16th GCC Closed Forum: ICH M10 implementation; NGS, qPCR/dPCR, flow cytometry validation; tissue biomarkers; IS response; immunogenicity harmonization; bioanalytical industry status.

The 16th GCC Closed Forum was held in Orlando, FL, USA, on 23 June 2023. Representatives from international bioanalytical Contract Research Organizations were in attendance in order to discuss scientific and regulatory issues specific to bioanalysis. The issues discussed at the meeting included: IS response, flow cytometry, changes to the bioanalytical industry, NGS assays, biomarker assay for tissues, dPCR validation, immunogenicity harmonization and ICH M10 implementation. Conclusions and consensus from discussions of these topics are included in this article.

Recommendations for the content and management of Certificates of Analysis for reference standards from the GCC for bioanalysis.

The 13th Global CRO Council (GCC) closed forum for bioanalysis was held in New Orleans, LA, USA on 5 April 2019. This GCC meeting was organized to discuss the contents of the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline published in February 2019 and consolidate the feedback of the GCC members. While ICH M10 will cover requirements for reference standards, one of the biggest challenges facing the CRO community is the lack of consistency and completeness of Certificates of Analysis for reference standards used in regulated bioanalysis. Similar challenges exist with critical reagents (e.g., capture and detection antibodies) used for assays supporting biologics. The recommendations provided in this publication are the minimum requirements for the content that GCC members believe should be included in Certificates of Analysis for reference standards obtained from commercial vendors, sponsors and compendial suppliers, for use in regulated bioanalytical studies. In addition, recommendations for internal standards, metabolites and critical reagents are discussed.

GCC Consolidated Feedback to ICH on the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline.

The 13th GCC Closed Forum for Bioanalysis was held in New Orleans, Louisiana, USA on April 5th, 2019. This GCC meeting was organized to discuss the contents of the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline published in February 2019 and consolidate the feedback of the GCC members. In attendance were 63 senior-level participants from eight countries representing 44 bioanalytical CRO companies/sites. This event represented a unique opportunity for CRO bioanalytical experts to share their opinions and concerns regarding the ICH M10 Bioanalytical Method Validation Draft Guideline and to build unified comments to be provided to the ICH.

Seminal Fluid-Mediated Inflammation in Physiology and Pathology of the Female Reproductive Tract.

Inflammation is a multifaceted process involving a host of resident and recruited immune cells that eliminate the insult or injury and initiate tissue repair. In the female reproductive tract (FMRT), inflammation-mediated alterations in epithelial, vascular, and immune functions are important components of complex physiological processes and many local and systemic pathologies. It is well established that intracoital and postcoital function of seminal fluid (SF) goes beyond nutritive support for the spermatozoa cells. SF, in particular, the inflammatory bioactive lipids, and prostaglandins present in vast quantities in SF, have a role in localized immune modulation and regulation of pathways that can exacerbate inflammation in the FMRT. In sexually active women SF-mediated inflammation has been implicated in physiologic processes such as ovulation, implantation, and parturition while also enhancing tumorigenesis and susceptibility to infection. This review highlights the molecular mechanism by which SF regulates inflammatory pathways in the FMRT and how alterations in these pathways contribute to physiology and pathology of the female reproductive function. In addition, based on findings from TaqMan® 96-Well Plate Arrays, on neoplastic cervical cells treated with SF, we discuss new findings on the role of SF as a potent driver of inflammatory and tumorigenic pathways in the cervix.

Early diagnosis of HIV among men who have sex with men in Western Australia: impact of a peer-led sexually transmissible infection testing service.

UNLABELLED: Background In July 2010, the Western Australian AIDS Council established the 'M Clinic', a peer-led STI testing service for MSM. This study describes trends in HIV notifications among MSM in WA from 2004 to 2013, particularly the impact of the M Clinic on newly acquired HIV diagnoses. METHODS: The number and proportion of MSM HIV cases with newly acquired infection were compared for the 2004-2006, 2007-2009 and 2011-2013 time periods. Data from 2010 were excluded as the M Clinic opened in July 2010. RESULTS: Between the 2004-2006 and 2007-2009 periods, the number of MSM with newly acquired HIV increased by 50% (23 to 33 cases) and the number of newly acquired cases as a proportion of all new HIV diagnoses among MSM increased from 27% to 35% (30% increase) (P=0.25). In the 2011-2013 period, the number of newly acquired HIV cases among MSM more than doubled to 70 cases and comprised 53% of all new HIV diagnoses among MSM (P<0.05). Of the 70 newly acquired HIV cases in the 2011-2013 period, 30% (n=21) were diagnosed at the M Clinic. CONCLUSIONS: The proportion of MSM HIV notifications that were newly acquired increased between 2004 and 2013 in WA, with the greatest increase seen after the M Clinic commenced operation. A peer-led approach to HIV testing should be considered in order to achieve early diagnosis and treatment of HIV among MSM.

Seminal plasma induces the expression of IL-1α in normal and neoplastic cervical cells via EP2/EGFR/PI3K/AKT pathway.

BACKGROUND: Cervical cancer is a chronic inflammatory disease of multifactorial etiology usually presenting in sexually active women. Exposure of neoplastic cervical epithelial cells to seminal plasma (SP) has been shown to promote the growth of cancer cells in vitro and tumors in vivo by inducing the expression of inflammatory mediators including pro-inflammatory cytokines. IL-1α is a pleotropic pro-inflammatory cytokine induced in several human cancers and has been associated with virulent tumor phenotype and poorer prognosis. Here we investigated the expression of IL-1α in cervical cancer, the role of SP in the regulation of IL-1α in neoplastic cervical epithelial cells and the molecular mechanism underlying this regulation. METHODS AND RESULTS: Real-time quantitative RT-PCR confirmed the elevated expression of IL-1α mRNA in cervical squamous cell carcinoma and adenocarcinoma tissue explants, compared with normal cervix. Using immunohistochemistry, IL-1α was localized to the neoplastically transformed squamous, columnar and glandular epithelium in all cases of squamous cell carcinoma and adenocarcinomas explants studied. We found that SP induced the expression of IL-α in both normal and neoplastic cervical tissue explants. Employing HeLa (adenocarcinoma) cell line as a model system we identified PGE2 and EGF as possible ligands responsible for SP-mediated induction of IL-1α in these neoplastic cells. In addition, we showed that SP activates EP2/EGFR/PI3kinase-Akt signaling to induce IL-1α mRNA and protein expression. Furthermore, we demonstrate that in normal cervical tissue explants the induction of IL-1α by SP is via the activation of EP2/EGFR/PI3 kinase-Akt signaling. CONCLUSION: SP-mediated induction of IL-1α in normal and neoplastic cervical epithelial cells suggests that SP may promote cervical inflammation as well as progression of cervical cancer in sexually active women.

The regulation of inflammatory pathways and infectious disease of the cervix by seminal fluid.

The connection between human papillomavirus (HPV) infection and the consequent sequelae which establishes cervical neoplastic transformation and invasive cervical cancer has redefined many aspects of cervical cancer research. However there is still much that we do not know. In particular, the impact of external factors, like seminal fluid in sexually active women, on pathways that regulate cervical inflammation and tumorigenesis, have yet to be fully understood. HPV infection is regarded as the initiating noninflammatory cause of the disease; however emerging evidence points to resident HPV infections as drivers of inflammatory pathways that play important roles in tumorigenesis as well as in the susceptibility to other infections such as human immunodeficiency virus (HIV) infection. Moreover there is emerging evidence to support a role for seminal fluid, in particular, the inflammatory bioactive lipids, and prostaglandins which are present in vast quantities in seminal fluid in regulating pathways that can exacerbate inflammation of the cervix, speed up tumorigenesis, and enhance susceptibility to HIV infection. This review will highlight some of our current knowledge of the role of seminal fluid as a potent driver of inflammatory and tumorigenic pathways in the cervix and will provide some evidence to propose a role for seminal plasma prostaglandins in HIV infection and AIDS-related cancer.

CCR5 expression is elevated in cervical cancer cells and is up-regulated by seminal plasma.

The interplay between inflammation, cervical cancer and HIV acquisition in women is poorly understood. We have previously shown that seminal plasma (SP) can promote cervical tumour cell growth in vitro and in vivo via the activation of potent inflammatory pathways. In this study, we investigated whether SP could regulate expression of chemokine receptors with known roles in HIV infection, in the cervix and in cervical cancer. The expression of CD4 and CCR5 was investigated by RT-PCR analysis and immunohistochemistry. CD4 and CCR5 expression was elevated in cervical cancer tissue compared with normal cervix. Ex vivo studies conducted on cervical tissues and HeLa cells showed that SP significantly increases the expression of CD4 and CCR5 transcripts. Furthermore, it was found that SP also up-regulates CCR5 protein in HeLa cells. The regulation of CCR5 expression was investigated following treatment of HeLa cells with SP in the presence/absence of chemical inhibitors of intracellular signalling, EP2 and EP4 antagonists, prostaglandin (PG) E2 and a cyclooxygenase (COX)-1 doxycycline-inducible expression system. These experiments demonstrated that the regulation of CCR5 expression by SP occurs via the epidermal growth factor receptor (EGFR)-COX-1-PGE2 pathway. This study provides a link between activation of inflammatory pathways and regulation of HIV receptor expression in cervical cancer cells.

Inflammatory pathways in cervical cancer - the UCT contribution.

Cervical cancer is the leading gynaecological malignancy in Southern Africa. The main causal factor for development of the disease is infection of the cervix with human papillomavirus. It is a multi-step disease with several contributing co-factors including multiple sexual partners, a compromised immune system and cervical inflammation caused by infections with Chlamydia trachomatis or Neisseria gonorrhoeae. Inflammation involves extensive tissue remodelling events which are orchestrated by complex networks of cytokines, chemokines and bio-active lipids working across multiple cellular compartments to maintain tissue homeostasis. Many pathological disorders or diseases, including cervical cancer, are characterised by the exacerbated activation and maintenance of inflammatory pathways. In this review we highlight our findings pertaining to activation of inflammatory pathways in cervical cancers, addressing their potential role in pathological changes of the cervix and the significance of these findings for intervention strategies.

Seminal plasma induces angiogenic chemokine expression in cervical cancer cells and regulates vascular function.

Cervical cancer is one of the leading gynecological malignancies in women. We have recently shown that seminal plasma (SP) can regulate the inflammatory cyclooxygenase-prostaglandin pathway and enhance the growth of cervical epithelial tumours in vivo by promoting cellular proliferation and alteration of vascular function. This study investigated the molecular mechanism whereby SP regulates vascular function using an in vitro model system of HeLa cervical adenocarcinoma cells and human umbilical vein endothelial cells (HUVECs). We found that SP rapidly enhanced the expression of the angiogenic chemokines, interleukin (IL)-8 and growth regulated oncogene alpha (GRO) in HeLa cells in a time-dependent manner. We investigated the molecular mechanism of SP-mediated regulation of IL-8 and GRO using a panel of chemical inhibitors of cell signalling. We found that treatment of HeLa cells with SP elevated expression of IL-8 and GRO by transactivation of the epidermal growth factor receptor, activation of extracellular signal-regulated kinase and induction of cyclooxygenase enzymes and nuclear factor kappa B. We investigated the impact of IL-8 and GRO, released from HeLa cells after treatment with SP, on vascular function using a co-culture model system of conditioned medium (CM) from HeLa cells, treated with or without SP, and HUVECs. We found that CM from HeLa cells induced the arrangement of endothelial cells into a network of tube-like structures via the CXCR2 receptor on HUVECs. Taken together our data outline a molecular mechanism whereby SP can alter vascular function in cervical cancers via the pro-angiogenic chemokines, IL-8 and GRO.

Seminal plasma enhances cervical adenocarcinoma cell proliferation and tumour growth in vivo.

Cervical cancer is one of the leading causes of cancer-related death in women in sub-Saharan Africa. Extensive evidence has shown that cervical cancer and its precursor lesions are caused by Human papillomavirus (HPV) infection. Although the vast majority of HPV infections are naturally resolved, failure to eradicate infected cells has been shown to promote viral persistence and tumorigenesis. Furthermore, following neoplastic transformation, exposure of cervical epithelial cells to inflammatory mediators either directly or via the systemic circulation may enhance progression of the disease. It is well recognised that seminal plasma contains an abundance of inflammatory mediators, which are identified as regulators of tumour growth. Here we investigated the role of seminal plasma in regulating neoplastic cervical epithelial cell growth and tumorigenesis. Using HeLa cervical adenocarcinoma cells, we found that seminal plasma (SP) induced the expression of the inflammatory enzymes, prostaglandin endoperoxide synthase (PTGS1 and PTGS2), cytokines interleukin (IL) -6, and -11 and vascular endothelial growth factor-A (VEGF-A). To investigate the role of SP on tumour cell growth in vivo, we xenografted HeLa cells subcutaneously into the dorsal flank of nude mice. Intra-peritoneal administration of SP rapidly and significantly enhanced the tumour growth rate and size of HeLa cell xenografts in nude mice. As observed in vitro, we found that SP induced expression of inflammatory PTGS enzymes, cytokines and VEGF-A in vivo. Furthermore we found that SP enhances blood vessel size in HeLa cell xenografts. Finally we show that SP-induced cytokine production, VEGF-A expression and cell proliferation are mediated via the induction of the inflammatory PTGS pathway.

Regulation of inflammatory pathways in cancer and infectious disease of the cervix.

Cervical cancer is one of the leading gynaecological malignancies worldwide. It is an infectious disease of the cervix, associated with human papillomavirus infection (HPV), infection with bacterial agents such as Chlamydia trachomatis and Neisseria gonorrhoea as well as human immunodeficiency virus (HIV). Furthermore, it is an AIDS-defining disease with an accelerated mortality in HIV-infected women with cervical cancer. With the introduction of robust vaccination strategies against HPV in the developed world, it is anticipated that the incidence of cervical cancer will decrease in the coming years. However, vaccination has limited benefit for women already infected with high-risk HPV, and alternative therapeutic intervention strategies are needed for these women. Many pathological disorders, including cervical cancer, are characterised by the exacerbated activation and maintenance of inflammatory pathways which are considered to be regulated by infectious agents. In cervical cancer, hyperactivation of these inflammatory pathways and regulation of immune infiltrate into tissues can potentially play a role not only in tumorigenesis but also in HIV infection. In this paper we will discuss the contribution of inflammatory pathways to cervical cancer progression and HIV infection and the role of HIV in cervical cancer progression.

A role for lipoxin A4 as an anti-inflammatory mediator in the human endometrium.

Lipoxin A(4) is a lipid mediator that elicits anti-inflammatory and pro-resolution actions via its receptor, formyl peptide receptor 2 (FPR2/ALX). In this study, we aimed to investigate the expression and potential role of lipoxin A(4) and FPR2/ALX in the regulation of inflammation associated with cyclical remodeling of the human endometrium across the menstrual cycle and during early pregnancy. Using quantitative RT-PCR analysis, we found that FPR2/ALX expression is upregulated during the menstrual phase of the cycle and in decidua tissue from the first trimester of pregnancy. We localized the site of expression of FPR2/ALX in menstrual phase endometrium and first-trimester decidua tissue to glandular epithelial cells and cells within the stromal compartment, including cells lining the blood vessels and immune cells. Measurement of serum lipoxin A(4) by ELISA revealed no difference in its levels across the menstrual cycle but an elevation in early pregnancy (P<0.001). We found that lipoxin A(4) was regulated by human chorionic gonadotrophin (hCG) during early pregnancy, because treatment of human decidua tissue with hCG increased lipoxin A(4) release (P<0.01). Finally, we have shown that lipoxin A(4) can suppress phorbol myristate acetate-induced expression of the inflammatory cytokines interleukin 6 and 8 in human endometrium and decidua tissue. These results demonstrate for the first time that lipoxin A(4) and its receptor FPR2/ALX can regulate inflammatory events in the human endometrium and decidua of early pregnancy.

Prokineticin 1 induces Dickkopf 1 expression and regulates cell proliferation and decidualization in the human endometrium.

Prokineticin 1 (PROK1) signalling via prokineticin receptor 1 (PROKR1) regulates the expression of several genes with important roles in endometrial receptivity and implantation. This study investigated PROK1 regulation of Dickkopf 1 (DKK1) expression, a negative regulator of canonical Wnt signalling, and its function in the non-pregnant endometrium and first trimester decidua. DKK1 mRNA expression is elevated during the mid-secretory phase of the menstrual cycle and expression increases further in first trimester decidua. DKK1 protein expression is localized to glandular epithelial and stromal cells during the proliferative, early- and mid-secretory phases, whereas expression is confined to the stroma in the late-secretory phase and first trimester decidua. PROK1 induces the expression of DKK1 in endometrial epithelial cells stably expressing PROKR1 and in first trimester decidua explants, via a Gq-calcium-calcineurin-nuclear factor of activated T-cells-mediated pathway. Endometrial epithelial cell proliferation is negatively regulated by PROK1-PROKR1 signalling. We demonstrate that this effect on cell proliferation occurs via DKK1 expression, as siRNA targeted against DKK1 reduces the PROK1-induced decrease in proliferation. Furthermore, decidualization of primary human endometrial stromal cells with progesterone and cyclic adenosine monophosphate is inhibited by miRNA knock down of PROK1 or DKK1. These data demonstrate important roles for PROK1 and DKK1 during endometrial receptivity and early pregnancy, which include regulation of endometrial cell proliferation and decidualization.

Hypoxia and prostaglandin E receptor 4 signalling pathways synergise to promote endometrial adenocarcinoma cell proliferation and tumour growth.

The prostaglandin endoperoxide synthase (PTGS) pathway is a potent driver of tumour development in humans by enhancing the biosynthesis and signalling of prostaglandin (PG) E(2). PTGS2 expression and PGE(2) biosynthesis is elevated in endometrial adenocarcinoma, however the mechanism whereby PTGS and PGE(2) regulate endometrial tumour growth is unknown. Here we investigated (a) the expression profile of the PGE synthase enzymes (PTGES, PTGES-2, PTGES-3) and PGE receptors (PTGER1-4) in endometrial adenocarcinomas compared with normal endometrium and (b) the role of PTGER4 in endometrial tumorigenesis in vivo. We found elevated expression of PTGES2 and PTGER4 and suppression of PTGER1 and PTGER3 in endometrial adenocarcinomas compared with normal endometrium. Using WT Ishikawa endometrial adenocarcinoma cells and Ishikawa cells stably transfected with the full length PTGER4 cDNA (PTGER4 cells) xenografted in the dorsal flanks of nude mice, we show that PTGER4 rapidly and significantly enhances tumour growth rate. Coincident with enhanced PTGER4-mediated tumour growth we found elevated expression of PTGS2 in PTGER4 xenografts compared with WT xenografts. Furthermore we found that the augmented growth rate of the PTGER4 xenografts was not due to enhanced angiogenesis, but regulated by an increased proliferation index and hypoxia. In vitro, we found that PGE(2) and hypoxia independently induce expression of PTGER4 indicating two independent pathways regulating prostanoid receptor expression. Finally we have shown that PGE(2) and hypoxia synergise to promote cellular proliferation of endometrial adenocarcinoma cells.

Molecular regulators of resolution of inflammation: potential therapeutic targets in the reproductive system.

Inflammatory processes are central to reproductive events including ovulation, menstruation, implantation and labour, while inflammatory dysregulation is a feature of numerous reproductive pathologies. In recent years, there has been much research into the endogenous mechanisms by which inflammatory reactions are terminated and tissue homoeostasis is restored, a process termed resolution. The identification and characterisation of naturally occurring pro-resolution mediators including lipoxins and annexin A1 has prompted a shift in the field of anti-inflammation whereby resolution is now observed as an active process, triggered as part of a normal inflammatory response. This review will address the process of resolution, discuss available evidence for expression of pro-resolution factors in the reproductive tract and explore possible roles for resolution in physiological reproductive processes and associated pathologies.

Chorionic gonadotrophin regulates CXCR4 expression in human endometrium via E-series prostanoid receptor 2 signalling to PI3K-ERK1/2: implications for fetal-maternal crosstalk for embryo implantation.

Murine knock-out models and blastocyst co-culture studies have identified prostaglandin-endoperoxide synthase (PTGS) 2, prostaglandin (PG) E receptor 2 (PTGER2) and the chemokine receptor CXCR4 as important regulators of early pregnancy events. In vitro studies and studies in non-human primates have shown that these proteins are regulated in the endometrium by the early embryonic signal, chorionic gonadotrophin (CG). Here we show that expressions of PTGER2 and CXCR4 are elevated during the mid-secretory phase of the menstrual cycle and decidua of early pregnancy in humans. Using first trimester decidua explants, we show that CG induces expression of PTGS2 and biosynthesis of PGE2, and expression of PTGER2. Subsequently, PGE2via PTGER2 induces expression of CXCR4. Using an in vitro model system of Ishikawa endometrial epithelial cells stably expressing PTGER2 and human first trimester decidua explants, we demonstrate that CXCR4 expression is regulated by PTGER2 via the epidermal growth factor receptor (EGFR)-phosphatidylinositol-3-kinase (PI3K)-extracellular signal-regulated kinase (ERK1/2) pathway.Taken together, our data suggest that early embryonic signals may regulate fetal-maternal crosstalk in the human endometrium by inducing CXCR4 expression via the PGE2-PTGER2-mediated induction of the EGFR, PI3K and ERK1/2 pathways.

PGF2α-F-prostanoid receptor signalling via ADAMTS1 modulates epithelial cell invasion and endothelial cell function in endometrial cancer.

BACKGROUND: An increase in cancer cell invasion and microvascular density is associated with a poorer prognosis for patients with endometrial cancer. In endometrial adenocarcinoma F-prostanoid (FP) receptor expression is elevated, along with its ligand prostaglandin (PG)F2α, where it regulates expression and secretion of a host of growth factors and chemokines involved in tumorigenesis. This study investigates the expression, regulation and role of a disintegrin and metalloproteinase with thrombospondin repeat 1 (ADAMTS1) in endometrial adenocarcinoma cells by PGF2α via the FP receptor. METHODS: Human endometrium and adenocarcinoma tissues were obtained in accordance with Lothian Research Ethics Committee guidance with informed patient consent. Expression of ADAMTS1 mRNA and protein in tissues was determined by quantitative RT-PCR analysis and immunohistochemistry. Signal transduction pathways regulating ADAMTS1 expression in Ishikawa cells stably expressing the FP receptor to levels seen in endometrial cancer (FPS cells) were determined by quantitative RT-PCR analysis. In vitro invasion and proliferation assays were performed with FPS cells and human umbilical vein endothelial cells (HUVECs) using conditioned medium (CM) from PGF2α-treated FPS cells from which ADAMTS1 was immunoneutralised and/or recombinant ADAMTS1. The role of endothelial ADAMTS1 in endothelial cell proliferation was confirmed with RNA interference. The data in this study were analysed by T-test or ANOVA. RESULTS: ADAMTS1 mRNA and protein expression is elevated in endometrial adenocarcinoma tissues compared with normal proliferative phase endometrium and is localised to the glandular and vascular cells. Using FPS cells, we show that PGF2α-FP signalling upregulates ADAMTS1 expression via a calmodulin-NFAT-dependent pathway and this promotes epithelial cell invasion through ECM and inhibits endothelial cell proliferation. Furthermore, we show that CM from FPS cells regulates endothelial cell ADAMTS1 expression in a rapid biphasic manner. Using RNA interference we show that endothelial cell ADAMTS1 also negatively regulates cellular proliferation. CONCLUSIONS: These data demonstrate elevated ADAMTS1 expression in endometrial adenocarcinoma. Furthermore we have highlighted a mechanism whereby FP receptor signalling regulates epithelial cell invasion and endothelial cell function via the PGF2α-FP receptor mediated induction of ADAMTS1.

F-Prostaglandin receptor regulates endothelial cell function via fibroblast growth factor-2.

BACKGROUND: Prostaglandin (PG) F(2alpha) is a key regulator of endometrial function and exerts its biological action after coupling with its heptahelical G protein-coupled receptor (FP receptor). In endometrial adenocarcinoma the FP receptor expression is elevated. We have shown previously that PGF(2alpha)-FP receptor signalling in endometrial adenocarcinoma cells can upregulate several angiogenic factors including fibroblast growth factor-2 (FGF2). In the present study, we investigated the paracrine effect of conditioned medium produced via PGF(2alpha)-FP receptor signalling in endometrial adenocarcinoma cells stably expressing the FP receptor (Ishikawa FPS cells), on endothelial cell function. RESULTS: Conditioned medium (CM) was collected from FPS cells after 24 hrs treatment with either vehicle (V CM) or 100 nM PGF(2alpha) (P CM). Treatment of human umbilical vein endothelial cells (HUVECs) with P CM significantly enhanced endothelial 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 PGF(2alpha) 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 PGF(2alpha) 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 F(2alpha)-FP signalling regulates endothelial cell network formation and proliferation. In addition we provide novel in vitro evidence to suggest that prostaglandin F(2alpha) 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 growth.

Prokineticin-1 (PROK1) modulates interleukin (IL)-11 expression via prokineticin receptor 1 (PROKR1) and the calcineurin/NFAT signalling pathway.

Prokineticin-1 (PROK1) is a multifunctional secreted protein which signals via the G-protein coupled receptor, PROKR1. Previous data from our laboratory using a human genome survey microarray showed that PROK1-prokineticin receptor 1 (PROKR1) signalling regulates numerous genes important for establishment of early pregnancy, including the cytokine interleukin (IL)-11. Here, we have shown that PROK1-PROKR1 induces the expression of IL-11 in PROKR1 Ishikawa cells and first trimester decidua via the calcium-calcineurin signalling pathway in a guanine nucleotide-binding protein (G(q/11)), extracellular signal-regulated kinases, Ca(2+) and calcineurin-nuclear factor of activated T cells dependent manner. Conversely, treatment of human decidua with a lentiviral miRNA to abolish endogenous PROK1 expression results in a significant reduction in IL-11 expression and secretion. Importantly, we have also shown a regulatory role for the regulator of calcineurin 1 isoform 4 (RCAN1-4). Overexpression of RCAN1-4 in PROKR1 Ishikawa cells using an adenovirus leads to a reduction in PROK1 induced IL-11 indicating that RCAN1-4 is a negative regulator in the calcineurin-mediated signalling to IL-11. Finally, we have shown the potential for both autocrine and paracrine signalling in the human endometrium by co-localizing IL-11, IL-11Ralpha and PROKR1 within the stromal and glandular epithelial cells of non-pregnant endometrium and first trimester decidua. Overall we have identified and characterized the signalling components of a novel PROK1-PROKR1 signalling pathway regulating IL-11.