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The ERK/MAPK pathway regulates the activity of the human tissue factor pathway inhibitor-2 promoter
Christina Kast1, Minglun Wang, Malcolm Whiteway
1Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Ave., Montreal, Quebec H4P 2R2, Canada. Christina.Kast@insel.ch
Abstract:
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.
Insights
Human tissue factor pathway inhibitor-2 (hTFPI-2) gene expression is activated by the Ras/Raf/MEK/ERK pathway. This study identifies key promoter regions and transcription factor binding sites involved in hTFPI-2 regulation.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Human tissue factor pathway inhibitor-2 (hTFPI-2) is an extracellular matrix-associated serine protease inhibitor.
- hTFPI-2 plays a potential role in tumor invasion and metastasis by inhibiting matrix-degrading proteases.
Purpose of the Study:
- To investigate the signal transduction pathway regulating hTFPI-2 gene activation.
- To identify the promoter regions and transcription factors involved in hTFPI-2 gene expression.
Main Methods:
- Cloning of the hTFPI-2 promoter region linked to a luciferase reporter gene.
- Induction studies using phorbol 12-myristate 13-acetate (PMA) and epidermal growth factor (EGF) in various cell lines (HEK293, A549, MCF7, HeLa).
- Analysis of promoter activity using deletion constructs and MEK inhibitor (UO126).
- Identification of transcription factor binding sites (AP-1, Sp1) using TFSEARCH and mutagenesis.
Main Results:
- PMA and EGF induced luciferase reporter gene expression, indicating activation of the hTFPI-2 promoter.
- MEK inhibitor UO126 blocked PMA-induced activation, implicating the MEK pathway.
- Constitutively active Ras/Raf/MEK/ERK components strongly induced reporter gene expression.
- Minimal basal promoter activity was localized between -89 and -384, while minimal inducible activity was between -89 and -222.
- AP-1 and Sp1 binding sites were identified at positions -156 and -134, respectively.
Conclusions:
- The Ras/Raf/MEK/ERK signaling pathway mediates the activation of the hTFPI-2 promoter.
- Specific promoter regions and transcription factors, including AP-1 and Sp1, are crucial for basal and inducible hTFPI-2 gene expression.