Stuart Adler

Estrogen decreases TNF gene expression by blocking JNK activity and the resulting production of c-Jun and JunD

Central to the bone-sparing effect of estrogen (E(2)) is its ability to block the monocytic production of the osteoclastogenic cytokine TNF-alpha (TNF). However, the mechanism by which E(2) downregulates TNF production is presently unknown. Transient transfection studies in HeLa cells, an E(2) receptor-negative line, suggest that E(2) inhibits TNF gene expression through an effect mediated by estrogen receptor beta (ERbeta). We also report that in RAW 264.7 cells, an E(2) receptor-positive murine monocytic line, E(2) downregulates cytokine-induced TNF gene expression by decreasing the activity of the Jun NH(2)-terminal kinase (JNK). The resulting diminished phosphorylation of c-Jun and JunD at their NH(2)-termini decreases the ability of these nuclear proteins to autostimulate the expression of the c-Jun and JunD genes, thus leading to lower production of c-Jun and JunD. The consequent decrease in the nuclear levels of c-Jun and JunD leads to diminished binding of c-Jun/c-Fos and JunD/c-Fos heterodimers to the AP-1 consensus sequence in the TNF promoter and, thus, to decreased transactivation of the TNF gene.

Site-directed mutagenesis identifies amino acid residues associated with the dehydrogenase and isomerase activities of human type I (placental) 3β-hydroxysteroid dehydrogenase/Isomerase

3beta-hydroxysteroid dehydrogenase/steroid delta5-->4-isomerase (3beta-HSD/isomerase) was expressed by baculovirus in Spodoptera fungiperda (Sf9) insect cells from cDNA sequences encoding human wild-type I (placental) and the human type I mutants - H261R, Y253F and Y253,254F. Western blots of SDS-polyacrylamide gels showed that the baculovirus-infected Sf9 cells expressed the immunoreactive wild-type, H261R, Y253F or Y253,254F protein that co-migrated with purified placental 3beta-HSD/isomerase (monomeric Mr=42,000 Da). The wild-type, H261R and Y253F enzymes were each purified as a single, homogeneous protein from a suspension of the Sf9 cells (5.01). In kinetic studies with purified enzyme, the H261R mutant enzyme had no 3beta-HSD activity, whereas the Km and Vmax values of the isomerase substrate were similar to the values obtained with the wild-type and native enzymes. The Vmax (88 nmol/min/mg) for the conversion of 5-androstene-3,17-dione to androstenedione by the Y253F isomerase activity was 7.0-fold less than the mean Vmax (620 nmol/min/mg) measured for the isomerase activity of the wild-type and native placental enzymes. In microsomal preparations, isomerase activity was completely abolished in the Y253,254F mutant enzyme, but Y253,254F had 45% of the 3beta-HSD activity of the wild-type enzyme. In contrast, the purified Y253F, wild-type and native enzymes had similar Vmax values for substrate oxidation by the 3beta-HSD activity. The 3beta-HSD activities of the Y253F, Y253,254F and wild-type enzymes reduced NAD+ with similar kinetic values. Although NADH activated the isomerase activities of the H261R and wild-type enzymes with similar kinetics, the activation of the isomerase activity of H261R by NAD+ was dramatically decreased. Based on these kinetic measurements, His261 appears to be a critical amino acid residue for the 3beta-HSD activity, and Tyr253 or Tyr254 participates in the isomerase activity of human type I (placental) enzyme.

Transcriptional regulation of estrogen-responsive genes by non-steroidal estrogens: Doisynolic and allenolic acids

Estrogen receptor (ER), a member of the nuclear receptor superfamily, exerts prominent physiological roles in both humans and other species by acting directly as a transcription factor, altering nuclear gene expression. One peculiarity of estrogenic regulation is that it is affected by a wide variety of non-steroidal compounds in addition to the natural hormone, estradiol. Doisynolic and allenolic acid compounds are non-steroidal compounds that act as potent estrogens in animal studies, yet bind to ER extremely poorly in competitive binding assays, raising the possibility of alternative molecular mechanisms for the observed estrogenic effects. In this work we demonstrate that (+/-)-Z-bisdehydrodoisynolic acid, (+/-)-Z-bisdehydrodoisynolic acid 3-methyl ether, and (-) allenolic acid can interact directly with ER. These compounds all serve as ligands for ER in mechanism-specific tissue culture-based reporter gene assays for both positive and negative gene regulation. We have also used a novel assay based on electromobility shift by ER for directly determining relative binding affinities for ER. In addition, we show cell-type-specific activity differences for (+/-)-Z-bisdehydrodoisynolic acid 3-methyl ether, supporting clinical observations indicating a higher potency of this compound in female animals than in humans.

Estrogenic activity of RU 486 (mifepristone) in rat uterus and cultured uterine myocytes

OBJECTIVE Our purpose was to determine whether RU 486 (mifepristone) has direct estrogenic activity in uterine myocytes. STUDY DESIGN Ovariectomized adult rats were treated with RU 486, and its effect on uterine oxytocin receptor concentration, as a marker of estrogenic activity, was measured. Results were compared with the induction by RU 486 of an estrogen-responsive reporter gene in a cultured Syrian hamster uterine myocyte cell line. RESULTS Baseline oxytocin receptor concentration was 58.8 +/- 7.2 fmol/mg protein (mean +/- SEM) and increased to 227 +/- 49 fmol/mg with 17 beta-estradiol (2.5 micrograms/kg) and to 145 +/- 18 fmol/mg after RU 486 (5 mg/kg) treatment, an effect that was inhibited by the antiestrogen ICI 182,780 (1.5 mg/kg). In the cultured Syrian hamster uterine myocyte cell line cells RU 486 (10(-6) mol/L) caused a 2.17 +/- 0.17-fold increase in the expression of the reporter gene versus 113.0 +/- 7.4-fold with 17 beta-estradiol (10(-8) mol/L). The estrogenic activity of RU 486 was dependent on the presence of both estrogen receptor and the promoters estrogen response element. CONCLUSION RU 486 has a weak estrogen-like activity in uterine myocytes. This activity may partly explain the therapeutic effects of RU 486 on this target organ.