11β-Hydroxysteroid Dehydrogenase 1 Transforms 11-Dehydrocorticosterone into Transcriptionally Active Glucocorticoid in Neonatal Rat Heart

0013-7227/02/$03.00/0 Printed in U.S.A. Endocrinology 143(1):198 –204 Copyright © 2002 by The Endocrine Society 11␤-Hydroxysteroid Dehydrogenase 1 Transforms 11-Dehydrocorticosterone into Transcriptionally Active Glucocorticoid in Neonatal Rat Heart KAREN E. SHEPPARD AND DOMINIC J. AUTELITANO Baker Medical Research Institute, Melbourne, Victoria, Australia 8008 The ability of cells to directly respond to glucocorticoids and aldosterone is a function of GR and MR expression, and coexpression of 11␤-hydroxysteroid dehydrogenases (11␤HSDs), which convert glucocorticoids and their 11-ketometabolites into either receptor inactive or active derivatives. The aim of the present study was to determine the cellular expression of GR, MR, 11␤HSD1, and 11␤HSD2 in neonatal rat heart and determine the role these enzymes play in modulating glucocorticoid and aldosterone action. Ribonuclease protection analysis and steroid binding assays showed that GR is expressed in both cardiac myocytes and fibroblasts, whereas MR is expressed only in myocytes. 11␤HSD2 was not detected in cardiac cells, but 11␤HSD1 was expressed at high levels in both cardiac myocytes and fibroblasts. Enzyme activity stud- ies demonstrated that 11␤HSD1 acted as a reductase only, converting biologically inactive 11-dehydrocorticosterone to corticosterone, which then stimulated serum and glucocorticoid-induced kinase gene transcription via GR. In both cardiac myocytes and fibroblasts, aldosterone stimulated serum and glucocorticoid-induced kinase gene expression exclusively via GR, but not MR, indicating that aldosterone can have glucocorticoid-like actions in heart. The ability of cardiac cells to use both circulating corticosterone and 11-dehydrocorticosterone as a source of glucocorticoid suggests that the heart is under tonic glucocorticoid control, implying that glucocorticoids play important homeostatic roles in the heart. (Endocrinology 143: 198 –204, 2002) C selective receptor. The enzyme 11␤-hydroxysteroid dehydrogenase (11␤HSD)2 converts endogenous glucocorticoids corticosterone (B) and cortisol to MR inactive 11-ketometabolites, 11-dehydrocorticosterone (11-DHB), and cortisone, thus enabling aldosterone to access MR in vivo (12, 13). In the absence of 11␤HSD2, MRs bind and can be activated by endogenous glucocorticoids (14, 15). The 11-ketometabolites of B and cortisol are also GR inactive, so that 11␤HSD2 also regulates steroid access to GR (16). In addition to 11␤HSD2, other 11␤HSD isoforms have been reported, of which one (11␤HSD1) has been cloned. In contrast to 11␤HSD2, which is NAD-dependent and operates as an exclusive dehydrogenase for B and cortisol, 11␤HSD1 in tissue homogenates is NADP/NADPH-dependent and catalyzes the reversible conversion of B and cortisol to 11DHB and cortisone, respectively (17). In intact cells, and in vivo, 11␤HSD1 is thought to act only as a reductase and thus can potentiate glucocorticoid action by increasing the local tissue concentration of endogenous glucocorticoids (18, 19). 11␤HSD isoforms thus play a critical role in modulating corticosteroid hormone action by interconverting endogenous glucocorticoids, B, and cortisol to GR- and MR-inert 11-ketometabolites. In human and rat heart homogenates, 11␤HSD activity is present (20, 21). Both cofactor dependence analysis (20, 22) and expression of specific mRNA suggest that 11␤HSD1 is the isoform predominantly expressed in heart, though 11␤HSD2 may be present at low levels (20, 23). The ability of the heart to respond to endogenous glucocorticoids and aldosterone is not only a function of the presence of GR and/or MR but also expression of the enzymes 11␤HSD1 and 11␤HSD2 in the same cells. Previous studies have suggested that both 11␤HSD1 and 11␤HSD2 are ARDIAC HYPERTROPHY IS an important compensatory mechanism of the heart in response to chronic increases in hemodynamic load; sustained hemodynamic load, however, eventually causes a transition from compensatory hypertrophy to heart failure. Elevated levels of both endogenous glucocorticoids and mineralocorticoids can induce increases in hemodynamic load by stimulating sodium retention, extravascular fluid expansion, and increasing total peripheral resistance (1). In addition to these effects, there is evidence for direct actions of these steroids on the heart that may contribute to the altered phenotypes associated with cardiac hypertrophy and failure (2, 3). Furthermore, elevated aldosterone has been implicated in the development of cardiac fibrosis (4); although, whether this is a direct aldosterone effect on cardiac fibroblast collagen synthesis is controversial (5–7). Direct effects of mineralocorticoids and glucocorticoids on cardiac cells require the presence of MRs and GRs. GRs are ubiquitously expressed and have been described in heart (8). Specific binding of aldosterone (9) and MR mRNA (10) have been demonstrated in rat heart, whereas both MR mRNA and MR protein have been detected in human cardiomyocytes (11). MR has equally high affinity for both endogenous glucocorticoids and aldosterone; and, given that circulating concentrations of glucocorticoids are usually three orders of magnitude higher than those of aldosterone, a cellular mechanism is required to allow aldosterone to bind to this non- Abbreviations: B, Corticosterone; BdU, bromodeoxyuridine; 11␤HSD, 11␤-hydroxysteroid dehydrogenase; 11-DHB, 11-dehydrocorticosterone; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; RNase, ribonuclease; SGK, serum and glucocorticoid-induced kinase. 198 Sheppard and Autelitano • GR, MR, and 11␤HSD in Cardiac Cells expressed in heart, although the cell(s) in which these enzymes is expressed, and the potential coexpression with MR and/or GR has not been reported. In addition, the role these enzymes play in modulating glucocorticoid and aldosterone action has not been addressed. Therefore, the present study has determined the expression of MR, GR, and 11␤HSD isoforms in cardiac myocytes and fibroblasts. In addition, 11␤HSD activity was assessed to determine whether access of steroids to these receptors is modulated and whether this affects receptor function. We demonstrate that cardiac myocytes and fibroblasts express functional GR, whereas MR expression is limited to myocytes. The absence of 11␤HSD2 and the presence of high levels of 11␤HSD1 reductase activity in both cardiac myocytes and fibroblasts allow these cells to use both 11-DHB and B as a source of transcriptionally active glucocorticoid and suggest that myocyte MRs mediate glucocorticoid effects in vivo. Materials and Methods Cells and tissues Primary cultures of cardiac myocytes and fibroblasts were prepared from 1- to 2-d-old Sprague Dawley rats as described (24). After preplating, to remove fibroblasts, nonattached myocytes were plated at an initial density of 750 cells/mm2 and incubated for 18 h in DMEM (ICN, Aurora, OH) containing 10% FCS ⫹ 5% horse serum and 0.1 mm bromodeoxyuridine (BdU). Cardio (...truncated)