Effects of corticotrophin releasing hormone (CRH) on cell viability and differentiation in the human BeWo choriocarcinoma cell line: a potential syncytialisation inducer distinct from cyclic adenosine monophosphate (cAMP)

Reproductive Biology and Endocrinology

Richard C Nicholson¹  Roger Smith²  Giavanna Angeli²  Kaushik Maiti²  Xin Pan²  Megan Allars²  YuXia Chen² 

[1] Department of Physiology, Second Military Medical University, Shanghai 200433, China;Mothers & Babies Research Centre, Hunter Medical Research Institute, John Hunter Hospital, University of Newcastle, Newcastle, NSW 2305, Australia

关键词: cAMP;    CRH;    Syncytiotrophoblast;    Syncytia;    Differentiation;    Cytotrophoblast;    Placenta;   

Others  :  812286
DOI  :  10.1186/1477-7827-11-30

 received in 2012-12-21, accepted in 2013-04-09,  发布年份 2013

【 摘 要 】

Background

Placental production of corticotrophin releasing hormone (CRH) rises exponentially as pregnancy progresses, and has been linked with the onset of normal and preterm labour. CRH is produced in syncytiotrophoblast cells and production is increased by glucocorticoids and cAMP. It remains unclear whether cAMP acts by inducing differentiation of cytotrophoblasts and/or through induction of syncytialisation. As CRH can stimulate cAMP pathways we have tested whether a feed-forward system may exist in placental cells during syncytialisation.

Methods

The choriocarcinoma BeWo cell line was treated with cAMP, CRH or vehicle. Cell viability was determined by MTT assay, while apoptosis was analysed by DAPI staining and by FACS. Differentiation was measured by assaying message for hCG and ERVW-1 (syncytin1) by qRT-PCR, as well as the respective protein by ELISA. Fusion of BeWo cells was assessed by co-staining cell membrane and nuclei with CellMask and Hoechst 33342. CRHR1 and CRHR2 mRNA levels were measured by qRT-PCR.

Results

We show that cAMP has an inductive effect on syncytialisation, as evidenced by induction of hCG secretion, by ERVW-1 mRNA expression and by formation of multinuclear cells. CRH mRNA expression was found to increase prior to the changes in the other syncytialisation markers. cAMP had an inhibitory effect on BeWo cell viability, but exogenous CRH did not. However, CRH did mimic the differentiation inducing effect of cAMP, suggesting a link between CRH and cAMP signalling in syncytialisation. We also found that treatment of BeWo cells with exogenous CRH resulted in elevated cellular CRHR1 levels.

Conclusions

This study suggests a positive feed-forward role exists for CRH in trophoblast cell differentiation, which may underlie the exponential rise in CRH observed as gestation advances.

【 授权许可】

   
2013 Chen et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Red-Horse K, Zhou Y, Genbacev O, Prakobphol A, Foulk R, McMaster M, Fisher SJ: Trophoblast differentiation during embryo implantation and formation of the maternal-fetal interface. J Clin Invest 2004, 114:744-754.
• [2]Hohn HP, Linke M, Ugele B, Denker HW: Differentiation markers and invasiveness: discordant regulation in normal trophoblast and choriocarcinoma cells. Exp Cell Res 1998, 244:249-258.
• [3]Handwerger S, Aronow B: Dynamic changes in gene expression during human trophoblast differentiation. Recent Prog Horm Res 2003, 58:263-281.
• [4]Vargas A, Moreau J, Le Bellego F, Lafond J, Barbeau B: Induction of trophoblast cell fusion by a protein tyrosine phosphatase inhibitor. Placenta 2008, 29:170-174.
• [5]Fogarty NM, Mayhew TM, Ferguson-Smith AC, Burton GJ: A quantitative analysis of transcriptionally active syncytiotrophoblast nuclei across human gestation. J Anat 2011, 219:601-610.
• [6]Gauster M, Huppertz B: Fusion of cytotrophoblast with syncytiotrophoblast in the human placenta: Factors involved in syncytialisation. J Reproduktionsmed Endokrinol 2008, 5:76-82.
• [7]Grino M, Chrousos GP, Margioris AN: The corticotrophin-releasing hormone gene is expressed in human placenta. Biochem Biophys Res Commun 1987, 148:1208-1214.
• [8]McLean M, Bisits A, Davies J, Woods R, Lowry P, Smith R: A placental clock controlling the length of human pregnancy. Nat Med 1995, 1:460-463.
• [9]King BR, Smith R, Nicholson RC: The regulation of human corticotropin-releasing hormone gene expression in the placenta. Peptides 2001, 22:1941-1947.
• [10]Grammatopoulos DK: Placental corticotrophin-releasing hormone and its receptors in human pregnancy and labour: Still a scientific enigma. J Neuroendocrinol 2008, 20:432-438.
• [11]Clifton VL, Read MA, Leitch IM, Boura AL, Robinson PJ, Smith R: Corticotropin-releasing hormone-induced vasodilatation in the human foetal placental circulation. J Clin Endocrinol Metab 1994, 79:666-669.
• [12]Smith R: Parturition. N Engl J Med 2007, 356:271-283.
• [13]Grammatopoulos DK: The role of CRH receptors and their agonists in myometrial contractility and quiescence during pregnancy and labour. Front Biosci 2007, 12:561-571.
• [14]Chen YX, Allars M, Maiti K, Angeli GL, Abou-Seif C, Smith R, Nicholson RC: Factors affecting cytotrophoblast cell viability and differentiation: Evidence of a link between syncytialisation and apoptosis. Int J Biochem Cell Biol 2011, 43:821-828.
• [15]Smith R, Nicholson RC: Corticotrophin-Releasing Hormone and the Timing of Birth. Front Biosci 2007, 12:912-918.
• [16]Sheng H, Xu Y, Chen Y, Ni X: Corticotropin-releasing hormone stimulates mitotic kinesin-like protein 1 expression via a PLC/PKC-dependent signaling pathway in hippocampal neurons. Mol Cell Endocrinol 2012, 362:157-164.
• [17]Gao L, Tao Y, Hu T, Liu W, Xu C, Liu J, You X, Gu H, Ni X: Regulation of estradiol and progesterone production by CRH-R1 and –R2 is through divergent signaling pathways in cultured human placental trophoblasts. Endocrinology 2012, 153:4918-4928.
• [18]Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001, 25:402-408.
• [19]Sehringer B, Zahradnik HP, Simon M, Ziegler R, Noethling C, Schaefer WR: mRNA expression profiles for corticotrophin-releasing hormone, urocortin, CRH-binding protein and CRH receptors in human term gestational tissues determined by real-time quantitative RT-PCR. J Mol Endocrinol 2004, 32:339-348.
• [20]Cheng Y-H, Nicholson RC, King B, Chan E-C, Fitter JT, Smith R: CRH gene expression in primary placental cells is modulated by cAMP. J Clin Endocrinol Metab 2000, 85:1239-1244.
• [21]Al-Nasiry S, Spitz B, Hanssens M, Luyten C, Pijnenborg R: Differential effects of inducers of syncytialisation and apoptosis on BeWo and JEG-3 choriocarcinoma cells. Hum Reprod 2006, 21:193-201.
• [22]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, 277:12280-12287.
• [23]Makrigiannakis A, Zoumakis E, Kalantaridou S, Mitsiades N, Margioris A, Chrousos GP, Gravanis A: Corticotropin-releasing hormone (CRH) and immunotolerance of the fetus. Biochem Pharmacol 2003, 65:917-921.
• [24]Minas V, Jeschke U, Kalantaridou SN, Richter DU, Reimer T, Mylonas I, Friese K, Makrigiannakis A: Abortion is associated with increased expression of FasL in decidual leukocytes and apoptosis of extravillous trophoblasts: a role for CRH and urocortin. Mol Hum Reprod 2007, 130:663-673.
• [25]Markovic D, Punn A, Lehnert H, Grammatopoulos DK: Molecular determinants and feedback circuits regulating type 2 CRH receptor signal integration. Biochim Biophys Acta 1813, 2011:896-907.
• [26]Hai T, Hartman MG: The molecular biology and nomenclature of the activating transcription factor/cAMP responsive element binding family of transcription factors: activating transcription factor proteins and homeostasis. Gene 2001, 273:1-11.
• [27]Shipman KL, Robinson PJ, King BR, Smith R, Nicholson RC: Identification of a Family of DNA-Binding Proteins with Homology to RNA Splicing Factors. Biochem Cell Biol 2006, 84:9-19.
• [28]Ni X, Hou Y, King BR, Tang X, Smith R, Nicholson RC: Estrogen receptor-mediated down-regulation of corticotropin-releasing hormone gene expression is dependent on a cyclic adenosine 3′,5′-monophosphate regulatory element in human placental syncytiotrophoblast cells. J Clin Endocrinol Metab 2004, 89:2312-2318.
• [29]Ni X, Nicholson RC: Steroid Hormone Mediated Regulation of Corticotropin-Releasing Hormone Gene Expression. Front Biosci 2006, 11:2909-2917.
• [30]King BR, Nicholson RC: Advances In Understanding Corticotrophin-Releasing Hormone Gene Expression. Front Biosci 2007, 12:581-590.
• [31]Saeed B, Fawcett M, Self C: Corticotropin-releasing hormone binding to the syncytiotrophoblast membranes. Eur J Clin Invest 2001, 31:125-130.
• [32]Wetzka B, Sehringer B, Schäfer WR, Biller S, Hör C, Benedek E, Deppert WR, Zahradnik HP: Expression patterns of CRH, CRH receptors, and CRH binding protein in human gestational tissue at term. Exp Clin Endocrinol Diabetes 2003, 111:154-161.
• [33]Wang W, Solc M, Ji P, Dow KE: Corticotropin-releasing hormone potentiates neural injury induced by oxygen-glucose deprivation: a possible involvement of microglia. Neurosci Lett 2004, 371:133-137.
• [34]Minas V, Rolaki A, Kalantaridou SN, Sidiropoulos J, Mitrou S, Petsas G, Jeschke U, Paraskevaidis EA, Fountzilas G, Chrousos GP, Pavlidis N, Makrigiannakis A: Intratumoral CRH modulates immuno-escape of ovarian cancer cells through FasL regulation. Br J Cancer 2007, 97:637-645.
• [35]Fahlbusch FB, Ruebner M, Volkert G, Offergeld R, Hartner A, Menendez-Castro C, Strick R, Rauh M, Rascher W, Doetsch J: Corticotropin-releasing hormone stimulates expression of leptin, 11beta-HSD2 and syncytin-1 in primary human trophoblasts. Reprod Biol Endocrinol 2012, 10:80. BioMed Central Full Text
• [36]Kao LC, Caltabiano S, Wu S, Strauss JF 3rd, Kliman HJ: The human villous cytotrophoblast: interactions with extracellular matrix proteins, endocrine function, and cytoplasmic differentiation in the absence of syncytium formation. Dev Biol 1988, 130:693-702.
• [37]Orendi K, Gauster M, Moser G, Meiri H, Huppertz B: The choriocarcinoma cell line BeWo: syncytial fusion and expression of syncytium-specific proteins. Reproduction 2010, 140:759-766.
• [38]Pidoux G, Gerbaud P, Cocquebert M, Segond N, Badet J, Fournier T, Guibourdenche J, Evain-Brion D: Review: Human trophoblast fusion and differentiation: lessons from trisomy 21 placenta. Placenta 2012, 33:S81-S86.
• [39]Malassine A, Cronier L: Hormones and human trophoblast differentiation: a review. Endocrine 2002, 19:3-11.
• [40]Insel PA, Zhang L, Murray F, Yokouchi H, Zambon AC: Cyclic AMP is both a pro-apoptotic and anti-apoptotic second messenger. Acta Physiol 2012, 204:277-287.