Homeobox gene transforming growth factor ß-induced factor-1 (TGIF-1) is a regulator of villous trophoblast differentiation and its expression is increased in human idiopathic fetal growth restriction.

Abnormal trophoblast function is associated with human fetal growth restriction (FGR). Targeted disruption of homeobox gene transforming growth ß-induced factor (TGIF-1) results in placental dysfunction in the mouse. The role of human TGIF-1 in placental cell function is unknown. The aims of this study were to determine the expression of TGIF-1 in human idiopathic FGR-affected placentae compared with gestation-matched controls (GMC), to elucidate the functional role of TGIF-1 in trophoblasts and to identify its downstream targets. Real-time PCR and immunoblotting revealed that TGIF-1 mRNA and protein expression was significantly increased in FGR-affected placentae compared with GMC (n = 25 in each group P < 0.05). Immunoreactive TGIF-1 was localized to the villous cytotrophoblasts, syncytiotrophoblast, microvascular endothelial cells and in scattered stromal cells in both FGR and GMC. TGIF-1 inactivation in BeWo cells using two independent siRNA resulted in significantly decreased mRNA and protein of trophoblast differentiation markers, human chorionic gonadotrophin (CGB/hCG), syncytin and 3ß-hydroxysteroid dehydrogenase/3ß-honest significant difference expression. Our data demonstrate that homeobox gene TGIF-1 is a potential up-stream regulator of trophoblast differentiation and the altered TGIF-1 expression may contribute to aberrant villous trophoblast differentiation in FGR.

Liver receptor homologue-1 expression in ovarian epithelial and granulosa cell tumours.

Granulosa cell tumours of the ovary (GCT) express aromatase and produce oestrogens. The ovarian-specific aromatase promoter (pII) is regulated by members of the group 5A nuclear receptor family, SF-1 and LRH-1. Since both SF-1 and LRH-1 are implicated in proliferation and cancer, we hypothesised that alteration in the expression of either or both receptors may be associated with GCT. We therefore determined the expression of LRH-1, SF-1 and aromatase in a cohort of GCT, mucinous and serous cystadenocarcinomas, and normal ovaries. LRH-1 mRNA was present at low level in normal ovary and serous cystadenocarcinoma, but was elevated approximately 30-fold in GCT, and 8-fold in mucinous cystadenocarcinoma, compared to normal ovary. LRH-1 protein expression was confirmed in GCT by immunohistochemistry. SF-1 mRNA was significantly lower that of LRH-1 in all samples and not significantly altered in GCT, compared to normal ovary. Aromatase mRNA was present at low level in normal ovary and serous and mucinous cystadenocarcinoma, and significantly elevated (18-fold) in GCT compared to normal ovary. Despite the coordinate over-expression of both LRH-1 and aromatase in GCT versus normal ovary, their levels did not correlate in individual patients; rather, aromatase expression correlated with that of SF-1. Finally, although both LRH-1 and SF-1 activated aromatase promoter activity in transient transfection studies, gel-shift and chromatin immunoprecipitation data indicated that SF-1, but not LRH-1, bound to the aromatase promoter. We conclude that SF-1 regulates aromatase expression in GCT; over-expression of LRH-1 suggests that this receptor may be involved in the pathogenesis of GCT by mechanisms other than the regulation of aromatase. Its role in this disease therefore warrants further investigation.

Homeobox gene Distal-less 3 is a regulator of villous cytotrophoblast differentiation and its expression is increased in human idiopathic foetal growth restriction.

Human idiopathic foetal growth restriction (FGR) is frequently associated with placental insufficiency. In our previous studies, we have reported the isolation and characterisation of the homeobox gene Distal-less 3 (DLX3) in the human placenta. In this study, we have investigated the level of DLX3 expression in idiopathic FGR-affected placentae and determined its functional role in villous trophoblast differentiation. FGR-affected placentae (n = 25) were collected based on well-defined clinical criteria and matched for gestation with control uncomplicated pregnancies (n = 25). Real-time polymerase chain reaction and immunoblotting showed increased DLX3 mRNA and protein expression in FGR-affected placentae compared with gestation-matched controls. Qualitative immunohistochemistry revealed DLX3 localisation in the syncytiotrophoblast, cytotrophoblasts and endothelial cells surrounding the foetal capillaries in both FGR-affected and control placentae. Down-regulation of DLX3 in primary villous trophoblast cells and a trophoblast-derived cell line showed decreased expression of differentiation markers, 3ßHSD, ßhCG and syncytin. Therefore, we conclude that increased DLX3 expression in FGR may contribute to trophoblast dysfunction observed in FGR.

Mesenchymal activin-A overcomes defective human trisomy 21 trophoblast fusion.

Placental development is markedly abnormal in trisomy 21 (T21) pregnancies. We hypothesized that abnormal paracrine cross talk between the fetal mesenchymal core and the trophoblast might be involved in the defect of syncytiotrophoblast formation and function. In a large series of primary cultured human cytotrophoblasts isolated from second-trimester control (n = 44) and T21 placentae (n = 71), abnormal trophoblast fusion and differentiation was observed in more than 90% of T21 cases. We then isolated and cultured villous mesenchymal cells from control (n = 10) and T21 placentae (n = 8) and confirmed their fetal origin. Conditioned medium of control mesenchymal cells overcame the abnormal trophoblast fusion of T21 cytotrophoblasts by activating the TGFß signaling pathway, as shown by the phosphospecific protein microarray analysis and the use of TGFß signaling pathway antagonists. Using protein arrays, we further analyzed the cytokines present in the conditioned medium from control and T21 mesenchymal cells. Activin-A was identified as strongly secreted by cells from both sources, but at a significantly (P < 0.01) lower level in the case of T21 mesenchymal cells. Recombinant activin-A stimulated T21 trophoblast fusion. Blocking activin-A antibody inhibited the fusion induced by conditioned medium and exogenous activin-A. Furthermore, follistatin, an activin-A binding protein largely secreted by T21 mesenchymal cells, inhibited the conditioned medium fusogenic activity. These results show that the defective trophoblast fusion and differentiation associated with T21 can be overcome in vitro and reveal the key role of the fetal mesenchymal core in human trophoblast differentiation.

Downstream targets of homeobox gene HLX show altered expression in human idiopathic fetal growth restriction.

Fetal growth restriction (FGR), a clinically significant pregnancy disorder, is poorly understood at the molecular level. This study investigates idiopathic FGR associated with placental insufficiency. Previously, we showed that the homeobox gene HLX is expressed in placental trophoblast cells and that HLX expression is significantly decreased in human idiopathic FGR. Here, we used the novel approach of identifying downstream targets of HLX in cell culture to detect potentially important genes involved in idiopathic FGR. Downstream targets were revealed by decreasing HLX expression in cultured trophoblast cells with HLX-specific small interfering RNAs to model human idiopathic FGR and comparing these levels with controls using a real-time PCR-based gene profiling system. Changes in candidate HLX target mRNA levels were verified in an independent trophoblast cell line, and candidate target gene expression was assessed in human idiopathic FGR-affected placentae (n = 25) compared with gestation-matched controls (n = 25). The downstream targets RB1 and MYC, cell cycle regulatory genes, showed significantly increased mRNA levels in FGR-affected tissues compared with gestation-matched controls, whereas CCNB1, ELK1, JUN, and CDKN1 showed significantly decreased mRNA levels (n = 25, P < 0.001, t-test). The changes for RB1 and CDKN1C were verified by Western blot analysis in FGR-affected placentae compared with gestation-matched controls (n = 6). We conclude that cell cycle regulatory genes RB1, MYC, CCNB1, ELK1, JUN, and CDKN1C, which control important trophoblast cell functions, are targets of HLX.

Expression of aromatase, estrogen receptors, and their coactivators in patients with endometrial cancer.

Quantitative polymerase chain reaction analysis of a panel of normal and malignant endometrial tissues revealed significant expression of both aromatase and estrogen receptor alpha in low-grade tumors, suggesting that local estrogen synthesis and action might contribute to the proliferation of these tumors.