The sexually differentiated delta 4-3-ketosteroid 5 beta-reductase of rat liver. Purification, characterization, and quantitation.

delta 4-3-Ketosteroid 5 beta-reductase was purified from male rat liver cytosol. The purification scheme consisted of column chromatographies on hydroxylapatite and DEAE-Sepharose, chromatofocusing, and Sephadex G-75 gel filtration followed by sodium dodecyl sulfate-gel electrophoresis. The column chromatography steps gave a 100-fold purification and resulted in a 90% pure preparation as judged by sodium dodecyl sulfate-gel electrophoresis. Kinetic properties with 4-androstene-3,17-dione as substrate were established for the enzyme, and its activity regarding three other delta 4-3-ketosteroids, testosterone, progesterone, and cortisol, was investigated. The relative rates of reduction of these steroids were 1.0, 0.8, 0.7, and 0.62, respectively. The electrophoretically purified 5 beta-reductase, with an Mr of 38,000, was used for immunization of rabbits. The antiserum was shown to be monospecific as judged from immunoblotting of electrophoretically separated rat liver cytosolic proteins. Immunological reactive protein and enzymatic 5 beta-reductase activity co-purified in the chromatographic steps. The sex difference in enzyme activity, 0.26 versus 0.10 nmol of product/mg of proteins/min for males and females, respectively, was shown to be due to a difference in concentration of enzyme protein. The 5 beta-reductase was calculated to constitute 1% of the total cytosolic proteins in male livers, whereas the corresponding figure for female livers was 0.3%.

Paracrine glucocorticoid activity produced by mouse thymic epithelial cells.

Previous data have suggested that glucocorticoids (GCs) are involved in the differentiation of thymocytes into mature T cells. In this report we demonstrate that the mouse thymic epithelial cells (TEC) express the cytochrome P450 hydroxylases Cyp11A1, Cyp21, and Cyp11B1. These enzymes, in combination with 3beta-hydroxysteroid dehydrogenase (3betaHSD), convert cholesterol into corticosterone, the major GC in rodents. In addition, when TEC were cocultured with 'reporter cells' containing the glucocorticoid receptor (GR) and a GR-dependent reporter gene, a specific induction of reporter gene activity was observed. Induction of reporter gene activity was blocked when the TEC and reporter cells were incubated in the presence of the Cyp11B1 inhibitor metyrapone or the 3betaHSD inhibitor trilostane, as well as by the GR antagonist RU486. Coculturing of TEC with thymocytes induced apoptosis in the latter, which was partially blocked by the enzyme inhibitors and RU486. We conclude that TEC secrete a GC hormone activity and suggest a paracrine role for this in thymocyte development.

Increased c-fos mRNA and binding to the AP-1 recognition sequence accompanies the proliferative response to deoxycholate of HT29 cells.

To further understand the molecular mechanisms of bile acid-mediated colon tumor promotion, we have examined the possible role of AP-1 activity in this process. The AP-1 complex has been reported to play an important role in control of cell growth. Our studies show that lithocholate, deoxycholate and ursodeoxycholate exhibited marked proliferative effects on a human adenocarcinoma cell line (HT29), while cholate was without effect. The proliferative effects appeared to be confined to narrow concentration windows which differed for the different bile acids. We demonstrate that deoxycholate caused an increase in expression of c-fos mRNA and increased binding to the AP-1 site, effects which were maximum at the concentration at which the bile acid induced the maximum proliferative effect on the cells. Cholate was without effect on AP-1 binding activity. In addition, we show that the AP-1 complex induced by treatment of the cells with the bile acid contained the c-fos protein. This could suggest that prolonged deregulated expression of AP-1 activity in colonic cells by certain bile acids may contribute to tumor promotion in the colon.

A new function for the C-terminal zinc finger of the glucocorticoid receptor. Repression of RelA transactivation.

Glucocorticoids inhibit NF-kappaB signaling by interfering with the NF-kappaB transcription factor RelA. Previous studies have identified the DNA-binding domain (DBD) in the glucocorticoid receptor (GR) as the major region responsible for this repressive activity. Using GR mutants with chimeric DBDs the repressive function was found to be located in the C-terminal zinc finger. As predicted from these results the mineralocorticoid receptor that contains a C-terminal zinc finger identical to that of the GR was also able to repress RelA-dependent transcription. Mutation of a conserved arginine or a lysine in the second zinc finger of the GR DBD (Arg-488 or Lys-490 in the rat GR) abolished the ability of GR to inhibit RelA activity. In contrast, C-terminal zinc finger GR mutants with mutations in the dimerization box or mutations necessary for full transcriptional GR activity were still able to repress RelA-dependent transcription. In addition, we found that the steroid analog ZK98299 known to induce GR transrepression of AP-1 had no inhibitory effect on RelA activity. In summary, these results demonstrate that the inhibition of NF-kappaB by glucocorticoids involves two critical amino acids in the C-terminal zinc finger of the GR. Furthermore, the results from the use of mineralocorticoid receptor and anti-glucocorticoids suggest that the mechanisms for GR-mediated repression of NF-kappaB and AP-1 are different.

Glucocorticoid effects on NF-kappaB binding in the transcription of the ICAM-1 gene.

Glucocorticoid hormones are potent antiinflammatory drugs. A key mechanism in the antiinflammatory action is repression of the nuclear factor kappa B (NF-kappaB) signaling pathway. This results in transcriptional repression of inflammatory genes controlled by NF-kappaB, including the intercellular adhesion molecule-1 (ICAM-1). We have investigated expression levels, nuclear translocation and DNA binding of NF-kappaB in vitro and in vivo in U937 cells during activation and repression. Repression of NF-kappaB signaling by glucocorticoids does not prevent NF-kappaB translocation or DNA binding. However interestingly, in vivo foot printing of the NF-kappaB site in the ICAM-1 gene indicates that glucocorticoids change the conformation of the protein complex binding to the NF-kappaB site. These results suggests that NF-kappaB interaction with the glucocorticoid receptor does not displace NF-kappaB from its DNA binding site but rather changes the complex into a transcriptionally inert form.