Induction of Endogenous Interferon Tau Gene Transcription by CDX2 and High Acetylation in Bovine Nontrophoblast Cells1

Sakurai, ﻿Toshihiro; Sakamoto, Atsushi; Muroi, Yoshikage; Bai, Hanako; Nagaoka, Kentaro; Tamura, Kazuhiro; Takahashi, Toshio; Hashizume, Kazuyoshi; Sakatani, Miki; Takahashi, Masashi; Godkin, James D.; Imakawa, Kazuhiko

doi:10.1095/biolreprod.108.073916

Cited by 56 publications

(75 citation statements)

References 39 publications

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“…The ligand for FGFR1, FGF1, is known to upregulate MMP13, resulting in EMT induction (Billottet et al 2008). It is possible that upregulation of MMP2 and MMP9 could be induced Reactions were performed in duplicate and the data were analyzed by using the change in cycle threshold value method (Sakurai et al 2009). ACTB mRNA served as the internal control.…”

Section: Discussionmentioning

confidence: 99%

“…(B) Levels of SNAI2, ZEB1, ZEB2, TWIST1, TWIST2, and KLF8 mRNAs in days 17, 20, and 22 conceptuses (nZ4 each day) were examined by quantitative RT-PCR. Reactions were performed in duplicate, and the data were analyzed by using the change in cycle threshold value method (Sakurai et al 2009). ACTB mRNA served as the internal control.…”

Section: Upregulation Of Emt Markers In Day 22 Bovine Conceptusesmentioning

confidence: 99%

“…The thermal profile for real-time PCR was at 95 8C for 10 min, followed by 40 cycles of 95 8C for 10 s, 60 8C for 20 s, and 72 8C for 40 s. Average threshold (C t ) values for mRNA were calculated and normalized to C t values for ACTB mRNA (Sakurai et al 2009). Each run was completed with a melting curve analysis to confirm the specificity of amplification and the absence of primer dimers.…”

Section: Quantitative Rt-pcrmentioning

confidence: 99%

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Expression of mesenchymal-related genes by the bovine trophectoderm following conceptus attachment to the endometrial epithelium

Yamakoshi¹,

Bai²,

Chaen³

et al. 2012

Reproduction

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In the course of experiments to identify and characterize the factors that function in bovine conceptuses during peri-attachment periods, various transcripts related to the epithelial-mesenchymal transition (EMT) were found. In this study, RNA was extracted from different sets of days 17, 20, and 22 (day 0Zday of estrous) bovine conceptuses and subjected to real-time PCR analysis as well as Western blotting, from which abundances of N-cadherin (CDH2), vimentin, matrix metalloproteinase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase) (MMP2), and matrix metallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase) (MMP9) mRNAs were determined on day 22, concurrent with (CDH1) mRNA and protein downregulation. Transcription factors in EMT processes were then analyzed and changes in snail homolog 2 (Drosophila) (SNAI), zinc finger E-box binding homeobox 1 (ZEB1), zinc finger E-box binding homeobox 2 (ZEB2), twist homolog 1 (Drosophila) (TWIST1), twist homolog 2 (Drosophila) (TWIST2), and Kruppel-like factor 8 (KLF8) transcripts were found in day 22 conceptuses, while confirming SNAI2 expression by Western blotting. Immunohistochemical analysis revealed that the day 22 trophectoderm expressed the mesenchymal markers N-cadherin and vimentin as well as the epithelial marker cytokeratin. In attempts to identify the molecular mechanisms by which the trophectoderm expressed EMT-related genes, growth factor receptors associated with EMT were analyzed. Upregulation of the growth factor receptor transcripts, fibroblast growth factor receptor 1 (FGFR1), platelet-derived growth factor receptor, alpha polypeptide (PDGFRA), platelet-derived growth factor receptor, beta polypeptide (PDGFRB), and transforming growth factor, beta receptor II (70/80 kDa) (TGFBR2) mRNAs, was found on day 22. The analysis was extended to determine the integrin (ITG) transcripts and found high levels of integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor) (ITGA4), integrin, alpha 8 (ITGA8), integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61) (ITGB3), and integrin, beta 5 (ITGB5) mRNAs on day 22. These observations indicate that after the conceptus-endometrium attachment, EMT-related transcripts as well as the epithelial marker cytokeratin were present in the bovine trophectoderm and suggest that the implantation process for noninvasive trophoblasts requires not only extracellular matrix expression but also partial EMT.

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Section: Discussionmentioning

confidence: 99%

Section: Upregulation Of Emt Markers In Day 22 Bovine Conceptusesmentioning

confidence: 99%

Section: Quantitative Rt-pcrmentioning

confidence: 99%

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Expression of mesenchymal-related genes by the bovine trophectoderm following conceptus attachment to the endometrial epithelium

Yamakoshi¹,

Bai²,

Chaen³

et al. 2012

Reproduction

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“…Each PCR product was subcloned, and their nucleotide structures verified by DNA sequencing. Quantitative PCR reactions were performed using a SYBR Green kit (Takara Biomedicals, Tokyo, Japan) and Applied Biosystems thermal cycle system (7900HT, Applied Biosystems, Tokyo, Japan), as previously described [29,30]. Average threshold (Ct) values for GATA2, GATA3 and trophoblast-specific factor mRNAs were calculated and normalized to the Ct value for GAPDH mRNA.…”

Section: Rna Extraction and Analysismentioning

confidence: 99%

Regulation of Trophoblast-Specific Factors by GATA2 and GATA3 in Bovine Trophoblast CT-1 Cells

et al. 2011

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Abstract. Numerous transcription factors that regulate trophoblast developmental processes have been identified; however, the regulation of trophoblast-specific gene expression has not been definitively characterized. While a new role of Gata3 in trophoblast development was being demonstrated in mice, we examined effects of GATA transcription factors on conceptus interferon tau (IFNT), a major trophectoderm factor in ruminants. In this study, expression patterns of trophoblast ASCL2, CDX2, CSH1, ELF5, HAND1, IFNT, and TKDP1 mRNAs were initially examined, from which ASCL2, CDX2, IFNT, and TKDP1 mRNAs were found to be similar to those of GATA2 and GATA3 in days 17, 20, and 22 (day 0=day of estrus) bovine conceptuses. A chromatin immunoprecipitation (ChIP) assay revealed that endogenous GATA2 and GATA3 occupied GATA binding sites on the upstream regions of CSH1, IFNT, and TKDP1 genes and on the intron 1 region of CDX2 gene in bovine trophoblast CT-1 cells. In transient transfection analyses of the upstream region of bovine CSH1, and IFNT or the intron 1 region of CDX2 gene, over-expression of GATA2 induced transactivation of these trophoblast-specific genes in bovine non-trophoblast ear fibroblast EF cells, but over-expression of GATA3 did not substantially affect their transactivation. In CT-1 cells, endogenous CDX2 and IFNT mRNAs were down-regulated by GATA2 siRNA, while endogenous ASCL2 and CDX2 mRNAs were down-regulated by GATA3 siRNA. Our results indicate that in addition to trophectoderm lineage specification, GATA2 and/or GATA3 are involved in the regulation of trophoblast-specific gene transcription in bovine trophoblast CT-1 cells. Key words: Bovine, GATA factors, Gene regulation, Trophoblast cells (J. Reprod. Dev. 57: [518][519][520][521][522][523][524][525] 2011) uring mammalian development, the first lineage decision is to establish the trophectoderm (TE) and inner cell mass (ICM). The outer TE layer attaches to and invades the maternal endometrium and eventually forms placental structures. It has been well established that caudal-type homeobox transcription factor 2 (Cdx2) serves as a lineage determining factor for the murine trophectoderm [1,2]. Quite recently, one of the GATA transcription factors, Gata3, was found to specify the trophectoderm lineage [3].In addition to these factors, there are numerous factors that are expressed in the trophoblasts during trophectoderm cell differentiation. These are E74-like factor 5 (Elf5), heart and neural crest derivatives expressed 1 (Hand1), a mammalian achaete scute-like homolog 2 (Ascl2), placental lactogens (PLs, CSHs) in many mammalian species, and interferon tau (IFNT) and trophoblast Kunitz domain proteins (TKDPs) in ruminant ungulates. Elf5 is necessary to maintain expression of the trophoblast stem cell genes Cdx2 and Eomes, and thereby reinforces trophoblast cell fate [4]. Hand1 and Ascl2, basic helix-loop-helix (bHLH) transcription factor genes, are essential for trophoblast proliferation and differentiation in mice [5,6]. CSHs belong to the growt...

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“…The thermal profile for real-time PCR was at 95 8C for 10 min, followed by 40 cycles of 95 8C for 10 s, 60 8C for 20 s, and 72 8C for 30 s. Average cycle threshold (C t ) values of each sample were calculated and normalized to C t values for ACTB using the 2 KDC t method. Each run was completed using a melting curve analysis to confirm the specificity of the amplification and the absence of primer dimers (Sakurai et al 2009). …”

Section: Rna Preparation and Quantitative Real-time Pcrmentioning

confidence: 99%

CD9 regulates transcription factor GCM1 and ERVWE1 expression through the cAMP/protein kinase A signaling pathway

Muroi¹,

Sakurai²,

Hanashi³

et al. 2009

Reproduction

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ERVWE1 (SYNCYTIN-1), a membrane protein originating from the envelope gene of human endogenous retrovirus-W (HERV-W), mediates the fusion of mononucleated cytotrophoblasts into multinucleated syncytiotrophoblast. Though ERVWE1 has been characterized since its discovery, regulatory mechanisms associated with ERVWE1 expression have not been firmly established. We hypothesized that membrane protein CD9, involved in cell-cell fusion of fertilization and myogenesis, could be involved in the regulation of ERVWE1 gene expression. In this study, regulatory mechanisms of ERVWE1 expression were studied using human choriocarcinoma BeWo cells. Forskolin is an activator of adenylate cyclase, which increased CD9 and ERVWE1 expression. The increase in CD9 expression was inhibited by a protein kinase A (PKA) inhibitor, Rp-cAMPS. These results indicate that CD9 expression is regulated by the cAMP/PKA signaling pathway. Overexpression of CD9 increased expression levels of ERVWE1 as well as GCM1 (hGCMa), which is a transcription factor known to activate ERVWE1 gene transcription. However, high ERVWE1 expression induced by CD9 overexpression did not result in the increase in chorionic gonadotropin, beta polypeptide production. Moreover, CD9-induced increase in ERVWE1 and GCM1 expressions were inhibited by Rp-cAMPS. These results suggest that CD9 increases GCM1 expression via the cAMP/PKA signaling pathway, resulting in the increase in ERVWE1 expression.

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Induction of Endogenous Interferon Tau Gene Transcription by CDX2 and High Acetylation in Bovine Nontrophoblast Cells1

Cited by 56 publications

References 39 publications

Expression of mesenchymal-related genes by the bovine trophectoderm following conceptus attachment to the endometrial epithelium

Expression of mesenchymal-related genes by the bovine trophectoderm following conceptus attachment to the endometrial epithelium

Regulation of Trophoblast-Specific Factors by GATA2 and GATA3 in Bovine Trophoblast CT-1 Cells

CD9 regulates transcription factor GCM1 and ERVWE1 expression through the cAMP/protein kinase A signaling pathway

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