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ABSTRACT

Corticosteroid regulation of Na/K-ATPase is of key importance in the modulation of Na⁺ transport across renal tubular epithelia. In amphibian renal cells, aldosterone induction of Na/K-ATPase α₁ and β₁ subunit gene transcription is mediated by an indirect mechanism dependent on the synthesis of a labile protein. In mammalian target cells, while both mineralo- and glucocorticoids increase the levels of Na/K-ATPase α₁ and β₁ subunit mRNA and enzyme activity, they are diminished by glycyrrhetinic acid (GE), the active ingredient of licorice.

To investigate the mechanisms underlying the regulation of mammalian renal Na/K-ATPase, levels of α₁ and β₁ mRNA were measured in rat kidney epithelial (NRK-52E) cells treated with a range of concentrations of aldosterone, corticosterone and GE in the presence of a specific inhibitor of mRNA synthesis, dichlororibofuranosylbenzimidazole (DRB), an inhibitor of total RNA synthesis, actinomycin D (ActD), and the protein synthesis inhibitor cycloheximide (CHX). In addition, GE was co-incubated with the sodium channel antagonist benzamiloride (BZ). The increase in both α₁ and β₁ mRNA levels following aldosterone and corticosterone was completely abolished by treatment with ActD and DRB, while CHX did not affect this response. Similarly, the GE-induced decrease in α₁ and β₁ mRNA was also completely abolished by ActD and DRB, but not by CHX or by BZ. The half-lives of α₁ and β₁ mRNA in these cells (means±s.e.m., n=4), estimated from the rate of mRNA decay in the presence of DRB, were 6·8±0·3 and 4·8±0·2 h respectively. This was unaffected by GE.

The inhibitory action of GE on α₁ and β₁ mRNA levels was accompanied by a dose-dependent decrease in levels of intracellular cAMP (means ± s.e.m., n=4) from 395±28 fmol cAMP/μg total cell protein to between 275 ± 19 fmol/μg total cell protein (0·1 μm GE) and 78±11 fmol/μg total cell protein (10 μm GE). This was abolished following down-regulation of protein kinase C by prolonged treatment with the phorbol ester tetradecanoylphorbol-13-acetate (TPA), and by pertussis toxin (PT), but not by cholera toxin (CT). Indeed, subunit mRNA levels were increased by 8-bromo-cAMP (2·2-fold) and stimulators of adenylate cyclase activity, i.e. forskolin (2·1-fold), PT (2·1-fold) and CT (1·9-fold), but not by TPA. In keeping with their effects on GE inhibition of cAMP synthesis, TPA and PT (but not CT) abolished the GE-induced decrease in subunit mRNA.

In conclusion, corticosteroid induction and GE inhibition of Na/K-ATPase subunit gene expression in rat kidney epithelial cells occur at the transcriptional level and do not require de novo synthesis of an intermediary protein. Furthermore, GE attenuation of subunit gene transcription may be mediated by both cAMP-dependent protein kinase A and diacylglycerol—protein kinase C pathways via interaction with a PT-sensitive G_i protein.

ABSTRACT

11β-Hydroxysteroid dehydrogenase (11β-HSD) catalyses the interconversion of biologically active cortisol to inactive cortisone in man, and corticosterone to 11-dehydrocorticosterone in rodents. As such, this enzyme has been shown to confer aldosterone-selectivity on the mineralocorticoid receptor and to modulate cortisol/corticosterone access to the glucocorticoid receptor (GR). Two kinetically distinct isoforms of this enzyme have been characterized in both rodents and man; a low-affinity NADP(H)-dependent enzyme (11β-HSD1) which predominantly acts as an oxo-reductase and, more recently, a high-affinity NAD-dependent uni-directional dehydrogenase (11β-HSD2). In this study we have analysed the expression of both 11β-HSD1 and 11β-HSD2 isoforms in rat adrenal cortex and medulla and have investigated their possible roles with respect to glucocorticoid-regulated enzymes mediating catecholamine biosynthesis in adrenal medullary chromaffin cells.

Using a rat 11β-HSD1 probe and a recently cloned in-house mouse 11β-HSD2 cDNA probe, Northern blot analyses revealed expression of mRNA species encoding both 11β-HSD1 (1·4kb) and 11β-HSD2 (1·9kb) in the whole adrenal. Consistent with this, 11β-dehydrogenase activity (pmol 11-dehydrocorticosterone formed/mg protein per h, mean ± s.e.m.) in adrenal homogenates, when incubated with 50 nm corticosterone in the presence of 200 μm NAD, was 97·0 ± 9·0 and with 500 nm corticosterone in the presence of 200 μm NADP, was 98·0 ± 1·4 11-Oxoreductase activity (pmol corticosterone formed/mg protein per h) with 500 nm 11-dehydrocorticosterone in the presence of 200 μm NADPH, was 187·7 ± 31·2. In situ hybridization studies of rat adrenal cortex and medulla using ³⁵S-labelled antisense 11β-HSD1 cRNA probe revealed specific localization of 11β-HSD1 mRNA expression predominantly to cells at the corticomedullary junction, most likely within the inner cortex. In contrast, 11β-HSD2 mRNA was more abundant in cortex versus medulla, and was more uniformly distributed over the adrenal gland. Negligible staining was detected using control sense probes.

Ingestion of the 11β-HSD inhibitor, glycyrrhizic acid (>100mg/kg body weight per day for 4 days) resulted in significant inhibition of adrenal NADP-dependent (98·0 ± 1·4 vs 42·5 ± 0·4) and NAD-dependent (97·0 ± 9·0 vs 73·2 ± 6·7) 11β-dehydrogenase activity and 11-oxoreductase activity (187·7 ± 31·2 vs 67·7 ± 15·3). However, while levels of 11β-HSD1 mRNA were similarly reduced (0·85 ± 0·07 vs 0·50 ± 0·05 arbitrary units), those for 11β-HSD2 remained unchanged (0·44 ± 0·03 vs 0·38 ± 0·01). Levels of mRNA encoding the glucocorticoid-dependent enzyme phenylethanolamine N-methyltransferase which catalyses the conversion of noradrenaline to adrenaline, were also significantly reduced in those rats given glycyrrhizic acid (1·12 ± 0·04 vs 0·78 ± 0·04), while those for the glucocorticoid-independent enzyme tyrosine hydroxylase (1·9 kb), which catalyses the conversion of tyrosine to DOPA, were unchanged (0·64 ± 0·04 vs 0·61 ± 0·04).

In conclusion, the rat adrenal gland expresses both 11β-HSD1 and 11β-HSD2 isoforms. 11β-HSDl gene expression is localized to the adrenal cortico-medullary junction, where it is ideally placed to regulate the supply of cortex-derived corticosterone to the medullary chromaffin cells. This, together with our in vivo studies, suggests that 11β-HSD1 may play an important role with respect to adrenocorticosteroid regulation of adrenaline biosynthesis. The role of 11β-HSD2 in the adrenal remains to be elucidated.

ABSTRACT

The type 2 isoform of 11 β-hydroxysteroid dehydrogenase (11β-HSD2), which catalyzes the conversion of cortisol to hormonally inactive cortisone in man, is principally expressed in the placenta and mineralocorticoid target tissues, kidney and colon. To date, few studies have addressed the regulation of this novel 11β-HSD2 isoform. We have characterized the nature and regulation of the 11β-HSD activity expressed in a human cytotrophoblastic cell line, the JEG-3 choriocarcinoma cell. The 11β-HSD activity in JEG-3 cell homogenates required NAD⁺ as cofactor with NADP ineffective and demonstrated a high affinity for cortisol (apparent K _m 31 nm). Incubation of JEG-3 cells with forskolin and dibutyryl cyclic AMP increased 11β-HSD2 activity several-fold in a time-dependent manner, while treatment with phorbol ester had little, if any, effect on 11β-HSD2 activity. Northern blot analysis of RNA isolated from JEG-3 cells after these treatments demonstrated a marked increase in a 1·9 kb 11β-HSD2 mRNA species in cells treated with forskolin for 24 h. We conclude that 11β-HSD2 is regulated by activation of the protein kinase A pathway, but not the protein kinase C pathway in human choriocarcinoma cells, and that this regulation occurs at a pretranslational level. JEG-3 cells provide an excellent model for further studies on the regulation of 11β-HSD2 gene expression in human trophoblast tissue.

ABSTRACT

A bovine trophoblast interferon (IFN-τ) gene promoter sequence (− 450 to +26 bp relative to the transcription start site) led to expression of reporter gene (CAT) constructs transfected into L929 (murine fibroblast) or JAR (human choriocarcinoma) cells. Expression depended on the presence of an exogenous (SV40) enhancer. Poly(I)(C) activated endogenous IFN production in L929 and JAR cells but had no consistent effect on CAT expression. Similar results were obtained in L929 cells with inactivated Newcastle disease virus. There was no 'priming' effect of exogenous Type I IFN. Deletion mutants revealed sites exerting negative control on expression between −338 and −247 bp, and between −150 and −71 bp; these regions contained sequences resembling previously identified negative regulatory domains. In the absence of viral inducibility it is proposed that negative regulation contributes towards the stringent control of expression characteristic of IFN-τ genes.

ABSTRACT

Expression of the RNA coding for the ACTH—β-lipotrophin precursor, pro-opiomelanocortin (POMC), has been demonstrated in five human small-cell lung cancer (SCLC) cell lines. Using Northern and slot-blot hybridization analysis of RNA and a bovine POMC cDNA as probe, the processed POMC RNA from SCLC cells was found to be approximately 1350 nucleotides in length, which is larger than that found in the normal human pituitary. Expression of the POMC gene was confirmed by measurement of ACTH precursors secreted by the cells, using a novel two-site immunoradiometric assay based on monoclonal antibodies, which directly quantifies both POMC and pro-ACTH but does not recognize ACTH. Levels of POMC in medium accumulated throughout the growth of the cells, in contrast to POMC RNA which showed a relatively constant level of expression. We conclude that human SCLC cell lines are valuable models for studying the aberrant expression and regulation of the human POMC gene.