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Vitiligo is common in the hereditary disorder autoimmune polyendocrine syndrome type I (APS I). Patients with APS I are known to have high titer autoantibodies directed against various tissue-specific antigens. Using sera from APS I patients for immunoscreening of a cDNA library from human scalp, we identified the transcription factors SOX9 and SOX10 as novel autoantigens related to this syndrome. Immunoreactivity against SOX9 was found in 14 (15%) and against SOX10 in 20 (22%) of the 91 APS I sera studied. All patients reacting with SOX9 displayed reactivity against SOX10, suggesting shared epitopes. Among the 19 patients with vitiligo, 12 (63%) were positive for SOX10 (p < 0.0001). Furthermore, three of 93 sera from patients with vitiligo unrelated to APS I showed strong reactivity against SOX10, which may indicate a more general role of SOX10 as an autoantigen in vitiligo.

Thioredoxin (Trx) is a small ubiquitous protein that displays different functions mainly via redox-mediated processes. We here report the cloning of a gene (trxC) coding for a novel thioredoxin in Escherichia coli as well as the expression and characterization of its product. The gene encodes a protein of 139 amino acids (Trx2) with a calculated molecular mass of 15.5 kDa. Trx2 contains two distinct domains: an N-terminal domain of 32 amino acids including two CXXC motifs and a C-terminal domain, with the conserved active site, Trp-Cys-Gly-Pro-Cys, showing high homology to the prokaryotic thioredoxins. Trx2 together with thioredoxin reductase and NADPH is an efficient electron donor for the essential enzyme ribonucleotide reductase and is also able to reduce the interchain disulfide bridges of insulin. The apparent Km value of Trx2 for thioredoxin reductase is similar to that of the previously characterized E. coli thioredoxin (Trx1). The enzymatic activity of Trx2 as a protein-disulfide reductase is increased by preincubation with dithiothreitol, suggesting that oxidation of cysteine residues other than the ones in the active site might regulate its activity. A truncated form of the protein, lacking the N-terminal domain, is insensitive to the presence of dithiothreitol, further confirming the involvement of the additional cysteine residues in modulating Trx2 activity. In addition, the presence of the N-terminal domain appears to confer heat sensitivity to Trx2, unlike Trx1. Finally, Trx2 is present normally in growing E. coli cells as shown by Western blot analysis.

Alternative splicing of the human glucocorticoid receptor (hGR) primary transcript generates two receptor isoforms, hGRalpha and hGRbeta, with different carboxyl termini diverging at amino acid 727. By reverse transcriptase-polymerase chain reactions it was previously demonstrated that the hGRbeta message had a widespread tissue distribution. To demonstrate the presence of hGRbeta as protein we produced specific rabbit antisera to hGRbeta, as well as a hGRbeta-specific mouse monoclonal IgM antibody, by peptide immunizations. By SDS-polyacrylamide gel electrophoresis and Western immunoblotting we showed that hGRbeta is endogenously expressed at the protein level in HeLa cells and human lymphatic leukemia cells. Using an antibody directed against an epitope shared by both isoforms we showed a relatively lower expression of the hGRbeta form. We also showed that hGRbeta bound to hsp90 by immunoprecipitation of in vitro translated hGRbeta in reticulocyte lysate with hsp90-specific antibodies, a coprecipitation occurring also in the presence of dexamethasone. We could not demonstrate that hGRbeta inhibited the effects of dexamethasone-activated hGRalpha on a glucocorticoid-responsive reporter gene. In conclusion, low hGRbeta expression levels and hGRbeta-hsp90 interaction maintained in the presence of ligand and lack of inhibition of hormone-activated hGRalpha effects challenge the concept of the hGRbeta isoform as a proposed dominant negative inhibitor of hGRalpha activity.

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.

Steroid hormone receptors and Stat factors comprise two distinct families of inducible transcription factors. Activation of a member of each family, namely the glucocorticoid receptor by glucocorticoids and Stat5 by prolactin, is required for the efficient induction of the expression of milk protein genes in the mammary epithelium. We have studied the mode of interaction between Stat5 and the glucocorticoid receptor in the activation of beta-casein gene transcription. The functional role of potential half-palindromic glucocorticoid receptor-binding sites mapped previously in the promoter region was investigated. beta-Casein gene promoter chloramphenicol acetyltransferase constructs containing mutations and deletions in these sites were tested for their responsiveness to the synergistic effect of prolactin and dexamethasone employing COS-7 cells or HC11 mammary epithelial cells. Synergism depended on promoter regions containing intact binding sites for the glucocorticoid receptor and Stat5. The carboxyl-terminal transactivation domains of Stat5a and Stat5b were not required for this synergism. Our results suggest that in lactogenic hormone response elements glucocorticoid receptor molecules bound to nonclassical half-palindromic sites gain competence as transcriptional activators by the interaction with Stat5 molecules binding to vicinal sites.

The glucocorticoid receptor (GR) is a ligand-activated transcription factor. In this study, we used the yeast two-hybrid system to isolate cDNAs encoding proteins that interact with the human GR ligand-binding domain (LBD) in a ligand-dependent manner. One isolated cDNA from a HeLa cell library encoded the COOH-terminal portion of the eta-isoform of the 14-3-3 protein (residues 187-246). Glucocorticoid agonists, triamcinolone acetonide and dexamethasone, induced the GR LBD/14-3-3eta protein fragment interaction, but an antagonist, RU486, did not. Glutathione S-transferase pull-down experiments in vitro showed that full-length 14-3-3eta protein also interacted with the activated GR. Transient transfection studies using COS-7 cells revealed a stimulatory effect of 14-3-3eta protein on transcriptional activation by the GR. The 14-3-3 family members have recently been found to associate with a number of important signaling proteins, such as protein kinase C and Raf-1, as functional modulators. Our findings suggest a novel regulatory role of 14-3-3eta protein in GR-mediated signaling pathways and also point to a mechanism whereby GR may cross-talk with other signal transduction systems.

We have isolated a 1276-base pair cDNA from a rat heart cDNA library that encodes a novel thioredoxin (Trx2) of 166 amino acid residues with a calculated molecular mass of 18.2 kDa. Trx2 possesses the conserved thioredoxin-active site, Trp-Cys-Gly-Pro-Cys, but lacks structural cysteines present in all mammalian thioredoxins. Trx2 also differs from the previously described rat thioredoxin (Trx1) by the presence of a 60-amino acid extension at the N terminus. This extension has properties characteristic for a mitochondrial translocation signal, and the cleavage at a putative mitochondrial peptidase cleavage site would give a mature protein of 12.2 kDa. Western blot analysis from cytosolic, peroxisomal, and mitochondrial rat liver cell fractions confirmed mitochondrial localization of Trx2. Northern blot and reverse transcriptase-polymerase chain reaction analyses revealed that Trx2 hybridized to a 1.3-kilobase message, and it was expressed in several tissues with the highest expression levels in heart, muscle, kidney, and adrenal gland. N-terminally truncated recombinant protein was expressed in bacteria and characterized biochemically. Trx2 possessed a dithiol-reducing enzymatic activity and, with mammalian thioredoxin reductase and NADPH, was able to reduce the interchain disulfide bridges of insulin. Furthermore, Trx2 was more resistant to oxidation than Trx1.

The present investigation has aimed at defining the factor responsible for the differences in microsomal metabolism of steroids between germ-free and conventional rats. Cholesterol, cholic acid, taurocholic acid, and chenodeoxycholic acid were fed to conventional and germ-free male rats and the effects on liver microsomal metabolism of 4-[4-14C]androstene-3,17-dione, 5alpha-[4-14C]androstane-3alpha,17beta-diol, [4-14C]-cholesterol, 7alpha-hydroxy-4-[6beta-3H]cholesten-3-one, and [24-14C]lithocholic acid were studied. The most consistent effects were found with dietary cholesterol that stimulated the activities of several of the hydroxylases active on 4-androstene-3,17-dione and 5alpha-androstane-3alpha,17beta-diol and that decreased the 5alpha reduction of 4-androstene-3,17-dione, increased the 7alpha hydroxylation of cholesterol, decreased the 12alpha hydroxylation of 7alpha-hydroxy-4-cholesten-3-one, and increased by 6beta hydroxylation of lithocholic acid. These effects of cholesterol feeding on the microsomal metabolism of steroids in conventional rats made the pattern of microsomal enzyme activities resemble that characteristic of germ-free rats. Cholesterol feeding led to a pronounced increase in the intestinal concentration of beta-muricholic acid in conventional rats. Furthermore, cholesterol feeding to conventional animals led to an intestinal ratio of chenodeoxycholic acid (including its metabolites alpha- and beta-muricholic acid and hyodeoxycholic acid) to cholic acid (including deoxycholic acid) that was almost identical to that in germ-free rats. Conventionalization of germ-free rats for a period of up to 56 days led only to a partial normalization of the liver microsomal metabolism of 5alpha-[4-14C]androstane-3alpha, 17beta-diol and 7alpha-hydroxy-4-[6beta-3H]cholesten-3-one and of the liver microsomal concentration of cytochrome P-450. The concentration of cholesterol was higher in both total liver homogenate and liver microsomal fraction of germ-free rats than in corresponding preparations from conventional rats. In conclusion, it is suggested that cholesterol is one of the factors responsible for the different microsomal metabolism of steroids in germ-free and conventional rats. It is also suggested that cholesterol may play a role as regulator of microsomal enzyme activities.

In vivo studies were carried out on three bile fistula patients to further elucidate the side chain oxidation pathways from C-27 bile acids to cholic acid in man. Two patients each received (25-R)- and (25-S)-3 alpha, 7 alpha,-12 alpha-trihydroxy-5 beta-[7 beta-3H]cholestanoic acid (THCA) on consecutive days and three patients wee administered 3 alpha, 7 alpha, 12 alhpa, 24 xi-tetrahydroxy-5 beta-[7 beta-3H]cholestanoic acid (varanic acid). The varanic acid was biosynthetically prepared with rat liver microsomes and was probably the 24 alpha isomer. The patients efficiently (84 to 97%) converted both (R)- and (S)-THCA to cholic acid. There was no apparent significant difference in the ability of either (R)- or (S)-THCA to form cholic acid. Varanic acid was poorly converted (20 to 27%) to cholic acid in all three patients. From 49 to 75% of the administered 3H activity was recovered in the bile as other labeled products. The bulk (30 to 35%) of this 3H activity was identified by thin layer chromatography as varanic acid. The rate of conversion of (R)-THCA, (S)-THCA, and varanic acid was extremely rapid in all three patients with a t 1/2 of 35 to 74 min. The findings suggest that (a) the stereospecific configuration at C-25 of THCA has no significant effect on the efficiency of side chain oxidation to cholic acid; and (b) side chain cleavage pathways may exist which do not pass through varanic acid, or the oxidation of varanic acid in man is highly stereospecific with respect to the hydroxyl group at C-24. To prove the latter, it will be necessary to compare the metabolism of the 24 alpha and 24 beta isomers of varanic acid.

The stereospecificity of microsomal "26" -hydroxylation in bile acid biosynthesis was studied. Cholesterol was biosynthesized from [2-14C] mevalonate by a rat liver preparation. The cholesterol was converted stepwise into 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestan-26-oic acid by microsomal and soluble fractions of rat liver homogenate. The 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestan-26-oic acid was decarboxylated chemically and the carbon dioxide was assayed for 14C. The amount of radioactivity in the liberated carbon dioxide was assayed for 14C. The amount of radioactivity in the liberated carbon dioxide was such as to indicate complete stereospecificity of the microsomal "26" -hydroxylase system. The system hydroxylates the methyl group in position C-26 (the 25-pro-R methyl group) and its stereospecificity is opposite that of the mitochondrial "26" -hydroxylase system which hydroxylates the methyl group in position C-27 (the 25-pro-S methyl group).

Conversion of 3alpha, 7alpha, 12alpha-trihydroxy-5beta-[7beta-3H]cholestanoic acid into 3alpha, 7alpha, 12alpha, 24-tetrahydroxy-5beta-cholestanoic acid in rat liver was catalyzed either by the mitochondrial fraction fortified with the 100,000 times g supernatant fluid or the microsomal fraction fortified with 100,000 times g supernatant fluid and ATP. The microsomal system was more active than the mitochondrial system. With the microsomal system the rate of reaction was considerably faster with free 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestanoic acid as substrate than with the corresponding coenzyme A ester. Addition of coenzyme A inhibited the activity. Addition of cofactors other than ATP and coenzyme A did not markedly influence the reaction. The 100,000 times g supernatant fluid could be substituted with a protein fraction obtained by ammonium sulfate precipitation and Sephadex chromatography of the 100,000 times g supernatant fluid. The reaction was not catalyzed by a mixed function oxidase since there was no incorporation of 18O into the product when the reaction was performed in an atmosphere containing 18O2. On the other hand, oxygen may be obligatory since there was almost complete inhibition when the reaction was performed in an atmosphere consisting of nitrogen. Carbon monoxide did not inhibit the reaction. One atom of deuterium was incorporated into the product when the reaction was performed in a medium containing deuterated water. It was concluded that microsomal 24-hydroxylation of 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestanoic acid involves the combined action of a desaturase and a hydratase. The reaction catalyzed by the hydratase appears to be stereospecific since the 24alpha epimer of 3alpha, 7alpha,12alpha-trihydroxy-5beta-cholestanoic acid was the predominant product. In contrast to the microsomal system, the mitochondrial system was not stimulated by the addition of ATP and was not inhibited by coenzyme A. The coenzyme A ester of 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestanoic acid was 24-hydroxylated more efficiently than the free acid.