Endogenously released or exogenously administered glucocorticosteroids are relevant hormones for controlling inflammation. Only 11β-hydroxy glucocorticosteroids, but not 11-keto glucocorticosteroids, activate glucocorticoid receptors. Since we found that glomerular mesangial cells (GMC) express 11β-hydroxysteroid dehydrogenase 1 (11β-OHSD1), which interconverts 11-keto glucocorticosteroids into 11β-hydroxy glucocorticosteroids (cortisone/cortisol shuttle), we explored whether 11β-OHSD1 determines the antiinflammatory effect of glucocorticosteroids. GMC exposed to interleukin (IL)-1β or tumor necrosis factor α (TNF-α) release group II phospholipase A2 (PLA2), a key enzyme producing inflammatory mediators. 11β-hydroxy glucocorticosteroids inhibited cytokine-induced transcription and release of PLA2 through a glucocorticoid receptor–dependent mechanism. This inhibition was enhanced by inhibiting 11β-OHSD1. Interestingly, 11-keto glucocorticosteroids decreased cytokine-induced PLA2 release as well, a finding abrogated by inhibiting 11β-OHSD1. Stimulating GMC with IL-1β or TNF-α increased expression and reductase activity of 11β-OHSD1. Similarly, this IL-1β– and TNF-α–induced formation of active 11β-hydroxy glucocorticosteroids from inert 11-keto glucocorticosteroids by the 11β-OHSD1 was shown in the Kiki cell line that expresses the stably transfected bacterial β-galactosidase gene under the control of a glucocorticosteroids response element. Thus, we conclude that 11β-OHSD1 controls access of 11β-hydroxy glucocorticosteroids and 11-keto glucocorticosteroids to glucocorticoid receptors and thus determines the anti-inflammatory effect of glucocorticosteroids. IL-1β and TNF-α upregulate specifically the reductase activity of 11β-OHSD1 and counterbalance by that mechanism their own proinflammatory effect.

No abstract

A semipermissive growth condition was defined for a Schizosaccharomyces pombe strain carrying a thermosensitive allele of DNA polymerase ␦ (pol␦ts03). Under this condition, DNA polymerase ␦ is semidisabled and causes a delay in S-phase progression. Using a genetic strategy, we have isolated a panel of mutants that enter premature mitosis when DNA replication is incomplete but which are not defective for arrest in G 2 /M following DNA damage. We characterized the aya14 mutant, which enters premature mitosis when S phase is arrested by genetic or chemical means. However, this mutant is sensitive to neither UV nor gamma irradiation. Two genomic clones, rad26؉ and cds1 ؉ , were found to suppress the hydroxyurea sensitivity of the aya14 mutant. Genetic analysis indicates that aya14 is a novel allele of the cell cycle checkpoint gene rad26 ؉ , which we have named rad26.a14. cds1؉ is a suppressor which suppresses the S-phase feedback control defect of rad26.a14 when S phase is inhibited by either hydroxyurea or cdc22, but it does not suppress the defect when S phase is arrested by a mutant DNA polymerase. Analyses of rad26.a14 in a variety of cdc mutant backgrounds indicate that strains containing rad26.a14 bypass S-phase arrest but not G 1 or late S/G 2 arrest. A model of how Rad26 monitors S-phase progression to maintain the dependency of cell cycle events and coordinates with other rad/hus checkpoint gene products in responding to radiation damage is proposed.Maintaining the genome stability of a cell requires a complex network of checkpoint mechanisms that ensure both that DNA replication cannot be initiated until completion of the previous mitosis and that mitosis does not occur until DNA replication is completed and damaged DNA is repaired (6,10,13,19,24,28,33,35). Checkpoints that control mitosis in response to DNA status have been investigated genetically in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe (1-4, 8, 12, 17, 29, 39, 40, 44-46, 48-50). DNA damage checkpoints respond to radiation damage by delaying cell cycle progression, thereby providing cells time to repair the DNA before entering mitosis (6,10,19,33,38). When DNA replication is delayed or blocked, the S phase to mitosis checkpoint (hereafter termed the S-M-phase checkpoint or S-phase feedback control) prevents cells from entering mitosis with incompletely replicated chromosomes. Although genetic evidence has indicated that the DNA damage and S-M-phase checkpoint rely on two distinct pathways (8,14), several mutants isolated from budding and fission yeasts that are defective in DNA damage checkpoints also show sensitivity to hydroxyurea (an S-phase inhibitor), suggesting that the Sphase feedback control and the G 2 /M-phase DNA damage checkpoint require many of the same gene products (3,12,25,26,40,50).Genetic evidence from studies of fission yeast has suggested that the replication complex is a possible source of the S-Mphase checkpoint (6,7,9,11,23,27,28,35,42). However, little is currently known about...

DNA uptake can be facilitated by addition of physiological amounts of llß-hydroxy glucocorticosteroids (such as cortisol) during transfection. In the presence of cortisol, but not of the inactive 11-keto glucocorticoid cortisone, twice as many cells uptake and express the reporter gene. The effect is specific and dose-dependent; the amounts of glucocorticosteroids needed to enhance transfection efficiency are in the nanomolar range, which corresponds to the dissociation constant of glucocorticoids for the glucocorticoid receptor in vitro. This effect can be abolished by an excess of the glucocorticoid antagonist RU486. We infer that the activated cytoplasmic glucocorticoid receptors enhance nuclear translocation of the incoming transfected DNA.

MSSP has been identified as a protein that binds to both single-and double-stranded sequences of a putative DNA replication origin sequence in the human c-myc gene. MSSP possesses versatile functions, including stimulation of DNA replication, transcriptional regulation, apoptosis induction, and cell transformation coordinated by c-Myc. MSSP contains two RNP domains, RNP1-A and RNP1-B, both of which are necessary for all of the functions of MSSP. In this study, we found that MSSP binds to the N-terminal region of a catalytic subunit of a human DNA polymerase K K via its RNP domains both in vitro and in human cells. Furthermore, MSSP was released from the putative DNA replication origin of the c-myc gene after it complexed with DNA polymerase K K, and MSSP stimulated DNA polymerase activity in vitro. ß