone and potential value as a tumor marker. J Clin Endocrinol Metab 70:115–121[Abstract]
  37. Camier B, Hedon B, Rochefort H, Maudelonde T 1996 Endometrial cathepsin D immunostaining throughout ovulatory and anovulatory menstrual cycles. Hum Reprod 11:392–397
  38. Falcon O, Chirino R, Leon L, Lopez-Bonilla A, Torres S, Fernandez L, Garcia-Hernandez JA, Valeron PF, Diaz-Chico JC 1999 Low levels of cathepsin D are associated with a poor prognosis in endometrial cancer. Br J Cancer 79:570–576[CrossRef][Medline]
  39. Denkert C, Kobel M, Pest S, Koch I, Berger S, Schwabe M, Siegert A, Reles A, Klosterhalfen B, Hauptmann S 2002 Expression of cyclooxygenase 2 is an independent prognostic factor in human ovarian carcinoma. Am J Pathol 160:893–903[Abstract/Free Full Text]
  40. Ferrandina G, Lauriola L, Distefano MG, Zannoni GF, Gessi M, Legge F, Maggiano N, Mancuso S, Capelli A, Scambia G, Ranelletti FO 2002 Increased cyclooxygenase-2 expression is associated with chemotherapy resistance and poor survival in cervical cancer patients. J Clin Oncol 20:973–981[Abstract/Free Full Text]
  41. O’Reilly MS, Holmgren L, Shing Y, Chen C, Rosenthal RA, Moses M, Lane WS, Cao Y, Sage EH, Folkman J 1994 Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 79:315–328[CrossRef][Medline]
  42. Ji WR, Castellino FJ, Chang Y, Deford ME, Gray H, Villarreal X, Kondri ME, Marti DN, Llinas M, Schaller J, Kramer RA, Trail PA 1998 Characterization of kringle domains of angiostatin as antagonists of endothelial cell migration, an important process in angiogenesis. FASEB J 12:1731–1738[Abstract/Free Full Text]
  43. Lucas R, Holmgren L, Garcia I, Jimenez B, Mandriota SJ, Borlat F, Sim BK, Wu Z, Grau GE, Shing Y, Soff GA, Bouck N, Pepper MS 1998 Multiple forms of angiostatin induce apoptosis in endothelial cells. Blood 92:4730–4741[Abstract/Free Full Text]
  44. Claesson-Welsh L, Welsh M, Ito N, Anand-Apte B, Soker S, Zetter B, O’Reilly M, Folkman J 1998 Angiostatin induces endothelial cell apoptosis and activation of focal adhesion kinase independently of the integrin-binding motif RGD. Proc Natl Acad Sci USA 95:5579–5583[Abstract/Free Full Text]
  45. Koshida R, Ou J, Matsunaga T, Chilian WM, Oldham KT, Ackerman AW, Pritchard Jr KA 2003 Angiostatin: a negative regulator of endothelial-dependent vasodilation. Circulation 107:803–806[Abstract/Free Full Text]
  46. Morikawa W, Yamamoto K, Ishikawa S, Takemoto S, Ono M, Fukushi J, Naito S, Nozaki C, Iwanaga S, Kuwano M 2000 Angiostatin generation by cathepsin D secreted by human prostate carcinoma cells. J Biol Chem 275:38912–38920[Abstract/Free Full Text]
  47. Patterson BC, Sang QA 1997 Angiostatin-converting enzyme activities of human matrilysin (MMP-7) and gelatinase B/type IV collagenase (MMP-9). J Biol Chem 272:28823–28825[Abstract/Free Full Text]
  48. Lijnen HR, Ugwu F, Bini A, Collen D 1998 Generation of an angiostatin-like fragment from plasminogen by stromelysin-1 (MMP-3). Biochemistry 37:4699–4702[CrossRef][Medline]
  49. Dong Z, Kumar R, Yang X, Fidler IJ 1997 Macrophage-derived metalloelastase is responsible for the generation of angiostatin in Lewis lung carcinoma. Cell 88:801–810[CrossRef][Medline]
  50. Gately S, Twardowski P, Stack MS, Cundiff DL, Grella D, Castellino FJ, Enghid J, Kwaan HC, Lee F, Kramer RA, Volpert O, Bouck N, Soff GA 1997 The mechanism of cancer-mediated conversion of plasminogen to the angiogenesis inhibitor angiostatin. Proc Natl Acad Sci USA 94:10868–10872[Abstract/Free Full Text]
  51. Seno H, Oshima M, Ishikawa TO, Oshima H, Takaku K, Chiba T, Narumiya S, Taketo MM 2002 Cyclooxygenase 2- and prostaglandin E2 receptor EP(2)-dependent angiogenesis in Apc({delta}716) mouse intestinal polyps. Cancer Res 62:506–511[Abstract/Free Full Text]
  52. Smith SK 1998 Angiogenesis, vascular endothelial growth factor and the endometrium. Hum Reprod Update 4:509–519[Abstract/Free Full Text]
  53. Ma L, del Soldato P, Wallace JL 2002 Divergent effects of new cyclooxygenase inhibitors on gastric ulcer healing: shifting the angiogenic balance. Proc Natl Acad Sci USA 99:13243–13247[Abstract/Free Full Text]
  54. Sennlaub F, Valamanesh F, Vazquez-Tello A, El-Asrar AM, Checchin D, Brault S, Gobeil F, Beauchamp MH, Mwaikambo B, Courtois Y, Geboes K, Varma DR, Lachapelle P, Ong H, Behar-Cohen F, Chemtob S 2003 Cyclooxygenase-2 in human and experimental ischemic proliferative retinopathy. Circulation 108:198–204[Abstract/Free Full Text]



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