Regular ArticleCharacterization and functional analysis of TFPI-2 gene promoter in a human choriocarcinoma cell line
Introduction
Tissue factor pathway inhibitor-2 (TFPI-2), a 32-kDa member of the Kunitz-type serine proteinase inhibitor family, is associated with extracellular matrices and plays a major role in cell migration and tumor invasion. Despite similarities in amino acid sequence and structure to TFPI-1, the main inhibitor of TF/VIIa complex, TFPI-2 weakly inhibits factor Xa and thrombin generation, and thus poorly regulates TF-mediated coagulation [1]. In contrast, TFPI-2 efficiently decreases activation of metalloproteinases MMP-1, MMP-3, MMP-9, and MMP-13, by inhibiting plasmin and trypsin [2], [3] and thus reduces tumor invasion and metastasis [4], [5]. In addition, TFPI-2 could also regulate plasmin in atherosclerotic plaques modulating extracellular proteolytic mechanisms [6].
TFPI-2 is synthesized and secreted by endothelial, mesenchymal and epithelial cells, monocytes/macrophages and the syncytiotrophoblast [7], [8], [9]. On the other hand, the expression of TFPI-2 has been reported to be undetectable or at a very low level in a variety of invasive tumor cell lines [4], [5], [10], [11], [12] and downregulation of TFPI-2 gene could thus favor cancer invasion.
The complete nucleotide sequences of human and murine TFPI-2 genes consist of five exons and four introns [13], [14]. The human TFPI-2 gene, located on chromosome 7q22 [15], spans approximately 7 kb and a recent analysis of the promoter region up to −3564 identified a single transcription initiation site without canonical TATA or CAAT box [14]. In contrast, the 5′-flanking region of the murine TFPI-2 gene contains a prototypic TATA box, a GC box and two CAAT boxes [13].
We recently reported that TFPI-2 mRNA synthesis by a human placenta cell line isolated from a choriocarcinoma (JEG-3) was stimulated by phorbol 12-myristate 13-acetate (PMA), whereas tumor necrosis factor (TNF-α) had no effect [16]. This cell line was therefore considered of interest to identify regulatory elements essential for transcriptional regulation of TFPI-2 expression.
By using a method for walking upstream in genomic DNA, the present study further defined the sequence of the human TFPI-2 promoter up to −4767 in the 5′-flanking region. We thus identified two new minor transcription initiation sites, and also compared the human TFPI-2 promoter to the murine sequence already published [13]. In addition, we studied TFPI-2 promoter activity with various promoter/luciferase reporter gene constructs transfected into human placenta choriocarcinoma JEG-3 cells. Finally, this approach allowed us to define further the regions required in the promoter for basal and inducible TFPI-2 expression in this trophoblastic cell line.
Section snippets
Cell culture and stimulation
Human trophoblast cell line JEG-3 was cultured in RPMI-1640 medium (Invitrogen Life Technologies, Cergy Pontoise, France) supplemented with 10% endotoxin-free heat-inactivated fetal calf serum (ATGC Biotechnologie, Noisy le Grand, France), 2 mM l-glutamine, 1 mM sodium pyruvate, 100 IU/ml penicillin, 100 mg/ml streptomycin, 15 mM sodium bicarbonate and 2 mM glucose. Cells were grown at 37 °C in an atmosphere of 95% air and 5% CO2.
Confluent cells were washed with Ca2+ and Mg2+-free Hanks'
Basal and inducible TFPI-2 mRNA expression
TFPI-2 mRNA expression was studied in 5×104 JEG-3 trophoblast cells using a specific RT-PCR method. The optimal TNF-α and PMA concentrations and incubation times defined for use for cell stimulation were 60 min in the presence of 20 ng/ml TNF-α and 4 h with 100 ng/ml of PMA. The obtained RT-PCR product appeared as a single band of 253 bp, the expected size of TFPI-2 cDNA fragment (Fig. 1). The sequencing of this product was similar to the previously published sequence [17] and confirmed the
Discussion
TFPI-2, a 32-kDa member of the Kunitz-type serine proteinase inhibitor family, is synthesised by many different cells, but is particularly abundant in the placenta [9], [16], [20]. It might therefore play a role in the regulation of hemostasis, and particularly of fibrinolysis within placenta, but it also could regulate trophoblast invasion and differentiation. Until recently, little was known about the regulation of the human TFPI-2 gene. In the present study, and in agreement with previous
Acknowledgements
The authors thank Doreen Raine for editing the English text. We particularly thank Antoine Touzé (EMI-U0010, Tours) for useful suggestions. This study was supported by the Institut pour la Recherche sur la Thrombose et l'Hémostase in Tours, France.
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2017, Molecular and Cellular EndocrinologyBeneficial role of overexpression of TFPI-2 on tumour progression in human small cell lung cancer
2013, FEBS Open BioCitation Excerpt :Unfortunately, TFPI-2 is downregulated in most aggressive tumours such as glioma [9], non-small cell lung cancer (NSCLC) [10,11], breast cancer [12], melanoma [13], colorectal cancer [14], pancreatic cancer [15] and hepatocellular carcinoma [16]. Such silencing is mainly due to epigenetic changes induced by TFPI-2 promoter hypermethylation and histone deacetylation [12,17,18,19]. Aberrant methylation of TFPI-2 is now being studied to differentiate benign from malign diseases and to evaluate disease progression [20,21,22].
Tissue factor pathway inhibitor 2 is induced by thrombin in human macrophages
2011, Biochimica et Biophysica Acta - Molecular Cell ResearchCitation Excerpt :Moreover, we observed that thrombin induced JNK phosphorylation after 15–30 min. Although the ERK1/2 and JNK pathways are essential for the activation of the transcription factor AP-1 [25], the three AP-1 binding sites identified in the TFPI2 promoter are involved in the negative control, but not in the induction of TFPI2 gene expression [26]. In consequence, inhibition of AP-1 by SR-11302 did not affect TFPI2 induction by thrombin.
Tissue Factor Pathway Inhibitor-2 gene methylation is associated with low expression in carotid atherosclerotic plaques
2009, AtherosclerosisCitation Excerpt :As the TFPI-2 gene methylation appears as an extensive process implicating a great part of the CpG islands in neoplasic tumors, we observed that this process shows a similar pattern in atherosclerotic plaques and homogenously modifies the 18 studied CpG. Furthermore, there was no significant difference in the methylation level of the transcription factor binding sites important in TFPI-2 induction, such as Sp1 (−214 bp) or egr1/Sp1 (−118 bp) [10,18]. These results suggest that this moderate but homogenous methylation is able to prevent TFPI-2 expression alone or in combination with other described mechanisms, such as activation of repression factors by other epigenetic mechanisms.
Influence of MMP-2 and MMP-9 promoter polymorphisms on gene expression and clinical outcome of non-small cell lung cancer
2007, Lung CancerCitation Excerpt :These fragments were then screened by a DHPLC method, and one variation corresponding to a G → A transition in position −167 was detected in the DNA of 3 healthy individuals (3%), but was not found in any of the patients with NSCLC. This −167G/A polymorphism is located in a region which has been shown to be critical for TFPI-2 gene expression [17] and has previously been described to have a frequency of 1.5% [18]. However, Siegling et al. also found other polymorphisms in the TFPI-2 promoter sequence, with frequencies ranging from 0.3 to 2.4%, but these were not detected in our patients or controls.