Selective inhibition of prostaglandin E2 receptors EP2 and EP4 inhibits invasion of human immortalized endometriotic epithelial and stromal cells through suppression of metalloproteinases☆
Introduction
Endometriosis is an estrogen-dependent inflammatory disease of reproductive-age women characterized by the presence of functional endometrial tissues outside the uterine cavity (Giudice and Kao, 2004, Bulun, 2009). The prevalence of the disease is ∼10–20% in childbearing-age women, increased to 20–30% in women with subfertility, and to 40–60% in women with dysmenorrhoea (Giudice and Kao, 2004, Bulun, 2009). The involvement of PGE2 in the pathogenesis of endometriosis has been considered for a long time; however, the underlying molecular and cellular mechanisms are largely unknown (Wu et al., 2007a). Cyclooxygenase-2 (COX-2) and prostaglandin E synthase-1 are the rate limiting enzymes regulate biosynthesis of PGE2. Multifaceted actions of PGE2 are mediated through G-protein coupled receptors designated EP1, EP2, EP3 and EP4. EP1 activates PKC pathway. EP2 and EP4 activate PKA pathway. Activation of EP3A-D produces a wide range of complex and opposite actions (Narumiya et al., 1999). Recently, we have found that PGE2 transactivates ERK1/2, AKT, NFκB, and β-catenin pathways through EP2 and EP4 in human endometriotic epithelial cells 12Z and stromal cells 22B (Banu et al., 2009a). We have shown that COX-2, EP2 and EP4 proteins are abundantly expressed in ectopic endometrium, highly expressed in eutopic endometrium of women with endometriosis compared to that of endometriosis-free women, and EP1 is expressed at very low levels and EP3 is undetectable in ectopic and eutopic endometria during proliferative phase of the menstrual cycle (Banu et al., 2008a, Banu et al., 2009a).
In endometriosis patients, concentrations of PGE2 in the peritoneal fluid are higher compared to that of endometriosis-free women (De Leon et al., 1986), and this increased PGE2 is thought to promote inflammation, invasion, survival, and pains of endometriosis (Wu et al., 2007a). Selective COX-2 inhibitor rofecoxib at 25 mg/day for six months effectively suppressed the pelvic pain symptoms in endometriosis patients (Cobellis et al., 2004). GnRH agonist leuprolide acetate at 3.75 mg for one month decreased expression of COX-2 along with p450 aromatase in eutopic endometria of endometriosis patients (Kim et al., 2009). PGE2 stimulates de novo estrogen biosynthesis from endometriotic lesions and in turn estradiol increases COX-2 expression and PGE2 production (Bulun, 2009). In vitro studies indicated that selective COX-2 inhibitor celecoxib prevented growth and survival of primary cultured eutopic endometrial epithelial cells from endometriosis patients (Olivares et al., 2008). In animal models for endometriosis, celecoxib decreased establishment of endometriosis and the number and size of endometriotic implants in rat (Matsuzaki et al., 2004), and selective COX-2 inhibitor NS-398 induced regression of endometriotic implants through caspase-3 dependent apoptosis in hamster (Laschke et al., 2007). Collectively, these studies strongly indicate an important role for COX-2/PGE2 in the pathogenesis of endometriosis and suggest that inhibition of COX-2-derived PGE2 production and action might emerge as a potential therapy for endometriosis.
The ability of the human endometriotic cells to invade the peritoneum is augmented by their increased proteolytic activity which is coordinately regulated by MMPs and TIMPs (Curry and Osteen, 2003, Osteen et al., 2003, Zhou and Nothnick, 2005, Pitsos and Kanakas, 2009). Severity and extent of endometriosis is primarily associated with degree of expression or activity of MMP2 and MMP9 at endometrial–peritoneal interface (Chung et al., 2002, Szamatowicz et al., 2002, Laudanski et al., 2005, Pan et al., 2008, Salata et al., 2008, Shaco-Levy et al., 2008, Han et al., 2009). Although spatial and temporal expression of MMPs and TIMPs are reported in eutopic and ectopic endometria during the menstrual cycle, the molecular endocrine control of MMPs in invasion of endometrial/endometriotic cells is not well understood. PGE2 regulates expression, secretion, and activation of MMP1 (Kim et al., 2005), MMP2 (Ito et al., 2004, Lee et al., 2007), MMP9 (Pavlovic et al., 2006, Lee et al., 2007, Gomez-Hernandez et al., 2008, Itatsu et al., 2009), MMP13 (Molloy et al., 2008), MT1-MMP (Alfranca et al., 2008), and EMMPRIN (Chang et al., 2008) in various cell types. We have recently found that inhibition of COX-2 decreased migration and invasion of endometriotic epithelial cells 12Z and stromal cells 22B through suppression of MMP2 and MMP9 activity and which is associated with decreased PGE2 production (Banu et al., 2008a). Unfortunately, clinical use of COX-2 selective inhibitors confers cardiovascular risk (Warner et al., 2002), therefore, identification of therapeutic targets down-stream of COX-2 becomes obligatory.
The objectives of the present study were to determine functional interaction between PGE2 signaling and invasion of human endometriotic epithelial and stromal cells and to unravel the underlying molecular and cellular mechanisms. Results of our present study for the first time indicate that loss-of-function of EP2 and EP4-mediated PGE2 signaling suppresses invasion of human endometriotic epithelial and stromal cells through MMP-mediated pathways in a stromal–epithelial cell specific manner.
Section snippets
Materials
The reagents used in this study were purchased from the following suppliers: prestained protein markers, Bio-Rad assay reagents, and protein standards (Bio-Rad Laboratories, Hercules, CA); Protran BA83 nitrocellulose membrane (Whatman Inc., Sanford, ME); Pierce ECL (Pierce Biotechnology, Rockford, IL); protease inhibitor cocktail tablets complete EDTA-free and PhosStop (Roche Applied Biosciences, Indianapolis, IN); antibiotic–antimycotic, and trypsin–EDTA (Invitrogen Life Technologies Inc.,
Selective inhibition of EP2 and EP4 decreased migration and invasion of human endometriotic cells
We determined whether selective inhibition of EP2 and EP4 inhibits migration and invasion of human endometriotic epithelial cells 12Z and stromal cells 22B. Results (Fig. 1A–H) indicated that selective pharmacological inhibition of either EP2 or EP4 decreased (P < 0.05) migration of 12Z cells ∼50–55% and 22B cells ∼35–40% and invasion of 12Z cells 40–42% and 22B cells 32–35%, respectively. Combined inhibition of EP2 and EP4 produced synergistic/additive effects and decreased (P < 0.05) migration of
Discussion
In the present study, we determined molecular and cellular mechanisms through which inhibition of EP2 and EP4-mediated PGE2 signaling decreased invasion of human endometriotic epithelial and stromal cells. Results indicate that human endometriotic epithelial and stromal cells have distinct migration and invasion potential. Selective inhibition of EP2 and EP4 decreases expression of MMP1, MMP2, MMP7, and MMP9 but not stromal-specific MMP3 proteins in 12Z cells and MMP1, MMP2, MMP3, MMP7, and
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This work was supported by program development award to JAA from the Department of Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University.