Proteinase Inhibitor 9, an Inhibitor of Granzyme B-mediated Apoptosis, Is a Primary Estrogen-inducible Gene in Human Liver Cells

• Published in: The Journal of biological chemistry 2000-02, Vol.275 (8), p.5867-5873
• Main Authors: Kanamori, Hiroshi, Krieg, Sacha, Mao, Chengjian, Di Pippo, Vincent A., Wang, Stanley, Zajchowski, Deborah A., Shapiro, David J.
• Format: Article
• Language: English
• Subjects: Apoptosis - drug effects; Base Sequence; Blotting, Northern; Carcinoma, Hepatocellular - metabolism; Dose-Response Relationship, Drug; Estrogen Antagonists - pharmacology; estrogen receptors; estrogens; Estrogens - pharmacology; Ethinyl Estradiol - analogs & derivatives; Ethinyl Estradiol - pharmacology; Gene Expression Regulation; granzyme B; Granzymes; Humans; Liver - metabolism; Molecular Sequence Data; moxestrol; Promoter Regions, Genetic; proteinase inhibitor 9; Response Elements; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - drug effects; Serine Endopeptidases - metabolism; Serine Proteinase Inhibitors - genetics; Serine Proteinase Inhibitors - physiology; Serpins - genetics; Serpins - physiology; Time Factors; Transcription, Genetic - drug effects; Tumor Cells, Cultured
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.275.8.5867

Although liver is an estrogen target tissue, the number of hepatic genes known to be directly induced by estrogen is very small. We identified proteinase inhibitor 9, or PI-9, as being rapidly and strongly induced by estrogen in an estrogen receptor-positive human liver cell line (HepG2-ER7). Since PI-9 mRNA was also induced by estrogen in a human liver biopsy sample, PI-9 is a genuine estrogen-regulated human gene. PI-9 is a potent inhibitor of granzyme B and of granzyme B-mediated apoptosis. Estrogens induced PI-9 mRNA within 2 h, PI-9 mRNA levels reached a plateau of 30–40-fold induction in 4 h, and induction was not blocked by cycloheximide, indicating that induction of PI-9 mRNA is a primary response. The antiestrogen trans-hydroxytamoxifen was a partial agonist for PI-9 mRNA induction, whereas the antiestrogen ICI 182,780 was a pure antagonist. Western blot analysis showed that estrogen strongly increases PI-9 protein levels. Inhibition of transcription with actinomycin D resulted in identical rates of PI-9 mRNA decay in the presence and absence of estrogen. We isolated genomic clones containing the PI-9 promoter region, identified a putative transcription start site, and carried out transient transfections of PI-9-luciferase reporter gene constructs. The estrogen, moxestrol, elicited a robust induction from the PI-9-luciferase reporter. Mutational inactivation of three potential imperfect estrogen response elements in the PI-9 5′-flanking region had no effect on moxestrol estrogen receptor induction.