Elsevier

Bone

Volume 27, Issue 3, September 2000, Pages 375-381
Bone

Original article
Expression and functional consequences of 11β-hydroxysteroid dehydrogenase activity in human bone

https://doi.org/10.1016/S8756-3282(00)00344-6Get rights and content

Abstract

Glucocorticoids have an essential role in skeletal development and function but are detrimental in excess. In several tissues, glucocorticoid action is dependent upon the expression of 11β-hydroxysteroid dehydrogenase (11β-HSD) isozymes, which interconvert active cortisol (F) and inactive cortisone (E). We previously demonstrated the expression of 11β-HSD isozymes in human osteosarcoma cell lines, osteoblast cultures, and fetal bone. We now characterize 11β-HSD expression in adult human bone using specific antihuman 11β-HSD antibodies, riboprobes, and enzyme activity studies. In addition, the effect of 11β-HSD on bone metabolism in vivo was assessed using the 11β-HSD inhibitor carbenoxolone in eight normal male volunteers. In fresh normal human bone tissue, both 11β-dehydrogenase (cortisol-to-cortisone conversion) and reductase (cortisone-to-cortisol conversion) activities were demonstrated. There was considerable interindividual variation in the dehydrogenase, but not reductase, activity. In bone homogenates, activity was NADP-dependent with a Km for F of 4.8 ± 1.2 μmol/L, suggesting the presence of 11β-HSD1. This was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Immunohistochemistry and in situ hybridization studies demonstrated 11β-HSD1 isozyme expression in cells of the osteoblast lineage and in osteoclasts. The 11β-HSD2 isozyme was expressed, but only in osteoblasts and at a low level. Ingestion of 300 mg of carbenoxolone by eight normal volunteers for 7 days resulted in a significant decrease in the bone resorption markers, pyridinoline (Pyr) and deoxypyridinoline (DPyr) (change in urinary Pyr/creatinine −1.55 ± 0.55 [mean ± SE], for DPyr/creatinine −0.4 ± 0.14 nmol/mmol; p < 0.05 for both), with no overall change in the bone formation markers C- and N-terminal propeptides of type I collagen (PICP and PINP). These data suggest that local tissue metabolism of glucocorticoids is likely to be important in determining the sensitivity of both osteoblasts and osteoclasts to glucocorticoids. In particular, variation in 11β-HSD isozyme expression and activity may explain individual variation in susceptibility to glucocorticoid-induced osteoporosis.

Introduction

Glucocorticoids have an essential role in normal skeletal development and function but are detrimental in excess. The effects of glucocorticoids in vitro depend upon corticosteroid concentration and receptor expression, timing of exposure, and stage of cellular differentiation, although little is known about the mechanisms underlying changes in cellular sensitivity to these agents. These mechanisms are particularly important in bone where glucocorticoid excess in vivo is associated with decreased bone formation and rapid decreases in bone formation markers17, 21 due to direct effects on cells of the osteoblast lineage.9, 16 Glucocorticoids may additionally cause a transient increase in bone resorption due to inappropriately increased osteoclastic activity. Despite this, susceptibility to the bone-specific effects of endogenous or exogenous glucocorticoids in individuals correlates only weakly with circulating levels of glucocorticoids7, 10 or variation in the affinity or concentration of the glucocorticoid receptor (GR).12 This suggests that there are intrinsic tissue-specific factors that modulate individual sensitivity to the effects of glucocorticoids. Recently, the functional importance of intracellular glucocorticoid metabolism in several tissues, such as kidney,27 liver,15 and omental fat,3, 4 has been demonstrated, but little is known about glucocorticoid metabolism in skeletal cells.

By catalyzing the interconversion of hormonally active cortisol (F) to inactive cortisone (E), 11β-hydroxysteroid dehydrogenase (11β-HSD) has been established as an important prereceptor signaling pathway in corticosteroid hormone action.31 Two isozymes of 11β-HSD, 11β-HSD1 and 11β-HSD2, have been shown to regulate glucocorticoid and mineralocorticoid hormone action.26 11β-HSD1 is a low-affinity bidirectional nicotinamide adenine dinucleotide phosphate (NADP[H])-dependent enzyme that can interconvert F and E,30 but, in intact tissues studied to date, acts predominantly as a reductase converting E to F. By contrast, 11β-HSD2 is a high-affinity nicotinamide adenine dinucleotine (NAD)-dependent dehydrogenase inactivating F to E28; reductase activity for physiological glucocorticoids has not been seen for 11β-HSD2. Both 11β-HSD isoenzymes are inhibited by liquorice and its derivatives, carbenoxolone and glycyrrhetinic acid. These inhibitors have been used in vivo to examine the functional role of 11β-HSD expression and activity.29 We have previously demonstrated the expression and activity of the 11β-HSD2 isozyme in rat and human osteosarcoma cell lines1 and expression in osteoblasts of normal human fetal bone,8 whereas the 11β-HSD1 isozyme is expressed in human osteoblast cultures generated from trabecular bone samples.1 A full and detailed analysis of the expression of 11β-HSD isozymes within human bone is required. In addition, by measuring well-established markers of bone formation (C- and N-terminal propeptides of type I collagen [PICP and PINP]) and resorption (pyridinoline and deoxypyridinoline) we investigated whether short-term inhibition of 11β-HSDs with the liquorice derivative, carbenoxolone,29 can affect bone metabolism in vivo.

Section snippets

Tissue preparation

Fresh human bone tissue was obtained from surgical resections with the approval of the local ethics committee. For enzyme activity and reverse transcription-polymerase chain reaction (RT-PCR) studies, specimens were obtained from four individuals (ages between 5 and 30 years) who were having lower limb amputation for localized malignancy. Normal tissue was obtained at least 10 cm distant from the site of pathology. For immunohistochemistry and in situ hybridization studies, tissue was obtained

Enzyme assays for glucocorticoid metabolism in normal human bone

Data shown in Figure 1 indicate the presence of intrinsic 11β-HSD activity in intact bone chips. For conversion of F to E, activity was considerably greater at 1 μmol/L F than at 50 nmol/L F, suggesting predominant 11β-HSD1 activity. Conversion of E to F was also evident again suggesting the presence of 11β-HSD1. Between individuals there was considerable variation in dehydrogenase activities, but the reductase activity was relatively constant. In bone homogenates, dehydrogenase activity was

Discussion

11β-HSD expression has been shown to be important in mediating glucocorticoid hormone action in several tissues.26 However, the extent to which this occurs in bone in vivo, and its functional role, is not yet clear. We have recently demonstrated the presence of 11β-HSD1 in primary cultures of human osteoblasts,1 and 11β-HSD2 in fetal human bone8 and human osteosarcoma cells.1 In this study, using primary human bone samples, we have shown that 11β-HSD1 is the predominant isozyme expressed in

Acknowledgements

The authors are grateful to Dr. D. C. Mangham, Maurice Adkins, and the staff at the Department of Musculoskeletal Pathology, Birmingham, UK, for the technical assistance in tissue sectioning and immunohistochemistry. We also thank Cedric Shackleton, Children’s Hospital, Oakland Research Institute, Oakland, CA, for performing the GC/MS on urine samples. Technical assistance for bone marker assays was provided by Brian Durham and Geoff Birkinshaw, Department of Clinical Chemistry, Royal Liverpool

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