RESUMO
BACKGROUND: During the in vitro 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-2alpha), a transcription factor that is critical for the terminal differentiation of these cells to a STB phenotype. METHODOLOGY/PRIMARY FINDINGS: 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). CONCLUSIONS/SIGNIFICANCE: 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.
Assuntos
Fator II de Transcrição COUP/genética , Diferenciação Celular/genética , Perfilação da Expressão Gênica , Trofoblastos/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Regiões Promotoras Genéticas/genética , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores do Ácido Retinoico/genética , Receptor alfa de Ácido Retinoico , Receptor X Retinoide alfa/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Fator de Transcrição AP-2/genética , Transfecção , Trofoblastos/citologiaRESUMO
Deletion analysis of the human growth hormone variant (GHV) promoter in transient transfection studies in BeWo choriocarcinoma and HepG2 cells indicated that the region extending from nt -158/+57 retained full transcriptional activity. DNase I footprint analysis of the fragment revealed a protected region at nt -82/-77, which is in a putative FOXF1/FOXF2 binding site. Supershift assays using an antiserum to human FOXF1 demonstrated that the protected region binds FOXF1. Overexpression of FOXF1 in BeWo and HepG2 cells induced the GHV promoter, whereas overexpression of FOXF2 was without effect. Mutagenesis of the FOXF1/FOXF2 site reduced basal promoter activity by 50-60% and markedly attenuated transactivation of the promoter by FOXF1. These studies indicate that FOXF1 induces GHV expression by interaction with a FOXF1/FOXF2 cis-element in the proximal promoter.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Hormônio do Crescimento Humano/genética , Placenta/fisiologia , Regiões 5' não Traduzidas/fisiologia , Coriocarcinoma , Pegada de DNA , Desoxirribonuclease I , Feminino , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Mutagênese Sítio-Dirigida , Gravidez , Regiões Promotoras Genéticas/fisiologia , Transfecção , Células Tumorais Cultivadas , Neoplasias UterinasRESUMO
Syncytin, a protein encoded by an envelope gene of a human endogenous retrovirus-W (HERV-W), plays a critical role in trophoblast differentiation. We isolated the 5'-flanking region of the syncytin gene from human genomic DNA by PCR and identified cis-acting elements on the promoter that are important for transcription. The major transcription initiation site identified by mung bean nuclease protection assays is 56 base pairs (bp) downstream from a putative CCAAT box. Deletion analysis of the 5'-flanking region of the syncytin gene indicated that the proximal 148 bp are essential for minimal promoter activity and that regions of the promoter from nt -1519 to -984 and nt -294 to -148 are required for maximal expression in normal trophoblast cells. DNase I footprint analysis of the region between nt -252 and +110 revealed three protected regions, FP1-FP3. Mutagenesis of a hepatocyte-specific nuclear protein-1 (HAPF1) binding site in FP1 and a TATA box in FP3 had no effects on basal promoter activity. However, mutation of the CCAAT motif and the octamer protein (Oct) binding site in FP2 decreased promoter activity by 88% and 76%, respectively. Mutation of the ecdysone receptor (EcR) response element in FP2, which may bind a nuclear hormone receptor, increased basal promoter activity by 2-fold. Gel shift and supershift assays indicated that CCAAT-binding factor (CBF) binds to the CCAAT motif and that Oct binds to the Oct binding site. Taken together, these findings indicate that the syncytin promoter is located in the 5' long terminal repeat (LTR) of the HERV-W gene and that binding sites for CBF and Oct in the proximal promoter are critical for transcriptional regulation of the gene in trophoblast cells.
