Involvement of Transcription Factor NR2F2 in Human Trophoblast Differentiation

differentiation of human villous cytotrophoblast (CTB) cells to a syncytiotrophoblast (STB) phenotype, mRNA levels for the nuclear hormone receptor NR2F2 (ARP-1, COUP-TFII) increase rapidly, reaching a peak at day 1 of differentiation that is 8.8-fold greater than that in undifferentiated CTB cells. To examine whether NR2F2 is involved in the regulation of villous CTB cell differentiation, studies were performed to determine whether NR2F2 regulates the expression of TFAP2A (AP-2α), a transcription factor that is critical for the terminal differentiation of these cells to a STB phenotype.Overexpression of NR2F2 in primary cultures of human CTB cells and JEG-3 human choriocarcinoma cells induced dose-dependent increases in TFAP2A promoter activity. Conversely, siRNA mediated silencing of the NR2F2 gene in villous CTB undergoing spontaneous differentiation blocked the induction of the mRNAs for TFAP2A and several STB cell specific marker genes, including human placental lactogen (hPL), pregnancy specific glycoprotein 1 (PSG1) and corticotropin releasing hormone (CRH) by 51–59%. The induction of TFAP2A promoter activity by NR2F2 was potentiated by the nuclear hormone receptors retinoic acid receptor alpha (RARA) and retinoid X receptor alpha (RXRA).Taken together, these results strongly suggest that NR2F2 is involved in villous CTB cell differentiation and that NR2F2 acts, at least in part, by directly activating TFAP2A gene expression and by potentiating the transactivation of TFAP2A by RARA and RXRA

Different Mechanisms of ras Proto-Oncogene Overexpression Detected by a Sib-Selection Tumorigenesis Assay

Human insulinoma and renal cell carcinoma DNAs were analysed by a sib-selection tumorigenesis assay to detect the presence of dominant acting oncogenes. Although no novel oncogenes were detected in the five tumor DNAs tested, the increased sensitivity of the tumorigenesis assay allowed detection of overexpressed ras proto-oncogenes activated during transfection. Tumorigenic overexpression of ras proteins occured by two different mechanisms: gene amplification, and increased translation efficiency of a fusion protein. Thirty to fifty fold amplification of a human N-ras gene was detected in primary and secondary mouse tumors. All tumors containing amplified copies of N-ras overexpressed p21 and the cloned gene had no coding sequence mutations. Since DNA from the original human tumor and several metastases did not show N-ras gene amplification, it was concluded that amplification had occured during the primary tumorigenesis assay. In another series of tumors co-integration of the mouse c-H -ras gene during the secondary transfection resulted in tumorigenic overexpression of ras protein without elevation of mRNA levels. Three c-H-ras cDNA clones from secondary tumor RNA contained no coding sequence mutations, but were divergent at the 3'end. Polymerase chain reaction amplification of RNA showed novel alternative splicing at intron E of mouse c-H-ras mRNA in both tumors and untransfected cells. An upstream ATG was identified that potentially initiates translation of an open reading frame (ORF) overlapping the H-ras p21 translation initiation site. A single base deletion within exon -1 of the cDNA clones placed this upstream ATG in frame with the ras coding sequence, creating a potential fusion protein. Translation of this mRNA in rabbit reticulocyte extracts and Xenopus oocytes showed exclusive production of a p23 ras fusion protein. When the upstream ATG was deleted, only p21 ras was translated in both systems. Based on these results it is proposed that in vivo recognition of the upstream ATG and translation of the ORF overlapping the p21 start site might serve to modulate the translation of p21. The single base deletion and resultant ras fusion protein may constitute a novel mechanism of ras overexpression by circumventing this translational regulation

The effect of RXRA on NR2F2-induced TFAP2A promoter activity.

<p>JEG-3 cells were co-transfected with pGL3β-TFAP2A-Luc and pMT2-NR2F2 in the presence and absence of pRSV-RXRA. An equivalent amount of the empty pRSV plasmid was co-transfected into the cells that were not co-transfected with pRSV-RXRA. Each bar represents the mean of triplicate wells; and the brackets enclose 1 SEM. NS = not significant; * = p<0.05; *** = p<0.001. Similar results were obtained in two other experiments.</p

The effect of RXRA and RARA on TFAP2A promoter activity.

<p>JEG-3 cells were co-transfected with pGL3β-TFAP2A-Luc and pRSV-RXRA or pRSV-RARA at the amounts indicated in the Figure. Each bar represents the mean of triplicate observations; and the bars enclose 1 SEM. Similar results were observed in two additional experiments.</p

The effects of NR2F2 gene silencing on TFAP2A mRNA levels and the mRNA levels of the syncytiotrophoblast cell markers, hPL, CRH, PSG1 and syncytin.

<p>Human cytotrophoblast cells were transfected in two separate experiments with a NR2F2 siRNA or a non-silencing (control) RNA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009417#s4" target="_blank">Methods</a>. The cultures were terminated 48 h later, total RNA was extracted, and quantitative real-time PCR was performed for the indicated genes. In each instance, the amount of mRNA for each gene was normalized to the amount of GAPDH mRNA in the same sample. The final results for each gene from the two experiments (n = 6) are expressed as the % change of the normalized mRNA in the NR2F2 siRNA-exposed cells relative to the silent (control) RNA ((silent RNA- siRNA)/silent RNA)x100)). The bars represent the % change in mRNA levels from the two experiments; the error bars represent ± 1 SEM (n = 6). Each of the mRNAs in the NR2F2 siRNA-exposed cells, with the exception of GAPDH mRNA, was significantly less than that in the silent RNA-exposed cells (P<0.001 to <0.005 in each instance). Similar results were observed in 2 other independent experiments. *<0.01</p

The effect of RARA on NR2F2-induced TFAP2A promoter activity.

<p>JEG-3 cells were co-transfected with pGL3β-TFAP2A-Luc and pMT2-NR2F2 in the presence and absence of pRSV-RARA. An equivalent amount of the empty pRSV plasmid was co-transfected into the cells that were not co-transfected with pRSV-RARA. Each bar represents the mean of triplicate wells; and the brackets enclose 1 SEM. NS = not significant; * = p<0.05; *** = p<0.001. Similar results were obtained in two other experiments.</p

Time course of TFAP2A and NR2F2 mRNA levels during villous CTB differentiation.

<p>An enriched fraction of enzymatically dispersed villous CTB cells were cultured <i>in vitro</i> for five days as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009417#s4" target="_blank">Methods</a>. TFAP2A and NR2F2 mRNA levels were determined by real-time PCR at the end of each day. The amounts of TFAP2A and NR2F2 mRNAs in each culture well were normalized to the amount of GAPDH mRNA in the same sample. Each point represents the mean ± SEM of triplicate observations from 3 different placenta cell preparations (n = 3 wells/placenta culture).</p