HUVECs were infected with scrambled adenovirus (scr) or sh478 for 24 hrs. independently of PGF2 secretion via an FGF2-FGFR1-ERK1/2 dependent mechanism involving activation of the mTOR pathway. Conclusions Taken together, we have shown a novel mechanism whereby epithelial prostaglandin F2-FP signalling regulates endothelial cell network formation and proliferation. In addition we provide novel in vitro evidence to suggest that prostaglandin F2 can directly regulate endothelial cell network formation but not endothelial cell proliferation. These findings have relevance for pathologies where the FP receptor is aberrantly expressed, such as endometrial adenocarcinoma, and provide in vitro evidence to suggest that targeting the FP receptor could provide an anti-angiogenic approach to reducing tumour vasculature and NGI-1 growth. Background Endometrial adenocarcinoma, originating from the glandular epithelial cells of the uterine endometrial lining, is one of the most prevalent cancers amongst women in the Western world [1,2]. It is a disease which particularly occurs in post menopausal women and recent evidence suggests that mutations in oncogene expression may play a role in the etiology of the disease [3]. Data generated in our laboratory and others have ascertained a role for the cyclooxygenase (COX)-prostaglandin (PG) axis in the regulation of endometrial adenocarcinomas by increasing cell proliferation and the secretion of angiogenic growth factors [4,5]. This is similar to other cancers where over-expression of COX enzymes and biosynthesis of prostaglandins has been shown to promote cellular proliferation [6], inhibit apoptosis [7] and enhance angiogenesis [8]. However, the molecular mechanisms mediating the role of prostaglandins in regulating vascular function and angiogenesis are still poorly defined. Angiogenesis is the process of endothelial cell sprouting from an existing vasculature towards cancer cells [9] and is required by any tumour larger than 2 mm in diameter [10]. The proposed mechanism of angiogenesis suggests that tumour cells secrete stimulatory factors which act in a paracrine manner on surrounding blood vessels, immune cells and fibroblasts to promote the proliferation, differentiation and migration of endothelial cells towards the stimulus [10,11]. These tumour stimulatory factors include vascular endothelial growth factor (VEGF-A) and fibroblast growth factor 2 (FGF2). In human endometrial adenocarcinomas VEGF-A and FGF2 expression and secretion are elevated [12-14] and both VEGF-A and FGF2 can stimulate angiogenesis in xenografts in vivo [15,16]. In a NGI-1 previous study we demonstrated elevated expression of the FP receptor, FGF2 and the FGF2 receptor 1 (FGFR1) in neoplastic endometrial epithelial and vascular cells and ascertained a role for the FGF2, produced by PGF2-FP receptor signalling, on epithelial cell proliferation [12]. In this study we have shown that conditioned medium from PGF2 treated Ishikawa cells stably expressing the FP receptor (Ishikawa FPS cells), can increase endothelial cell differentiation (network formation) and proliferation. Treatment of Ishikawa FPS cells with PGF2 increases FGF2 secretion which in turn activates FGFR1 signalling in endothelial cells and induces the phosphorylation of extracellular signal-regulated kinase (ERK1/2), COX-2 expression and secretion of PGF2. Following its release from endothelial cells, we show for the first time that, PGF2 promotes endothelial cell network formation in an autocrine/paracrine manner, via the endothelial FP receptor. By contrast, PGF2 is not involved in endothelial cell proliferation which we show to be regulated by FGF2-FGFR1 signalling via the mammalian target of rapamycin (mTOR) pathway. Taken together, our data highlight two molecular pathways by which PGF2-FP receptor signalling can regulate endothelial cell function in endometrial adenocarcinomas. Results PGF2-FP signalling mediates endothelial cell network formation and proliferation via FGF2-FGFR1 signalling We previously demonstrated elevated expression of the FP receptor, FGF2 and FGFR1 in endometrial adenocarcinoma [12]. Using a neoplastic epithelial cell line stably expressing the FP receptor to the levels observed in endometrial adenocarcinoma (Ishikawa FPS cells), we ascertained a role for FGF2, produced by PGF2-FP receptor signalling, on epithelial cell proliferation [12]. In addition, we found that FP receptor, FGF2 and FGFR1 co-localised within the vascular endothelial cells in endometrial adenocarcinomas suggesting that PGF2 may directly and indirectly regulate endothelial cell function [12]. To determine if the effects of PGF2-FP receptor interaction in endometrial adenocarcinoma cells on endothelial cell function were mediated by FGF2, we used conditioned medium (CM) from Ishikawa FPS cells treated with vehicle or 100 nM PGF2 for.By contrast, PGF2 is not involved in endothelial cell proliferation which we show to be regulated by FGF2-FGFR1 signalling via the mammalian target of rapamycin (mTOR) pathway. cell differentiation (network formation) and proliferation. Using chemical inhibitors of intracellular signalling, we found that P CM-stimulated endothelial cell network formation was mediated by secretion of endothelial PGF2 and activation of endothelial FP receptors, following FGF2-FGFR1 signalling, phosphorylation of ERK1/2 and induction of COX-2. Whereas, P CM stimulation of endothelial cell proliferation occurred independently of PGF2 secretion via an FGF2-FGFR1-ERK1/2 dependent mechanism involving activation of the mTOR pathway. Conclusions Taken together, we have shown a novel mechanism whereby epithelial prostaglandin F2-FP signalling regulates endothelial cell network formation and proliferation. In addition we provide novel in vitro evidence to suggest that prostaglandin F2 can directly regulate endothelial cell network formation but not endothelial cell proliferation. These findings have relevance for pathologies where the FP receptor is aberrantly expressed, such as endometrial adenocarcinoma, and Rabbit Polyclonal to Thyroid Hormone Receptor beta provide in vitro evidence to suggest that targeting the FP receptor could provide an anti-angiogenic approach to reducing tumour vasculature and growth. Background Endometrial adenocarcinoma, originating from the glandular epithelial cells of the uterine endometrial lining, is one of the most prevalent cancers amongst women in the Western world [1,2]. It is a disease which NGI-1 particularly occurs in post menopausal women and recent evidence suggests that mutations in oncogene expression may play a role in the etiology of the disease [3]. Data generated in our laboratory and others have ascertained a role for the cyclooxygenase (COX)-prostaglandin (PG) axis in the regulation of endometrial adenocarcinomas by increasing cell proliferation and the secretion of angiogenic growth factors [4,5]. This is similar to other cancers where over-expression of COX enzymes and biosynthesis of prostaglandins has been shown to promote cellular proliferation [6], inhibit apoptosis [7] and enhance angiogenesis [8]. However, the molecular mechanisms mediating the role of prostaglandins in regulating vascular function and angiogenesis are still poorly defined. Angiogenesis is the process of endothelial cell sprouting from an existing vasculature towards cancer cells [9] and is required by any tumour larger than 2 mm in diameter [10]. The proposed mechanism of angiogenesis suggests that tumour cells secrete stimulatory factors which act in a paracrine manner on surrounding blood vessels, immune cells and fibroblasts to promote the proliferation, differentiation and migration of endothelial cells towards the stimulus [10,11]. These tumour stimulatory factors include vascular endothelial growth factor (VEGF-A) and fibroblast growth factor 2 (FGF2). In human endometrial adenocarcinomas VEGF-A and FGF2 expression and secretion are elevated [12-14] and both VEGF-A and FGF2 can stimulate angiogenesis in xenografts in vivo [15,16]. In a previous study we demonstrated elevated expression of the FP receptor, FGF2 and the FGF2 receptor 1 (FGFR1) in neoplastic endometrial epithelial and vascular cells and ascertained a role for the FGF2, produced by PGF2-FP receptor signalling, on epithelial cell proliferation [12]. In this study we have shown that conditioned medium from PGF2 treated Ishikawa cells stably expressing the FP receptor (Ishikawa FPS cells), can increase endothelial cell differentiation (network formation) and proliferation. Treatment of Ishikawa FPS cells with PGF2 increases FGF2 secretion which in turn activates FGFR1 signalling in endothelial cells and induces the phosphorylation of extracellular signal-regulated kinase (ERK1/2), COX-2 manifestation and secretion of PGF2. After its launch from endothelial cells, we display for the very first time that, PGF2 promotes endothelial cell network development within an autocrine/paracrine way, via the endothelial FP receptor. In comparison, PGF2 isn’t involved with endothelial cell proliferation which we display to become controlled by FGF2-FGFR1 signalling via the mammalian focus on of rapamycin (mTOR) pathway. Used collectively, our data focus on two molecular pathways where PGF2-FP receptor signalling can control endothelial cell function in endometrial adenocarcinomas. Outcomes PGF2-FP signalling mediates endothelial cell network development and proliferation via FGF2-FGFR1 signalling We previously proven elevated manifestation from the FP receptor, FGF2 and FGFR1 in endometrial adenocarcinoma [12]. Utilizing a neoplastic epithelial cell range stably expressing the FP receptor towards the levels seen in endometrial adenocarcinoma (Ishikawa FPS cells), we ascertained a job for FGF2, made by PGF2-FP receptor signalling, on epithelial cell proliferation [12]. Furthermore, we discovered that FP receptor, FGF2 and FGFR1 co-localised inside the vascular endothelial cells in endometrial adenocarcinomas recommending that PGF2 may straight and indirectly regulate endothelial cell function [12]. To see whether the consequences of PGF2-FP receptor discussion in endometrial adenocarcinoma cells on endothelial cell function had been mediated by FGF2, we utilized conditioned moderate (CM) from Ishikawa FPS cells treated with automobile or 100 nM PGF2 every day and night. The current presence of FGF2 in CM from automobile (V CM) and PGF2-treated (P CM) Ishikawa FPS cells was verified by ELISA. Immunoneutralisation of P CM with FGF2 antibody.
