Prostaglandin E2 induces proliferation of glandular epithelial cells of the human endometrium via extracellular regulated kinase 1/2-mediated pathway.

In this study, we investigated the effect of prostaglandin E(2) (PGE(2)) on MAPK ERK1/2 protein phosphorylation and on proliferation of epithelial cells of the human endometrium. Treatment of proliferative phase endometrium with PGE(2) induced rapid phosphorylation of ERK1/2 proteins in glandular epithelial and endothelial cells. Treatment of human endometrial tissue with PGE(2) for 24 h resulted in increased incorporation of 5-bromo-2'-deoxyuridine (a marker of cellular proliferation) in glandular epithelial cells. To investigate further the effect of PGE(2) on proliferation of epithelial cells, we used an endometrial epithelial cell line (HES). HES cells express functional EP4 (with absence of expression of EP1, EP2, and EP3) receptors and stimulate cAMP release and rapid phosphorylation of ERK1/2 proteins in response to PGE(2) or forskolin. Treatment of HES cells with PGE(2) or forskolin alone resulted in a significant increase in HES cell proliferation compared with control untreated cells (P < 0.05). Cotreatment of the cells with PGE(2) or forskolin and PD98059 abolished the increase in cellular proliferation. These data demonstrate ERK1/2 phosphorylation in response to PGE(2) in the human endometrium and suggest that PGE(2) via EP4 receptor may induce glandular epithelial cell proliferation in ERK1/2- dependent manner during the proliferative phase of the menstrual cycle.

Expression of functional prolactin receptors in nonpregnant human endometrium: janus kinase-2, signal transducer and activator of transcription-1 (STAT1), and STAT5 proteins are phosphorylated after stimulation with prolactin.

PRL is synthesized by decidualized endometrial stromal cells from the midsecretory phase in a nonconception cycle and throughout pregnancy. The exact role of PRL in the human endometrium remains to be elucidated; however, the pattern of expression supports a role for PRL during implantation and placentation. This study investigated the site and pattern of expression of PRL receptors in the nonpregnant human endometrium. In situ hybridization and immunohistochemistry localized expression of the receptor in the glandular epithelium and a subset of stromal cells of the endometrium. As judged by the intensity of staining, expression of the receptor was dramatically up-regulated during the secretory phase. Expression of the PRL receptor gene in the endometrium from the secretory phase of the menstrual cycle was confirmed by ribonuclease protection assay using 50 micrograms total ribonucleic acid. Phosphorylation of Janus kinase-2 (JAK2), STAT1 (signal transducer and activator of transcription-1), and STAT5 proteins in response to PRL was investigated to establish the signaling pathway of PRL in the human endometrium. Endometrial tissue was collected during the secretory phase of the menstrual cycle and incubated in the presence of 100 ng/mL human PRL for 0, 5, 10, and 20 min. JAK2 phosphorylation was induced by PRL at 5 min, whereas STAT1 and STAT5 phosphorylation was apparent 20 min after stimulation with PRL. Immunohistochemistry localized the JAK/STAT proteins in the glandular epithelial cells and a subset of stromal cells, as was observed for the PRL receptor. Secretory phase stromal and glandular cells cultured separately and in the presence or absence of 100 ng/mL PRL confirmed the PRL-induced phosphorylation of JAK2/STAT proteins, at least in the glandular compartment. These studies demonstrate an up-regulation of expression of functional PRL receptors during the secretory phase of the menstrual cycle. Further, decidual PRL through a paracrine mechanism may influence glandular epithelial function/secretions and direct gene transcription through the JAK/STAT pathway. The target genes activated by PRL in the glandular epithelium of the nonpregnant human endometrium remain to be elucidated.

Expression, localization, and signaling of PGE(2) and EP2/EP4 receptors in human nonpregnant endometrium across the menstrual cycle.

This study was designed to elucidate the sites of synthesis and action of PGE(2) in the nonpregnant human uterus across the menstrual cycle. The sites of expression of PGE synthase and synthesis of PGE(2) were investigated by immunohistochemistry using full thickness uterine biopsies. Expression of PGE synthase and synthesis of PGE(2) were localized to glandular epithelial and endothelial cells in both basalis and functionalis regions of the human endometrium. By contrast, stromal staining was predominantly localized in the functionalis layer. Some cyclical variation in expression of PGE synthase and PGE(2) synthesis was observed, with reduced expression/synthesis detected in the stromal compartment of the functionalis during the late secretory phase of the menstrual cycle. Subsequently, we assessed the site of action of PGE(2) by investigating the expression of two PGE(2) receptor isoforms, namely EP2 and EP4. Cyclical variation in endometrial EP2 and EP4 receptor mRNA expression was quantified by TaqMan quantitative RT-PCR using RNA isolated from endometrial tissue collected across the menstrual cycle. No differences in EP2 receptor mRNA expression were detected; however, EP4 receptor mRNA expression was significantly higher in late proliferative stage (P < 0.05) than in early, mid, and late secretory stage endometrium. Expression patterns of EP2 and EP4 receptors were localized by nonradioactive in situ hybridization using fluorescein isothiocyanate end- labeled oligonucleotide probes. Expression of both receptors was observed in endometrial glandular epithelial and vascular cells, with no notable spatial or temporal variation. Finally, signaling of EP2/EP4 receptors was assessed by investigating cAMP generation in vitro after stimulation with PGE(2). Endometrial cAMP generation in response to PGE(2) was significantly greater in proliferative tissue compared with early and midsecretory stage tissue (3.77 +/- 0.85 vs. 1.96 +/- 0.28 and 1.38 +/- 0.23, respectively; P < 0.05). In conclusion, this study demonstrates glandular and vascular coexpression of PGE synthase, PGE(2), EP2, and EP4 receptors and suggests an autocrine/paracrine role for PGE(2) in epithelial/endothelial cell function in the human endometrium.

Localization of interferon regulatory factor-1 (IRF-1) in nonpregnant human endometrium: expression of IRF-1 is up-regulated by prolactin during the secretory phase of the menstrual cycle.

PRL expression in the human uterus is up-regulated during the mid to late secretory phase of the menstrual cycle. This coincides with up-regulation of the expression of the PRL receptor, which is localized primarily to the endometrial glandular epithelial cells. Recent data have demonstrated activation of the Jak (Janus kinase)/Stat (signal transducer and activator of transcription) signaling pathway in the secretory endometrium after stimulation with exogenous PRL. However, the target genes for the action of PRL on the endometrial epithelial cells have not been elucidated. In this study we have investigated the pattern/site of expression of the transcription factor interferon regulatory factor-1 (IRF-1) as well as the effect of exogenous PRL on the transcription of IRF-1 in the human endometrium during the mid to late secretory phase of the menstrual cycle. Expression of the IRF-1 gene was confirmed by RNase protection assays using a 260-bp homologous [alpha-32P]UTP-labeled IRF-1 complementary ribonucleic acid (RNA) probe and 10 microg total RNA extracted from human endometrium (n = 5) collected between days 19 and 26 of the menstrual cycle. Northern and Western blot analyses were conducted on secretory phase human endometrium (n = 3) using human [alpha-32P]dCTP-labeled IRF-1 complementary DNA and antihuman IRF-1 antibody. Expression of the IRF-1 gene in the secretory phase endometrium was encoded by a RNA transcript of approximately 2.1 kb and a protein of 48 kDa. Furthermore, expression of the IRF-1 gene in the secretory phase endometrium was localized by immunohistochemistry predominantly to the glandular epithelial cells as has been shown previously for the PRL receptor. To investigate the effect of PRL on expression of IRF-1, human endometrial biopsies (n = 3) collected between days 24-26 of the menstrual cycle were cultured in the presence of cycloheximide with or without 100 ng/mL human PRL for 2 and 4 h. Culture of endometrial tissue with PRL for 2 and 4 h resulted in 2.9 +/- 0.3-fold (P < 0.01) and 1.7 +/- 0.1-fold induction of expression of the IRF-1 gene, respectively. These data demonstrate the expression of the transcription factor IRF-1 in the glandular epithelium of the endometrium and its regulation by PRL during the secretory phase of the menstrual cycle. Previous observations of the temporal up-regulation of expression of both PRL and PRL receptors in the secretory human endometrium and their localization to the stromal and glandular compartments, respectively, suggest that endometrial PRL mediates transcription of the IRF-1 gene in a paracrine fashion.

Pattern and localisation of expression of vascular endothelial growth factor and its receptor flt-1 in the ovine pituitary gland: expression is independent of hypothalamic control.

The pituitary gland, a highly vascularised endocrine organ, contains permeable fenestrated endothelium that allows direct access of endocrine cells to the hemal milieu. Vascular endothelial growth factor (VEGF) has a mitogenic effect on endothelial cells and renders the endothelium more permeable. The following study investigated the expression of VEGF and its receptor flt-1 mRNA and protein in the pituitary gland of sheep. VEGF expression was localised, by in situ hybridisation and immunocytochemistry, mainly to the pars tuberalis/zona tuberalis (PT/ZT) region of the gland. No hybridisation signal was observed in the pars intermedia or pars nervosa. Reverse transcriptase-polymerase chain reaction (RT-PCR) Southern blotting confirmed the predominant expression of VEGF in the PT/ZT compared with the pars distalis (PD). Western blot analysis with the VEGF antibody revealed major (48 kDa) and minor (24 kDa) bands representing the monomer and dimer forms of VEGF and also confirmed the differential expression of VEGF in the PT/ZT compared with the PD. Double immunocytochemistry with VEGF and prolactin or luteinising hormone-beta (LH-beta) antibodies demonstrated that the VEGF-secreting cells are not lactotrophs or gonadotrophs. However, co-localisation of VEGF with S-100 was observed in a proportion of cells suggesting that some VEGF secreting cells are follicular stellate. Immunocytochemistry with a flt-1 antibody confirmed the expression of this high affinity receptor for VEGF in endothelial cells across the pituitary gland. Immunocytochemistry with the VEGF antibody using pituitary glands from intact and hypothalamo-pituitary disconnected sheep demonstrated comparable expression patterns suggesting that the regulation of blood flow and vascular permeability in the pituitary gland is under local regulation and is independent of hypothalamic input.

Autocrine/paracrine regulation of apoptosis in epithelial cells by prostaglandin E2.

This study was designed to investigate the effect of IL-1alpha-induced up-regulation of cyclooxygenase-2 (COX-2) on prostaglandin E(2) (PGE(2)) secretion and the subsequent phenotypic effects of PGE(2) on epithelial cells. The effect of IL-1alpha on COX-2 expression was investigated in the T24 bladder epithelial cell line following treatment with 0, 0.05, 0.5, 1 or 10 ng/ml IL-1alpha for 1, 2, 4 or 6 h. Quantitative PCR confirmed up-regulation of expression of COX-2 with maximal expression observed following treatment with 0.5 ng/ml IL-1alpha for 1 h. Co-treatment of the cells with 0.5 ng/ml IL-1alpha in the presence or absence of 100 ng/ml IL-1 receptor antagonist (RA) abolished the up-regulation in COX-2 expression confirming that the effect of IL-1alpha is mediated via its membrane-bound receptors. Treatment with 0.5 ng/ml IL-1alpha resulted in a time-dependent increase in PGE(2) secretion with maximal secretion detected at 24 and 48 h after stimulation with IL-1alpha. Co-treatment of the cells with IL-1alpha and IL-1RA or the COX-2 enzyme inhibitor NS398 abolished the IL-1alpha mediated secretion of PGE(2). Treatment of T24 cells with 100 nM PGE(2) resulted in a significant elevation in cAMP generation confirming the expression of functional PGE(2) receptors. Finally, the effect of exogenous treatment with PGE(2) on apoptosis of T24 cells was assessed using cell death detection ELISA. T24 cells were treated with camptothecin to induce apoptosis in the presence or absence of 50 or 100 nM PGE(2) or 10 microM forskolin. Treatment of T24 cells with increasing doses of camptothecin alone resulted in a significant increase in the induction of apoptosis (P<0.01). However, co-treatment of the cells with 50 or 100 nM PGE(2) or 10 microM forskolin resulted in the inhibition of induction of the apoptotic pathway by camptothecin. These data demonstrate that PGE(2) inhibits apoptosis of epithelial cells possibly via cAMP-dependent pathway.

F-prostanoid receptor alters adhesion, morphology and migration of endometrial adenocarcinoma cells.

Cellular adhesion to extracellular matrix is a central phenomenon for the maintenance of tissue integrity and cellular movement. Collectively, these processes are regulated by a fine-tuned balance between the formation and loosening of adhesive contacts, a process involving integrins, and the elevation and diminution of cytoplasmic signalling molecules. We demonstrate that prostaglandin (PG) F(2alpha) stimulation rapidly increases the capacity of Ishikawa cells stably expressing the F-prostanoid receptor (FPS) to adhere to vitronectin. Coincident with this elevation in matrix adhesion, we demonstrate a profound PGF(2alpha)-induced alteration in cytoskeletal remodelling, characterized by polymerization of the actin cytoskeleton and recruitment of focal adhesion kinase at focal adhesions and enhanced cell migration. Moreover, we show that these PGF(2alpha)-induced alterations in adhesion and morphology on vitronectin and migration could be abolished by cultivating FPS cells in the presence of integrin alphavbeta3 antibody or alphavbeta3-directed tetrapeptide arg-gly-asp-ser or inhibition of FP receptor signalling with the FP receptor antagonist, chemical disruptors of the phospholipase C-beta, protein kinase A, c-Src and epidermal growth factor receptor kinase pathways or inhibition of the monomeric G proteins Rho, Rac and CDC42. These results reveal a mechanism by which prostanoids regulate cell movement, which may be relevant to pathologies of the endometrium.

Expression of COX-2 and PGE synthase and synthesis of PGE(2)in endometrial adenocarcinoma: a possible autocrine/paracrine regulation of neoplastic cell function via EP2/EP4 receptors.

This study was designed to investigate the possible role of cyclo-oxygenase-2 (COX-2) and prostaglandin E(2)(PGE(2)) in endometrial adenocarcinoma. COX-2 RNA expression was confirmed in various grades of adenocarcinoma by ribonuclease protection assay. COX-2 and microsomal glutathione-dependent prostaglandin E synthase (mPGES) expression and PGE(2)synthesis were localised to the neoplastic epithelial cells and endothelial cells. In order to establish whether PGE(2)has an autocrine/paracrine effect in adenocarcinomas, we investigated the expression of 2 subtypes of PGE(2)receptors, namely EP2 and EP4, by real time quantitative PCR. Expression of EP2 and EP4 receptors was detected in adenocarcinomas from all grades of differentiation and was significantly higher than that detected in normal secretory phase endometrium (P< 0.01). The fold induction of expression in adenocarcinoma compared with normal secretory phase endometrium was 28.0 +/- 7.4 and 52.5 +/- 10.1 for EP2 and EP4 receptors respectively. Immunohistochemistry localised the site of expression of EP4 receptor in neoplastic epithelial cells and in the endothelium of carcinomas of all grades of differentiation. Finally, the functionality of the EP2/EP4 receptors was assessed by investigating cAMP generation following in vitro culture of adenocarcinoma tissue in the presence or absence of 300 nM PGE(2). cAMP production in response to PGE(2)was significantly higher in carcinoma tissue than that detected in normal secretory phase endometrium (3.42 +/- 0.46 vs 1.15 +/- 0.05 respectively; P< 0.001). In conclusion, these data suggest that PGE(2)may regulate neoplastic cell function in an autocrine/paracrine manner via the EP2/EP4 receptors.