Corticotropin releasing hormone and Urocortin 2 activate inflammatory pathways in cultured trophoblast cell lines

European Journal of Obstetrics & Gynecology and Reproductive Biology 195 (2015) 200–205

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Corticotropin releasing hormone and Urocortin 2 activate inflammatory pathways in cultured trophoblast cell lines Romina Novembri a, Caterina De Clemente b, Lucia Funghi a, Michela Torricelli a, Chiara Voltolini a, John R. Challis c, Felice Petraglia a,* a b c

Department of Molecular and Developmental Medicine, University of Siena Viale Bracci, Siena, Italy Department of Biotechnology, University of Siena, via Fiorentina 1, 53100 Siena, Italy The University of Western Australia M460A, 35 Stirling Highway, Crawley, WA 6009, Australia

A R T I C L E I N F O

A B S T R A C T

Article history: Received 17 January 2015 Received in revised form 26 June 2015 Accepted 28 October 2015

Objective: Embryo implantation and parturition are recognized as inflammatory events involving endocrine and immune system. NF-kB and MAPK are two transcription factor families involved in inflammation. A possible role of neuroendocrine mechanism in early pregnancy and delivery was proposed for the neuropeptides related to corticotropin releasing hormones (CRH), named Urocortins (Ucns). Experimental and clinical studies support a role for CRH, Ucn, Ucn2 and Ucn3 in the endocrine/ immune modulation of inflammation in human trophoblast; however the intracellular mechanisms are not yet recognized. The aim of the present study was to evaluate which of these neuropeptides modulate NF-kB or MAPKs pathways. Study design: In Jeg-3 placental cell line the effect of CRH, Ucn, Ucn2 or Ucn3 on NF-kB and MAPKs pathways were evaluated using Western blot analysis. Results: CRH induced the phosphorylation of MAPK subunits; Ucn2 was able to induce the phosphorylation of both NF-kB and MAPK subunits. Ucn and Ucn3 had no effects on these pathways. Conclusions: These data provide novel information on inflammatory process in trophoblast cells: Ucn2 is a potent pro-inflammatory neuropeptide via NF-kB and MAPK pathways and CRH via MAPK, and CRH and Ucn2 network participates in the inflammatory mechanisms of pregnancy and parturition. ß 2015 Elsevier Ireland Ltd. All rights reserved.

Keywords: CRH Urocortin 2 MAPK NF-kB Jeg 3

Introduction Accumulating evidences suggest that embryo implantation and parturition are inflammatory events involving endocrine and immune system [1–3]. In this context, an influx of inflammatory cells into endometrium, myometrium and fetal membranes occurs [4–6] increasing vascular and leukocyte adhesion molecule expression and pro-inflammatory cytokines (TNF-a, IL-1b and IL-6) [7,8] prostaglandins and matrix-degrading enzymes production representing the final mechanism. Among transcription factors, both Nuclear factor-kappa B (NF-kB) [9] and mitogenactivated protein kinases (MAPK) [10] have a crucial role in intrauterine inflammatory process activated at time of embryo

* Corresponding author at: Obstetrics and Gynecology, Department of Molecular and Developmental Medicine, University of Siena, Policlinico ‘‘Le Scotte’’ Viale Bracci, 53100 Siena, Italy. Tel.: +39 0577 233 453; fax: +39 0577 233 454. E-mail address: [email protected] (F. Petraglia). http://dx.doi.org/10.1016/j.ejogrb.2015.10.027 0301-2115/ß 2015 Elsevier Ireland Ltd. All rights reserved.

implantation or delivery [11,12] following the binding of bacteria, cytokines, or reactive oxygen species [13]. The NF-kB family comprises at least five proteins: NF-kB1 (p50/ p105), NF-kB2 (p52/p100), RelA (p65), RelB and RelC present in cell cytoplasm in an inactive form, bound to a family of inhibitory proteins of which IkB-a represents the most important subunit [14]. This transcription family has been identified as an important factor in the process of human labor and delivery [15,16] and in vitro studies conducted on Jeg-3 cell lines demonstrated that NFkB is highly inducible by pro-inflammatory stimuli such as TNF-a, LPS [14] and IL-1b [17]. MAP kinases (extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinases (JNK) and p38), are members of a serine/ threonine kinases family required for the enzymes phosphorylation and activate a signaling cascade the downstream effect of which modulates the inflammatory response [18]. Few data are available regarding the role of MAPKs in human gestation and labor, however an activation of this pathway has been observed in preterm fetal membranes and an induction has been demonstrated

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under pro-inflammatory products such as IL-8, TNF-a [19,20] and LPS [18]. Recently a relationship between stress hormones and inflammatory mediators was identified; among stress hormones corticotrophin releasing hormone (CRH) represents the most important factor secreted by placenta and gestational tissues during pregnancy influencing inflammatory processes [21]. Three urocortins (Ucns: Ucn, Ucn2 and Ucn3) are also known and these peptides show a high sequence homology with CRH and are expressed in gestational tissues where they take part to control of immune and endocrine function [22]. CRH and Ucns act by binding two receptors CRH-R1 and CRH-R2; CRH and Ucn can bind, with different affinity, to both receptors, whereas Ucn2 and Ucn3 bind exclusively to CRH-R2. Despite the involvement of CRH-family members on inflammatory/infective processes in placenta and fetal membranes has been described [22,23], the intracellular mechanism by which they act does not yet recognize. The present study investigated the effect of CRH and Ucns on NF-kB and MAPK pathways in placental trophoblast cell line. Materials and methods Cell culture and treatments Jeg-3 cells, a steroidogenic human placental cell line derived from choriocarcinoma, were used for this study maintained in DMEM medium supplemented with 10% fetal calf serum, and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin) (Sigma–Aldrich, Milan, Italy). Cells were plated in 25 cm3 flasks under standard cell culture conditions until 80–90% of confluence. Cell cultures were starved overnight and treatments were done in serum-free DMEM medium. Initial control experiments examined the ability of TNF-a to stimulate the expression of components of the NF-kB and MAPK pathways in our model. Subsequently to positive results obtained in control experiments, cells were cultured with different doses of CRH, Ucn, Ucn2 or Ucn3 (10 7, 10 8 M) (kindly donate by Salk Institute, La Jolla, CA, USA) while control group received serum free cultured medium alone or vehicle alone dimethylsulphoxide (Euroclone, Milan, Italy). MAPK (total and phosphorylated ERK and cytosolic and phosphorylated p38) were evaluated after 5 and 10 min of treatment, while NF-kB proteins (NF-kB1 and RelA) and the inhibitory protein IkB-a were evaluated after 30 min and 1 h of treatment. Experiments were also done in presence of a pretreatment (30 min) with a CRH-receptors antagonist: antalarmin (Ant) or astressin 2b (Ast-2b) (kindly donate by Salk Institute, La Jolla, CA, USA) to specifically block CRH-R1 and CRH-R2 respectively. The study was replicated 5 times and each treatment was performed in triplicate. Jeg-3 cell line has been used because this cell line is frequently used for placental functional studies, moreover previous studies on NF-kB and MAPK pathways used this model [14,24,25], peptides concentrations used for treatments were chosen on the bases of data present in literature and after dose response preliminary experiments with 10 6, 10 7, 10 8, 10 9 and 10 10 M [26,27]; these concentrations reflect the maternal or intrauterine tissue concentrations found in human pregnancy at term that range from 10 12 to 10 6 M [28]. Treatment time was chosen after timecourse experiments; times of incubation were been 3, 5, 10, 15, 20, 30 min 1 and 3 h. Analysis of NF-kB and MAPK proteins In the present study NF-kB and MAPK proteins were analyzed by Western blotting.

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For NF-kB family we evaluated two proteins NF-kB1 (p50/105) and RelA (p65) and the inhibitory proteins IkB-a. Antibodies for NF-kB1 p65 (id. BK3987S), p105/50 (id. BK3035), IkB-a (id. BK4812) and Phospho IkB-a (id. BK9246S) were taken by Cell Signaling (Euroclone, Milan, Italy). Regarding MAPKs, ERK and phospho ERK and p38 and phospho p38 were analyzed. Antibody for ERK1/2 and p38 were taken by Cell Signaling (Euroclone, Milan, Italy). Protein extraction and Western blotting Following stimulations, cells were lysed in RIPA plus buffer (50 mM Tris–HCl [pH 7.2], 100 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% Na-deoxycholate, 50 mM NaF, 2 mM Na-orthovanadate, 1 mm DTT), antiprotease and antiphosphatase cocktails (Sigma–Aldrich, Milan, Italy), and cell debris were eliminated by centrifugation at 21,000  g at 4 8C for 10 min. Equal amounts of proteins were subjected to SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose filters, and probed by immunoblot. The following antibodies were used for immunoblotting: anti-p38 MAPKpThr180/Tyr182 (#9211), anti-p38 MAPK (#9212), anti-Erk1/2 p44/42 pThr202/Tyr204 (#4370), anti-Erk1/2 p44/42 MAPK (#9102), anti-phospho-IkBa (Ser32/36) (#9246), anti-IkBa (#4812), anti-NF-k p105/p50 (#3035), anti-NF-k p65 (#3987). All antibodies were purchased from Cell Signaling Technology, Beverly, MA. Protein expression of each component was identified by co-migration of a positive control and by comparison of the mobility of protein standard. Films were quantified using ImageQuant LAS 4000 (GE Healthcare, Milan, Italy). Statistical analysis All data were assessed for normality of distribution using computer software (Prism 4; Graphpad Software, CA, USA). Where the data were normally distributed, differences among three or more groups were analyzed by ANOVA with Tukey’s multiple comparison test. The statistical significance was achieved when p < 0.05. Data were expressed as mean  SD (standard deviation). Results Effects on NF-kB pathway By Western blot experiments expression of NF-kB family proteins NF-kB1 (p105/p50) and RelA (p65) after treatment with CRH and Ucns was analyzed. The addition of Ucn2 10 8 and 10 7 M to JEG-3 cells for 1 h significantly increased the expression of p50/ p105 ratio (p < 0.01), and of p65 protein (p < 0.05). When Jeg-3 cells were pretreated with CRH-R2 antagonist Ast-2b, the Ucn2induced p50/p105 and p65 stimulation was significantly reduced (p < 0.05 and p < 0.01 respectively) (Fig. 1, panels B and C). Treatments with Ucn2 for 30 min and with CRH, Ucn and Ucn3 both for 30 min and for 1 h did not induce significant activation of NF-kB subunits (data not shown). Effects on IkB-a Total and phosphorylated NF-kB inhibitory subunit IkB-a was analyzed with Western blot. Ucn2 10 8 and 10 7 M increased the pIkB-a/IkB-a ratio (p < 0.05); 5 min of treatment resulting in a significantly phosphorylation of IkB-a and leading to the activation of NF-kB pathway; the signal was lost with 10 min of treatment (data not shown). Incubation of JEG-3 with CRH-R2 antagonist Ast-2b significantly decreased Ucn2 stimulated protein expression of pIkB-a/IkB-a ratio (p < 0.01) (Fig. 2). Since only Ucn2 demonstrated to have effect on activation NF-kB pathways

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Fig. 1. Effects on NF-kB pathway: Nf-kB1 and RelA. Represents Western blot of NF-kB1(p105/p50) and Rel A (p65) in jeg 3 after 1 h of treatment with or without CRH, Ucn, Ucn2 and Ucn3 and with or without CRH-R2 astressin 2b (Ast) (panel A). The addition of Ucn2 10 8 and 10 7 M for 1 h significantly increased the p50/p105 ratio (**p < 0.01) (panel B), and of p65 protein (*p < 0.05) (panel C). In presence of pretreatment with CRH-R2 antagonist astressin 2b (Ast) the Ucn2-induced p50/p105 and p65 stimulation was significantly reduced (8p < 0.05 and 88p < 0.01) (panels B and C, respectively). CRH, Ucn and Ucn3 did not induce any effect on NF-kB pathway (data not shown).

we did not performed treatments with CRH, Ucn and Ucn3 for the NF-kB inhibitory subunit. Effects on MAPKs Western blot analysis for MAPKs detected the expression of cytosolic total (p38) and phosphorylated (pp38) p38 (Fig. 3, panel A), and of cytosolic total (ERK) and phosphorylated (pERK) ERK (Fig. 4, panel A). 5 min of treatment with CRH 10 8 and 10 7 M or Ucn2 10 7 M significantly increased the ratio pp38/p38 (p < 0.01 for CRH 10 8 M and Ucn2 10 7 M and p < 0.05 for CRH 10 7 M) (Fig. 3, Panels B and C), and of pERK protein (p < 0.05) (Fig. 4, panels B and C). Preincubation with CRH-Rs antagonists Ast-2b and Ant significantly decreased the effects induced by Ucn2 or CRH on p38 and ERK pathway (p < 0.01). Both CRH and Ucn2 signals were lost with 10 min of treatment; no effect was induced by Ucn and Ucn3 at 5 and 10 min (data not shown).

Comments The present results corroborate a relationship between stress peptides and intracellular pathways leading to inflammatory cascade, showing that in Jeg-3 cell line Ucn2 activates NF-kB and MAPK pathways and that CRH activates only MAPK pathway. No significant activations of these two transcription factors were observed after Ucn or Ucn3 treatments. NF-kB and MAPK play an important role in various tissues in the regulation of immune/inflammatory response and are essentials for the expression of a wide variety of genes including proinflammatory cytokines, chemokines and adhesion molecules; this pathway modulates the physiological cycle associated with inflammation and our results agree with data obtained in mouse thymocytes [29] and human colonic epithelial cells [30]. An inappropriate activation by these regulators is associated with pathological reproductive disorders like miscarriage and preterm

Fig. 2. Effects on NF-kB pathway: inhibitory subunit IkB-a. Representative Western blot analysis in which total and phosphorylated IKB-a are expressed after 1 h of treatment with or without Ucn2 and with or without CRH-R2 astressin 2b (Ast) (panel A). Ucn2 10 8 and 10 7 M for 1 h increased the pIKB-a/IKB-a ratio (*p < 0.05) (panel B). Preincubation of JEG-3 with Ast significantly decreased Ucn2 stimulated protein expression of pIkB-a (p < 0.01) (panel B).

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Fig. 3. Effects on MAPK pathway: protein p38. Western blot analysis detected the protein expression of cytosolic total (p38) and phosphorylated (pp38) p38 after 5 min of treatment with or without CRH, Ucn, Ucn2 and Ucn3 and with or without CRH receptors antagonists astressin 2b (Ast) an antalarmin (Ant) (panel A). CRH 10 7 and 10 8 M and Ucn2 10 7 M (5 min of treatments) significantly increased the ratio pp38/p38 (**p < 0.01 and *p < 0.05) (panels A and B, respectively). Pretreatment with CRH-Rs antagonist Ast and Ant significantly decreased the effects induced by Ucn2 or CRH on p38 pathway (88p < 0.01) (panels A and B). Ucn and Ucn3 did not induce any effect on this pathway (data not shown).

birth [9,31] and inflammatory/infective agents may activate NF-kB and MAPK leading to increased production cytokines and chemokines, stimulating prostaglandins release, ECM remodeling, and recruitment of immune cells in gestational tissues [14]. Indeed

NF-kB and MAPK proteins are rapidly activated by pro-inflammatory products in human intrauterine tissues: IL-1b activates NF-kB and MAPK respectively in amnion [32] and in myometrial cells [33], and TNF-a activates NF-kB and MAPK respectively in human

Fig. 4. Effects on MAPK pathway: protein ERK. Western blot analysis detected the protein expression of cytosolic total (ERK) and phosphorylated (pERK) ERK after 5 min of treatment with or without CRH, Ucn and Ucn2 with or without CRH receptors antagonists astressin 2b (Ast) an antalarmin (Ant) (panel A). CRH 10 7 and 10 8 M and Ucn2 10 7 M (5 min of treatments) significantly increased the expression of pERK (*p < 0.05) (panels B and C). Preincubation with Ast 2b and Ant significantly decreased the effects induced by Ucn2 or CRH on ERK pathway (88p < 0.01) (panels B and C). Ucn and Ucn3 did not induce any effect on NF-kB this (data not shown).

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third trimester placental cell line Jeg-3 [14] and in endometrial stromal cells [34] and LPS in Jeg-3 [35] and in human placenta and fetal membranes [35]. It is well known that CRH stimulates NF-kB activation and translocation from cytoplasm to nucleus in human normal epidermal keratinocytes [36] and in mouse thymocytes [22] while has an opposite effect in line of HaCaT keratinocytes [37], in human immortalized epidermal (PIG1) melanocytes [38] and in pituitary corticotroph cell line AtT20 [39]. The effect of CRH on MAPK pathways is stimulatory in PC12 cells [40] and in cultured rat microglia [41] while no effect vas shown on myometrium [42]. The present study demonstrated that in human placental Jeg-3 cell line CRH significantly stimulates the phosphorylation and the activation of p38 and ERK acting as a pro-inflammatory molecule, an effect reversed by CRH-R1 or CRH-R2 antagonists. It is very important to consider that among MAPK, p38 is involved in signaling mechanisms when inflammation causes preterm birth [43]. With a specific tissues function, a strong relationship between transcription factors and stress hormones was shown. A study conducted on primary mouse macrophages demonstrated that using specific CRH-R1 and CRH-R2 antagonists the effects exerted by CRH on inflammatory pathways was reduced suggesting that both CRH-Rs are involved in this mechanisms [44]. In some study was shown that CRH acts as an inflammatory mediator binding the CRH-R1 [45–47]. In vivo experiment on mouse demonstrated that CRH play a crucial role in implantation, in fact higher concentration of the CRH transcript and its peptide product at the early implantation sites of pregnant rats compared to the interimplantation uterine areas [48]; intra-peritoneal injections of CRH antibodies at day 2 of pregnancy decrease the number of fetuses within the uterus by 60% [49], whereas administration of antalarmin to early pregnant rats results up to a 70% reduction in the number of implantation sites [50]. Moreover defective CRH/ CRH-R1 system during early pregnancy may be implicated in the pathophysiology of recurrent miscarriage, and preeclampsia [51]. Our present results partially agree with a recent study made on first trimester placenta cells in which a strong activation of p38/ MAPK was observed after CRH treatment but no effect was observed on ERK activation [45]. Ucn2 activation of NF-kB and MAPK correlates with data obtained in human colonocytes [29], as shown by degradation of IKBa and phosphorylation of the p65 and p50/p105 subunits of NFkB and ERK. A pro-inflammatory role of Ucn2 in mechanisms leading to parturition was suggested by the evidence that Ucn2 stimulates TNF-a secretion in trophoblast explants [27] and Ucn2 mRNA is highly expressed in trophoblast tissues collected from women delivering preterm with histological chorioamnionitis [23]. Any activations of NF-kB and MAPK pathways were observed after Ucn and Ucn3 stimulation and this data fit with previous observations that Ucn and Ucn3 stimulate the trophoblast secretion of anti-inflammatory cytokines, such as IL-4 and IL-10, reducing the secretion of TNF-a, through CRH-R2 receptor [26,27]. Despite Ucn2 and Ucn3 exert they action by binding the same CRH receptors, an opposite effects of these two peptides was previously described [27]; the present study corroborate these findings showing that Ucn3 and did not activated NF-kB and MAPKs while Ucn2 induced the activation of these pathway. The fact that these two opposite effects are both mediated by the link with the same receptor (CRH-R2) led us to hypothesized that CRH-R2 activate different signaling pathways depending on its ligands. These data provide novel information on inflammatory process suggesting that CRH and Ucn2 in trophoblast act as inflammatory molecules via transcription factor Nf-kB and MAPK modulating the hormonal/immune mechanisms occurring during gestation.

Acknowledgements This work was supported by MIUR Grant entitled ‘‘Preterm birth: molecular, biochemical and biophysical markers of the fetoplacental unit’’ (prot. 20102CHST5) and partially from Accademia Dei Lincei. References [1] Rice GE. Cytokines and the initiation of parturition. Front Horm Res 2001;27: 113–46. [2] Romero R, Espinoza J, Goncalves LF, Kusanovic JP, Friel LA, Nien JK. Inflammation in preterm and term labour and delivery. Semin Fetal Neonatal Med 2006;11:317–26. [3] Osman I, Young A, Ledingham MA, et al. Leukocyte density and pro-inflammatory cytokine expression in human fetal membranes, decidua, cervix and myometrium before and during labour at term. Mol Hum Reprod 2003;9: 41– 5. [4] Bokstrom H, Brannstrom M, Alexandersson M, Norstrom A. Leukocyte subpopulations in the human uterine cervical stroma at early and term pregnancy. Hum Reprod 1997;12:586–90. [5] Ledingham MA, Thomson AJ, Jordan F, Young A, Crawford M, Norman JE. Cell adhesion molecule expression in the cervix and myometrium during pregnancy and parturition. Obstet Gynecol 2001;97:235–42. [6] Thomson AJ, Telfer JF, Young A, et al. Leukocytes infiltrate the myometrium during human parturition: further evidence that labour is an inflammatory process. Hum Reprod 1999;14:229–36. [7] Cox SM, King MR, Casey ML, MacDonald PC. Interleukin-1 beta, -1 alpha, and -6 and prostaglandins in vaginal/cervical fluids of pregnant women before and during labor. J Clin Endocrinol Metab 1993;77:805–15. [8] Romero R, Mazor M, Brandt F, et al. Interleukin-1 alpha and interleukin-1 beta in preterm and term human parturition. Am J Reprod Immunol 1992;27:117–23. [9] Lindstrom M. Social capital, the miniaturisation of community and consumption of homemade liquor and smuggled liquor during the past year. A population-based study. Eur J Public Health 2005;15:593–600. [10] Shoji T, Yoshida S, Mitsunari M, et al. Involvement of p38 MAP kinase in lipopolysaccharide-induced production of pro- and anti-inflammatory cytokines and prostaglandin E(2) in human choriodecidua. J Reprod Immunol 2007;75:82–90. [11] Allport VC, Pieber D, Slater DM, Newton R, White JO, Bennett PR. Human labour is associated with nuclear factor-kappaB activity which mediates cyclo-oxygenase-2 expression and is involved with the ‘functional progesterone withdrawal’. Mol Hum Reprod 2001;7:581–6. [12] Elliott CL, Allport VC, Loudon JA, Wu GD, Bennett PR. Nuclear factor-kappa B is essential for up-regulation of interleukin-8 expression in human amnion and cervical epithelial cells. Mol Hum Reprod 2001;7:787–90. [13] Challis JR, Lockwood CJ, Myatt L, Norman JE, Strauss JF3rd, Petraglia F. Inflammation and pregnancy. Reprod Sci 2009;16:206–15. [14] Lappas M, Yee K, Permezel M, Rice GE. Lipopolysaccharide and TNF-alpha activate the nuclear factor kappa B pathway in the human placental JEG-3 cells. Placenta 2006;27:568–75. [15] Cookson VJ, Chapman NR. NF-kappaB function in the human myometrium during pregnancy and parturition. Histol Histopathol 2010;25:945–56. [16] Stjernholm-Vladic Y, Stygar D, Mansson C, et al. Factors involved in the inflammatory events of cervical ripening in humans. Reprod Biol Endocrinol 2004;2:74. [17] Ackermann LW, Denning GM. Nuclear factor-kappaB contributes to interleukin-4- and interferon-dependent polymeric immunoglobulin receptor expression in human intestinal epithelial cells. Immunology 2004;111:75–85. [18] Lappas M, Permezel M, Rice GE. Mitogen-activated protein kinase proteins regulate LPS-stimulated release of pro-inflammatory cytokines and prostaglandins from human gestational tissues. Placenta 2007;28:936–45. [19] Bartlett SR, Sawdy R, Mann GE. Induction of cyclooxygenase-2 expression in human myometrial smooth muscle cells by interleukin-1beta: involvement of p38 mitogen-activated protein kinase. J Physiol 1999;520(Pt 2):399–406. [20] Yazlovitskaya EM, Pelling JC, Persons DL. Association of apoptosis with the inhibition of extracellular signal-regulated protein kinase activity in the tumor necrosis factor alpha-resistant ovarian carcinoma cell line UCI 101. Mol Carcinog 1999;25:14–20. [21] Zoumakis E, Kalantaridou SN, Makrigiannakis A. CRH-like peptides in human reproduction. Curr Med Chem 2009;16:4230–5. [22] Petraglia F, Imperatore A, Challis JR. Neuroendocrine mechanisms in pregnancy and parturition. Endocr Rev 2010;31:783–816. [23] Torricelli M, Novembri R, Bloise E, De Bonis M, Challis JR, Petraglia F. Changes in placental CRH, urocortins, and CRH-receptor mRNA expression associated with preterm delivery and chorioamnionitis. J Clin Endocrinol Metab 2011;96:534–40. [24] Wagener J, Yang W, Kazuschke K, Winterhager E, Gellhaus A. CCN3 regulates proliferation and migration properties in Jeg3 trophoblast cells via ERK1/2, Akt and Notch signalling. Mol Hum Reprod 2013;19:237–49. [25] Yu Y, Wang L, Tang W, Zhang D, Shang T. RNA interference-mediated knockdown of Notch-1 inhibits migration and invasion, down-regulates matrix metalloproteinases and suppresses NF-kappaB signaling pathway in trophoblast cells. Acta Histochem 2014;116:911–9.

R. Novembri et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 195 (2015) 200–205 [26] Torricelli M, Voltolini C, Bloise E, et al. Urocortin increases IL-4 and IL-10 secretion and reverses LPS-induced TNF-alpha release from human trophoblast primary cells. Am J Reprod Immunol 2009;62:224–31. [27] Novembri R, Torricelli M, Bloise E, et al. Effects of urocortin 2 and urocortin 3 on IL-10 and TNF-alpha expression and secretion from human trophoblast explants. Placenta 2011;32:969–74. [28] Li W, Challis JR. Corticotropin-releasing hormone and urocortin induce secretion of matrix metalloproteinase-9 (MMP-9) without change in tissue inhibitors of MMP-1 by cultured cells from human placenta and fetal membranes. J Clin Endocrinol Metab 2005;90:6569–74. [29] Zhao J, Karalis KP. Regulation of nuclear factor-kappaB by corticotropinreleasing hormone in mouse thymocytes. Mol Endocrinol 2002;16:2561–70. [30] Moss AC, Anton P, Savidge T, et al. Urocortin II mediates pro-inflammatory effects in human colonocytes via corticotropin-releasing hormone receptor 2alpha. Gut 2007;56:1210–7. [31] Gomez R, Romero R, Edwin SS, David C. Pathogenesis of preterm labor and preterm premature rupture of membranes associated with intraamniotic infection. Infect Dis Clin North Am 1997;11:135–76. [32] Lee Y, Allport V, Sykes A, Lindstrom T, Slater D, Bennett P. The effects of labour and of interleukin 1 beta upon the expression of nuclear factor kappa B related proteins in human amnion. Mol Hum Reprod 2003;9:213–8. [33] Belt AR, Baldassare JJ, Molnar M, Romero R, Hertelendy F. The nuclear transcription factor NF-kappaB mediates interleukin-1beta-induced expression of cyclooxygenase-2 in human myometrial cells. Am J Obstet Gynecol 1999;181: 359–66. [34] Kim YA, Kim JY, Kim MR, Hwang KJ, Chang DY, Jeon MK. Tumor necrosis factoralpha-induced cyclooxygenase-2 overexpression in eutopic endometrium of women with endometriosis by stromal cell culture through nuclear factorkappaB activation. J Reprod Med 2009;54:625–30. [35] Lappas M, Permezel M, Georgiou HM, Rice GE. Nuclear factor kappa B regulation of proinflammatory cytokines in human gestational tissues in vitro. Biol Reprod 2002;67:668–73. [36] Zbytek B, Pfeffer LM, Slominski AT. Corticotropin-releasing hormone stimulates NF-kappaB in human epidermal keratinocytes. J Endocrinol 2004;181:R1–7. [37] Zbytek B, Pfeffer LM, Slominski AT. Corticotropin-releasing hormone inhibits nuclear factor-kappaB pathway in human HaCaT keratinocytes. J Invest Dermatol 2003;121:1496–9. [38] Zbytek B, Pfeffer LM, Slominski AT. CRH inhibits NF-kappa B signaling in human melanocytes. Peptides 2006;27:3276–83. [39] Karalis KP, Venihaki M, Zhao J, van Vlerken LE, Chandras C. NF-kappaB participates in the corticotropin-releasing, hormone-induced regulation of the pituitary proopiomelanocortin gene. J Biol Chem 2004;279:10837–40.

205

[40] Dermitzaki E, Tsatsanis C, Gravanis A, Margioris AN. Corticotropin-releasing hormone induces Fas ligand production and apoptosis in PC12 cells via activation of p38 mitogen-activated protein kinase. J Biol Chem 2002;277: 12280–87. [41] Wang W, Ji P, Dow KE. Corticotropin-releasing hormone induces proliferation and TNF-alpha release in cultured rat microglia via MAP kinase signalling pathways. J Neurochem 2003;84:189–95. [42] Grammatopoulos DK, Randeva HS, Levine MA, Katsanou ES, Hillhouse EW. Urocortin, but not corticotropin-releasing hormone (CRH), activates the mitogen-activated protein kinase signal transduction pathway in human pregnant myometrium: an effect mediated via R1alpha and R2beta CRH receptor subtypes and stimulation of Gq-proteins. Mol Endocrinol 2000;14: 2076–91. [43] Lappas M, Riley C, Lim R, et al. MAPK and AP-1 proteins are increased in term pre-labour fetal membranes overlying the cervix: regulation of enzymes involved in the degradation of fetal membranes. Placenta 2011;32:1016–25. [44] Tsatsanis C, Androulidaki A, Dermitzaki E, Gravanis A, Margioris AN. Corticotropin releasing factor receptor 1 (CRF1) and CRF2 agonists exert an antiinflammatory effect during the early phase of inflammation suppressing LPSinduced TNF-alpha release from macrophages via induction of COX-2 and PGE2. J Cell Physiol 2007;210:774–83. [45] Wang W, Nan X, Ji P, Dow KE. Corticotropin releasing hormone modulates endotoxin-induced inflammatory cytokine expression in human trophoblast cells. Placenta 2007;28:1032–8. [46] Webster EL, Tracey DE, Jutila MA, Wolfe Jr SA, De Souza EB. Corticotropinreleasing factor receptors in mouse spleen: identification of receptor-bearing cells as resident macrophages. Endocrinology 1990;127:440–52. [47] Singh VK, Fudenberg HH. Binding of [125I] corticotropin releasing factor to blood immunocytes and its reduction in Alzheimer’s disease. Immunol Lett 1988;18:5–8. [48] Makrigiannakis A, Margioris AN, Le Goascogne C, et al. Corticotropin-releasing hormone (CRH) is expressed at the implantation sites of early pregnant rat uterus. Life Sci 1995;57:1869–75. [49] Athanassakis I, Farmakiotis V, Aifantis I, Gravanis A, Vassiliadis S. Expression of corticotrophin-releasing hormone in the mouse uterus: participation in embryo implantation. J Endocrinol 1999;163:221–7. [50] Makrigiannakis A, Zoumakis E, Kalantaridou S, et al. Corticotropin-releasing hormone promotes blastocyst implantation and early maternal tolerance. Nat Immunol 2001;2:1018–24. [51] Kalantaridou SN, Zoumakis E, Makrigiannakis A, Godoy H, Chrousos GP. The role of corticotropin-releasing hormone in blastocyst implantation and early fetal immunotolerance. Horm Metab Res 2007;39:474–7.