Urocortin 2 role in placental and myometrial inflammatory mechanisms at parturition.

The purpose of the study was to investigate urocortin (Ucn)2 involvement in placental and myometrial inflammatory pathways associated with parturition by evaluating: 1) Ucn2 and its receptor, CRH-receptor type 2 (CRH-R2), expression in laboring/nonlaboring human gestational tissues and in mouse utero-placental tissues approaching delivery; and 2) Ucn2 effect on myometrial contractility and on the expression of inflammatory mediators (prostaglandin F2α receptor and cytokines) and regulation of Ucn2 by TNF-α in cultured myometrial cell line. Placenta (n = 16), fetal membranes (n = 16), and myometrium (n = 22) were obtained from healthy pregnant women delivering at term by vaginal/elective caesarean delivery and from timed-pregnant mice on days 16-19. Expression of Ucn2/CRH-R2 in human/mouse tissues and inflammatory mediators in myometrial cell lines were measured by RT-PCR or ELISA, mouse Ucn2/CRH-R2 protein localization by immunohistochemistry. Ucn2 but not CRH-R2 was up-regulated (P < .05) in all human tissues in labor (compared with before labor) and increased significantly (P < .01) in mouse placenta approaching delivery. Ucn2 was up-regulated by TNF-α via nuclear factor-κB (NF-kB) in myometrium cell lines (P < .05 or P < .01 on the basis of treatment doses) and increased proinflammatory mediators and prostaglandin F (PGF2α) receptor expression (P < .05) via CRH-R2, without a direct effect on contractility. Placental and myometrial Ucn2 may play a role in the endocrine-inflammatory processes of parturition, representing a potential target for treating inflammation-induced obstetric complications.

A novel antiinflammatory role for the short-chain fatty acids in human labor.

Human parturition is an inflammatory process that can be activated prematurely by pathological stimuli. This study investigated the expression of G protein-coupled receptors GPR43 and GPR41 receptors in human uteroplacental tissues and the role of short-chain fatty acids (SCFA) in modulating inflammatory pathways in fetal membranes. Expression of GPR43 and GPR41 was investigated in uteroplacental tissues collected from women delivering at term or preterm after ethical approval and patient informed consent. The effect of SCFA on expression of inflammatory genes was assessed in amnion explants after culture with a mimetic of infection (lipopolysaccharide, LPS). Sodium propionate effect on LPS-induced neutrophil chemotaxis was evaluated by transwell assay. GPR43 and GPR41 mRNA expression was higher in myometrium and fetal membranes collected from women after the onset of labor. GPR43 protein expression localized to immune cells and vascular endothelium in the myometrium and epithelium of fetal membranes. Treatment with LPS significantly increased mRNA expression of GPR43 and inflammatory genes. Cotreatment with LPS and sodium propionate decreased LPS-induced expression of inflammatory genes including IL-6, IL-8, cyclooxygenase-2, IL-1α, intercellular adhesion molecule-1, and platelet endothelial cell adhesion molecule-1 but not IL-1ß or lymphocyte function-associated antigen-1. Sodium propionate reduced LPS-induced neutrophil chemotaxis and protein secretion of the neutrophil chemoattractant IL-8. Finally, fetal membrane expression of GPR43 was significantly higher in women delivering preterm with evidence of infection. GPR43-SCFA interactions may represent novel pathways that regulate inflammatory processes involved in human labor. Suppression of inflammatory pathways by SCFA may be therapeutically beneficial for pregnant women at risk of pathogen-induced preterm delivery.

The expression and regulation of adrenomedullin in the human endometrium: a candidate for endometrial repair.

After menstruation, the endometrium has a remarkable capacity for repair, but the factors involved remain undefined. We hypothesize adrenomedullin (AM) plays a role in this process. Premenstrually progesterone levels decline, stimulating prostaglandin (PG) synthesis, vasoconstriction, and hypoxia. This study aimed to determine 1) AM expression throughout the menstrual (M) cycle and 2) its regulation by PG and hypoxia. Human endometrial biopsies (n = 51) were collected with ethical approval and consent. AM mRNA expression was examined by quantitative RT-PCR and was found to be selectively elevated in endometrium from the menstrual (M) phase (P < 0.001). AM immunohistochemical staining was maximal in M and proliferative (P) endometrium. Culture of secretory, but not P, explants with 100 nm PGF(2α) or hypoxia (0.5% O2) increased AM mRNA (P < 0.05). P explants were induced to increase AM expression using in vitro progesterone withdrawal but required the presence of hypoxia (P < 0.05). Short hairpin sequences against hypoxia-inducible factor-1α (HIF-1α) inhibited AM hypoxic up-regulation but did not alter PGF(2α)-induced expression. The AM receptor was immunolocalized to endothelial cells in both lymphatic and blood vessels. Conditioned medium from PGF(2α)-treated cells increased endothelial cell proliferation and branching (P < 0.05). This was abolished by AM receptor antagonists. In conclusion, AM is elevated at the time of endometrial repair and induces both angiogenesis and lymphangiogenesis by stimulating endothelial cell proliferation and tube formation. In the human endometrium, AM expression is up-regulated by two mechanisms: a HIF-1α-mediated hypoxic induction and a HIF-1α-independent PGF(2α) pathway. These physiological mechanisms may provide novel therapeutic targets for disorders such as heavy menstrual bleeding.

Prokineticin-1: a novel mediator of the inflammatory response in third-trimester human placenta.

Prokineticin-1 (PK1) is a recently described protein with a wide range of functions, including tissue-specific angiogenesis, modulation of inflammatory responses, and regulation of hemopoiesis. The aim of this study was to investigate the localization and expression of PK1 and PK receptor-1 (PKR1), their signaling pathways, and the effect of PK1 on expression of the inflammatory mediators cyclooxygenase (COX)-2 and IL-8 in third-trimester placenta. PK1 and PKR1 were highly expressed in term placenta and immunolocalized to syncytiotrophoblasts, cytotrophoblasts, fetal endothelium, and macrophages. PK1 induced a time-dependent increase in expression of IL-8 and COX-2, which was significantly reduced by inhibitors of Gq, cSrc, epidermal growth factor receptor (EGFR), and MAPK kinase. Treatment of third-trimester placenta with 40 nm PK1 induced a rapid phosphorylation of cSrc, EGFR, and ERK1/2. Phosphorylation of ERK1/2 in response to PK1 was dependent on sequential phosphorylation of cSrc and EGFR. Using double-immunofluorescent immunohistochemistry, PKR1 colocalized with IL-8 and COX-2 in placenta. These data suggest that PK1 may have a novel role as a mediator of the inflammatory response in placenta.

Reciprocal cross talk between gonadotropin-releasing hormone (GnRH) and prostaglandin receptors regulates GnRH receptor expression and differential gonadotropin secretion.

The asynchronous secretion of gonadotrope LH and FSH under the control of GnRH is crucial for ovarian cyclicity but the underlying mechanism is not fully resolved. Because prostaglandins (PG) are autocrine regulators in many tissues, we determined whether they have this role in gonadotropes. We first demonstrated that GnRH stimulates PG synthesis by induction of cyclooxygenase-2, via the protein kinase C/c-Src/phosphatidylinositol 3'-kinase/MAPK pathway in the LbetaT2 gonadotrope cell line. We then demonstrated that PGF(2alpha) and PGI2, but not PGE2 inhibited GnRH receptor expression by inhibition of phosphoinositide turnover. PGF(2alpha), but not PGI2 or PGE2, reduced GnRH-induction of LHbeta gene expression, but not the alpha-gonadotropin subunit or the FSHbeta subunit genes. The prostanoid receptors EP1, EP2, FP, and IP were expressed in rat gonadotropes. Incubations of rat pituitaries with PGF(2alpha), but not PGI2 or PGE2, inhibited GnRH-induced LH secretion, whereas the cyclooxygenase inhibitor, indomethacin, stimulated GnRH-induced LH secretion. None of these treatments had any effect on GnRH-induced FSH secretion. The findings have thus elaborated a novel GnRH signaling pathway mediated by PGF(2alpha)-FP and PGI2-IP, which acts through an autocrine/paracrine modality to limit autoregulation of the GnRH receptor and differentially inhibit LH and FSH release. These findings provide a mechanism for asynchronous LH and FSH secretions and suggest the use of combination therapies of GnRH and prostanoid analogs to treat infertility, diseases with unbalanced LH and FSH secretion and in hormone-dependent diseases such as prostatic cancer.

Prostaglandin receptors are mediators of vascular function in endometrial pathologies.

Prostaglandins are bioactive lipids produced from arachidonic acid by cyclooxygenase enzymes and specific terminal prostanoid synthase enzymes. Following biosynthesis, prostaglandins exert an autocrine/paracrine function by coupling to specific prostanoid G protein-coupled receptors to activate intracellular signaling and gene transcription. For many years prostaglandins have been recognised as key molecules in reproductive biology by regulating ovulation, endometrial physiology and proliferation of endometrial glands and menstruation. More recently a role for COX enzymes and prostaglandins has been ascertained in reproductive tract pathology, including dysmenorrhea, endometriosis, menorrhagia and cancer. Emerging evidence supports a role for COX enzymes, prostaglandins and prostaglandin receptor signaling pathways in a multitude of phenotypic changes in reproductive tissues including the promotion of angiogenesis and vascular function. Here we provide an overview of some of the findings from these studies with specific emphasis on the role of cyclooxygenase enzymes, prostaglandins and their receptors in benign and neoplastic pathologies of the human endometrium.

Prostacyclin receptor up-regulates the expression of angiogenic genes in human endometrium via cross talk with epidermal growth factor Receptor and the extracellular signaling receptor kinase 1/2 pathway.

Prostacyclin (PGI) is a member of the prostanoid family of lipid mediators that mediates its effects through a seven-transmembrane G protein-coupled receptor (IP receptor). Recent studies have ascertained a role for prostanoid-receptor signaling in angiogenesis. In this study we examined the temporal-spatial expression of the IP receptor within normal human endometrium and additionally explored the signaling pathways mediating the role of IP receptor in activation of target angiogenic genes. Quantitative RT-PCR analysis demonstrated the highest endometrial expression of the IP receptor during the menstrual phase compared with all other stages of the menstrual cycle. Immunohistochemical analysis localized the site of IP receptor expression to the glandular epithelial compartment with stromal and perivascular cell immunoreactivity. Expression of the immunoreactive IP receptor protein was greatest during the proliferative and early secretory phases of the menstrual cycle. To explore the role of the IP receptor in glandular epithelial cells, we used the Ishikawa endometrial epithelial cell line. Stimulation of Ishikawa cells and human endometrial biopsy explants with 100 nm iloprost (a PGI analog) rapidly activated ERK1/2 signaling and induced the expression of proangiogenic genes, basic fibroblast growth factor, angiopoietin-1, and angiopoietin-2, in an epidermal growth factor receptor (EGFR)-dependent manner. Furthermore, EGFR colocalized with IP receptor in the glandular epithelial compartment. These data suggest that PGI-IP interaction within glandular epithelial cells can promote the expression of proangiogenic genes in human endometrium via cross talk with the EGFR.

Prostaglandin E2 mediates phosphorylation and down-regulation of the tuberous sclerosis-2 tumor suppressor (tuberin) in human endometrial adenocarcinoma cells via the Akt signaling pathway.

Prostaglandin (PG) E2 promotes tumor growth via interaction with its G protein-coupled receptors and activation of intracellular signaling. Tuberous sclerosis 2 (tuberin) is a tumor suppressor, which negatively regulates cell growth. Its phosphorylation results in its inactivation and targeted down- regulation, thus lifting the growth inhibition effects. This study investigated the expression and localization of tuberin in neoplastic and normal endometrium and the effect of PGE2 on phosphorylation of tuberin via the Akt pathway. Quantitative RT-PCR and Western blot analysis demonstrated reduced expression of tuberin in neoplastic tissue, compared with normal endometrial tissue. Tuberin expression was localized by immunohistochemistry to the glandular epithelial compartment in neoplastic and normal endometrium. We investigated the effect of PGE2 on phosphorylation of tuberin via the Akt pathway. Treatment of neoplastic and normal endometrium with 100 nm PGE2 enhanced phosphorylated tuberin immunoreactivity in the glandular epithelium. PGE2 also phosphorylated Akt and tuberin in Ishikawa endometrial adenocarcinoma cells, leading to a reduction in expression of total tuberin protein. Cotreatment of cells with wortmannin or LY294002 inhibited the PGE2-induced phosphorylation of Akt and tuberin. These data suggest that PGE2 signaling may promote endometrial tumorigenesis by inactivation of tuberin after its phosphorylation via the Akt signaling pathway.

Serotonin transporter 5HTTLPR polymorphism and affective disorders: no evidence of association in a large European multicenter study.

The available data from preclinical and pharmacological studies on the role of the serotonin transporter (5-HTT) support the hypothesis that a dysfunction in brain serotonergic system activity contributes to the vulnerability to affective disorders (AD). 5-HTT is the major site of serotonin reuptake into the presynaptic neuron, and it has been shown that the polymorphic repeat polymorphism in the 5-HTT promotor region (5-HTTLPR) may affect gene-transcription activity. 5-HTT maps to chromosome 17 at position 17q11.17-q12, and the 5-HTTLPR polymorphisms have been extensively investigated in AD with conflicting results. The present study tested the genetic contribution of the 5-HTTLPR polymorphism in a large European multicenter case-control sample, including 539 unipolar (UPAD), 572 bipolar patients (BPAD), and 821 controls (C). Our European collaboration has led to efforts to optimize a methodology that attenuates some of the major limitations of the case-control association approach. No association was found with primary psychiatric diagnosis (UPAD and BPAD) and with phenotypic traits (family history of AD, suicidal attempt, and presence of psychotic features). Our negative findings are not attributable to the lack of statistical power, and may contribute to clarify the role of 5-HTTLPR polymorphism in AD.

Expression and localization of endothelial monocyte-activating polypeptide II in the human endometrium across the menstrual cycle: regulation of expression by prostaglandin E(2).

A role for prostaglandins (PGs) in the regulation of endometrial functions, such as menstruation, has been established, although the mechanisms by which this is achieved are not fully elucidated. In the present study, cDNA array analysis has identified endothelial monocyte-activating polypeptide II (EMAP II) as a PGE(2)-regulated gene in endometrial epithelial cells. Incubation of endometrial epithelial cells with 100 nM PGE(2) for 4 and 24 h resulted in a 2.3- and 16-fold decrease in EMAP II expression, respectively. In endometrial tissue collected across the menstrual cycle, a significant increase in EMAP II mRNA was observed during the late secretory phase, compared with the proliferative and early-midsecretory phases. The temporal pattern of EMAP II expression was confirmed further by Western blotting; EMAP II protein expression was detected as a 43-kDa band. In situ hybridization and immunohistochemistry localized EMAP II mRNA and protein expression in glandular epithelial, endothelial, and stromal cells in the functionalis and basalis layers of the endometrium. Finally, the role of PGE(2) in the regulation of EMAP II expression in the endometrium was assessed. Incubation of fresh endometrial tissue (n = 5) with 3 micro g/ml indomethacin resulted in an increase in EMAP II protein expression, compared with control untreated tissue. However, cotreatment of the cells with 100 nM PGE(2) resulted in a significant decrease in EMAP II protein expression, compared with tissue incubated with indomethacin alone (P < 0.05). These data confirm temporal variation in EMAP II expression in the human endometrium across the menstrual cycle and localize expression to glandular epithelial, endothelial, and stromal cells. Moreover, EMAP II expression is negatively regulated by PGE(2).