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Molecular Medicine

, Volume 21, Issue 1, pp 645–656 | Cite as

An EG-VEGF-Dependent Decrease in Homeobox Gene NKX3.1 Contributes to Cytotrophoblast Dysfunction: A Possible Mechanism in Human Fetal Growth Restriction

  • Padma Murthi
  • Sophie Brouillet
  • Anita Pratt
  • Anthony Borg
  • Bill Kalionis
  • Frederic Goffin
  • Vassilis Tsatsaris
  • Carine Munaut
  • Jean-Jacques Feige
  • Mohamed Benharouga
  • Thierry Fournier
  • Nadia Alfaidy
Research Article

Abstract

Idiopathic fetal growth restriction (FGR) is frequently associated with placental insufficiency. Previous reports have provided evidence that endocrine gland-derived vascular endothelial growth factor (EG-VEGF), a placental secreted protein, is expressed during the first trimester of pregnancy, controls both trophoblast proliferation and invasion, and its increased expression is associated with human FGR. In this study, we hypothesize that EG-VEGF-dependent changes in placental homeobox gene expressions contribute to trophoblast dysfunction in idiopathic FGR. The changes in EG-VEGF-dependent homeobox gene expressions were determined using a homeobox gene cDNA array on placental explants of 8–12 wks gestation after stimulation with EG-VEGF in vitro for 24 h. The homeobox gene array identified a greater-than-five-fold increase in HOXA9, HOXC8, HOXC10, HOXD1, HOXD8, HOXD9 and HOXD11, while NKX3.1 showed a greater-than-two-fold decrease in mRNA expression compared with untreated controls. Homeobox gene NKX3.1 was selected as a candidate because it is a downstream target of EG-VEGF and its expression and functional roles are largely unknown in control and idiopathic FGR-affected placentae. Real-time PCR and immunoblotting showed a significant decrease in NKX3.1 mRNA and protein levels, respectively, in placentae from FGR compared with control pregnancies. Gene inactivation in vitro using short-interference RNA specific for NKX3.1 demonstrated an increase in BeWo cell differentiation and a decrease in HTR-8/SVneo proliferation. We conclude that the decreased expression of homeobox gene NKX3.1 downstream of EG-VEGF may contribute to the trophoblast dysfunction associated with idiopathic FGR pregnancies.

Notes

Acknowledgments

The authors wish to thank the consenting patients and the clinical and research midwives at the Pregnancy Research Centre, Department of Perinatal Medicine, The Royal Women’s Hospital for the supply of FGR and gestation-matched control placental tissues. INSERM (U1036), University Joseph Fourier, Commissariat à L’Energie Atomique (DSV/iRTSV/BCI). Funding support was provided from Groupement des Entreprises Françaises pour la Lutte contre le Cancer Comité Isère to N Alfaidy. Funding support for this work was provided from the Australian National Health and Medical Research Council (NHMRC New Investigator project grant #509140) award to P Murthi. We also thank F Balboni and QY Zhou for their collaboration. The human placental trophoblast-derived choriocarcinoma BeWo cell line (B30 clone) was a kind gift from Stephen Rogerson, The University of Melbourne, Department of Medicine, the Royal Melbourne Hospital, Victoria, Australia. The HTR-8/SVneo cells were a kind gift from Charles Graham, Queens University, Canada.

References

  1. 1.
    Godfrey KM, Barker DJ. (2000) Fetal nutrition and adult disease. Am. J. Clin. Nutr. 71:1344S–52S.CrossRefGoogle Scholar
  2. 2.
    Illanes S, Soothill P. (2004) Management of fetal growth restriction. Semin. Fetal Neonatal Med. 9:395–401.CrossRefGoogle Scholar
  3. 3.
    Brodsky D, Christou H. (2004) Current concepts in intrauterine growth restriction. J. Intensive Care Med. 19:307–19.CrossRefGoogle Scholar
  4. 4.
    Gagnon R. (2003) Placental insufficiency and its consequences. Eur. J. Obstet. Gynecol. Reprod. Biol. 110 Suppl 1:S99–107.CrossRefGoogle Scholar
  5. 5.
    Kingdom J, Huppertz B, Seaward G, Kaufmann P. (2000) Development of the placental villous tree and its consequences for fetal growth. Eur. J. Obstet. Gynecol. Reprod. Biol. 92:35–43.CrossRefGoogle Scholar
  6. 6.
    Chaddha V, Viero S, Huppertz B, Kingdom J. (2004) Developmental biology of the placenta and the origins of placental insufficiency. Semin. Fetal Neonatal Med. 9:357–69.CrossRefGoogle Scholar
  7. 7.
    Pidoux G, et al. (2012) Review: Human trophoblast fusion and differentiation: lessons from trisomy 21 placenta. Placenta. 33 Suppl: S81–6.CrossRefGoogle Scholar
  8. 8.
    Pidoux G, et al. (2014) A PKA-ezrin-Cx43 signaling complex controls gap junction communication and thereby trophoblast cell fusion. J. Cell Sci. 127:4172–85.CrossRefGoogle Scholar
  9. 9.
    Ishihara N, et al. (2002) Increased apoptosis in the syncytiotrophoblast in human term placentas complicated by either preeclampsia or intrauterine growth retardation. Am. J. Obstet. Gynecol. 186:158–66.CrossRefGoogle Scholar
  10. 10.
    Knofler M. (2010) Critical growth factors and signalling pathways controlling human trophoblast invasion. Int. J. Dev. Biol. 54:269–80.CrossRefGoogle Scholar
  11. 11.
    Burton GJ, Jauniaux E, Charnock-Jones DS. (2007) Human early placental development: potential roles of the endometrial glands. Placenta. 28 Suppl A:S64–9.CrossRefGoogle Scholar
  12. 12.
    Cartwright JE, et al. (2002) Trophoblast invasion of spiral arteries: a novel in vitro model. Placenta. 23:232–5.CrossRefGoogle Scholar
  13. 13.
    Graham CH, Lala PK. (1992) Mechanisms of placental invasion of the uterus and their control. Biochem. Cell Biol. 70:867–74.CrossRefGoogle Scholar
  14. 14.
    LeCouter J, et al. (2001) Identification of an angiogenic mitogen selective for endocrine gland endothelium. Nature. 412:877–84.CrossRefGoogle Scholar
  15. 15.
    Lin DC, et al. (2002) Identification and molecular characterization of two closely related G proteincoupled receptors activated by prokineticins/endocrine gland vascular endothelial growth factor. J. Biol. Chem. 277:19276–80.CrossRefGoogle Scholar
  16. 16.
    Brouillet S, et al. (2010) Molecular characterization of EG-VEGF-mediated angiogenesis: differential effects on microvascular and macrovascular endothelial cells. Mol. Biol. Cell. 21:2832–43.CrossRefGoogle Scholar
  17. 17.
    Hoffmann P, Feige JJ, Alfaidy N. (2006) Expression and oxygen regulation of endocrine gland-derived vascular endothelial growth factor/prokineticin-1 and its receptors in human placenta during early pregnancy. Endocrinology. 147:1675–84.CrossRefGoogle Scholar
  18. 18.
    Hoffmann P, Feige JJ, Alfaidy N. (2007) Placental expression of EG-VEGF and its receptors PKR1 (prokineticin receptor-1) and PKR2 throughout mouse gestation. Placenta. 28:1049–58.CrossRefGoogle Scholar
  19. 19.
    Hoffmann P, et al. (2009) Role of EG-VEGF in human placentation: Physiological and pathological implications. J. Cell. Mol. Med. 13:2224–35.CrossRefGoogle Scholar
  20. 20.
    Brouillet S, et al. (2013) EG-VEGF controls placental growth and survival in normal and pathological pregnancies: case of fetal growth restriction (FGR). Cell. Mol. Life Sci. 70:511–25.CrossRefGoogle Scholar
  21. 21.
    Rajaraman G, Murthi P, Brennecke SP, Kalionis B. (2010) Homeobox gene HLX is a regulator of HGF/c-met-mediated migration of human trophoblast-derived cell lines. Biol. Reprod. 83:676–83.CrossRefGoogle Scholar
  22. 22.
    Murthi P, et al. (2013) Homeobox genes and down-stream transcription factor PPARgamma in normal and pathological human placental development. Placenta. 34:299–309.CrossRefGoogle Scholar
  23. 23.
    Murthi P, Kalionis B, Rajaraman G, Keogh RJ, Da Silva Costa F. (2012) The role of homeobox genes in the development of placental insufficiency. Fetal Diagn. Ther. 32:225–30.CrossRefGoogle Scholar
  24. 24.
    Quinn LM, Latham SE, Kalionis B. (2000) The homeobox genes MSX2 and MOX2 are candidates for regulating epithelial-mesenchymal cell interactions in the human placenta. Placenta. 21 Suppl A:S50–4.CrossRefGoogle Scholar
  25. 25.
    Murthi P, et al. (2006) Homeobox gene HLX1 expression is decreased in idiopathic human fetal growth restriction. Am. J. Pathol. 168:511–8.CrossRefGoogle Scholar
  26. 26.
    Murthi P, et al. (2006) Homeobox gene ESX1L expression is decreased in human pre-term idiopathic fetal growth restriction. Mol. Hum. Reprod. 12:335–40.CrossRefGoogle Scholar
  27. 27.
    Guaran RL, Wein P, Sheedy M, Walstab J, Beischer NA. (1994) Update of growth percentiles for infants born in an Australian population. Aust. N. Z. J. Obstet. Gynaecol. 34:39–50.CrossRefGoogle Scholar
  28. 28.
    Tsatsaris V, et al. (2003) Overexpression of the soluble vascular endothelial growth factor receptor in preeclamptic patients: pathophysiological consequences. J. Clin. Endocrinol. Metab. 88:5555–5563.CrossRefGoogle Scholar
  29. 29.
    Handschuh K, et al. (2007) Human chorionic gonadotropin produced by the invasive trophoblast but not the villous trophoblast promotes cell invasion and is down-regulated by peroxisome proliferator-activated receptor-gamma. Endocrinology. 148:5011–9.CrossRefGoogle Scholar
  30. 30.
    Graham CH, et al. (1993) Establishment and characterization of first trimester human trophoblast cells with extended lifespan. Exp. Cell Res. 206:204–11.CrossRefGoogle Scholar
  31. 31.
    Murthi P, et al. (2006) Homeobox gene DLX4 expression is increased in idiopathic human fetal growth restriction. Mol. Hum. Reprod. 12:763–9.CrossRefGoogle Scholar
  32. 32.
    Livak KJ, Schmittgen TD. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 25:402–8.CrossRefGoogle Scholar
  33. 33.
    Chui A, et al. (2010) Homeobox gene distal-less 3 is expressed in proliferating and differentiating cells of the human placenta. Placenta. 31:691–7.CrossRefGoogle Scholar
  34. 34.
    Pathirage NA, et al. (2013) Homeobox gene transforming growth factor beta-induced factor-1 (TGIF-1) is a regulator of villous trophoblast differentiation and its expression is increased in human idiopathic fetal growth restriction. Mol Hum. Reprod. 19:665–75.CrossRefGoogle Scholar
  35. 35.
    Evseenko DA, Paxton JW, Keelan JA. (2007) Independent regulation of apical and basolateral drug transporter expression and function in placental trophoblasts by cytokines, steroids, and growth factors. Drug Metab. Dispos. 35:595–601.CrossRefGoogle Scholar
  36. 36.
    Chui A, et al. (2011) Homeobox gene Distal-less 3 (DLX3) is a regulator of villous cytotrophoblast differentiation. Placenta. 32:745–51.CrossRefGoogle Scholar
  37. 37.
    Balboni G, et al. (2008) Triazine compounds as antagonists at Bv8-prokineticin receptors. J. Med. Chem. 51:7635–7639.CrossRefGoogle Scholar
  38. 38.
    Curtis VF, et al. (2013) A PK2/Bv8/PROK2 antagonist suppresses tumorigenic processes by inhibiting angiogenesis in glioma and blocking myeloid cell infiltration in pancreatic cancer. PloS One. 8:e54916.CrossRefGoogle Scholar
  39. 39.
    Keogh RJ. (2010) New technology for investigating trophoblast function. Placenta. 31:347–50.CrossRefGoogle Scholar
  40. 40.
    Garcia MG, et al. (2007) High expression of survivin and down-regulation of Stat-3 characterize the feto-maternal interface in failing murine pregnancies during the implantation period. Placenta. 28:650–7.CrossRefGoogle Scholar
  41. 41.
    Regnault TR, Galan HL, Parker TA, Anthony RV. (2002) Placental development in normal and compromised pregnancies— a review. Placenta. 23 Suppl A:S119–29.CrossRefGoogle Scholar
  42. 42.
    Salafia CM. (1997) Placental pathology of fetal growth restriction. Clin. Obstet. Gynecol. 40:740–9.CrossRefGoogle Scholar
  43. 43.
    Buckberry S, Bianco-Miotto T, Bent SJ, Dekker GA, Roberts CT. (2014) Integrative transcriptome meta-analysis reveals widespread sex-biased gene expression at the human fetal-maternal interface. Mol. Hum. Reprod. 20:810–9.CrossRefGoogle Scholar
  44. 44.
    Bhatia-Gaur R, et al. (1999) Roles for Nkx3.1 in prostate development and cancer. Genes. Dev. 13:966–77.CrossRefGoogle Scholar
  45. 45.
    Schneider A, Brand T, Zweigerdt R, Arnold H. (2000) Targeted disruption of the Nkx3.1 gene in mice results in morphogenetic defects of minor salivary glands: parallels to glandular duct morphogenesis in prostate. Mech. Dev. 95:163–74.CrossRefGoogle Scholar
  46. 46.
    Meeks JJ, Schaeffer EM. (2011) Genetic regulation of prostate development. J. Androl. 32:210–7.CrossRefGoogle Scholar
  47. 47.
    Amesse LS, Moulton R, Zhang YM, Pfaff-Amesse T. (2003) Expression of HOX gene products in normal and abnormal trophoblastic tissue. Gynecol. Oncol. 90:512–8.CrossRefGoogle Scholar
  48. 48.
    Zhang YM, Xu B, Rote N, Peterson L, Amesse LS. (2002) Expression of homeobox gene transcripts in trophoblastic cells. Am. J. Obstet. Gynecol. 187:24–32.CrossRefGoogle Scholar
  49. 49.
    Orendi K, Gauster M, Moser G, Meiri H, Huppertz B. (2010) The choriocarcinoma cell line BeWo: syncytial fusion and expression of syncytium-specific proteins. Reproduction. 140:759–66.CrossRefGoogle Scholar
  50. 50.
    Fitzgerald JS, et al. (2010) Governing the invasive trophoblast: current aspects on intra- and extracellular regulation. Am. J. Reprod. Immunol. 63:492–505.CrossRefGoogle Scholar
  51. 51.
    Gerbaud P, Pidoux G. (2014) An overview of molecular events occurring in human trophoblast fusion. Placenta. 36 Suppl 1:S35–42.PubMedGoogle Scholar
  52. 52.
    Wice B, Menton D, Geuze H, Schwartz AL. (1990) Modulators of cyclic AMP metabolism induce syncytiotrophoblast formation in vitro. Exp. Cell Res. 186:306–16.CrossRefGoogle Scholar
  53. 53.
    Chui A, et al. (2012) Homeobox gene Distal-less 3 is a regulator of villous cytotrophoblast differentiation and its expression is increased in human idiopathic foetal growth restriction. J. Mol. Med. (Berl) 90:273–84.CrossRefGoogle Scholar
  54. 54.
    Huppertz B, Frank HG, Kingdom JC, Reister F, Kaufmann P. (1998) Villous cytotrophoblast regulation of the syncytial apoptotic cascade in the human placenta. Histochem. Cell Biol. 110:495–508.CrossRefGoogle Scholar
  55. 55.
    Agata KB, Anita S, Urszula KK, Agnieszka NK, Grzegorz B. (2009) Expression of caspase-3, Bax nad Bcl-2 in placentas from pregnancies complicated by treated and non-treated fetal growth restriction. Ginekol. Pol. 80:652–6.PubMedGoogle Scholar
  56. 56.
    Karowicz-Bilinska A, Szczerba A, Kowalska-Koprek U, Nawrocka-Kunecka A. (2007) The evaluation of selected indices of apoptosis in placentas from pregnancies complicated by fetal growth restriction. Ginekol. Pol. 78:521–6.PubMedGoogle Scholar

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Authors and Affiliations

  • Padma Murthi
    • 1
    • 2
  • Sophie Brouillet
    • 3
    • 4
    • 5
    • 6
  • Anita Pratt
    • 1
  • Anthony Borg
    • 1
  • Bill Kalionis
    • 1
  • Frederic Goffin
    • 7
  • Vassilis Tsatsaris
    • 8
  • Carine Munaut
    • 7
  • Jean-Jacques Feige
    • 3
    • 4
    • 5
  • Mohamed Benharouga
    • 9
  • Thierry Fournier
    • 10
  • Nadia Alfaidy
    • 3
    • 4
    • 5
  1. 1.Department of Perinatal Medicine Pregnancy Research Centre, The Royal Women’s Hospital and The University of Melbourne Department of Obstetrics and GynaecologyThe Royal Women’s HospitalMelbourneAustralia
  2. 2.Department of MedicineMonash UniversityMonashAustralia
  3. 3.Institut National de la Santé et de la Recherche Médicale, Unité 1036Laboratoire BCI -iRTSVParisFrance
  4. 4.Université Grenoble-AlpesGrenobleFrance
  5. 5.Commissariat à L’Energie Atomique (CEA)iRTSV-Biology of Cancer and InfectionGrenobleFrance
  6. 6.Centre Hospitalier Universitaire de Grenoble, Hôpital Couple-EnfantCentre Clinique et Biologique d’Assistance Médicale à la ProcréationLa TroncheFrance
  7. 7.Laboratory of Tumor and Developmental BiologyUniversity of LiègeLiègeBelgium
  8. 8.Department of Obstetrics and Gynecology, Hôpital Cochin, Maternité Port-RoyalUniversité Rene DescartesParisFrance
  9. 9.Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5249Laboratoire de Chimie et Biologie des MétauxGrenobleFrance
  10. 10.INSERM, U1139; Universite Paris Descartes, UMR-S1139; and PremUp FoundationParisFrance

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