In vivo footprinting of the human 11beta-hydroxysteroid dehydrogenase type 2 promoter: evidence for cell-specific regulation by Sp1 and Sp3.
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11beta-Hydroxysteroid dehydrogenase type 2 is selectively expressed in aldosterone target tissues, where it confers aldosterone selectivity for the mineralocorticoid receptor by inactivating 11beta-hydroxyglucocorticoids with a high affinity for the mineralocorticoid receptor. The present investigation aimed to elucidate the mechanisms accounting for the rigorous control of the HSD11B2 gene in humans. Using dimethyl sulfate in vivo footprinting via ligation-mediated PCR, we identified potentially important regions for HSD11B2 regulation in human cell lines: two GC-rich regions in the first exon (I and II) and two upstream elements (III and IV). The footprints suggest a correlation between the extent of in vivo protein occupancy at three of these regions (I, II, and III) and the rate of HSD11B2 transcription in cells with high (SW620), intermediate (HCD, MCF-7, and HK-2), or low HSD11B2 mRNA levels (SUT). Moreover, gel shift assays with nuclear extracts from these cell lines revealed that decreased HSD11B2 expression is related to a decreased binding activity with oligonucleotides containing the putative regulatory elements. Antibody supershifts identified the majority of the components of the binding complexes as the transcription factors Sp1 and Sp3. Finally, transient transfections with various deletion mutant reporters define positive regulatory elements that might account for basal and selective expression of 11beta-hydroxysteroid dehydrogenase type 2. (+info)
Myometrial expression of 11 beta-hydroxysteroid dehydrogenase type 2 in rat pregnancy.
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The enzyme 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2), which reduces glucocorticoid potency in target cells by metabolism of active glucocorticoids, is expressed in the non-pregnant rat uterus in an oestrogen-dependent manner. Because glucocorticoids appear to facilitate parturition in many species, expression of 11 beta-HSD2 in pregnant myometrium is likely to influence pregnancy maintenance and possibly the onset and progression of labour. The present study therefore examined myometrial 11 beta-HSD2 mRNA, protein and bioactivity across rat pregnancy, with emphasis on the peripartum period. A single 1.9 kb transcript of 11 beta-HSD2 mRNA was evident in myometrium at all stages, with maximal (P<0.05) levels observed at day 16 (term=day 23). Consistent with this pattern of mRNA expression, Western blot analysis showed the presence of a 40 kDa 11 beta-HSD2 protein at all stages, with the maximal immunoreactive signal also observed on day 16. The 11 beta-HSD2 signal was immunolocalized to myometrial smooth muscle cells and endometrial stromal cells. Bioactivity specific to 11 beta-HSD2 was detectable in myometrium at all stages, but in contrast to the patterns of 11 beta-HSD2 mRNA and protein, the V(max) decreased at the beginning of pregnancy and remained stable until term. The apparent K(m) of 11 beta-HSD2 for corticosterone increased from 47 +/- 11 nM in non-pregnant myometrium to 75 +/- 7 nM by day 10 of pregnancy, and remained high until returning to an intermediate level on the day of delivery (60 +/- 8 nM). Progesterone competitively inhibited 11 beta-HSD2 bioactivity (K(i)=1.75 muM) whereas 20 alpha-hydroxypregn-4-en-3-one, the other major progestin present during rat pregnancy, had no such effect. In conclusion, these data suggest that local levels of active glucocorticoid in the myometrium are determined by the net effects of myometrial 11 beta-HSD-1 and -2 expression across pregnancy. Because the previously reported increase in myometrial 11 beta-HSD-1 near term occurs with little change in myometrial 11 beta-HSD2 bioactivity, this is likely to facilitate parturition by increasing local concentrations of active glucocorticoid. (+info)
Regulation of 11 beta-hydroxysteroid dehydrogenase type 2 activity in ovine placenta by fetal cortisol.
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The effect of fetal cortisol on the activity of the type 2 isoform of the enzyme, 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD2), was examined in ovine placenta and fetal kidney by measuring tissue 11 beta-HSD2 activity during late gestation when endogenous fetal cortisol levels rise and after exogenous cortisol administration to immature fetuses before the prepartum cortisol surge. Placental 11 beta-HSD2 activity decreased between 128-132 days and term (approximately 145 days of gestation) in association with the normal prepartum increase in fetal plasma cortisol. Raising fetal cortisol levels to prepartum values in the immature fetus at 128--132 days of gestation reduced placental 11 beta-HSD2 activity to term values. In contrast, 11 beta-HSD2 activity in the fetal renal cortex was unaffected by gestational age or cortisol infusion. When all the data were combined, there was an inverse correlation between the log fetal plasma cortisol level at delivery and placental 11 beta-HSD2 activity, expressed both on a weight-specific basis and per mg placental protein. Fetal cortisol therefore appears to be a physiological regulator of placental, but not renal, 11 beta-HSD2 activity in fetal sheep during late gestation. These findings have important implications, not only for glucocorticoid exposure in utero, but also for the local actions of cortisol within the placental tissues that are involved in initiating parturition in the sheep. (+info)
The 11 beta-hydroxysteroid dehydrogenase type 2 activity in human placental microsomes is inactivated by zinc and the sulfhydryl modifying reagent N-ethylmaleimide.
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Proper glucocorticoid exposure in utero is vital to normal fetal organ growth and maturation. The human placental 11 beta-hydroxysteroid dehydrogenase type 2 enzyme (11 beta-HSD2) catalyzes the unidirectional conversion of cortisol to its inert metabolite cortisone, thereby controlling fetal exposure to maternal cortisol. The present study examined the effect of zinc and the relatively specific sulfhydryl modifying reagent N-ethylmaleimide (NEM) on the activity of 11 beta-HSD2 in human placental microsomes. Enzyme activity, reflected by the rate of conversion of cortisol to cortisone, was inactivated by NEM (IC(50)=10 microM), while the activity was markedly increased by the sulfhydryl protecting reagent dithiothreitol (DTT; EC(50)=1 mM). Furthermore, DTT blocked the NEM-induced inhibition of 11 beta-HSD2 activity. Taken together, these results suggested that the sulfhydryl (SH) group(s) of the microsomal 11 beta-HSD2 may be critical for enzyme activity. Zn(2+) also inactivated enzyme activity (IC(50)=2.5 microM), but through a novel mechanism not involving the SH groups. In addition, prior incubation of human placental microsomes with NAD(+) (cofactor) but not cortisol (substrate) resulted in a concentration-dependent increase (EC(50)=8 microM) in 11 beta-HSD2 activity, indicating that binding of NAD(+) to the microsomal 11 beta-HSD2 facilitated the conversion of cortisol to cortisone. Thus, this finding substantiates the previously proposed concept that a compulsorily ordered ternary complex mechanism may operate for 11 beta-HSD2, with NAD(+) binding first, followed by a conformational change allowing cortisol binding with high affinity. Collectively, the present results suggest that cellular mechanisms of SH group modification and intracellular levels of Zn(2+) may play an important role in regulation of placental 11 beta-HSD2 activity. (+info)
Association studies between the HSD11B2 gene (encoding human 11beta-hydroxysteroid dehydrogenase type 2), type 1 diabetes mellitus and diabetic nephropathy.
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OBJECTIVE: Mutations in the HSD11B2 gene (encoding human 11beta-hydroxysteroid dehydrogenase type 2) explain the syndrome of apparent mineralocorticoid excess where cortisol acts as a mineralocorticoid. A microsatellite marker within the HSD11B2 gene associates with salt sensitivity and hypertension--phenotypes characterising diabetic nephropathy. Here, we evaluate the HSD11B2 gene as a susceptibility locus for diabetic nephropathy. DESIGN: 150 patients with type 1 diabetes and nephropathy (DN), 145 patients with type 1 diabetes with a long duration of non-nephropathy (LDNN) and 151 normal controls were studied. METHODS: We determined allele frequencies for the (CA)n repeat marker within intron I of the HSD11B2 gene. Duration of type 1 diabetes, diabetic status and renal function were recorded. RESULTS: 11 alleles (138-158) for the marker were observed. Allele 152 was significantly increased in controls compared with LDNN (70.5% vs 57.6%, P(c)<0.05 where P(c) is the P value corrected for multiple comparisons) but no difference was observed between DN and LDNN subjects. Allele 154 was significantly increased in the LDNN compared with the DN subjects (15.9% vs 7.0%, P(c)<0.01) but no difference was observed between DN and controls. A greater proportion of subjects carried at least 1 allele <152 in DN compared with control subjects (47.3% vs 28.5%, P(c)<0.01), but no difference was observed in LDNN compared with control and DN subjects. CONCLUSIONS: Weak associations are reported between the HSD11B2 gene, type 1 diabetes mellitus and nephropathy. The increased frequency of HSD11B2 short alleles in the diabetic groups may reflect reduced renal 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) activity and may, in part, explain the enhanced salt sensitivity observed in patients with type 1 diabetes. (+info)
Increased cortisol metabolites and reduced activity of 11beta-hydroxysteroid dehydrogenase in patients on hemodialysis.
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BACKGROUND: Patients with renal failure have symptoms assumed to be attributable to the accumulation of toxic endo- or xenobiotics. Most of these molecules, especially those with a molecular weight>300 D, have not been identified. In addition to excretion, the kidney is involved in some defined metabolic processes. In the cortical collecting duct, the enzyme 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) interconverts cortisol (F) and cortisone (E), and the metabolites of these glucocorticoids, tetrahydrocortisol (THF), 5alpha-tetrahydrocortisol (5alpha-THF) and tetrahydrocortisone (THE), are excreted in urine. We hypothesized that first, these metabolites accumulate and second, their concentration pattern changes in patients on hemodialysis. METHODS: THF, 5alpha-THF, THE, F and E were measured in plasma of 63 patients on dialysis and in 34 healthy controls by gas-chromatography-mass spectrometry (GC/MS). In 11 patients, the metabolite clearance was determined during high flux hemodialysis by using a population pharmacokinetic approach. RESULTS: Mean plasma concentrations of THF, 5alpha-THF and THE were more than five times higher and those of E lower in patients than in controls. The ratios of (THF + 5alpha-THF)/THE and F/E were increased in patients, indicating a reduced activity of 11beta-HSD2. Intradialytic clearances were between 120 and 300 mL/min and not sufficient to normalize the steroid concentrations. CONCLUSION: Patients on hemodialysis exhibit pronounced increases in THF, 5alpha-THF and THE concentrations in plasma with insufficient removal during dialysis. Due to a reduced 11beta-HSD2 activity, an abnormal pattern of the concentrations of these cortisol and cortisone metabolites is observed. Since many signs and symptoms in uremic patients resemble those observed in subjects with glucocorticoid excess, the clinical relevance of the high concentrations of these glucocorticoid metabolites deserves further investigation. (+info)
Chenodeoxycholic acid and deoxycholic acid inhibit 11 beta-hydroxysteroid dehydrogenase type 2 and cause cortisol-induced transcriptional activation of the mineralocorticoid receptor.
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Inappropriate activation of the mineralocorticoid receptor (MR) results in renal sodium retention and potassium loss in patients with liver cirrhosis. Recent evidence suggested that this MR activation is, at least in part, a result of bile acid-dependent reduction in 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta HSD2) activity, an enzyme preventing cortisol-dependent activation of MR by converting cortisol to cortisone. Here, we investigated the molecular mechanisms underlying bile acid-mediated MR activation. Analysis of urinary bile acids from 12 patients with biliary obstruction revealed highly elevated concentrations of chenodeoxycholic acid (CDCA), cholic acid (CA), and deoxycholic acid (DCA), with average concentrations of 50-80 microm. Although CDCA and DCA both mediated nuclear translocation of MR in the absence of 11 beta HSD2 and steroids in transiently expressing HEK-293 cells, the transcriptional activity of MR was not stimulated. In contrast, CDCA and DCA both inhibited 11 beta HSD2 with IC(50) values of 22 and 38 microm, respectively and caused cortisol-dependent nuclear translocation and increased transcriptional activity of MR. LCA, the bile acid that most efficiently inhibited 11 beta HSD2, was present at very low concentrations in cholestatic patients, whereas the weak inhibitor CA did not cause MR activation. In conclusion, these findings indicate that CDCA, and to a lesser extent DCA, by inhibiting 11 beta HSD2, mediate cortisol-dependent nuclear translocation and transcriptional activation of MR and are responsible at least for a part of the sodium retention and potassium excretion observed in patients with biliary obstruction. (+info)
Modulation of renal calcium handling by 11 beta-hydroxysteroid dehydrogenase type 2.
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Reduced concentration of serum ionized calcium and increased urinary calcium excretion have been reported in primary aldosteronism and glucocorticoid-treated patients. A reduced activity of the 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta HSD2) results in overstimulation of the mineralocorticoid receptor by cortisol. Whether inhibition of the 11 beta HSD2 by glycyrrhetinic acid (GA) may increase renal calcium excretion is unknown. Serum and urinary electrolyte and creatinine, serum ionized calcium, urinary calcium excretion, and the steroid metabolites (THF+5 alpha THF)/THE as a parameter of 11 beta HSD2 activity were repeatedly measured in 20 healthy subjects during baseline conditions and during 1 wk of 500 mg/d GA. One week of GA induced a maximal increment of 93% in (THF+5 alpha THF)/THE. Ambulatory BP was significantly higher at day 7 of GA than at baseline (126/77 +/- 10/7 versus 115/73 +/- 8/6 mmHg; P < 0.001 for systolic; P < 0.05 for diastolic). During GA administration, serum ionized calcium decreased from 1.26 +/- 0.05 to 1.18 +/- 0.04 mmol/L (P < 0.0001), and absolute urinary calcium excretion was enhanced from 29.2 +/- 3.6 to 31.9 +/- 3.1 micromol/L GFR (P < 0.01). Fractional calcium excretion increased from 2.4 +/- 0.3 to 2.7 +/- 0.3% (P < 0.01) and was negatively correlated to the fractional sodium excretion during GA (R = -0.35; P < 0.001). Moreover, serum potassium correlated positively with serum ionized calcium (R = 0.66; P < 0.0001). Inhibition of 11 beta HSD2 activity is sufficient to significantly increase the fractional excretion of calcium and decrease serum ionized calcium, suggesting decreased tubular reabsorption of this divalent cation under conditions of renal glucocorticoid/mineralocorticoid excess. The likely site of steroid-regulated renal calcium handling appears to be the distal tubule. (+info)