Tumor necrosis factor suppresses NR5A2 activity and intestinal glucocorticoid synthesis to sustain chronic colitis.

Intestinal crypt epithelial cells synthesize glucocorticoids, steroid hormones that protect against inflammatory bowel disease. To investigate how intestinal glucocorticoids are regulated during chronic inflammation, we induced chronic colitis in mice by exposing them to the chemical dextran sulfate sodium (DSS). We found that intestinal glucocorticoid secretion and expression of the genes Cyp11a1 and Cyp11b1 (which encode enzymes that synthesize glucocorticoids) were initially stimulated, but declined during the chronic phase, whereas tumor necrosis factor (TNF) and inflammatory cytokines secreted by T helper type 1 (TH1) and TH17 cells continuously increased in abundance in the inflamed colon. This suggested that inadequate intestinal glucocorticoid synthesis is a feature of chronic intestinal inflammation. We screened for cytokines that regulated intestinal glucocorticoid synthesis and found that TNF suppressed corticosterone secretion and Cyp11a1 and Cyp11b1 expression in an intestinal crypt epithelial cell line. TNF suppressed steroidogenesis by activating the transcription factors c-Jun and nuclear factor κB (NF-κB), which both interacted with the transcription factor NR5A2 and repressed Cyp11a1 reporter activity. This repression was relieved by expression of a dominant-negative form of c-Jun amino-terminal kinase 1 (JNK1), inhibitor of NF-κB, or by a JNK inhibitor. Furthermore, the dominant-negative TNF inhibitor XPro1595 inhibited c-Jun and NF-κB activation in mice, restored intestinal Cyp11a1 and Cyp11b1 expression, reduced colonic cell death, and rescued chronic colitis caused by DSS. Thus, during chronic colitis, TNF suppresses intestinal steroidogenic gene expression by inhibiting the activity of NR5A2, thus decreasing glucocorticoid synthesis and sustaining chronic inflammation.

Differential expression of 11ß-hydroxysteroid dehydrogenase type 1 and 2 in mild and moderate/severe persistent allergic nasal mucosa and regulation of their expression by Th2 cytokines: asthma and rhinitis.

BACKGROUND: Glucocorticoids are used to treat allergic rhinitis, but the mechanisms by which they induce disease remission are unclear. 11ß-hydroxysteroid dehydrogenase (11ß-HSD) is a tissue-specific regulator of glucocorticoid responses, inducing the interconversion of inactive and active glucocorticoids. OBJECTIVE: We analysed the expression and distribution patterns of 11ß-HSD1, 11ß-HSD2, and steroidogenic enzymes in normal and allergic nasal mucosa, and cytokine-driven regulation of their expression. The production levels of cortisol in normal, allergic nasal mucosa and in cultured epithelial cells stimulated with cytokines were also determined. METHODS: The expression levels of 11ß-HSD1, 11ß-HSD2, steroidogenic enzymes (CYP11B1, CYP11A1), and cortisol in normal, mild, and moderate/severe persistent allergic nasal mucosa were assessed by real-time PCR, Western blot, immunohistochemistry, and ELISA. The expression levels of 11ß-HSD1, 11ß-HSD2, CYP11B1, CYP11A1, and cortisol were also determined in cultured nasal epithelial cell treated with IL-4, IL-5, IL-13, IL-17A, and IFN-γ. Conversion ratio of cortisone to cortisol was evaluated using siRNA technique, 11ß-HSD1 inhibitor, and the measurement of 11ß-HSD1 activity. RESULTS: The expression levels of 11ß-HSD1, CYP11B1, and cortisol were up-regulated in mild and moderate/severe persistent allergic nasal mucosa. By contrast, 11ß-HSD2 expression was decreased in allergic nasal mucosa. In cultured epithelial cells treated with IL-4, IL-5, IL-13, and IL-17A, 11ß-HSD1 expression and activity increased in parallel with the expression levels of CYP11B1 and cortisol, but the production of 11ß-HSD2 decreased. CYP11A1 expression level was not changed in allergic nasal mucosa or in response to stimulation with cytokines. SiRNA technique or the measurement of 11ß-HSD1 activity showed that nasal epithelium activates cortisone to cortisol in a 11ß-HSD-dependent manner. CONCLUSIONS AND CLINICAL RELEVANCE: These results indicate that the localized anti-inflammatory effects of glucocorticoids are regulated by inflammatory cytokines, which can modulate the expression of 11ß-HSD1, 11ß-HSD2, and CYP11B1, and by the intracellular concentrations of bioactive glucocorticoids.

Development of monoclonal antibodies against human CYP11B1 and CYP11B2.

1. The final enzymes in the biosynthesis of aldosterone and cortisol are by the cytochrome P450 CYP11B2 and CYP11B1, respectively. The enzymes are 93% homologous at the amino acid level and specific antibodies have been difficult to generate. 2. Mice and rats were immunized with multiple peptides conjugated to various immunogenic proteins and monoclonal antibodies were generated. The only peptide sequences that generated specific antibodies were amino acids 41-52 for the CYP11B2 and amino acids 80-90 for the CYP11B1 enzyme. 3. The mouse monoclonal CYP11B2-41 was specific and sensitive for use in western blots and produced specific staining of the zona glomerulosa of normal adrenal glands. The rat monoclonal CYP11B1-80 also detected a single band by western blot and detected only the zona fasciculata. Triple immunofluorescence of the adrenal demonstrated that the CYP11B1 and the CYP11B2 did not co-localize, while as expected the CYP11B1 co-localized with the 17α-hydroxylase.

A novel stage-specific antigen is expressed only in early stages of spermatogonia in Japanese eel, Anguilla japonica testis.

We produced an antibody that recognized only early stages of spermatogonia in Japanese eel testis. This antibody (anti-spermatogonia-specific antigen-1, anti-SGSA-1) recognized a band of about 38 kDa in Western blot analysis of extracts from eel testis. This antigen was observed by immunohistochemistry only in type-A and early type-B spermatogonia and could not be seen in the late type-B spermatogonia, which appeared after the initiation of spermatogenesis by a single injection of human chorionic gonadotropin. Immunoreactive SGSA-1 was absent in spermatocytes, spermatids, spermatozoa, Sertoli cells, and interstitial Leydig cells. Similarly, this antigen was also detected only in type-A/primary spermatogonia in the testes of two species of teleosts, medaka (Oryzias latipes) and tilapia (Oreochromis niloticus), as well as a toad (Xenopus laevis). These results imply that the disappearance of SGSA-1 in late type-B/secondary spermatogonia is a critical step in the progression of spermatogenesis, and indicate that anti-SGSA-1 is a useful marker for analysis of the molecular mechanism controlling the differentiation of spermatogonia in lower vertebrates.

Immunochemical characterization of steroid hydroxylases of adrenocortical mitochondria. 1. Antibodies as inhibitors of cytochrome P450scc (CYP11A1) and P450(11 beta) (CYP11B1) activities.

The effects of antibodies against protein components of the monooxygenase systems of adrenocortical mitochondria on the reactions of hydroxylation of cholesterol and 11 beta-deoxycorticosterone were investigated in a reconstituted system containing cytochromes P450scc (CYP11A1) and P450(11 beta) (CYP11B1) as the terminal oxidases and the electron-transfer proteins adrenodoxin reductase and adrenodoxin. It has been shown that affinity-purified antibodies to cytochromes P450scc and P450(11) beta are efficient modulators of the activity of these systems, and their inhibiting effect is mainly due to interference with the interaction of heme proteins and adrenodoxin. The antibodies against polypeptide fragments of the cytochrome P450scc molecule F1 (Ile1-Arg256), F2 (Asn257-Ala481), and F3 (Asn257-Arg399) were used to demonstrate that the interaction of heme protein with adrenodoxin has a multisite character and involves regions located in the N- and C-terminal sequences of cytochrome P450scc.

Clara cell heterogeneity in differentiation: correlation with proliferation, ultrastructural composition, and cell position in the rat bronchiole.

Postnatal differentiation of nonciliated bronchiolar epithelial (Clara) cells occurs in a wave-like pattern beginning in the upper airways and ending in the terminal bronchiole. The heterogeneity of Clara cell differentiation observed during postnatal development in rats may be due to both cell turnover rate and cell position in the airways. To test the importance of these two factors in Clara cell differentiation, terminal bronchioles were examined in rats from gestational day 21 through postnatal day 100. The volume fraction of smooth endoplasmic reticulum (SER), a marker of differentiation, was seen to increase with age, while the epithelial cell labeling index of terminal bronchioles decreased over the same period. This represented a significant inverse correlation between SER volume density and cell proliferation rates (r2 = 0.80, P < 0.02). To evaluate the importance of cell position as a factor in cellular differentiation, the abundance of SER and secretory granules and the expression of cytochrome P450 isozyme 2B in Clara cells were examined along the entire length of the terminal bronchiole in animals 1, 21, and 100 days of age. For all three characteristics, Clara cells showed a similar degree of maturation from the proximal bronchiolar bifurcation to the bronchiole-alveolar duct junction (BADJ) (a span of approximately 35 cells). We conclude that during prenatal and postnatal bronchiolar development in rats: (1) the Clara cell is the most actively dividing cell type for the lower airways; (2) the stage of Clara cell differentiation is inversely related to Clara cell mitotic activity; and (3) the heterogeneity of Clara cell maturation and mitotic activity is not influenced by position within the terminal bronchiole.

Immunological analysis of developmental changes in ecdysone 20-mono-oxygenase expression in the cotton leafworm, Spodoptera littoralis.

The developmental changes in ecdysone 20-mono-oxygenase during the sixth larval instar of the cotton leafworm, Spodoptera littoralis, were investigated. The specific activity of mitochondrial ecdysone 20-mono-oxygenase in the fat-body exhibited a distinct peak at 72 h, at which time the larvae stop feeding. Immunoblot analyses, using antibodies raised against components of vertebrate mitochondrial steroidogenic enzyme systems [anti-(cytochrome P-450scc), anti-(cytochrome P-450(11) beta), anti-adrenodoxin and anti-(adrenodoxin reductase) antibodies], revealed the presence of specific immunoreactive polypeptides in fat-body mitochondrial extracts. In addition, these antibodies effectively inhibited fat-body mitochondrial ecdysone 20-mono-oxygenase activity. This suggests that the S. littoralis steroid-hydroxylating system(s) may contain polypeptide components analogous to those present in vertebrates. A close correlation between developmental changes in mitochondrial ecdysone 20-mono-oxygenase activity and the abundance of polypeptides (approx. 66 kDa and 50 kDa) recognized by the anti-(cytochrome P-450(11) beta) antibody and a polypeptide (approx. 52 kDa) recognized by the anti-(adrenodoxin reductase) antibody were observed in both fat-body and midgut. These results suggest that developmental changes in the abundance of components of the ecdysone 20-mono-oxygenase system may play an important role in the developmental regulation of the enzyme expression and, hence, of 20-hydroxyecdysone titre.

Cytochrome P-45011 beta in rat brain.

The presence of cytochrome P-45011 beta in rat brain was studied by immunohistochemistry using polyclonal rabbit antibodies raised against purified bovine adrenocortical P-45011 beta, which is involved in the steroid 11 beta-hydroxylation and glucocorticoid formation. The results showed that cytochrome P-45011 beta immunoreactivity is selectively localized to the tracts of myelinated fibers throughout the brain. The specificity of immunohistochemical stainings with P-45011 beta antibodies was established by control tests including nonimmune rabbit immunoglobulin Gs and P-45011 beta antibodies absorbed with purified antigen. Western immunoblots of homogenates from different brain areas with P-45011 beta antibodies, together with biochemical enzymatic assays for cytochrome P-45011 beta monooxygenase activity in these homogenates, confirmed the selective localization of this enzyme observed with immunohistochemistry. Cytochrome P-45011 beta and 11 beta-hydroxylase activity were detected in a homogenate from the cortical white matter (brain area rich in myelinated fibers) as in that from the rat adrenal, but were not detectable in a homogenate from the cerebral cortex (brain area poor in myelinated fibers). Furthermore, quantitation of the P-45011 beta bands on the immunoblots by the areal density revealed that the cortical white matter contains approximately 1.4 pmol of cytochrome P-45011 beta/mg of tissue protein, the value of which was about one sixth of the corresponding value estimated in the rat adrenal. This relatively high content of cytochrome P-45011 beta was also reflected in a relatively high level of 11 beta-hydroxylase activity measured in a homogenate of this brain area by biochemical enzymatic assays using [4-14C]-11-deoxycorticosterone.(ABSTRACT TRUNCATED AT 250 WORDS)