Tissue Factor Pathway Inhibitor-2 Promoter Research Articles

Promoter of TFPI-2 is Hypermethylated in Chinese Pediatric Acute Myeloid Leukemia

Human tissue factor pathway inhibitor-2 (TFPI-2) has been implicated as a metastasis-associated gene in many types of tumors. In this study, we investigated whether TFPI-2 was inactivated epigenetically in pediatric acute myeloid leukemia (AML). Methylation status was investigated by methylation-specific polymerase chain reaction and bisulfate genomic sequencing. TFPI-2 was aberrantly methylated in 50% (3/6) of AML cell lines. Aberrant methylation of TFPI-2 promoter was detected in 71.6% (48/67) of the Chinese pediatric AML patients. TFPI-2 transcript was significantly lower in AML group compared with controls (3.44 vs. 32.8, P<0.001). Patients with methylated TFPI-2 gene had significantly lower TFPI-2 transcript than those patients without methylated TFPI-2 (P=0.04). Promoter hypermethylation of TFPI-2 is frequent and specific event in pediatric AML.

Tissue factor pathway inhibitor 2 ( TFPI2) is frequently silenced by aberrant promoter hypermethylation in gastric cancer

Aberrant methylation of promoter CpG islands is associated with transcriptional inactivation of tumor-suppressor genes in cancer. TFPI2, a Kunitz-type serine proteinase inhibitor, has been identified as a putative tumor-suppressor gene from genome-wide screening for aberrant methylation, using a microarray combined with the methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-aza-dCyd) in various types of tumors. We assessed the methylation status of TFPI2 and investigated its expression pattern in human primary gastric cancer (GC) tissues and in GC cell lines. Hypermethylation of the promoter CpG island, which was observed in more or less all of GC cell lines, was prevalent in a high proportion of primary GC tissues (15/18, or 83%), compared with noncancerous (4/18, or 22%) or normal (0/3, or 0%) stomach tissues, and expression of TFPI2 mRNA was reduced in 7 of the 17 primary GC tissues (41%). Moreover, immunohistochemical analyses showed decreased levels of TFPI-2 protein, compared with adjacent noncancerous tissues in 8 of the 20 primary GC tissues examined (40%). TFPI2 mRNA expression was restored in gene-silenced GC cells after treatment with 5-aza-dCyd. Aberrant methylation of TFPI2 promoter CpG island occurred not only in GC cells but also in primary GC tissues at a high frequency, suggesting that epigenetic silencing of TFPI2 may contribute to gastric carcinogenesis.

Tissue Factor Pathway Inhibitor-2 gene methylation is associated with low expression in carotid atherosclerotic plaques

The tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine-protease inhibitor which is expressed in atherosclerotic plaques. Epigenetic regulation of the TFPI-2 gene, through methylation of CpG islands, has been advocated in cancer. We hypothesized that TFPI-2 gene methylation could regulate TFPI-2 expression in atherosclerosis. We used Methylation Specific PCR (MSP) and pyrosequencing in order to identify 18 CpG of the TFPI-2 promoter, in 59 carotid atherosclerotic plaques and 26 control mammary arteries. MSP showed methylation of the TFPI-2 gene (MSP+) in 16 plaques (27%), while no methylation (MSP-) was found in control arteries. Pyrosequencing confirmed that MSP+ plaques presented higher methylation levels than MSP- ones and arteries (p=0.03 and 0.01). Moreover, the TFPI-2 mRNA levels were lower in methylated plaques than in unmethylated ones and than in arteries (p=0.04 and <0.0001). The methylated plaques contained less lipids and macrophage infiltration than unmethylated ones. Their TFPI-2 immunoreactivity was mainly detected in the macrophages located in the media on the adventitial side, rather than in the lipid-rich core. Methylation of the TFPI-2 gene takes place in atherosclerotic plaques and is associated with decreased TFPI-2 expression. The place of this process in atherosclerosis progression remains to be investigated.

Analysis of a T-287C polymorphism in the tissue factor pathway inhibitor gene and identification of a repressor element in the promoter

Tissue factor pathway inhibitor (TFPI) is the principle regulator of tissue factor induced blood coagulation. The aim of this study was to investigate the potential association of a T-287C polymorphism in the promoter of the TFPI gene, which we described previously, with deep vein thrombosis (DVT) and to analyse its effect on promoter function. Low TFPI levels were shown to be associated with elevated risk of DVT and we present evidence for a possible association between DVT and T-287C. Any association between T-287C and plasma TFPI levels was masked by environmental effects. We studied the TFPI promoter region between bases − 1999 and + 229 around the transcription start site and the biological effects of the T-287C polymorphism in a human endothelial cell line using a Dual Luciferase-Reporter Gene Assay. Luciferase activity driven by the 2.2 kb TFPI promoter sequence was low for both allelic variants, but levels of expression from the -287C allele were significantly lower than -287T. Truncated TFPI promoter fragments from both alleles generated much higher transcriptional activity, but differences in reporter gene expression between allelic variants were not significant. Our results indicate the presence of potent repressor sequences between − 1999 and − 345 in the TFPI promoter and suggest that the TFPI T-287C promoter polymorphism might be a risk factor for DVT.

The Role of Tissue Factor Pathway Inhibitor-2 in Cancer Biology

Tissue factor pathway inhibitor-2 (TFPI-2), a member of the Kunitz-type serine proteinase inhibitor family, is a structural homologue of tissue factor pathway inhibitor (TFPI). The expression of TFPI-2 in tumors is inversely related to an increasing degree of malignancy, which may suggest a role for TFPI-2 in the maintenance of tumor stability and inhibition of the growth of neoplasms. TFPI-2 inhibits the tissue factor/factor VIIa (TF/VIIa) complex and a wide variety of serine proteinases including plasmin, plasma kallikrein, factor XIa, trypsin, and chymotrypsin. Aberrant methylation of TFPI-2 promoter cytosine-phosphorothioate-guanine (CpG) islands in human cancers and cancer cell lines was widely documented to be responsible for diminished expression of mRNA encoding TFPI-2 and decreased or inhibited synthesis of TFPI-2 protein during cancer progression. Furthermore, an aberrantly spliced variant of TFPI-2 mRNA (designated asTFPI-2) was detected, which represents an untranslated form of TFPI-2. The levels of asTFPI-2 were very low or undetectable in normal cells but markedly upregulated in neoplastic tissue. TFPI-2 functions in the maintenance of the stability of the tumor environment and inhibits invasiveness and growth of neoplasms, as well as metastases formation. TFPI-2 has also been shown to induce apoptosis and inhibit angiogenesis, which may contribute significantly to tumor growth inhibition. Restoration of TFPI-2 expression in tumor tissue inhibits invasion, tumor growth, and metastasis, which creates a novel possibility of cancer patient treatment. However, more information is still needed to define the precise role of TFPI-2 in human tumor biology.

Tissue factor pathway inhibitor-2 as a frequently silenced tumor suppressor gene in hepatocellular carcinoma

In HCC, inactivation of tumor suppressor genes plays a significant role in carcinogenesis. Apart from deletions and mutations, growing evidence has indicated that epigenetic alterations including aberrant promoter methylation and histone deacetylation are also implicated in inactivation of tumor suppressor genes. The goal of this study was to identify epigenetically silenced candidate tumor suppressor genes in human HCC by comparing the changes in oligonucleotide microarray gene expression profiles in HCC cell lines upon pharmacological treatment with the demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-dC). By analyzing the gene expression profiles, we selected tissue factor pathway inhibitor-2 (TFPI-2), a Kunitz-type serine protease inhibitor, for validation and further characterization. Our results showed that TFPI-2 was frequently silenced in human HCC and HCC cell lines. TFPI-2 was significantly underexpressed in approximately 90% of primary HCCs when compared with their corresponding nontumorous livers. TFPI-2 promoter methylation was detected in 80% of HCC cell lines and 47% of human HCCs and was accompanied by reduced TFPI-2 messenger RNA expression. In addition, TFPI-2 expression in HCC cell lines can be robustly restored by combined treatment with 5-Aza-dC and histone deacetylase inhibitor trichostatin A. These findings indicate that TFPI-2 is frequently silenced in human HCC via epigenetic alterations, including promoter methylation and histone deacetylation. Moreover, ectopic overexpression of TFPI-2 significantly suppressed the proliferation and invasiveness of HCC cells. Our findings suggest that TFPI-2 is a candidate tumor suppressor gene in human HCC.

Influence of MMP-2 and MMP-9 promoter polymorphisms on gene expression and clinical outcome of non-small cell lung cancer

Matrix metalloproteinases (MMP) including MMP-2 and MMP-9 play a major role in tumour invasion by proteolysing the extracellular matrix. Their activation, particularly that of MMP-9, is partly dependent on plasmin that is inhibited by TFPI-2 (tissue factor pathway inhibitor-2), a serine protease inhibitor whose gene expression is decreased in about one-third of non-small cell lung cancers (NSCLC). In addition, MMP-2 and MMP-9 are essential in the development of NSCLC and can be regulated by functional promoter polymorphisms. In this study, the -1306C/T MMP-2, -735C/T MMP-2 and -1562C/T MMP-9 polymorphisms were analysed in 90 NSCLC patients and 90 controls. In addition, the promoter region of the TFPI-2 gene was screened for sequence variations in both groups by DHPLC. A -167G/A polymorphism was identified in 3% of controls whereas none of the 90 patients exhibited this genetic variation in the TFPI-2 promoter region. Moreover, no difference in -1306C/T MMP-2, -735C/T MMP-2 and -1562C/T MMP-9 genotypes was found between cases and controls. However, the homozygous -1562CC MMP-9 genotype was more frequent in patients with squamous cell carcinoma than in controls (p=0.018). When genotype distributions were compared to MMP-2 and MMP-9 gene expression in tumours, no relationship was found with the -1306 MMP-2 and -1562 MMP-9 polymorphisms. In contrast, tumour MMP-2 gene expression was lower in homozygous -735CC patients than in those with CT or TT genotypes. In addition, the survival time was longer in patients with the MMP-2 -735T allele than in those with the CC genotype (p=0.02). The relative risk of death was increased 2.6-fold in -735CC patients (p=0.045; 95% CI=1.0-6.7). The results of this study suggest that the -735C/T MMP-2 polymorphism might be an independent prognostic marker in NSCLC, but this should be confirmed in a larger cohort of patients.

Analysis of sequence variations in the promoter region of the human tissue factor pathway inhibitor 2 gene in apoplectic patients and blood donors

Tissue factor pathway inhibitor 2 (TFPI-2) is a Kunitz-type serine protease inhibitor with homology to TFPI-1, an important regulator of the extrinsic pathway of blood coagulation. Recent studies have focused on TFPI-2 and its implications for atherosclerosis. The promoter region and the exons of the human TFPI-2 gene were screened for sequence variations in 41 apoplectic patients and 140 blood donors with no history of ischemic stroke. The sequence variations -567T>C, -546T>C, -353A>G, -161G>C, -167G>A, -47C>A, and -18C>A, which are located in the TFPI-2 promoter, were discovered in both cohorts with allelic frequencies ranging from 0.3 to 2.4%. The influence of these sequence variations on the transcriptional activity of the TFPI-2 gene was investigated in HEK-293 cells using a promoter test system. A wild-type TFPI-2 promoter fragment 716 bp upstream of the translation start site was cloned into a secreted alkaline phosphatase expression vector, and the sequence variations were introduced by site-directed mutagenesis. Interestingly, the promoter activity of the tested mutants was reduced by 1.3- to 2.8-fold compared to that of wild-type control. The variation -18C>A, where a putative binding site of the transcription factor Sp-1 is located, had the strongest effect on transcriptional activity. In conclusion, our present study shows that the transcription of TFPI-2 is changed by single nucleotide polymorphisms and that the sequence variations in transcription factor binding sites of the TFPI-2 promoter may influence the regulation of this gene.

A Locus on Chromosome 2 Influences Levels of Tissue Factor Pathway Inhibitor

Levels of tissue factor pathway inhibitor (TFPI) have been associated with arteriosclerosis and thrombotic disease. Although a genetic component to variation in TFPI levels is well-documented, no systematic genome-wide screens have been conducted to localize genes influencing levels of TFPI. We studied TFPI levels in 397 individuals in 21 Spanish families participating in the Genetic Analysis of Idiopathic Thrombosis (GAIT) study. Twelve families were selected through a proband with idiopathic thrombosis and 9 were ascertained without regard to phenotype. A genome scan was performed using microsatellite markers spaced at approximately 10 cM intervals. Standard multipoint variance component linkage methods were used. The heritability of TFPI levels was 0.52 (P<0.0001), with no evidence for shared household effects. In the genome screen, only 1 LOD score >2 was observed. On chromosome 2q, the maximum multipoint LOD score was 3.52 near marker D2S1384. This is near the structural gene for TFPI, which is located at 2q32. In follow-up association analyses, marginal evidence of association (P=0.04) was observed with the TFPI promoter variant C-399T. These results suggest that polymorphisms in and around the TFPI structural gene may be the major genetic determinants of variation in TFPI levels.

Sequential 5-Aza 2′-deoxycytidine/depsipeptide FK228 treatment induces tissue factor pathway inhibitor 2 (TFPI-2) expression in cancer cells

cDNA arrays were used to examine gene induction in CALU-6 and H460 lung cancer cells mediated by sequential 5-aza 2'-deoxycytidine (DAC)/depsipeptide FK228 (DP) exposure in order to identify translational end points for clinical trials evaluating these agents. In both cell lines, sequential DAC/DP treatment induced expression of tissue factor pathway inhibitor-2 (TFPI-2), an inhibitor of Factor VII: tissue factor signal transduction known to diminish the malignant phenotype of cancer cells. TFPI-2 expression was diminished or absent in 16 of 32 cell lines established from thoracic malignancies. Sequential DAC/DP treatment induced TFPI-2 in cancer cells deficient for TFPI-2 expression in the basal state. Promoter methylation coincided with loss of TFPI-2 expression in a number of cancer lines. TFPI-2 promoter methylation was observed in one of five pulmonary adenocarcinomas, and seven of seven esophageal adenocarcinomas, but not corresponding normal tissues. DP enhanced acetylation of TFPI-2-associated histones in CALU-6 cells. DP or PDBU, alone, induced TFPI-2 expression in cancer cells deficient for TFPI-2 expression in the absence of promoter methylation. In these cells, DP-mediated TFPI-2 induction was abrogated by calphostin. Induction of TFPI-2 by distinct, yet cooperative mechanisms involving chromatin remodeling and PKC signaling strengthens the preclinical rationale for sequential administration of DNA demethylating agents and HDAC inhibitors in cancer patients. Furthermore, induction of TFPI-2 may be a useful surrogate marker of treatment response in individuals receiving sequential DAC/DP infusions.

Epigenetic inactivation of TFPI-2 as a common mechanism associated with growth and invasion of pancreatic ductal adenocarcinoma

Using microarrays, we have screened for genes reactivated by drugs that modify epigenetic mechanisms in pancreatic cancer cells. One of the genes identified was tissue factor pathway inhibitor 2 (TFPI-2), which encodes for a broad-spectrum serine proteinase inhibitor that negatively regulates the extracellular matrix degradation, an essential step in tumor invasion and metastasis. We therefore investigated the expression and methylation patterns of the TFPI-2 gene in pancreatic adenocarcinoma, and determined its role in tumor growth and invasion. In contrast to its abundant expression in normal pancreas, TFPI-2 mRNA was undetectable in a high fraction of pancreatic cancer cell lines and in primary pancreatic ductal neoplasms (IPMNs). Loss of TFPI-2 expression was associated with aberrant hypermethylation of its promoter CpG island. Treatment with the phorbol ester (PMA), known to stimulate the TFPI-2 promoter activity, augmented the TFPI-2 expression in cell lines with unmethylated or partially methylated TFPI-2, but failed to induce the expression in cell lines that harbored fully methylated TFPI-2. Aberrant methylation of TFPI-2 was also detected in 73% (102/140) of pancreatic cancer xenografts and primary pancreatic adenocarcinomas, was more likely in older patients with pancreatic cancer, and significantly correlated with progression of IPMNs (P=0.0002). Restored expression of the TFPI-2 gene in nonexpressing pancreatic cancer cells resulted in marked suppression in their proliferation, migration, and invasive potential in vitro. We thus conclude that epigenetic inactivation of TFPI-2 is a common mechanism that contributes to the aggressive phenotype of pancreatic ductal adenocarcinoma.

Promoter methylation and silencing of the tissue factor pathway inhibitor-2 (TFPI-2), a gene encoding an inhibitor of matrix metalloproteinases in human glioma cells.

We have shown previously that the tissue factor pathway inhibitor-2 (TFPI-2), a broad range proteinase inhibitor, is highly expressed in low-grade gliomas, but, minimally expressed or undetectable in glioblastomas, and that enforced expression of this gene reduces the invasive properties of brain tumor cells. Here, we examined the role of promoter methylation as a mechanism of TFPI-2 gene silencing. In SNB19 glioblastoma cells, which have no detectable TFPI-2 expression, 5-aza-2'-deoxycytidine (5aC), an inhibitor of DNA methyltransferase, induced TFPI-2 mRNA in a dose-dependent manner. Trichostatin A (TSA), the histone deacetylase (HDAC) inhibitor, by itself, was more efficient than 5aC in inducing TFPI-2 transcripts, and the 5aC+TSA combination resulted in highly synergistic reactivation of the gene, both at the transcript and protein levels. In Hs683 glioma cells, which express the TFPI-2 gene at high levels, transfection of the in vitro methylated TFPI-2 promoter constructs resulted in a drastic decrease of promoter activity compared to the unmethylated promoter. Further, the methylation-specific PCR in SNB19 and Hs683 cells showed that TFPI-2 gene repression was closely linked with methylation of the CpG islands in the promoter. Finally, the chromatin immunoprecipitation assays in SNB19 cells showed that the methylated and repressed TFPI-2 promoter was associated with the methyl-CpG binding protein 2 (MeCP2), and that gene reactivation resulted in the loss of MeCP2 from this site. These studies establish that TFPI-2 is transcriptionally silenced through promoter methylation in SNB19 cells.

Methylation of TFPI-2 gene is not the sole cause of its silencing

TFPI-2 (tissue factor pathway inhibitor-2) is a serine protease inhibitor that may suppress tumor cell invasion and metastasis. TFPI-2 expression is often lost in cells derived from tumors of diverse organs. We have examined whether aberrant hypermethylation of the 5' end of the TFPI-2 gene is associated with its loss of expression. After 5-azacytidine treatment of three cell lines lacking TFPI-2 expression (HT1080 fibrosarcoma cells, MCF-7 breast carcinoma cells, and LNCaP prostate carcinoma cells), TFPI-2 transcripts could be detected by RT-PCR. In these three cell lines, methylation of the 5' end of the TFPI-2 gene was detected, while two prostate carcinoma cell lines in which the TFPI-2 gene was expressed, PC-3 and DU-145, showed no methylation. However, all the three cell lines which lacked TFPI-2 expression also contained unmethylated TFPI-2 alleles. Furthermore, a transiently transfected TFPI-2 promoter was non-functional in the three cell lines, but function was attained following treatment with 5-azacytidine. Our results indicate that while methylation of the TFPI-2 gene is associated with its silencing, it is not the sole cause, and we suggest that one or more components of pathways regulating TFPI-2 expression have also undergone methylation-associated silencing in these cell lines.

The ERK/MAPK Pathway Regulates the Activity of the Human Tissue Factor Pathway Inhibitor-2 Promoter

Human tissue factor pathway inhibitor-2 (hTFPI-2) is a 32-kDa serine protease inhibitor that is associated with the extracellular matrix. hTFPI-2 inhibits several extracellular matrix-degrading serine proteases and may play a role in tumor invasion and metastasis. To study the signal transduction pathway that leads to the activation of the hTFPI-2, we cloned the potential promoter region of this gene adjacent to a heterologous luciferase reporter gene. Phorbol 12-myristate 13-acetate (PMA) induced the luciferase reporter gene in HEK293 cells and other epithelial cell lines, such as the human lung carcinoma A549 cells, the breast carcinoma MCF7 cells, and the cervical HeLa cells. This PMA induction was blocked with the MEK inhibitor UO126, suggesting that the PMA-induced activation of the hTFPI-2 promoter is mediated through MEK. Furthermore, epidermal growth factor induced the luciferase reporter gene in HeLa cells. Cotransfection of the luciferase construct with constitutively active components of the Ras/Raf/MEK/ERK pathway in EcR-293 cells lead to a 7- to 92-fold induction of the luciferase reporter gene, indicating that regulation of hTFPI-2 is mediated through this pathway. A series of luciferase reporter gene constructs with progressive deletions of the 5'-flanking region suggested that the minimal basal promoter activity is located between nucleotide positions -89 and -384, whereas the minimal inducible promoter activity is between -89 and -222. We have used the computer program TFSEARCH and mutagenesis to analyze potential transcription factor binding sites. We identified an AP-1 binding site at nucleotide position -156 (inducible activity) and a Sp1 site at position -134 (basal activity) as potential cis-acting elements in the promoter region of the hTFPI-2.

Characterization and functional analysis of TFPI-2 gene promoter in a human choriocarcinoma cell line

Tissue factor pathway Inhibitor-2 (TFPI-2) is associated with extracellular matrices and plays a major role in cell migration and tumor invasion. In this study, a 4.8-kb human TFPI-2 gene 5′-flanking region was isolated, cloned and sequenced. Promoter region analysis revealed a high GC-rich content without canonical TATA and CAAT boxes but three transcription initiation sites were identified. Moreover, several putative binding sites for transcription factors were identified (MyoD, LYF1, NF-Y, GATA, oct-1, AP-1, Sp1, NF1, NF-κB and egr-1). To characterize potential regulatory regions, TFPI-2/luciferase promoter constructs were then transfected in human choriocarcinoma JEG-3 cells. We first showed that the minimal TFPI-2 promoter is located between −166 and −111 from the translation start site. Luciferase activity consistently increased after stimulation of JEG-3 cells by phorbol 12-myristate 13-acetate indicating that NF1, NF-κB and egr-1/Sp1 binding sites are crucial in inducible TFPI-2 expression. Moreover, negative regulatory regions included AP-1 binding sites were identified. This study demonstrates that the TFPI-2 gene promoter exhibits typical features of a housekeeping gene.

Transcriptional Silencing of the TFPI-2 Gene by Promoter Hypermethylation in Choriocarcinoma Cells

Tissue factor pathway inhibitor-2 (TFPI-2), a Kunitz-type serine proteinase inhibitor associated with the extracellular matrix, has been shown to reduce tumor invasion. In the present study we identified the presence of a complete CpG island region spanning exon 1 and the three transcription initiation sites. We demonstrate that DNA demethylation by 5'-aza-2'-deoxycytidine restores TFPI-2 transcription in JAR choriocarcinoma cells. The effect of in vitro DNA methylation on TFPI-2 promoter function was also confirmed with TFPI-2/luciferase promoter constructs. Finally, we determined the precise methylation status of CpG sites of the TFPI-2 promoter in normal and tumor trophoblast cells using the bisulfite genomic sequencing method. We conclude that hypermethylation of the TFPI-2 gene is correlated with transcriptional silencing and that the TFPI-2 gene may be a candidate tumor suppressor gene.

Role of GATA Motifs in Tissue Factor Pathway Inhibitor Gene Expression in Malignant Cells

Tissue factor pathway inhibitor (TFPI) is the primary physiologic inhibitor of tissue factor-induced clotting. The TFPI gene contains three GATA motifs in the region flanking its transcription initiation sites. GATA motifs present in promoters of other genes bind GATA-2 transcription factor and thereby regulate their transcriptional expression. Both TFPI and GATA-2 transcription factor are synthesized by a variety of normal as well as malignant cells including hepatocellular carcinoma HepG2 and bladder carcinoma ECV304. Here, we studied whether the three GATA motifs flanking the transcription initiation sites regulate TFPI gene expression in HepG2 and ECV304 cells by binding to the GATA-2 transcription factor. Synthetic oligonucleotides containing GATA sequences from the TFPI regulatory region formed DNA–protein complexes with HepG2 and ECV304 nuclear extracts in an electrophoretic mobility shift assay. Using a 740-bp fragment (−496/+244) from TFPI regulatory region, the effect of base substitutions at each of the three GATA motifs was studied in a luciferase reporter gene system. TFPI promoter activity in HepG2 cells was increased 3-fold with mutation in one of the three GATA motifs and in ECV304 cells was essentially unchanged with mutations in all three GATA motifs. Thus, GATA motifs appear to serve a tissue-specific regulatory role in TFPI gene expression in malignant cells.

The Promoter of Human Tissue Factor Pathway Inhibitor Gene: Identification of Potential Regulatory Elements

Tissue factor pathway inhibitor is the major potent physiologic inhibitor of tissue factor-induced coagulation. Several potential binding sites for transcription factors have been described in the 750 bp of the 5′ flanking region of the human tissue factor pathway inhibitor gene reported earlier. To identify elements that regulate the expression of tissue factor pathway inhibitor in endothelial, hepatocyte, and monocyte cells, the sequence of an additional 770 bp of tissue factor pathway inhibitor was determined. Comparison of this new sequence as well as that reported earlier with consensus sequences for transcription factor binding sites provided matches for GATA-2, SP1, and c-Myc sequences. Moreover, plasmids containing deletion mutants of the 5′ tissue factor pathway inhibitor promoter region and the luciferase reporter gene were transfected into HepG2, ECV304, and THP1 cells. Three negative regulatory elements were localized between −548 to −390, − 390 to −75, and −1158 to −796 relative to the transcriptional start, respectively, in HepG2, ECV304 and THP-1 cells.