cis-Elements Required for Expression of Human Protein C Inhibitor Gene in HepG2 Cells and Its Androgen-Dependent Expression in Rat Reproductive Organs

Koji Suzuki; Tatsuya Hayashi

doi:10.1055/s-2000-9807

Seminars in Thrombosis and Hemostasis, Table of Contents

Semin Thromb Hemost 2000; Volume 26(Number 01): 075-084
DOI: 10.1055/s-2000-9807

cis-Elements Required for Expression of Human Protein C Inhibitor Gene in HepG2 Cells and Its Androgen-Dependent Expression in Rat Reproductive Organs

Koji Suzuki, Tatsuya Hayashi

Supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture and Sports of Japan (nos.06771216 and 07780509). Department of Molecular Pathobiology, Mie University School of Medicine, Tsu-city, Mie, Japan

Abstract

ABSTRACT

Protein C inhibitor (PCI) is a plasma serine protease inhibitor of activated protein C, which is the main protease of the anticoagulant protein C pathway. Human PCI is synthesized in the liver, kidney, and several reproductive organs (testis, seminal vesicle, and prostate). In the present study, we characterized cis-elements of the human PCI gene required for expression in the hepatoma-derived cell line, HepG2 cells, and also evaluated rat PCI mRNA expression, particularly on the effect of androgen in rat reproductive tissues. On the PCI gene expression in HepG2 cells, transient expression assays using several deletion mutants and site-directed mutants of the human PCI gene and gel mobility shift assays using several synthetic oligonucleotides showed that the Sp1-binding site (residues -302 to -294) and the upstream AP2-binding site (residues -350 to -343) play roles as the promoter and the enhancer, respectively. Both the A-activator-binding site (residues -422 to -414) and the interferon-γ response element (residues -164 to -157) serve as the silencer. In the study on PCI mRNA expression in the reproductive organs, we first cloned rat PCI cDNA and then evaluated the effect of androgen on the PCI mRNA expression. The isolated rat PCI cDNA contained a 1,218-bp coding region of a 406-amino acid precursor protein. The deduced amino acid sequence of rat PCI showed an 85.7% and 62.2% homology with that of mouse and human PCIs, respectively. Northern blot analysis showed that rat PCI mRNA was strongly expressed in the seminal vesicles, moderately in the testes, but not in the liver. PCI mRNA expression in seminal vesicles and testes increased during the process of development. The PCI mRNA expression in seminal vesicles was significantly decreased after castration or after 17β-estradiol treatment. Treatment with testosterone in the castrated rats significantly enhanced its mRNA expression. These findings suggest that the PCI gene expression in rat seminal vesicles is under androgen control.

KEYWORD

Protein C inhibitor gene - transcriptional gene regulation - cis-elements - reproductive tissues - androgen control

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