Minimal and inducible regulation of tissue factor pathway inhibitor-2 in human gliomas

Abstract

Tissue factor pathway inhibitor-2 (TFPI-2), a serine protease inhibitor abundant in the extra cellular matrix, is highly expressed in non-invasive cells but undetectable levels in highly invasive human glioma cells. The mechanisms responsible for its transcriptional regulation are not well elucidated. In this study, we made several deletion constructs from a 3.6 kb genomic fragment from Hs683 cells containing the 5'-¯anking region of the TFPI-2 gene, transiently transfected with these constructs into non-invasive (Hs683) and highly invasive (SNB19) human glioma cells, and assessed their expression by using a luciferase reporter gene. Three constructs showed high promoter activity (pTF5, 7670 to +1; pTF6, 7312 to +1; pTF2, 71511 to +1). Another construct, pTF8 (781 to +1), showed no activity. PTF9, a variant of pTF5 in which a further 231 bp fragment (7312 to 781) was deleted, from the [7670 to +1] pTF5 region, also showed no promoter activity. Hence, (7312 to 781) this region is essential for the transcription of TFPI-2 in glioma cells. Sequencing of this promoter region revealed that it has a high G+C content, contains potential SP1 and AP1 binding motifs, and lacks canonical TATA and CAAT boxes immediately upstream of the major transcriptional initiation site, although CAAT boxes were found about 73000 bp upstream of the transcription start site. We also found a strong repressor in the region between 7927 to ± 1181, upstream of the major transcriptional initiation site, followed by positive elements or enhancers between ± 1511 to 71181. These positive elements masked the silencer eect. Finally TFPI-2 was induced in Hs683 cells transfected with the pTF6 construct (7312 to +1) and stimulated with phorbol-12-myristate-13-acetate (PMA). We conclude that the 7312 to +1 region is critical for the minimal and inducible regulation of TFPI-2 in non-invasive (Hs683) and highly invasive (SNB19) human glioma cell lines. Oncogene (2002) 21, 921 ± 928.

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

“…Apicidin induces p21 expression by PKC pathways, which are mediated via SP-1 (Han et al, 2001); recent experiments from our lab indicate that DP mediates p21 induction in cancer cells by similar mechanisms (Nguyen et al, 2004). Although our experiments have not directly demonstrated that the effects of PKC signaling on TFPI-2 expression are mediated via SP-1, it is intriguing that the two SP-1 sites believed to mediate induction of TFPI-2 promoter activity by PMA or DAG lie within in the CpG island of the TFPI-2 promoter (Kamei et al, 2001;Konduri et al, 2002). Conceivably, methylation within or adjacent to these SP-1 sites modulates PKC signaling at the level of the TFPI-2 promoter.…”

“…The TFPI-2 promoter lacks a TATA box; however, two CAAT boxes are located approximately 3 kb proximal to the transcription initiation site. Konduri et al (2002) reported that region À312 to þ 1 of the TFPI-2 promoter was critical for basal as well as PMA-induced expression of this gene in glioma cells; region À312 to À81, which contains several positive regulatory sequences including AP-1, SP-1, NFkB, NFkB-like, and Lyf-1 binding sites appears essential for TFPI-2 transcription in these cells. In a subsequent study, Konduri et al (2003b) observed that PMA, DAG, IFNa, and IFNg induced TFPI-2 expression in low-grade glioma cells, and that mutations involving two AP-1 or two SP-1 sites (À310 to À300 and À165 to À154, respectively), abrogated TFPI-2 promoter activation by these agents.…”

“…Several investigators have observed that TFPI-2 reporter gene expression is markedly lower in cultured cells that lack endogenous TFPI-2 expression relative to those which retain expression of this gene (Kamei et al, 2001;Konduri et al, 2002;Rao et al, 2003). Whereas these observations suggest a strong selective pressure to inactivate TFPI-2 in some cancer cells, the data do not exclude the possibility that silencing of this gene occurs simply as a manifestation of altered DNMT activity in these cells (Robertson, 2001); furthermore, the data do not exclude the possibility that TFPI-2 expression is governed by other genes that are regulated by DNA methylation.…”

Promoter methylation and silencing of the tissue factor pathway inhibitor-2 (TFPI-2), in human glioma cells

“…The wild-type pTF6 construct used in this study has been described previously by Konduri et al (2002). From this construct, the promoter region was excised using KpnI and XhoI restriction enzymes, gel purified and incubated with SsssI methylase and then ligated into the PGL-3 basic luciferase reporter vector.…”

“…Most of the TFPI-2 protein is associated with the extracellular matrix (Kaludov and Wolffe, 2000;Rao et al, 1998). TFPI-2 inhibits trypsin, plasmin, chymotrypsin, cathepsin G, plasma kallikerin and factor VIIa-tissue factor complex but not uPA, tissue plasminogen activator (tPA), or thrombin (Bauer et al, 1994;Konduri et al, 2001aKonduri et al, , 2002. The TFPI-2 gene has been mapped to chromosome 6q22 by fluorescence in situ hybridization (Petersen et al, 1996;Miyagi et al, 1996).…”