Original Article
SLIT3 is Increased in Supracervical Human Foetal Membranes and in Labouring Myometrium and Regulates Pro-Inflammatory Mediators
Ratana Lim,
Gillian Barker,
Martha Lappas,
Ratana Lim
Mercy Perinatal Research Centre, Mercy Hospital for Women, Heidelberg, Vic., Australia
Department of Obstetrics and Gynaecology, University of Melbourne, Heidelberg, Vic., Australia
Search for more papers by this authorGillian Barker
Mercy Perinatal Research Centre, Mercy Hospital for Women, Heidelberg, Vic., Australia
Department of Obstetrics and Gynaecology, University of Melbourne, Heidelberg, Vic., Australia
Search for more papers by this authorCorresponding Author
Martha Lappas
Mercy Perinatal Research Centre, Mercy Hospital for Women, Heidelberg, Vic., Australia
Department of Obstetrics and Gynaecology, University of Melbourne, Heidelberg, Vic., Australia
Correspondence
Martha Lappas, Department of Obstetrics and Gynaecology, University of Melbourne, Mercy Hospital for Women, Level 4/163 Studley Road, Heidelberg, 3084 Vic., Australia.
E-mail: [email protected]
Search for more papers by this authorRatana Lim,
Gillian Barker,
Martha Lappas,
Ratana Lim
Mercy Perinatal Research Centre, Mercy Hospital for Women, Heidelberg, Vic., Australia
Department of Obstetrics and Gynaecology, University of Melbourne, Heidelberg, Vic., Australia
Search for more papers by this authorGillian Barker
Mercy Perinatal Research Centre, Mercy Hospital for Women, Heidelberg, Vic., Australia
Department of Obstetrics and Gynaecology, University of Melbourne, Heidelberg, Vic., Australia
Search for more papers by this authorCorresponding Author
Martha Lappas
Mercy Perinatal Research Centre, Mercy Hospital for Women, Heidelberg, Vic., Australia
Department of Obstetrics and Gynaecology, University of Melbourne, Heidelberg, Vic., Australia
Correspondence
Martha Lappas, Department of Obstetrics and Gynaecology, University of Melbourne, Mercy Hospital for Women, Level 4/163 Studley Road, Heidelberg, 3084 Vic., Australia.
E-mail: [email protected]
Search for more papers by this authorAbstract
Problem
Inflammation is associated with preterm birth, a worldwide healthcare issue. SLIT3 has a role in inflammation, and thus, the purpose of this study was to determine the effect of SLIT3 on labour mediators in human gestational tissues.
Method of study
SLIT3 protein expression was performed using immunohistochemistry in foetal membranes and myometrium with no labour and after labour. Foetal membranes were also obtained from a distal site (DS) and supracervical site (overlying the cervix; SCS). SLIT3 gene silencing was achieved using siRNA in primary amnion and myometrial cells. Pro-inflammatory and pro-labour mediators were evaluated by qRT-PCR, ELISA and gelatin zymography.
Results
SLIT3 expression was greater in foetal membranes from the SCS compared with DS and in myometrium after term spontaneous labour onset. SLIT3 siRNA in primary amnion and myometrial cells decreased IL-1β-induced pro-inflammatory cytokine gene expression and release (IL-6 and IL-8) and MMP-9 gene expression and release. In amnion cells, SLIT3 siRNA knockdown decreased IL-1β-induced COX-2 expression and prostaglandin PGE2 release. There was no effect of SLIT3 siRNA on IL-1β-induced NF-κB transcriptional activity.
Conclusion
Our results demonstrate that SLIT3 is increased with labour, and both our amnion and our myometrial studies describe a pro-inflammatory effect of SLIT3 in these tissues.
References
- 1 Institute of Medicine (US) Committee on Understanding Premature Birth and Assuring Healthy Outcomes: Societal costs of preterm birth. In Preterm Birth: Causes, Consequences, and Prevention, Behrman RE, Butler AS (eds). National Academies Press, Washington, DC, 2007, pp 398–429.
- 2Liu L, Johnson HL, Cousens S, Perin J, Scott S, Lawn JE, Rudan I, Campbell H, Cibulskis R, Li M, Mathers C, Black RE; Child Health Epidemiology Reference Group of WHO and UNICEF: Global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since 2000. Lancet 2012; 379: 2151–2161.
- 3Goldenberg RL, Culhane JF, Iams JD, Romero R: Epidemiology and causes of preterm birth. Lancet 2008; 371: 75–84.
- 4Malak TM, Bell SC: Structural characteristics of term human fetal membranes: a novel zone of extreme morphological alteration within the rupture site. Br J Obstet Gynaecol 1994; 101: 375–386.
- 5McLaren J, Malak TM, Bell SC: Structural characteristics of term human fetal membranes prior to labour: identification of an area of altered morphology overlying the cervix. Hum Reprod 1999; 14: 237–241.
- 6Malak TM, Mulholland G, Bell SC: Morphometric characteristics of the decidua, cytotrophoblast, and connective tissue of the prelabor ruptured fetal membranes. Ann N Y Acad Sci 1994; 734: 430–432.
- 7Romero R, Mazor M, Munoz H, Gomez R, Galasso M, Sherer DM: The preterm labor syndrome. Ann N Y Acad Sci 1994; 734: 414–429.
- 8Romero 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: 317–326.
- 9Bowen JM, Chamley L, Mitchell MD, Keelan JA: Cytokines of the placenta and extra-placental membranes: biosynthesis, secretion and roles in establishment of pregnancy in women. Placenta 2002; 23: 239–256.
- 10Christiaens I, Zaragoza DB, Guilbert L, Robertson SA, Mitchell BF, Olson DM: Inflammatory processes in preterm and term parturition. J Reprod Immunol 2008; 79: 50–57.
- 11Olson DM: The role of prostaglandins in the initiation of parturition. Best Pract Res Clin Obstet Gynaecol 2003; 17: 717–730.
- 12Weiss A, Goldman S, Shalev E: The matrix metalloproteinases (MMPS) in the decidua and fetal membranes. Front Biosci 2007; 12: 649–659.
- 13Lappas 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: 668–673.
- 14Lappas M, Rice GE: The role and regulation of the nuclear factor kappa B signalling pathway in human labour. Placenta 2007; 28: 543–556.
- 15Lindstrom TM, Bennett PR: The role of nuclear factor kappa B in human labour. Reproduction 2005; 130: 569–581.
- 16Pirianov G, Waddington SN, Lindstrom TM, Terzidou V, Mehmet H, Bennett PR: The cyclopentenone 15-deoxy-delta 12,14-prostaglandin J(2) delays lipopolysaccharide-induced preterm delivery and reduces mortality in the newborn mouse. Endocrinology 2009; 150: 699–706.
- 17Brose K, Bland KS, Wang KH, Arnott D, Henzel W, Goodman CS, Tessier-Lavigne M, Kidd T: Slit proteins bind Robo receptors and have an evolutionarily conserved role in repulsive axon guidance. Cell 1999; 96: 795–806.
- 18Dickinson RE, Duncan WC: The SLIT–ROBO pathway: a regulator of cell function with implications for the reproductive system. Reproduction 2010; 139: 697–704.
- 19Ballard MS, Hinck L: A roundabout way to cancer. Adv Cancer Res 2012; 114: 187–235.
- 20Narayan G, Goparaju C, Arias-Pulido H, Kaufmann AM, Schneider A, Dürst M, Mansukhani M, Pothuri B, Murty VV: Promoter hypermethylation-mediated inactivation of multiple Slit-Robo pathway genes in cervical cancer progression. Mol Cancer 2006; 5: 16.
- 21Duncan WC, McDonald SE, Dickinson RE, Shaw JL, Lourenco PC, Wheelhouse N, Lee KF, Critchley HO, Horne AW: Expression of the repulsive SLIT/ROBO pathway in the human endometrium and Fallopian tube. Mol Hum Reprod 2010; 16: 950–959.
- 22Dickinson RE, Dallol A, Bieche I, Krex D, Morton D, Maher ER, Latif F: Epigenetic inactivation of SLIT3 and SLIT1 genes in human cancers. Br J Cancer 2004; 91: 2071–2078.
- 23Latil A, Chêne L, Cochant-Priollet B, Mangin P, Fournier G, Berthon P, Cussenot O: Quantification of expression of netrins, slits and their receptors in human prostate tumors. Int J Cancer 2003; 103: 306–315.
- 24Geutskens SB, Hordijk PL, van Hennik PB: The chemorepellent Slit3 promotes monocyte migration. J Immunol 2010; 185: 7691–7698.
- 25Liao WX, Laurent LC, Agent S, Hodges J, Chen DB: Human placental expression of SLIT/ROBO signaling cues: effects of preeclampsia and hypoxia. Biol Reprod 2012; 86: 111.
- 26Lappas M, Rice GE. Transcriptional regulation of the processes of human labour and delivery. Placenta 2009; 30(Suppl A): S90–S95.
- 27Lee 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: 213–218.
- 28Lappas M, Mitton A, Lim R, Barker G, Riley C, Permezel M: SIRT1 is a novel regulator of key pathways of human labor. Biol Reprod 2011; 84: 167–178.
- 29Lappas M, Riley C, Rice GE, Permezel M: Increased expression of ac-FoxO1 protein in prelabor fetal membranes overlying the cervix: possible role in human fetal membrane rupture. Reprod Sci 2009; 16: 635–641.
- 30Lim R, Barker G, Lappas M. A novel role for FOXO3 in human labor: increased expression in laboring myometrium, and regulation of pro-inflammatory and pro-labor mediators in pregnant human myometrial cells. Biol Reprod, 2013; 88: 156.
- 31Lappas M, Odumetse TL, Riley C, Reti NG, Holdsworth-Carson SJ, Rice GE, Permezel M: Pre-labour fetal membranes overlying the cervix display alterations in inflammation and NF-kappaB signalling pathways. Placenta 2008; 29: 995–1002.
- 32Moore RM, Novak JB, Kumar D, Mansour JM, Mercer BM, Moore JJ: Alpha-lipoic acid inhibits tumor necrosis factor-induced remodeling and weakening of human fetal membranes. Biol Reprod 2009; 80: 781–787.
- 33Lim R, Barker G, Lappas M: SIRT6 is decreased with preterm labor and regulates key terminal effector pathways of human labor in fetal membranes. Biol Reprod 2013; 88: 17.
- 34Lim R, Barker G, Riley C, Lappas M. Apelin is decreased with human preterm and term labor and regulates prolabor mediators in human primary amnion cells. Reprod Sci 2013; 20: 957–967.
- 35Lappas M. Forkhead box O1 (FOXO1) in pregnant human myometrial cells: a role as a pro-inflammatory mediator in human parturition. J Reprod Immunol 2013; 99: 24–32.
- 36Lappas M, Permezel M, Rice GE: 15-Deoxy-Delta(12,14)-prostaglandin J(2) and troglitazone regulation of the release of phospholipid metabolites, inflammatory cytokines and proteases from human gestational tissues. Placenta 2006; 27: 1060–1072.
- 37Romero R, Espinoza J, Kusanovic JP, Gotsch F, Hassan S, Erez O, Chaiworapongsa T, Mazor M: The preterm parturition syndrome. BJOG 2006; 113(Suppl 3): 17–42.
- 38French JI, McGregor JA: The pathobiology of premature rupture of membranes. Semin Perinatol 1996; 20: 344–368.
- 39Parry S, Strauss JF 3rd: Premature rupture of the fetal membranes. N Engl J Med 1998; 338: 663–670.
- 40Moore RM, Mansour JM, Redline RW, Mercer BM, Moore JJ: The physiology of fetal membrane rupture: insight gained from the determination of physical properties. Placenta 2006; 27: 1037–1051.
- 41El Khwad M, Pandey V, Stetzer B, Mercer BM, Kumar D, Moore RM, Fox J, Redline RW, Mansour JM, Moore JJ: Fetal membranes from term vaginal deliveries have a zone of weakness exhibiting characteristics of apoptosis and remodeling. J Soc Gynecol Investig 2006; 13: 191–195.
- 42Reti NG, Lappas M, Riley C, Wlodek ME, Permezel M, Walker S, Rice GE: Why do membranes rupture at term? Evidence of increased cellular apoptosis in the supracervical fetal membranes. Am J Obstet Gynecol 2007; 196: 484 e1–484 e10.
- 43Cox SM, Casey ML, MacDonald PC: Accumulation of interleukin-1beta and interleukin-6 in amniotic fluid: a sequela of labour at term and preterm. Hum Reprod Update 1997; 3: 517–527.
- 44Maymon E, Ghezzi F, Edwin SS, Mazor M, Yoon BH, Gomez R, Romero R: The tumor necrosis factor alpha and its soluble receptor profile in term and preterm parturition. Am J Obstet Gynecol 1999; 181: 1142–1148.
- 45Kumar D, Fung W, Moore RM, Pandey V, Fox J, Stetzer B, Mansour JM, Mercer BM, Redline RW, Moore JJ: Proinflammatory cytokines found in amniotic fluid induce collagen remodeling, apoptosis, and biophysical weakening of cultured human fetal membranes. Biol Reprod 2006; 74: 29–34.
- 46Bryant-Greenwood GD, Yamamoto SY: Control of peripartal collagenolysis in the human chorion-decidua. Am J Obstet Gynecol 1995; 172: 63–70.
- 47Fortunato SJ, Menon R: IL-1 beta is a better inducer of apoptosis in human fetal membranes than IL-6. Placenta 2003; 24: 922–928.
- 48Ledingham 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: 235–242.
- 49Osman I, Young A, Ledingham MA, Thomson AJ, Jordan F, Greer IA, Norman JE: 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: 41–45.
- 50Slater DM, Berger LC, Newton R, Moore GE, Bennett PR: Expression of cyclooxygenase types 1 and 2 in human fetal membranes at term. Am J Obstet Gynecol 1995; 172: 77–82.
- 51Lappas M, Permezel M, Ho PW, Moseley JM, Wlodek ME, Rice GE: Effect of nuclear factor-kappa B inhibitors and peroxisome proliferator-activated receptor-gamma ligands on PTHrP release from human fetal membranes. Placenta 2004; 25: 699–704.
- 52Khanjani S, Kandola MK, Lindstrom TM, Sooranna SR, Melchionda M, Lee YS, Terzidou V, Johnson MR, Bennett PR: NF-kappaB regulates a cassette of immune/inflammatory genes in human pregnant myometrium at term. J Cell Mol Med 2011; 15: 809–824.
- 53Elliott 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: 787–790.
- 54Zhang B, Dietrich UM, Geng JG, Bicknell R, Esko JD, Wang L: Repulsive axon guidance molecule Slit3 is a novel angiogenic factor. Blood 2009; 114: 4300–4309.
- 55Shimada H, Rajagopalan LE: Rho kinase-2 activation in human endothelial cells drives lysophosphatidic acid-mediated expression of cell adhesion molecules via NF-kappaB p65. J Biol Chem 2010; 285: 12536–12542.
- 56Montaner S, Perona R, Saniger L, Lacal JC: Multiple signalling pathways lead to the activation of the nuclear factor kappaB by the Rho family of GTPases. J Biol Chem 1998; 273: 12779–12785.
- 57Denk AE, Braig S, Schubert T, Bosserhoff AK: Slit3 inhibits activator protein 1-mediated migration of malignant melanoma cells. Int J Mol Med 2011; 28: 721–726.
- 58Denk AE, Kaufmann S, Stark K, Schedel J, Lowin T, Schubert T, Bosserhoff AK: Slit3 inhibits Robo3-induced invasion of synovial fibroblasts in rheumatoid arthritis. Arthritis Res Ther 2010; 12: R45.
- 59Bollapragada S, Youssef R, Jordan F, Greer I, Norman J, Nelson S: Term labor is associated with a core inflammatory response in human fetal membranes, myometrium, and cervix. Am J Obstet Gynecol 2009; 200: 104 e1–104 e11.
- 60Keelan JA, Blumenstein M, Helliwell RJ, Sato TA, Marvin KW, Mitchell MD. Cytokines, prostaglandins and parturition–a review. Placenta 2003; 24(Suppl A): S33–S46.
- 61Murphy G, Reynolds JJ, Bretz U, Baggiolini M: Partial purification of collagenase and gelatinase from human polymorphonuclear leucocytes. Analysis of their actions on soluble and insoluble collagens. Biochem J 1982; 203: 209–221.
- 62Tanno T, Fujiwara A, Sakaguchi K, Tanaka K, Takenaka S, Tsuyama S: Slit3 regulates cell motility through Rac/Cdc42 activation in lipopolysaccharide-stimulated macrophages. FEBS Lett 2007; 581: 1022–1026.
- 63Chen B, Blair DG, Plisov S, Vasiliev G, Perantoni AO, Chen Q, Athanasiou M, Wu JY, Oppenheim JJ, Yang D: Cutting edge: bone morphogenetic protein antagonists Drm/Gremlin and Dan interact with Slits and act as negative regulators of monocyte chemotaxis. J Immunol 2004; 173: 5914–5917.
- 64Wu JY, Feng L, Park HT, Havlioglu N, Wen L, Tang H, Bacon KB, Jiang ZH, Zhang XC, Rao Y: The neuronal repellent Slit inhibits leukocyte chemotaxis induced by chemotactic factors. Nature 2001; 410: 948–952.
- 65Guan H, Zu G, Xie Y, Tang H, Johnson M, Xu X, Kevil C, Xiong WC, Elmets C, Rao Y, Wu JY, Xu H: Neuronal repellent Slit2 inhibits dendritic cell migration and the development of immune responses. J Immunol 2003; 171: 6519–6526.
- 66Ye BQ, Geng ZH, Ma L, Geng JG: Slit2 regulates attractive eosinophil and repulsive neutrophil chemotaxis through differential srGAP1 expression during lung inflammation. J Immunol 2010; 185: 6294–6305.
- 67London NR, Zhu W, Bozza FA, Smith MC, Greif DM, Sorensen LK, Chen L, Kaminoh Y, Chan AC, Passi SF, Day CW, Barnard DL, Zimmerman GA, Krasnow MA, Li DY: Targeting Robo4-dependent Slit signaling to survive the cytokine storm in sepsis and influenza. Sci Transl Med 2010; 2: 23ra19.
- 68Zhou WJ, Geng ZH, Chi S, Zhang W, Niu XF, Lan SJ, Ma L, Yang X, Wang LJ, Ding YQ, Geng JG: Slit-Robo signaling induces malignant transformation through Hakai-mediated E-cadherin degradation during colorectal epithelial cell carcinogenesis. Cell Res 2011; 21: 609–626.
- 69MacIntyre DA, Sykes L, Teoh TG, Bennett PR: Prevention of preterm labour via the modulation of inflammatory pathways. J Matern Fetal Neonatal Med 2012; 25(Suppl 1): 17–20.
