Stanniocalcin-1 in the female reproductive system and pregnancy.

BACKGROUND: Stanniocalcin-1 (STC-1) is a widely expressed glycoprotein hormone involved in a diverse spectrum of physiological and pathophysiological processes including angiogenesis, mineral homeostasis, cell proliferation, inflammation and apoptosis. Over the last 20 years, numerous studies have reported STC-1 expression within female reproductive tissues including the uterus, ovaries and placenta and implicated STC-1 in processes such as ovarian follicular development, blastocyst implantation, vascular remodelling in early pregnancy and placental development. Notably, dysregulation of STC-1 within reproductive tissues has been linked to the onset of severe reproductive disorders including endometriosis, polycystic ovary syndrome, poor trophoblast invasion and placental perfusion in early pregnancy. Furthermore, significant changes in tissue expression and in maternal systemic concentration take place throughout pregnancy and further substantiate the vital role of this protein in reproductive health and disease. OBJECTIVE AND RATIONALE: Our aim is to provide a comprehensive overview of the existing literature, to summarise the expression profile and roles of STC-1 within the female reproductive system and its associated pathologies. We highlight the gaps in the current knowledge and suggest potential avenues for future research. SEARCH METHODS: Relevant studies were identified through searching the PubMed database using the following search terms: 'stanniocalcin-1', 'placenta', 'ovary', 'endometrium', 'pregnancy', 'reproduction', 'early gestation'. Only English language papers published between 1995 and 2020 were included. OUTCOMES: This review provides compelling evidence of the vital function that STC-1 plays within the female reproductive system. The literature presented summarise the wide expression profile of STC-1 within female reproductive organs, as well as highlighting the putative roles of STC-1 in various functions in the reproductive system. Moreover, the observed link between altered STC-1 expression and the onset of various reproductive pathologies is presented, including those in pregnancy whose aetiology occurs in the first trimester. This summary emphasises the requirement for further studies on the mechanisms underlying the regulation of STC-1 expression and function. WIDER IMPLICATIONS: STC-1 is a pleiotropic hormone involved in the regulation of a number of important biological functions needed to maintain female reproductive health. There is also growing evidence that dysregulation of STC-1 is implicated in common reproductive and obstetric disorders. Greater understanding of the physiology and biochemistry of STC-1 within the field may therefore identify possible targets for therapeutic intervention and/or diagnosis.

Regulation of stanniocalcin-1 secretion by BeWo cells and first trimester human placental tissue from normal pregnancies and those at increased risk of developing preeclampsia.

Stanniocalcin-1 (STC-1) is a multi-functional glycosylated peptide present in the plasma of healthy women postpartum and increased further in pregnancies complicated by preeclampsia. Although the STC-1 gene is expressed by the placenta what regulates its secretion and from which cells at the feto-maternal interface is unknown. Here, we demonstrate for the first time that the syncytiotrophoblast and cytotrophoblast are a major site of STC-1 protein expression in first trimester placental tissue. Further, in response to low oxygen, first trimester chorionic villous tissue from pregnancies at increased risk of developing preeclampsia secreted significantly more STC-1 than normal tissue under the same conditions. Using the human trophoblast cell line BeWo we have shown that low oxygen increased the secretion of STC-1 but it required co-stimulation with the Adenosine-3', 5'-cyclic monophosphate (cAMP) analogue, 8-Bromo adenosine-3', 5'-cyclic monophosphate cAMP (8 Br-cAMP) to reach significance. Inhibition of Hypoxia inducible factor 2α (HIF-2α) and the Phosphatidylinositol-3 kinase (PI3 -Kinase)/AKT/Serum and glucocorticoid-induced kinase-1(SGK-1) pathway resulted in significant inhibition of STC-1 secretion. As both low oxygen and cAMP are known to play a central role in placental function, their regulation of STC-1 points to a potentially important role in the maintenance of a normal healthy pregnancy and we would hypothesize that it may act to protect against prolonged placental hypoxia seen in preeclampsia.

The phenotype of decidual CD56+ lymphocytes is influenced by secreted factors from decidual stromal cells but not macrophages in the first trimester of pregnancy.

During the first trimester of pregnancy the decidua is comprised of decidual stromal cells (DSC), invading fetal trophoblast cells and maternal leukocytes, including decidual natural killer (dNK) cells and macrophages. dNK cells are distinct from peripheral blood NK cells and have a role in regulating trophoblast invasion and spiral artery remodelling. The unique phenotype of dNK cells results from the decidual environment in which they reside, however the interaction and influence of other cells in the decidua on dNK phenotype is unknown. We isolated first trimester DSC and decidual macrophages and investigated the effect that DSC and decidual macrophage secreted factors have on CD56+ decidual lymphocyte receptor expression and cytokine secretion (including dNK cells). We report that DSC secreted factors induce the secretion of the cytokines IL-8 and IL-6 from first trimester CD56+ cells. However, neither DSC nor decidual macrophage secreted factors changed CD56+ cell receptor expression. These results suggest that secreted factors from DSC influence CD56+ decidual lymphocytes during the first trimester of pregnancy and therefore may play a role in regulating the unique phenotype and function of dNK cells during placentation.

Glial Cells Missing 1 Regulates Equine Chorionic Gonadotrophin Beta Subunit via Binding to the Proximal Promoter.

Equine chorionic gonadotrophin (eCG) is a placental glycoprotein critical for early equine pregnancy and used therapeutically in a number of species to support reproductive activity. The factors in trophoblast that transcriptionally regulate eCGß-subunit (LHB), the gene which confers the hormones specificity for the receptor, are not known. The aim of this study was to determine if glial cells missing 1 regulates LHB promoter activity. Here, studies of the LHB proximal promoter identified four binding sites for glial cells missing 1 (GCM1) and western blot analysis confirmed GCM1 was expressed in equine chorionic girdle (ChG) and surrounding tissues. Luciferase assays demonstrated endogenous activity of the LHB promoter in BeWo choriocarcinoma cells with greatest activity by a proximal 335 bp promoter fragment. Transactivation studies in COS7 cells using an equine GCM1 expression vector showed GCM1 could transactivate the proximal 335 bp LHB promoter. Chromatin immunoprecipitation using primary ChG trophoblast cells showed GCM1 to preferentially bind to the most proximal GCM1-binding site over site 2. Mutation of site 1 but not site 2 resulted in a loss of endogenous promoter activity in BeWo cells and failure of GCM1 to transactivate the promoter in COS-7 cells. Together, these data show that GCM1 binds to site 1 in the LHB promoter but also requires the upstream segment of the LHB promoter between -119 bp and -335 bp of the translation start codon for activity. GCM1 binding partners, ETV1, ETV7, HOXA13, and PITX1, were found to be differentially expressed in the ChG between days 27 and 34 and are excellent candidates for this role. In conclusion, GCM1 was demonstrated to drive the LHB promoter, through direct binding to a predicted GCM1-binding site, with requirement for another factor(s) to bind the proximal promoter to exert this function. Based on these findings, we hypothesize that ETV7 and HOXA13 act in concert with GCM1 to initiate LHB transcription between days 30 and 31, with ETV1 partnering with GCM1 to maintain transcription.

Effects of normal and high circulating concentrations of activin A on vascular endothelial cell functions and vasoactive factor production.

OBJECTIVES: Activin A, a TGFß family member, circulates in the maternal blood at increasing concentrations throughout gestation during a healthy pregnancy. The circulating concentration of activin A is further increased in pre-eclampsia (PE), a hypertensive disorder of pregnancy that is marked by systemic maternal vascular endothelial cell dysfunction. The effect of increasing activin A concentrations on the maternal vascular endothelium is unknown. The study aim was to investigate the effect of physiological and pathological activin A concentrations observed in normotensive and PE pregnancies respectively, on vascular endothelial cell function. METHODS AND RESULTS: Immunostaining demonstrated the presence of the activin A receptor, ACVR2A, in SGHEC-7 cells used to model the vascular endothelium. SGHEC-7 cells were treated with activin A concentrations representative of concentrations throughout gestation in normotensive (0-10ng/ml) and PE (50ng/ml) pregnancies. xCELLigence functional assays revealed that normotensive activin A concentrations increased SGHEC-7 proliferation and migration, which was inhibited by PE concentrations. Additionally, fluorescence based assays showed that PE concentrations increased endothelial permeability. None of the tested activin A concentrations affected cell apoptosis. PE concentrations also resulted in an imbalance of the vasoactive factors eNOS, PTGIS and EDN1, as determined by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assays. CONCLUSION: Compared with normotensive activin A concentrations, the higher PE activin A concentrations resulted in abnormal endothelial functions, which may contribute to the systemic maternal vascular endothelial cell dysfunction observed in the disorder.

Decidual natural killer cells regulate vessel stability: implications for impaired spiral artery remodelling.

Decidual NK (dNK) cells are present during uterine spiral artery remodelling, an event that is crucial for successful placentation and the provision of an adequate blood supply to the developing fetus. Spiral artery remodelling is impaired in the pregnancy complication pre-eclampsia. Although dNK cells are known to play active roles at the maternal-fetal interface, little is known about their effect on endothelial integrity, an important component of vessel stability. We present a study in which we have modelled dNK-endothelium interactions, using first-trimester dNK cells isolated from both normal pregnancies and those with impaired spiral artery remodelling. dNK cells were isolated from first-trimester pregnancies, screened by uterine artery Doppler ultrasound to determine resistance indices (RI) that relate to the extent of spiral artery remodelling. dNK culture supernatant from normal-RI pregnancies (but not high-RI pregnancies) destabilised endothelial tube-like structures in Matrigel, and normal-RI dNK cells induced endothelial intercellular adhesion molecule-1 and tumour necrosis factor-α expression to a greater extent than high-RI dNK cells. We have established a functional role for dNK cells in the disruption of endothelial structures and have suggested how impairment of this process may be contributing to the reduced vessel remodelling in pregnancies with a high uterine artery resistance index. These findings have implications for our understanding of the pathology of pre-eclampsia and other pregnancy disorders where remodelling is impaired.

Decidual natural killer cell interactions with trophoblasts are impaired in pregnancies at increased risk of preeclampsia.

Transformation of the uterine spiral arteries (SAs) during pregnancy is critical to support the developing fetus, and is impaired in some pregnancy disorders, including preeclampsia. Decidual natural killer (dNK) cells play a role in SA remodeling, although their interactions with fetal trophoblast remain unclear. A uterine artery Doppler resistance index (RI) in the first trimester of pregnancy can be used as a proxy measure of the extent of SA remodeling; we have used this technique to characterize dNK cells from pregnancies with normal (normal RI) and impaired (high RI) SA remodeling, which display least and highest risk of developing preeclampsia, respectively. We examined the impact of dNK cell secreted factors on trophoblast motility, chemoattraction, and signaling pathways to determine the contribution of dNK cells to SA transformation. We demonstrated that the chemoattraction of the trophoblast by dNK cells is impaired in pregnancies with high RI, as is the ability to induce trophoblast outgrowth from placental villous explants. These processes are dependent on activation of the extracellular signal-regulated kinase 1/2 and phosphatidylinositol 3-kinase-Akt signaling pathways, which were altered in trophoblasts incubated with secreted factors from dNK cells from high RI pregnancies. Therefore, by characterizing pregnancies using uterine artery Doppler RI before dNK cell isolation, we have identified that impaired dNK-trophoblast interactions may lead to poor placentation. These findings have implications for pregnancy pathological conditions, such as preeclampsia.

Role of the eIF4E binding protein 4E-BP1 in regulation of the sensitivity of human pancreatic cancer cells to TRAIL and celastrol-induced apoptosis.

BACKGROUND INFORMATION: Tumour cells can be induced to undergo apoptosis after treatment with the tumour necrosis factor α-related death-inducing ligand (TRAIL). Although human pancreatic cancer cells show varying degrees of response they can be sensitised to the pro-apoptotic effects of TRAIL in the presence of celastrol, a natural compound extracted from the plant Tripterygium wilfordii Hook F. One important aspect of the cellular response to TRAIL is the control of protein synthesis, a key regulator of which is the eukaryotic initiation factor 4E-binding protein, 4E-BP1. RESULTS: We examined the effects of celastrol and TRAIL in several pancreatic cancer cell lines. In cells that are normally resistant to TRAIL, synergistic effects of TRAIL plus celastrol on commitment to apoptosis and inhibition of protein synthesis were observed. These were associated with a strong up-regulation and dephosphorylation of 4E-BP1. The enhancement of 4E-BP1 expression, which correlated with a threefold increase in the level of the 4E-BP1 transcript, was blocked by inhibitors of reactive oxygen species and the JNK protein kinase. When the expression of 4E-BP1 was reduced by an inducible micro-RNA, TRAIL-mediated apoptosis was inhibited. CONCLUSION: These results suggest that 4E-BP1 plays a critical role in the mechanism by which TRAIL and celastrol together cause apoptotic cell death in human pancreatic tumour cells.

Caffeine inhibits EGF-stimulated trophoblast cell motility through the inhibition of mTORC2 and Akt.

Impaired trophoblast invasion is associated with pregnancy disorders such as early pregnancy loss and preeclampsia. There is evidence to suggest that the consumption of caffeine during pregnancy may increase the risk of pregnancy loss; however, little is known about the direct effect of caffeine on normal trophoblast biology. Our objectives were to examine the effect of caffeine on trophoblast migration and motility after stimulation with epidermal growth factor (EGF) and to investigate the intracellular signaling pathways involved in this process. Primary first-trimester extravillous trophoblasts (EVT) and the EVT-derived cell line SGHPL-4 were used to study the effect of caffeine on EGF-stimulated cellular motility using time-lapse microscopy. SGHPL-4 cells were further used to study the effect of caffeine and cAMP on EGF-stimulated invasion of fibrin gels. The influence of caffeine and cAMP on EGF-stimulated intracellular signaling pathways leading to the activation of Akt were investigated by Western blot analysis. Caffeine inhibits both EGF-stimulated primary EVT and SGHPL-4 cell motility. EGF stimulation activates phosphatidylinositol 3-kinase, and Akt and caffeine inhibit this activation. Although cAMP inhibits both motility and invasion, it does not inhibit the activation of Akt, indicating that the effects of caffeine seen in this study are independent of cAMP. Further investigation indicated a role for mammalian target of rapamycin complex 2 (mTORC2) as a target for the inhibitory effect of caffeine. In conclusion, we demonstrate that caffeine inhibits EGF-stimulated trophoblast invasion and motility in vitro and so could adversely influence trophoblast biology in vivo.

Impaired decidual natural killer cell regulation of vascular remodelling in early human pregnancies with high uterine artery resistance.

During human pregnancy, natural killer (NK) cells accumulate in the maternal decidua, but their specific roles remain to be determined. Decidual NK (dNK) cells are present during trophoblast invasion and uterine spiral artery remodelling. These events are crucial for successful placentation and the provision of an adequate blood supply to the developing fetus. Remodelling of spiral arteries is impaired in the dangerous pregnancy complication pre-eclampsia. We studied dNK cells isolated from pregnancies at 9-14 weeks' gestation, screened by uterine artery Doppler ultrasound to determine resistance indices which relate to the extent of spiral artery remodelling. dNK cells were able to promote the invasive behaviour of fetal trophoblast cells, partly through HGF. Cells isolated from pregnancies with higher resistance indices were less able to do this and secreted fewer pro-invasive factors. dNK cells from pregnancies with normal resistance indices could induce apoptotic changes in vascular smooth muscle and endothelial cells in vitro, events of importance in vessel remodelling, partly through Fas signalling. dNK cells isolated from high resistance index pregnancies failed to induce vascular apoptosis and secreted fewer pro-apoptotic factors. We have modelled the cellular interactions at the maternal-fetal interface and provide the first demonstration of a functional role for dNK cells in influencing vascular cells. A potential mechanism contributing to impaired vessel remodelling in pregnancies with a higher uterine artery resistance is presented. These findings may be informative in determining the cellular interactions contributing to the pathology of pregnancy disorders where remodelling is impaired, such as pre-eclampsia.

Altered placental expression of kisspeptin and its receptor in pre-eclampsia.

Kisspeptin, originally identified as metastatin, important in preventing cancer metastasis, has more recently been shown to be important in pregnancy. Roles indicated for kisspeptin in pregnancy include regulating trophoblast invasion and migration during placentation. The pregnancy-specific disorder pre-eclampsia (PE) is now accepted to begin with inadequate trophoblast invasion and the current study therefore sets out to characterise placental expression of both kisspeptin (KISS1) and its receptor (KISS1R) throughout pregnancy and in PE. Placental tissue was obtained from women undergoing elective surgical termination of early pregnancy (n=10) and from women following Caesarean section at term in normal pregnancy (n=10) and with PE (n=10). Immunohistochemistry of paraffin embedded sections and western immunoblotting were performed to assess protein localisation and expression. Quantitative real-time PCR was carried out to evaluate mRNA expression of both KISS1 and KISS1R. Protein and mRNA expression was found to mirror each other with KISS1 expression found to be reduced in PE compared with that in normal term pregnancy. Interestingly, KISS1R expression at both the mRNA and protein levels was found to be increased in PE compared with that in normal term pregnancy. The current findings of increased KISS1R expression may represent a mechanism by which functional activity of KISS1 is higher in PE than in normal pregnancy. Higher levels of activity of KISS1R may be involved in inhibition of trophoblast invasion and angiogenesis, which are associated with PE.

Extravillous trophoblast and decidual natural killer cells: a remodelling partnership.

BACKGROUND: During pregnancy, maternal uterine spiral arteries (SAs) are remodelled from minimal-flow, high-resistance vessels into larger diameter vessels with low resistance and high flow. Fetal extravillous trophoblasts (EVT) have important roles in this process. Decidual natural killer cells (dNK cells) are the major maternal immune component of the decidua and accumulate around SAs before trophoblast invasion. A role for dNK cells in vessel remodelling is beginning to be elucidated. This review examines the overlapping and dissimilar mechanisms used by EVT and dNK cells in this process and how this may mirror another example of tissue remodelling, namely cancer development. METHODS: The published literature was searched using Pubmed focusing on EVT, dNK cells and SA remodelling. Additional papers discussing cancer development are also included. RESULTS: Similarities exist between actions carried out by dNK cells and EVT. Both interact with vascular cells lining the SA, as well as with each other, to promote transformation of the SA. EVT differentiation has previously been likened to the epithelial-mesenchymal transition in cancer cells, and we discuss how dNK-EVT interactions at the maternal-fetal interface can also be compared with the roles of immune cells in cancer. CONCLUSIONS: The combined role that dNK cells and EVT play in SA remodelling suggests that these interactions could be described as a partnership. The investigation of pregnancy as a multicellular system involving both fetal and maternal components, as well as comparisons to similar examples of tissue remodelling, will further identify the key mechanisms in SA remodelling that are required for a successful pregnancy.

Shear stress and spiral artery remodelling: the effects of low shear stress on trophoblast-induced endothelial cell apoptosis.

AIMS: In the first trimester of pregnancy, placental trophoblasts migrate along uterine spiral arteries (SA) and replace endothelial cells (EC) lining these vessels by mechanisms involving EC apoptosis. Inadequate SA remodelling has been associated with pre-eclampsia and intra-uterine growth restriction. Until 10-12 weeks of gestation, trophoblasts plug SA, preventing maternal blood flow into the intervillous space and resulting in high resistance, low shear flow in these vessels. We aimed to determine the effect of shear stress on trophoblast-induced EC apoptosis. METHODS AND RESULTS: Trophoblasts and human umbilical vein endothelial cells (HUVECs) were cultured for 13-31 h under shear stresses from 0.02 to 7 dyne/cm². Apoptosis was quantified by time-lapse microscopy. Treatment with tumour necrosis factor α/actinomycin D increased apoptotic death in all cell types (P < 0.01). However, trophoblasts cultured in 3 dyne/cm² underwent less apoptosis than those in 0.5 dyne/cm² cultures (P < 0.05). Trophoblasts cultured on HUVEC monolayers at 0.5 or 3 dyne/cm² significantly induced apoptosis in directly adjacent HUVECs, in comparison to HUVECs >2 cells away or HUVEC only controls (P < 0.05), and this was mediated by Fas/Fas-ligand mechanisms. The amount of induced apoptosis decreased from 0.5 to 3 dyne/cm² cultures (P < 0.05), and trophoblasts failed to significantly induce HUVEC apoptosis in 5 or 7 dyne/cm² cultures. CONCLUSION: The survival advantage of trophoblasts over EC at low shear stress, and the inhibition of trophoblast-induced EC apoptosis with increasing shear stress, suggests that low shear stresses in plugged first-trimester SA may aid trophoblast-induced EC apoptosis involved in SA remodelling.

Trophoblast-mediated spiral artery remodelling: a role for apoptosis.

In the first 20 weeks of pregnancy a number of important changes take place in the maternal uterine vasculature. Vascular endothelial and smooth muscle cells are lost from the spiral arteries and are replaced by fetal trophoblast cells. The resulting increase in blood flow to the intervillous space ensures that the fetus receives the nutrients and respiratory gases required for growth. Failure of the vessels to remodel sufficiently is a common feature of pregnancy pathologies such as early pregnancy loss, intrauterine growth restriction and pre-eclampsia. Although there is evidence to suggest that some vascular changes occur prior to trophoblast invasion, it is clear that in the absence of trophoblast invasion the remodelling of the spiral arteries is reduced. The cellular and molecular mechanisms by which trophoblasts influence vessel structure have been little studied. Trophoblasts synthesize and release a plethora of cytokines and growth factors including members of the tumour necrosis factor family such as tumour necrosis factor alpha, Fas-ligand and tumour necrosis factor-related apoptosis-inducing ligand. Recent studies suggest that these factors may be important in regulating the remodelling process by inducing both endothelial cell and vascular smooth muscle cell apoptosis.

Soluble HLA-G regulates motility and invasion of the trophoblast-derived cell line SGHPL-4.

BACKGROUND: Soluble human leucocyte antigen-G (sHLA-G) is secreted by extravillous trophoblast (EVT) and has roles in regulating immune cells within the decidua. HLA-G expression on EVT increases as they approach uterine spiral arteries and we have suggested that sHLA-G may be important in the remodelling of these vessels. The autocrine role of sHLA-G in regulating trophoblast function at this critical phase has not been studied. We aimed to investigate the effects of sHLA-G on trophoblast motility, invasion and survival. METHODS: The human EVT line, SGHPL-4, was stably transfected to over-express sHLA-G (SGHPL-4sG1). Motility and apoptosis were assessed by time-lapse microscopy. Cells were cultured on microcarrier beads embedded in fibrin gels to assess invasion. The effect of sHLA-G expression on motility, invasion and apoptosis in response to stimulation with either hepatocyte growth factor (HGF) or epidermal growth factor (EGF) was determined. RESULTS: There was no difference in the motility of either SGHPL-4 cells or SGHPL-4sG1 cells in the absence of stimulation. However, sHLA-G inhibited HGF-induced EVT motility. HGF- and EGF-induced invasions were significantly inhibited in SGHPL-4sG1 compared with SGHPL-4 cells. Increased expression of HLA-G had no significant effect on tumour necrosis factor (TNF)-alpha/actinomycin-induced apoptosis. CONCLUSIONS: Growth factor-stimulated trophoblast motility and invasion are regulated by sHLA-G, indicating a novel autocrine role. The inhibition of trophoblast invasion at the spiral artery may be important to allow interactions leading to vascular remodelling.

The androgen and progesterone receptors regulate distinct gene networks and cellular functions in decidualizing endometrium.

Progesterone is indispensable for differentiation of human endometrial stromal cells (HESCs) into decidual cells, a process that critically controls embryo implantation. We now show an important role for androgen receptor (AR) signaling in this differentiation process. Decreased posttranslational modification of the AR by small ubiquitin-like modifier (SUMO)-1 in decidualizing cells accounted for increased responsiveness to androgen. By combining small interfering RNA technology with genome-wide expression profiling, we found that AR and progesterone receptor (PR) regulate the expression of distinct decidual gene networks. Ingenuity pathway analysis implicated a preponderance of AR-induced genes in cytoskeletal organization and cell motility, whereas analysis of AR-repressed genes suggested involvement in cell cycle regulation. Functionally, AR depletion prevented differentiation-dependent stress fiber formation and promoted motility and proliferation of decidualizing cells. In comparison, PR depletion perturbed the expression of many more genes, underscoring the importance of this nuclear receptor in diverse cellular functions. However, several PR-dependent genes encode for signaling intermediates, and knockdown of PR, but not AR, compromised activation of WNT/beta-catenin, TGFbeta/SMAD, and signal transducer and activator of transcription (STAT) pathways in decidualizing cells. Thus, the nonredundant function of the AR in decidualizing HESCs, centered on cytoskeletal organization and cell cycle regulation, implies an important role for androgens in modulating fetal-maternal interactions. Moreover, we show that PR regulates HESC differentiation, at least in part, by reprogramming growth factor and cytokine signal transduction.

S-nitrosylation of proteins at the leading edge of migrating trophoblasts by inducible nitric oxide synthase promotes trophoblast invasion.

Nitric oxide regulates many important cellular processes including motility and invasion. Many of its effects are mediated through the modification of specific cysteine residues in target proteins, a process called S-nitrosylation. Here we show that S-nitrosylation of proteins occurs at the leading edge of migrating trophoblasts and can be attributed to the specific enrichment of inducible nitric oxide synthase (iNOS/NOS2) in this region. Localisation of iNOS to the leading edge is co-incidental with a site of extensive actin polymerisation and is only observed in actively migrating cells. In contrast endothelial nitric oxide synthase (eNOS/NOS3) shows distribution that is distinct and non-colocalised with iNOS, suggesting that the protein S-nitrosylation observed at the leading edge is caused only by iNOS and not eNOS. We have identified MMP-9 as a potential target for S-nitrosylation in these cells and demonstrate that it co-localises with iNOS at the leading edge of migrating cells. We further demonstrate that iNOS plays an important role in promoting trophoblast invasion, which is an essential process in the establishment of a successful pregnancy.

Fas ligand-induced apoptosis is regulated by nitric oxide through the inhibition of fas receptor clustering and the nitrosylation of protein kinase Cepsilon.

Apoptosis induced by the death-inducing ligand FasL (CD95L) is a major mechanism of cell death. Trophoblast cells express the Fas receptor yet survive in an environment that is rich in the ligand. We report that basal nitric oxide (NO) production is responsible for the resistance of trophoblasts to FasL-induced apoptosis. In this study we demonstrate that basal NO production resulted in the inhibition of receptor clustering following ligand binding. In addition NO also protected cells through the selective nitrosylation, and inhibition, of protein kinase Cepsilon (PKCepsilon) but not PKCalpha. In the absence of NO production PKCepsilon interacted with, and phosphorylated, the anti-apoptotic protein cFLIP. The interaction is predominantly with the short form of cFLIP and its phosphorylation reduces its recruitment to the death-inducing signaling complex (DISC) that is formed following binding of a death-inducing ligand to its receptor. Inhibition of cFLIP recruitment to the DISC leads to increased activation of caspase 8 and subsequently to apoptosis. Inhibition of PKCepsilon using siRNA significantly reversed the sensitivity to apoptosis induced by inhibition of NO synthesis suggesting that NO-mediated inhibition of PKCepsilon plays an important role in the regulation of Fas-induced apoptosis.

Fetal-derived trophoblast use the apoptotic cytokine tumor necrosis factor-alpha-related apoptosis-inducing ligand to induce smooth muscle cell death.

Remodeling of the uterine spiral arteries during pregnancy transforms them from high to low resistance vessels that lack vasoconstrictive properties. This process is essential to meet the demand for increased blood flow imposed by the growing fetus. Loss of endothelial and smooth muscle cells (SMC) is evident in remodeled arteries but the mechanisms underlying this transformation remain unknown. This study investigated the hypothesis that fetal trophoblast invading from the placenta instigate remodeling by triggering cell death in vascular SMC. Specifically, a role for trophoblast-derived death inducing cytokine tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) was investigated. Expression of the activating TRAIL receptors R1 and R2 was detected by flow cytometry on human aortic SMC and by immunohistochemistry on spiral artery SMC. Recombinant human TRAIL induced human aortic SMC apoptosis, which was inhibited by antibodies against TRAIL-R1 or -R2. Perfusion of denuded spiral artery segments with recombinant human TRAIL also induced SMC apoptosis. Trophoblasts isolated from first trimester placenta expressed membrane-associated TRAIL and induced apoptosis of human aortic SMC; apoptosis was significantly inhibited by a recombinant human TRAIL-R1:Fc construct. Trophoblast within the first trimester placental bed also expressed TRAIL. These data show that: 1) TRAIL causes SMC death; 2) trophoblast produce the apoptotic cytokine TRAIL; and 3) trophoblast induce SMC apoptosis via a TRAIL-dependent mechanism. We conclude that TRAIL produced by trophoblast causes apoptosis of SMC and thus may contribute to SMC loss during spiral artery remodeling in pregnancy.

Hypoxia and placental remodelling.

In the first trimester of pregnancy fetal trophoblast cells invade the maternal uterine spiral arteries leading to loss of the vascular cells from the vessel wall and remodelling of the extracellular matrix. This is crucial to ensure that sufficient blood can reach the developing fetus. Impaired arterial remodelling is a feature of the major pregnancy pathologies pre-eclampsia and fetal growth restriction. Despite its importance, little is known about the regulation of this process. We have shown, using in vitro culture models and ex vivo explant models, that trophoblast cells play an active role in remodelling spiral arteries, and have implicated apoptotic events in this process. Further we have shown that trophoblast-derived factors such as Fas-ligand, tumor necrosis factor-related apoptosis inducing ligand (TRAIL) are important regulators of this process. The oxygen tension within the uteroplacental environment will vary with gestational age and will depend on the extent of trophoblast invasion and artery remodelling. Fluctuations in oxygen tension may be an important determinant of cellular events both during invasion towards uterine vessels and during the remodelling process. The components of this process known to be regulated by oxygen are reviewed, including lessons that can be learned from pregnancies at high altitude. In addition, data on the effect of varying oxygen tension on trophoblast production of pro-apoptotic factors and susceptibility of vascular smooth muscle cells to induction of apoptosis are described.