Characterization of 11beta-hydroxysteroid dehydrogenase activity and corticosteroid receptor expression in human osteosarcoma cell lines.
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Studies in vitro and in vivo have shown that corticosteroids play an important role in bone physiology and pathophysiology. It is now established that corticosteroid hormone action is regulated, in part, at the pre-receptor level through the expression of isozymes of 11beta-hydroxysteroid dehydrogenase (11beta-HSD), which are responsible for the interconversion of hormonally active cortisol to cortisone. In this report we demonstrate 11beta-HSD activity in human osteoblast (OB) cells. Osteosarcoma-derived OB cell lines TE-85, MG-63 and SaOS-2 and fibrosarcoma Hs913T cells express the type 2 isoform of 11beta-HSD, as determined by reverse transcription polymerase chain reaction (RT-PCR) and specific enzyme assays. Enzyme activity was shown to be strictly NAD dependent with a Km of approximately 71 nM; 11beta-HSD type 1 mRNA expression and enzyme activity were not detected. All four cell lines expressed mRNA for the glucocorticoid receptor (GR) and mineralocorticoid receptor, but specific binding was only detectable with radiolabelled dexamethasone (Kd=10 nM) and not aldosterone. MG-63 cells had two to three times more GR than the other OB cells, which correlated with the higher levels of 11beta-HSD 2 activity in these cells. In contrast to the osteosarcoma cell studies, RT-PCR analysis of primary cultures of human OB cells revealed the presence of mRNA for 11beta-HSD 1 as well as 11beta-HSD 2. However, enzyme activity in these cells remained predominantly oxidative, i.e. inactivation of cortisol to cortisone (147 pmol/h per mg protein at 500 nM cortisol) was greater than cortisone to cortisol (10.3 pmol/h per mg protein at 250 nM cortisone). Data from normal human OB and osteosarcoma cells demonstrate the presence of an endogenous mechanism for inactivation of glucocorticoids in OB cells. We postulate that expression of the type 1 and type 2 isoforms of 11beta-HSD in human bone plays an important role in normal bone homeostasis, and may be implicated in the pathogenesis of steroid-induced osteoporosis. (+info)
11beta-hydroxysteroid dehydrogenase in cultured human vascular cells. Possible role in the development of hypertension.
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11beta-Hydroxysteroid dehydrogenases (11beta-HSD) interconvert cortisol, the physiological glucocorticoid, and its inactive metabolite cortisone in humans. The diminished dehydrogenase activity (cortisol to cortisone) has been demonstrated in patients with essential hypertension and in resistance vessels of genetically hypertensive rats. 11beta-Hydroxysteroid dehydrogenase type 2 (11beta-HSD2) catalyzes only 11beta-dehydrogenation. However, a functional relationship between diminished vascular 11beta-HSD2 activity and elevated blood pressure has been unclear. In this study we showed the expression and enzyme activity of 11beta-HSD2 and 11beta-HSD type 1 (which is mainly oxoreductase, converting cortisone to cortisol) in human vascular smooth muscle cells. Glucocorticoids and mineralocorticoids increase vascular tone by upregulating the receptors of pressor hormones such as angiotensin II. We found that physiological concentrations of cortisol-induced increase in angiotensin II binding were significantly enhanced by the inhibition of 11beta-HSD2 activity with an antisense DNA complementary to 11beta-HSD2 mRNA, and the enhancement was partially but significantly abolished by a selective aldosterone receptor antagonist. This may indicate that impaired 11beta-HSD2 activity in vascular wall results in increased vascular tone by the contribution of cortisol, which acts as a mineralocorticoid. In congenital 11beta-HSD deficiency and after administration of 11beta-HSD inhibitors, suppression of 11beta-HSD2 activity in the kidney has been believed to cause renal mineralocorticoid excess, resulting in sodium retention and hypertension. In the present study we provide evidence for a mechanism that could link impaired vascular 11beta-HSD2 activity, increased vascular tone, and elevated blood pressure without invoking renal sodium retention. (+info)
Regulation of 11beta-hydroxysteroid dehydrogenase type 2 by diuretics and the renin-angiotensin-aldosterone axis.
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In the kidney and colon 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) inactivates cortisol to cortisone, thereby protecting the non-selective mineralocorticoid receptor from cortisol. Deficiency of 11beta-HSD2 results in cortisol-mediated sodium retention and hypertension, suggesting that the physiological regulation of 11beta-HSD2 in mineralocorticoid target tissues may be important in modulating sodium homoeostasis and blood pressure control. Using the human epithelial colon cell line SW-620, reverse transcriptase-polymerase chain reaction and enzyme kinetic analysis indicated expression of only 11beta-HSD2 (Km for cortisol 66 nmol/l). Bradykinin (10(-8) to 10(-12) mol/l), frusemide (10(-4) to 10(-9) mol/l), benzamiloride hydrochloride (10(-5) to 10(-10) mol/l) and atrial natriuretic peptide (10(-6) to 10(-10) mol/l) had no effect on 11beta-HSD2 expression. Using a range of concentrations of angiotensin II (2x10(-8) to 2x10(-5) mol/l) a significant reduction in activity was seen but only at supra-physiological concentrations, [e.g. 2x10(-6) mol/l at 4 h pretreatment: 36.7+/-2.0 pmol cortisone. h-1.mg-1 (mean+/-S.E.M.) compared with 45.1+/-1.7 pmol.h-1.mg-1 in control; P<0.05]. The angiotensin-converting enzyme inhibitors captopril, enalapril, lisinopril, perindopril, quinapril and trandolapril at 10(-7) mol/l, but not fosinopril, significantly increased 11beta-HSD2 activity after pretreatment for 16 or 24 h (P<0.05-P<0.01 compared with control). No effects were seen at 4 h pretreatment. Hydrochlorothiazide (10(-7) mol/l) significantly decreased 11beta-HSD2 activity (P<0.05 compared with control) at 4 h pretreatment. Commonly used diuretics, atrial natriuretic peptide and physiological concentrations of angiotensin II and bradykinin do not alter 11beta-HSD2 activity. In contrast, a series of angiotensin-converting enzyme inhibitors significantly increase 11beta-HSD2 activity in vitro. This may explain how intrarenal infusions of angiotensin-converting enzyme inhibitors increase renal sodium excretion independent of circulating concentrations of angiotensin II. The interaction between angiotensin-converting enzyme inhibitors and 11beta-HSD2 may be an additional mechanism by which the former can lower blood pressure. (+info)
Cortisol effects on body mass, blood pressure, and cholesterol in the general population.
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The effects of excess cortisol secretion on blood pressure and fat deposition are well documented, but the importance of this glucocorticoid in controlling these processes in normal individuals is less clear. We studied the relationship between cortisol excretion rate (tetrahydrocortisol [THF]+allo-THF+tetrahydrocortisone [THE]) and a range of important cardiovascular risk factors in 439 normal subjects (238 male) sampled from the North of Glasgow (Scotland) population. There were marked gender differences: female subjects were lighter and had lower blood pressures and cortisol levels, whereas HDL cholesterol was higher. The pattern of cortisol metabolism was also different; the index of 11beta-hydroxysteroid dehydrogenase activity (THF+allo-THF/THE) was lower and that of 5alpha-reductase (allo-THF/THF) was higher. There was a strong correlation of blood pressure (positive), cholesterol (positive), and HDL cholesterol (negative in women, positive in men) with age. Cortisol excretion rate did not correlate with blood pressure but correlated strongly with parameters of body habitus (body mass index and waist and hip measurements [positive]) and HDL cholesterol (negative). With multiple regression analysis, there remained a significant association of cortisol excretion rate with HDL cholesterol in men and women and with body mass index in men. These results suggest that glucocorticoids regulate key components of cardiovascular risk. (+info)
Maternal hypertension and progeny blood pressure: role of aldosterone and 11beta-HSD.
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Epidemiological and experimental evidence suggests that gestational events modulate the level of blood pressure that will be "normal" for the individual as an adult. Glucocorticoid excess during gestation is associated with low birth weight, a large placenta, and adult hypertension in humans and animals. It has been proposed that the deficiency in placental 11beta-hydroxysteroid dehydrogenase activity in humans produces a gestational hormonal milieu, notwithstanding normal circulating levels of glucocorticoids, that predisposes the adult progeny to hypertension. Animal studies indicate that maternal hypertension, excess glucocorticoids, and hydroxysteroid dehydrogenase inhibition program adult blood pressure. Blood pressures of Sprague-Dawley rat dams were manipulated during gestation with continuous intracerebroventricular infusions of vehicle, aldosterone, 11alpha-hydroxyprogesterone, or carbenoxolone at doses known to produce hypertension with no renal effects or with subcutaneous infusions of larger, equally hypertensinogenic doses that produce systemic effects. Blood pressures of all treated dams were significantly greater (P<0.01) during gestation than those of the vehicle ICV control rats but not significantly different from each other. The blood pressures of both male and female progeny (n>/=6 per group, comprising representatives from at least 4 litters) were measured after 6 weeks of age. No significant difference was found in the blood pressure of the pups regardless of the maternal gestational blood pressure or treatment with an enzyme inhibitor, even after high-salt diet challenge. (+info)
Prostaglandins and leukotriene B4 are potent inhibitors of 11beta-hydroxysteroid dehydrogenase type 2 activity in human choriocarcinoma JEG-3 cells.
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The 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) is responsible for the inactivation of glucocorticoids. This is the predominant isozyme in the human placenta, where it is proposed to protect the fetus from high levels of maternal cortisol. In the present study, we examined the effects of eicosanoids on the activity of 11beta-HSD2 in human choriocarcinoma JEG-3 cells, a well-established model for placental trophoblasts. Treatment of JEG-3 cells for 24 h with either prostaglandin (PG) E2 or F2alpha attenuated 11beta-HSD2 activity ( approximately 40%). Paradoxically, indomethacin, an inhibitor of cyclooxygenases, inhibited (approximately 40%) rather than stimulated the activity of this enzyme. This indicated that the arachidonic acid metabolism may be diverted to other pathway(s), the products of which may inhibit 11beta-HSD2 activity. To determine whether the lipoxygenase pathways were involved, the cells were treated with nordihydroguaretic acid (NDGA), a blocker of all three (5-, 12-, and 15-) lipoxygenases. NDGA caused a 3-fold increase in 11beta-HSD2 activity. To further delineate which specific lipoxygenase pathway was involved, the cells were incubated with zileuton, a selective inhibitor of 5-lipoxygenase. This resulted in a similar increase in 11beta-HSD2 activity, suggesting that the products of this pathway (e.g., leukotrienes) may be involved. Given that leukotriene B4 (LTB4) is the most biologically active product of the 5-lipoxygenase pathway, we treated the cells with LTB4, which inhibited 11beta-HSD2 activity in a time- and dose-dependent manner with a maximal effect (60% reduction) at 10 nM for 9 h. Semiquantitative reverse transcription-polymerase chain reaction analysis revealed that 11beta-HSD2 mRNA levels were not altered by the addition of LTB4, PGE2, or PGF2alpha, indicating an effect at the posttranscriptional level. In conclusion, these results demonstrate that prostaglandins and LTB4 are potent inhibitors of 11beta-HSD2 activity in JEG-3 cells, suggesting that placental 11beta-HSD2 activity is modulated by these locally produced eicosanoids. This is the first time that the products of arachidonic acid metabolism have been found to regulate the activity of 11beta-HSD2. (+info)
Cortisol metabolism, cortisol sensitivity and the pathogenesis of leprosy reactions.
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The concentration of cortisol in a tissue is regulated by a reversible enzyme 'shuttle' that can deactivate cortisol by converting it to cortisone, or activate cortisone by converting it to cortisol. The activity of this shuttle, and the direction in which it operates, is regulated by numerous factors including cytokines. This results in large swings in the effective cortisol concentration in sites of inflammation at different phases of an inflammatory response. Thus changes in local cortisol concentration can be largely independent of circulating cortisol levels. The relevant shuttle enzymes are present in skin, blood vessels and nervous tissue, and inhibition of the enzymes in skin enhances the local anti-inflammatory effect of cortisol. We therefore suggest that changes in the activity or direction of action of the shuttle in leprosy lesions may predispose to reactions, requiring exogenous steroid supplements to regain control of the inflammation. (+info)
Corticosteroid receptors and 11beta-hydroxysteroid dehydrogenase isoforms in rat intestinal epithelia.
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To evaluate the potential roles that both receptors and enzymes play in corticosteroid regulation of intestinal function, we have determined glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and 11beta-hydroxysteroid dehydrogenase (11beta-HSD) expression in intestinal epithelial cells. GR and MR mRNA and receptor binding were ubiquitously expressed in epithelial cells, with receptor levels higher in ileum and colon than jejunum and duodenum. RNase protection analysis showed that 11beta-HSD1 was not expressed in intestinal epithelial cells, and enzyme activity studies detected no 11-reductase activity. 11beta-HSD2 mRNA and protein were demonstrated in ileal and colonic epithelia; both MR and GR binding increased when enzyme activity was inhibited with carbenoxolone. Duodenal and jejunal epithelial cells showed very little 11beta-HSD2 mRNA and undetectable 11beta-HSD2 protein; despite minor (<7%) dehydrogenase activity in these cells, enzyme activity did not alter binding of corticosterone to either MR or GR. These findings demonstrate the ubiquitous but differential expression of MR and GR in intestinal epithelia and that 11beta-HSD2 modulates corticosteroid binding to both MR and GR in ileum and proximal and distal colon but not in duodenum or jejunum. (+info)