Estrogen regulation of proteinase inhibitor 9 gene expression and interleukin-1beta production

Estrogen has long been known to protect against diseases of inflammatory origin such as atherosclerosis and osteoporosis. We recently identified an estrogen inducible inhibitor of caspase 1, a primary effector of inflammation. This inhibitor is proteinase inhibitor-9 (PI-9). PI-9 is induced by estrogen in ER positive cultured human liver cells and in human liver. To analyze the role of PI-9 in inflammatory disease, we are investigating ethanol-induced inflammation in liver cell culture. Caspase-1 catalyzes the proteolytic maturation of inactive prointerleukin-1β (IL-1β) to the potent pro-inflammatory cytokine IL-1β. ELISA analysis of liver cells exposed to ethanol showed a marked decrease in IL-1β secretion in response to estrogen. These findings suggest that PI-9 expression in response to estrogen can ultimately inhibit IL-1β maturation in HepG2 cells, thereby preventing a crucial event in both rapid and chronic inflammatory disease.; To dissect the mechanism of estrogen-induced PI-9 transcription, I isolated a human lambda genomic clone containing the PI-9 promoter. When this promoter was placed upstream of a luciferase reporter a 10 to 15-fold estrogen induction of luciferase activity was seen. Deletion analysis of various potential promoter elements led to the identification of a novel downstream estrogen responsive unit (ERU) consisting of an imperfect estrogen response element and two half site direct repeats. Electrophoretic mobility shift assays demonstrated that this ERU interacts with 2 ER dimers. This finding extends the range of DNA sequences that can function as estrogen responsive elements.; The ability of several estrogens and antiestrogens to activate PI-9 gene expression was assessed by transient transfection. Of interest, the mixed agonist/antagonist tamoxifen (OHT) was an agonist on the transiently transfected reporter gene but behaved as an antagonist on the cellular gene. To begin to assess how OHT-ER interacts with the native PI-9 promoter we carded out chromatin immunoprecipitation (ChIP) assays. The ChIP assays demonstrated that moxestrol-ER complex bound to the PI-9 promoter, but that OHT-ER (and unliganded ER) did not bind. These data provide the first evidence that the agonist activity of OHT-ER stems from the inability of OHT to elicit conformational changes in chromatin which allow ER to bind to ERE's.