Proteinase inhibitor 9, an inhibitor of granzyme B-mediated apoptosis, is a primary estrogen-inducible gene in human liver cells

Although liver is an estrogen target tissue, the number of hepatic genes kn own to be directly induced by estrogen is very small. We identified protein ase 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 mRN A 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 induc tion of PI-9 mRNA is a primary response. The antiestrogen trans-hydroxytamo xifen was a partial agonist for PI-9 mRNA induction, whereas the antiestrog en ICI 182,780 was a pure antagonist. Western blot analysis showed that est rogen strongly increases PI-9 protein levels. Inhibition of transcription w ith actinomycin D resulted in identical rates of PI-B mRNA decay in the pre sence and absence of estrogen, We isolated genomic clones containing the PI -9 promoter region, identified a putative transcription start site, and car ried out transient transfections of PI-g-luciferase reporter gene construct s. The estrogen, moxestrol, elicited a robust induction from the PI-9-lucif erase reporter, Mutational inactivation of three potential imperfect estrog en response elements in the PI-9 5'-flanking region had no effect on moxest rol estrogen receptor induction.