|Matsuda, Fuko - UNIV TOKYO, JAPAN|
|Xu, Ningchun - UNIV TOKYO, JAPAN|
|Kijima, Sho - UNIV TOKYO, JAPAN|
|Tachi, Chikashi - MITSUBISHI-KAGAKU CO JPN|
|Sakai, Senkiti - UNIV TOKYO, JAPAN|
|Imakawa, Kazuhiko - UNIV TOKYO, JAPAN|
Submitted to: Placenta
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 27, 2003
Publication Date: February 20, 2004
Citation: Matsuda, F., Xu, N., Kijima, S., Tachi, C., Christenson, R.K., Sakai, S., Imakawa, K. 2004. Analysis of transcriptional control elements in the 5'-upstream region of ovine interferon-tau gene using feeder-independent caprine trophoblast cell line, HTS-1. Placenta. 25(2-3):166-175. Interpretive Summary: Reproductive loss during early pregnancy is one of the most costly factors influencing the livestock industry. A large majority of reproductive failure in sheep occur as a result of an impaired signal from the developing conceptus to the maternal system during implantation. In sheep, the gene controlling the conceptus signaling product (ovine interferon-tau, oIFNtau) is known, but molecular mechanisms of tissue-specific gene expression and down-regulation of the IFNtau gene are not known. The objectives of this study were to identify the 5'-upstream region of the oIFNtau gene required for transcription in a newly developed Shiba-goat placental cell line. Comparison to a commonly used human choriocarcinoma cell line was conducted. Substantial differences were found in oIFNtau gene transcription in goat and human cell lines. Co-transfection with expression vectors revealed that different binding sites in the promoter region were activated in goat versus human cell lines. Thorough understanding of conceptus gene regulation should enable improvement in survival throughout gestation.
Technical Abstract: Interferon-tau (IFNtau) is a protein secreted from the embryonic trophectoderm of ruminant ungulates during peri-implantation period. This protein acts on the uterine endometrium, which indirectly maintains corpus luteum function, and is therefore considered essential for the process of maternal recognition of pregnancy. Transcriptional regulation of IFNtau genes had been examined using human choriocarcinoma cell lines, JEG-3 or JAR, however, molecular mechanisms by which cell and term specific IFNtau expression are regulated have not been elucidated. Recently, a feeder cell free-trophoblast cell line derived from Shiba-goat placenta, termed HTS-1, was established. In the present investigation, the 5'-upstream region of ovine IFNtau (IFNtau gene was analyzed using this cell line, which would provide more suitable system for studies of the ovine trophoblast specific gene than human choriocarcinoma cells. Variously modified 5'-upstream sequences of the IFNtau gene fused to a luciferase reporter gene were transiently transfected into HTS-1 cells, and human JEG-3 cells were used as a control. These results and co-transfection with expression vectors revealed that Ets-2 binding site in the promoter region was important in HTS-1, whereas AP-1 that binds to the enhancer region was a major activator in JEG-3. By electrophoretic mobility shift assay, a nuclear protein from HTS-1 cells was confirmed to bind specifically to the Ets-2 site of IFNtau promoter region. Differences in amounts of AP-1 and Ets-2 protein were demonstrated in nuclear extracts from HTS-1, JEG-3 and ovine conceptuses. Substantial differences on IFNtau gene transcriptions found between caprine HTS-1 and human JEG-3 cells suggest that this cell line could be valuable in the elucidation of a molecular mechanism(s) by which IFNtau gene expression is regulated in a cell specific manner.