To inhibit arthritis upstream of inflammatory cytokine release and matrix metalloproteinase (MMP) action, we designed de novo a small-molecule inhibitor of c-Fos/activator protein-1 (AP-1) using three-dimensional (3D) pharmacophore modeling. This model was based on the 3D structure of the basic region-leucine zipper domain of AP-1-DNA complex. Administration of this inhibitor prevented type II collagen-induced arthritis from day 21, before the onset of arthritis, or from day 27, resolved arthritis after its onset. Suppression of disease was accomplished by reducing the amounts of inflammatory cytokines and MMPs in vivo in sera and joints and in vitro in synovial cell and chondrocyte cultures. The primary action of this molecule was the inhibition of matrix-degrading MMPs and inflammatory cytokines including interleukin 1beta; this molecule also synergized with anti-tumor necrosis factor alpha to inhibit arthritis. Thus, selective inhibition of c-Fos/AP-1 resolves arthritis in a preclinical model of the disease.
A functional cDNA clone for the mouse prostaglandin I PG) E receptor EP 2 subtype was isolated from a mouse cDNA library. The mouse EP2 receptor consists of 362 amino acid residues with seven putative transmembrane domains. [3H]PGE2 bound specifically to the membrane of Chinese hamster ovary tells stably expressing the cloned receptor. This binding was displaced by unlabeled prostanoids in the order of PGEz = I'GEt >> iloprost, a stable PGI 2 agonist > PGFz~ > PGDz. Binding was also inhibited by butaprost (an EP2 agonist) and to a lesser extent by M&B 28767 (an EP 3 agonist), but not by sulprostone (an EP~ and EP 3 agonis0 or SC-19220 (an EP~ antagonist). PGE 2 and butaprost increased the cAMP level in the Chinese hamster ovary cells in a concentration-dependent manner. Northvrn blot analysis revealed that EP 2 mRNA is expressed most abundantly in the uterus, followed by the spleen, lung, thymus, ileum, liver, and stomach.
Regional and cellular distribution of mRNAs for prostaglandin E (PGE) receptor subtypes was investigated in the mouse gastrointestinal tract by in situ hybridization. Strong signals for EP1 transcripts were detected in cells of the muscularis mucosae layer, especially in the body of the stomach. Intense signals for EP3 transcripts were detected in neurons of the myenteric ganglia throughout the tract. Moderate EP3 mRNA expression was also observed in fundic gland epithelial cells, except for surface mucous cells in the stomach. Expression of EP4 mRNA was moderate in surface epithelial cells of the corpus and in glands from the surface to the base of the antrum. Strong EP4 signals were observed in the epithelium in the duodenum, jejunum, and ileum. In the ileum, signals were only observed in the upper part of the villi. However, no or weak signals for EP2 transcripts were detected. These findings suggest that PGE2 modulates various gastric or intestinal functions via at least three different PGE receptors.
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