Identification of pathway-selective estrogen receptor ligands that inhibit NF-kappaB transcriptional activity

Abstract

Inflammation is now recognized as a key component in a number of diseases such as atherosclerosis, rheumatoid arthritis, and inflammatory bowel disease. The transcription factor NF-kappaB has been shown to be involved in both the early and late stages of the inflammatory-proliferative process. In this report, we describe the identification of the pathway-selective estrogen receptor (ER) ligand, WAY-169916, that inhibits NF-kappaB transcriptional activity but is devoid of conventional estrogenic activity. This pathway-selective ligand does not promote the classic actions of estrogens such as stimulation of uterine proliferation or ER-mediated gene expression, but is a potent antiinflammatory agent, as demonstrated in the HLA-B27 transgenic rat model of inflammatory bowel disease. Our results indicate the potential utility of pathway-selective ER ligands such as WAY-169916 in the treatment of chronic inflammatory diseases.

Fig. 1.

The in vitro activity of WAY-169916. (A) The structure of WAY-169916. (B) Effect of WAY-169916 on NF-κB-mediated luciferase reporter activity in HAECT-1 cells expressing ERα. The cells were stimulated with IL-1β and cotreated with increasing concentrations of WAY-169916 (▵), E2 (▪), or raloxifene (•). (C) IL-6 expression from the same experiment was determined by ELISA, using the culture media. (D) The remaining lysate from B was used to determine CK activity. (E) The experiment was performed as in B, except the cells were treated with 1 μM WAY-169916, raloxifene, or ICI as indicated. (F) The experiment was performed as in D, except the cells were treated with 2 μM WAY-169916 or 30 nM E2 as indicated. *, P < 0.1 vs. vehicle treatment; **, P < 0.1 vs. E2 treatment.

Fig. 2.

WAY-169916 inhibits IL-6 promoter activity through ERα or ERβ. (A) Effect of WAY-169916 on the human IL-6 promoter luciferase reporter activity in HAECT-1 cells expressing ERβ. The cells were stimulated with IL-1β and cotreated with increasing concentrations of either WAY-169916 (▵) or E2 (▪). (B) Experiments were performed as above, except the cells were expressing ERα instead of ERβ.

Fig. 3.

Selective inhibition of diet-induced inflammatory gene expression by WAY-169916. Ovariectomized C57BL/6 mice were fed a chow diet with vehicle treatment (hatched boxes), a high-fat diet with vehicle (black boxes), WAY-169916 (10 mg/kg orally, white boxes), or EE (10 μg/kg orally, gray boxes). Steady-state liver mRNA levels for vascular cell adhesion molecule 1 (VCAM-1), TNFα, and regulated upon activation, normal T cell-expressed and secreted factor (RANTES) (A) or intestinal trefoil factor (ITF) and myo-inositol-1-phosphate synthase (IPS) (B) are shown. Uterine wet weights were also recorded.

Fig. 4.

Effect of WAY-169916 on stool scores in the HLA-B27 transgenic rat. (A) Rats demonstrating chronic diarrhea were treated with vehicle or WAY-169916 (0.5, 0.15, and 0.05 mg/kg orally) for 8 days. (B) Rats demonstrating chronic diarrhea were treated with vehicle, WAY-169916 (0.5 mg/kg orally, once a day), or WAY-169916 plus ICI (25 mg/kg s.c., twice a day) for 8 days. Stool scoring was as follows: 3, diarrhea; 2, soft stool; 1, normal stool, and was plotted as mean ± SD.