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MS Cooper, M Hewison, and PM Stewart

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R Bland, CA Worker, BS Noble, LJ Eyre, IJ Bujalska, MC Sheppard, PM Stewart, and M Hewison

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.

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ML Ricketts, KJ Shoesmith, M Hewison, A Strain, MC Eggo, and PM Stewart

Two isozymes of the enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) are responsible for the interconversion of the active glucocorticoid, cortisol in man, (corticosterone in the rodent), to the inactive 11-keto metabolite, cortisone (11-dehydrocorticosterone). We have examined the regulation of type 1 11 beta-HSD (11 beta-HSD1) using primary cultures of rat and human hepatocytes, both of which express only 11 beta-HSD1. Only 11 oxo-reductase activity could be demonstrated in cultured hepatocytes (apparent Km for cortisone 382 +/- 43 nM in human hepatocytes, apparent Km for 11-dehydrocorticosterone 14.6 +/- 1.5 microM in rat hepatocytes). There exists a marked discrepancy between 11 beta-HSD oxo-reductase activity and 11 beta-HSD1 mRNA levels in cultured human hepatocytes and human liver. Thus oxo-reductase specific activity is much higher in the cultured hepatocytes (7.2 +/- 0.01 nmoles cortisol/mg/h vs 0.89 +/- 0.06 for whole liver homogenates) whilst the converse is true for steady state 11 beta-HSD1 mRNA levels (0.78 +/- 0.02 vs 1.94 +/- 0.07 in whole liver, 11 beta-HSD1/18S expressed as arbitrary units). Carbenoxolone has a significant inhibitory effect on 11 oxo-reductase activity in both rat and human hepatocytes. However, there is clear species-specific regulation of 11 oxo-reductase activity by thyroid hormone (tri-iodothyronine (T3)), which increases 11 oxo-reductase activity in rat hepatocytes but has no effect on activity in human hepatocytes, and progesterone which inhibits activity in human hepatocytes, but has no effect on activity in rat hepatocytes. Neither T3 nor progesterone altered 11 beta-HSD1 mRNA levels. A series of growth factors (hepatocyte growth factor, epidermal growth factor, basic fibroblast growth factor, transforming growth factor beta 1) were without effect on 11 oxo-reductase activity in cultured rat hepatocytes. In contrast to homogenates of human liver, cultured hepatocytes express only 11 beta-HSD oxo-reductase activity. This is inhibited by carbenoxolone and shows species-specific regulation by T3 and progesterone. Growth factors do not appear to regulate activity or expression of 11 beta-HSD1. The discrepant enzyme activity data and 11 beta-HSD1 mRNA expression in hepatocytes and whole liver could reflect unstable 11 beta-HSD1 oxo-reductase activity or, alternatively, an additional 11 beta-HSD oxo-reductase isoform in cultured hepatocytes.

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LM Thurston, E Chin, KC Jonas, IJ Bujalska, PM Stewart, DR Abayasekara, and AE Michael

In a range of tIssues, cortisol is inter-converted with cortisone by 11beta-hydroxysteroid dehydrogenase (11betaHSD). To date, two isoforms of 11betaHSD have been cloned. Previous studies have shown that human granulosa cells express type 2 11betaHSD mRNA during the follicular phase of the ovarian cycle, switching to type 1 11betaHSD mRNA expression as luteinization occurs. However, it is not known whether protein expression, and 11betaHSD enzyme activities reflect this reported pattern of mRNA expression. Hence, the aims of the current study were to investigate the expression and activities of 11betaHSD proteins in luteinizing human granulosa-lutein (hGL) cells. Luteinizing hGL cells were cultured for up to 3 days with enzyme activities (11beta-dehydrogenase (11betaDH) and 11-ketosteroid reductase (11 KSR)) and protein expression (type 1 and type 2 11betaHSD) assessed on each day of culture. In Western blots, an immunopurified type 1 11betaHSD antibody recognized a band of 38 kDa in hGL cells and in human embryonic kidney (HEK) cells stably transfected with human type 1 11betaHSD. The type 2 11betaHSD antibody recognized a band of 48 kDa in HEK cells transfected with human type 2 11betaHSD cDNA but the type 2 protein was not expressed in hGL cells throughout the 3 days of culture. While the expression of type 1 11betaHSD protein increased progressively by 2.7-fold over 3 days as hGL cells luteinized, both 11betaDH and reductase activities declined (by 52.9% and 34.2%; P<0.05) over this same period. Changes in enzyme expression and activity were unaffected by the suppression of ovarian steroid synthesis.