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