Elizabeth Rabbitt

Modulation of 11β-Hydroxysteroid Dehydrogenase Isozymes by Proinflammatory Cytokines in Osteoblasts: An Autocrine Switch from Glucocorticoid Inactivation to Activation

Tissue damage by proinflammatory cytokines is attenuated at both systemic and cellular levels by counter anti‐inflammatory factors such as corticosteroids. Target cell responses to corticosteroids are dependent on several factors including prereceptor regulation via local steroidogenic enzymes. In particular, two isozymes of 11β‐hydroxysteroid dehydrogenase (11β‐HSD), by interconverting hormonally active cortisol (F) to inactive cortisone (E), regulate the peripheral action of corticosteroids 11β‐HSD1 by converting E to F and 11β‐HSD2 by inactivating F to E. In different in vitro and in vivo systems both 11β‐HSD isozymes have been shown to be expressed in osteoblasts (OBs). Using the MG‐63 human osteosarcoma cell‐line and primary cultures of human OBs, we have studied the regulation of osteoblastic 11β‐HSD isozyme expression and activity by cytokines and hormones with established roles in bone physiology. In MG‐63 cells, interleukin‐1β (IL‐1β) and tumor necrosis factor α (TNF‐α) potently inhibited 11β‐HSD2 activity (cortisol‐cortisone conversion) and messenger RNA (mRNA) levels in a dose‐dependent manner while stimulating reciprocal expression of 11β‐HSD1 mRNA and activity (cortisone‐cortisol conversion). A similar rise in 11β‐HSD1 reductase activity also was observed in primary cultures of OBs treated with 10 ng/ml TNF‐α. Pretreatment of MG‐63 cells with 0.1 ng/ml IL‐1β resulted in increased cellular sensitivity to physiological glucocorticoids as shown by induction of serum and glucocorticoid‐inducible kinase (SGK; relative increase with 50 nM F but no IL‐1β pretreatment 1.12 ± 0.34; with pretreatment 2.63 ± 0.50; p < 0.01). These results highlight a novel mechanism within bone cells whereby inflammatory cytokines cause an autocrine switch in intracellular corticosteroid metabolism by disabling glucocorticoid inactivation (11β‐HSD2) while inducing glucocorticoid activation (11β‐HSD1). Therefore, it can be postulated that some of the effects of proinflammatory cytokines within bone (e.g., periarticular erosions in inflammatory arthritis) are mediated by this mechanism.

Osteoblastic 11β‐Hydroxysteroid Dehydrogenase Type 1 Activity Increases With Age and Glucocorticoid Exposure

The risk of glucocorticoid‐induced osteoporosis increases substantially with age but there is considerable individual variation. In recent studies we have shown that the effects of glucocorticoids on bone are dependent on autocrine actions of the enzyme 11β‐hydroxysteroid dehydrogenase type 1 (11β‐HSD1); expression of 11β‐HSD1 in osteoblasts (OBs) facilitates local synthesis of active glucocorticoids with consequent effects on osteoblastic proliferation and differentiation. Using primary cultures of human OBs, we have now characterized the age‐specific variation in osteoblastic 11β‐HSD1 and defined enzyme kinetics and regulation using natural and therapeutic glucocorticoids. 11β‐HSD1 reductase activity (cortisone to cortisol conversion) was recognized in all OB cultures and correlated positively with age (r = 0.58 with all cultures, p < 0.01, and n = 18; r = 0.87 with calcaneal‐derived cultures, p < 0.001, and n = 14). Glucocorticoid treatment caused a time‐ and dose‐dependent increase in 11β‐HSD1 activity over control (e.g., dexamethasone [DEX; 1 μM], 2.6‐fold ± 0.5 (mean ± SE), p < 0.001, and n = 16; cortisol (100 nM), 1.7‐fold ± 0.1, p < 0.05, and n = 14). Similar increases in 11β‐HSD1 mRNA expression were indicated using real‐time quantitative reverse‐transcription polymerase chain reaction (RT‐PCR) analyses (3.5‐fold with DEX, p < 0.01; 2.5‐fold with cortisol, p < 0.05). The capacity of 11β‐HSD1 to metabolize the synthetic glucocorticoids prednisone and prednisolone was investigated in human OBs (hOBs) and fetal kidney‐293 cells stably transfected with human 11β‐HSD1 cDNA. Transfected cells and hOBs were able to interconvert prednisone and prednisolone with reaction kinetics indistinguishable from those for cortisone and cortisol. To assess the in vivo availability of substrates for osteoblastic 11β‐HSD1, plasma cortisone and prednisone levels were measured in normal males before and after oral prednisolone (5 mg). The 9:00 a.m. serum cortisone levels were 110 ± 5 nmol/liter and prednisone levels peaked at 78 ± 23 nmol/liter 120 minutes after administration of prednisolone. Thus, therapeutic use of steroids increases substrate availability for 11β‐HSD1 in bone. These studies indicate that activation of glucocorticoids at an autocrine level within bone is likely to play an important role in the age‐related decrease in bone formation and increased risk of glucocorticoid‐induced osteoporosis.

Local and systemic glucocorticoid metabolism in inflammatory arthritis

Background: Isolated, primary synovial fibroblasts generate active glucocorticoids through expression of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). This enzyme produces cortisol from inactive cortisone (and prednisolone from prednisone). Objective: To determine how intact synovial tissue metabolises glucocorticoids and to identify the local and systemic consequences of this activity by examination of glucocorticoid metabolism in patients with rheumatoid arthritis (RA). Methods: Synovial tissue was taken from patients with RA during joint replacement surgery. Glucocorticoid metabolism in explants was assessed by thin-layer chromatography and specific enzyme inhibitors. RT-PCR and immunohistochemistry were used to determine expression and distribution of 11β-HSD enzymes. Systemic glucocorticoid metabolism was examined in patients with RA using gas chromatography/mass spectrometry. Results: Synovial tissue synthesised cortisol from cortisone, confirming functional 11β-HSD1 expression. In patients with RA, enzyme activity correlated with donor erythrocyte sedimentation rate (ESR). Synovial tissues could also convert cortisol back to cortisone. Inhibitor studies and immunohistochemistry suggested this was owing to 11β-HSD2 expression in synovial macrophages, whereas 11β-HSD1 expression occurred primarily in fibroblasts. Synovial fluids exhibited lower cortisone levels than matched serum samples, indicating net local steroid activation. Urinary analyses indicated high 11β-HSD1 activity in untreated patients with RA compared with controls and a significant correlation between total body 11β-HSD1 activity and ESR. Conclusions: Synovial tissue metabolises glucocorticoids, the predominant effect being glucocorticoid activation, and this increases with inflammation. Endogenous glucocorticoid production in the joint is likely to have an impact on local inflammation and bone integrity.

Prereceptor regulation of glucocorticoid action by 11β-hydroxysteroid dehydrogenase: a novel determinant of cell proliferation

Isozymes of 11β‐hydroxysteroid dehydrogenase (11β‐HSD) act at a prereceptor level to regulate the tissue‐specific availability of active glu‐cocorticoids. To examine the effect of this on cell proliferation and differentiation, we have developed transfectant variants of a rat osteosarcoma cell line that express cDNA for 11β‐HSD1 (ROS 17/2.8β1) or 11β‐HSD2 (ROS 17/2.8β2). ROS 17/2.8β1 showed net conversion of cortisone to cortisol whereas ROS 17/2.8β2 showed only inactivation of cortisol to cortisone. There was no significant difference in glucocorticoid receptor (GR) expression between the different clones. However, in proliferation and differentiation studies, ROS 17/2.8β2 cells were completely resistant to cortisol. In contrast, ROS 17/2.8β1 were sensitive to both cortisone and cortisol. Expression of 11β‐HSD1 de‐creased cell proliferation whereas 11β‐HSD2 increased proliferation. These responses appear to be due to metabolism of endogenous serum glucocorticoids; proliferation of ROS 17/2.8β1 decreased further with exogenous cortisone or cortisol whereas ROS 17/2.8β2 were resistant to both compounds. The pro‐prolifera‐tive effects of 11β‐HSD2 were abrogated by 18β‐glycyrrhetinic acid, an 11β‐HSD inhibitor, and in cells transfected with cDNA encoding inactive 11β‐HSD 2. Data indicate that differential regulation of 11β‐HSD1 and 2 (rather than GR expression) is a key determinant of cell proliferation. Dysregulated expression of 11β‐HSD2 may be a novel feature of tumorigenesis.—Rabbitt, E. H., Lavery, G. G., Walker, E. A., Cooper, M. S., Stewart, P. M., Hewison, M. Prereceptor regulation of glucocorticoid action by 11β‐hydroxysteroid dehydrogenase: a novel determinant of cell proliferation. FASEB J. 16, 36–44 (2002)

11β-Hydroxysteroid dehydrogenase blockade prevents age-induced skin structure and function defects

Glucocorticoid (GC) excess adversely affects skin integrity, inducing thinning and impaired wound healing. Aged skin, particularly that which has been photo-exposed, shares a similar phenotype. Previously, we demonstrated age-induced expression of the GC-activating enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in cultured human dermal fibroblasts (HDFs). Here, we determined 11β-HSD1 levels in human skin biopsies from young and older volunteers and examined the aged 11β-HSD1 KO mouse skin phenotype. 11β-HSD1 activity was elevated in aged human and mouse skin and in PE compared with donor-matched photo-protected human biopsies. Age-induced dermal atrophy with deranged collagen structural organization was prevented in 11β-HSD1 KO mice, which also exhibited increased collagen density. We found that treatment of HDFs with physiological concentrations of cortisol inhibited rate-limiting steps in collagen biosynthesis and processing. Furthermore, topical 11β-HSD1 inhibitor treatment accelerated healing of full-thickness mouse dorsal wounds, with improved healing also observed in aged 11β-HSD1 KO mice. These findings suggest that elevated 11β-HSD1 activity in aging skin leads to increased local GC generation, which may account for adverse changes occurring in the elderly, and 11β-HSD1 inhibitors may be useful in the treatment of age-associated impairments in dermal integrity and wound healing.

Differential expression, function and response to inflammatory stimuli of 11β-hydroxysteroid dehydrogenase type 1 in human fibroblasts: a mechanism for tissue-specific regulation of inflammation

Stromal cells such as fibroblasts play an important role in defining tissue-specific responses during the resolution of inflammation. We hypothesized that this involves tissue-specific regulation of glucocorticoids, mediated via differential regulation of the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). Expression, activity and function of 11β-HSD1 was assessed in matched fibroblasts derived from various tissues (synovium, bone marrow and skin) obtained from patients with rheumatoid arthritis or osteoarthritis. 11β-HSD1 was expressed in fibroblasts from all tissues but mRNA levels and enzyme activity were higher in synovial fibroblasts (2-fold and 13-fold higher mRNA levels in dermal and synovial fibroblasts, respectively, relative to bone marrow). Expression and activity of the enzyme increased in all fibroblasts following treatment with tumour necrosis factor-α or IL-1β (bone marrow: 8-fold and 37-fold, respectively, compared to vehicle; dermal fibroblasts: 4-fold and 14-fold; synovial fibroblasts: 7-fold and 31-fold; all P < 0.01 compared with vehicle). Treatment with IL-4 or interferon-γ was without effect, and there was no difference in 11β-HSD1 expression between fibroblasts (from any site) obtained from patients with rheumatoid arthritis or osteoarthritis. In the presence of 100 nmol/l cortisone, IL-6 production – a characteristic feature of synovial derived fibroblasts – was significantly reduced in synovial but not dermal or bone marrow fibroblasts. This was prevented by co-treatment with an 11β-HSD inhibitor, emphasizing the potential for autocrine activation of glucocorticoids in synovial fibroblasts. These data indicate that differences in fibroblast-derived glucocorticoid production (via the enzyme 11β-HSD1) between cells from distinct anatomical locations may play a key role in the predeliction of certain tissues to develop persistent inflammation.

Late-Onset Apparent Mineralocorticoid Excess Caused by Novel Compound Heterozygous Mutations in the HSD11B2 Gene

Abstract—Mutations in the gene encoding 11&bgr;-hydroxysteroid dehydrogenase type 2, 11&bgr;-HSD2 (HSD11B2), explain the molecular basis for the syndrome of apparent mineralocorticoid excess (AME), characterized by severe hypertension and hypokalemic alkalosis. Cortisol is the offending mineralocorticoid in AME, as the result of a lack of 11&bgr;-HSD2–mediated cortisol to cortisone inactivation. In this study, we describe mutations in the HSD11B2 gene in 3 additional AME kindreds in which probands presented in adult life, with milder phenotypes including the original seminal case reported by Stewart and Edwards. Genetic analysis of the HSD11B2 gene revealed that all probands were compound heterozygotes, for a total of 7 novel coding and noncoding mutations. Of the 7 mutations detected, 6 were investigated for their effects on gene expression and enzyme activity by the use of mutant cDNA and minigene constructs transfected into HEK 293 cells. Four missense mutations resulted in enzymes with varying degrees of activity, all <10% of wild type. A further 2 mutations generated incorrectly spliced mRNA and predicted severely truncated, inactive enzyme. The mothers of 2 probands heterozygous for missense mutations have presented with a phenotype indistinguishable from “essential” hypertension. These genetic and biochemical data emphasize the heterogeneous nature of AME and the effects that heterozygosity at the HSD11B2 locus can have on blood pressure in later life.

Abnormal expression of 11 beta-hydroxysteroid dehydrogenase type 2 in human pituitary adenomas: a prereceptor determinant of pituitary cell proliferation.

The physiological effects of glucocorticoids (GCs) are, at least in part, mediated by inhibition of cell proliferation. Two isozymes of 11β-hydroxysteroid dehydrogenase (11β-HSD) interconvert cortisol (F) and inactive cortisone (E), and are thus able to modulate GC action at an autocrine level. Previously, we have demonstrated absent expression of 11β-HSD2 in normal pituitaries; however, in a small number of pituitary tumors analysed, 11β-HSD2 was readily demonstrable. Here we have used real-time RT–PCR to quantify expression of mRNA for 11 β-HSD1 and 2 in 105 human pituitary tumors and have performed enzyme expression and activity studies in primary pituitary cultures. Overall, pituitary tumors expressed lower levels of 11β-HSDl mRNA compared with normals (0.2-fold, P<0.05). In contrast, expression of 11β-HSD2 mRNA was 9.8-fold greater in tumors than in normals (P<0.001). Enzyme assays showed significant 11β-HSD2 activity (71.9±22.3 pmol/h/mg protein (mean±s.d.)) but no detectable 11β-HSDl activity. Proliferation assays showed that addition of glycyrrhetinic acid (an 11β-HSD2 inhibitor) resulted in a 30.3±7.7% inhibition of cell proliferation. In summary, we describe a switch in expression from 11β-HSDl to 11β-HSD2 in neoplastic pituitary tissue. We propose that abnormal expression of 11β-HSD2 acts as a proproliferative prereceptor determinant of pituitary cell growth, and may provide a novel target for future tumor therapy.

Lack of Significant Metabolic Abnormalities in Mice with Liver-Specific Disruption of 11β-Hydroxysteroid Dehydrogenase Type 1.

Glucocorticoids (GC) are implicated in the development of metabolic syndrome, and patients with GC excess share many clinical features, such as central obesity and glucose intolerance. In patients with obesity or type 2 diabetes, systemic GC concentrations seem to be invariably normal. Tissue GC concentrations determined by the hypothalamic-pituitary-adrenal (HPA) axis and local cortisol (corticosterone in mice) regeneration from cortisone (11-dehydrocorticosterone in mice) by the 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme, principally expressed in the liver. Transgenic mice have demonstrated the importance of 11β-HSD1 in mediating aspects of the metabolic syndrome, as well as HPA axis control. In order to address the primacy of hepatic 11β-HSD1 in regulating metabolism and the HPA axis, we have generated liver-specific 11β-HSD1 knockout (LKO) mice, assessed biomarkers of GC metabolism, and examined responses to high-fat feeding. LKO mice were able to regenerate cortisol from cortisone to 40% of control and had no discernible difference in a urinary metabolite marker of 11β-HSD1 activity. Although circulating corticosterone was unaltered, adrenal size was increased, indicative of chronic HPA stimulation. There was a mild improvement in glucose tolerance but with insulin sensitivity largely unaffected. Adiposity and body weight were unaffected as were aspects of hepatic lipid homeostasis, triglyceride accumulation, and serum lipids. Additionally, no changes in the expression of genes involved in glucose or lipid homeostasis were observed. Liver-specific deletion of 11β-HSD1 reduces corticosterone regeneration and may be important for setting aspects of HPA axis tone, without impacting upon urinary steroid metabolite profile. These discordant data have significant implications for the use of these biomarkers of 11β-HSD1 activity in clinical studies. The paucity of metabolic abnormalities in LKO points to important compensatory effects by HPA activation and to a crucial role of extrahepatic 11β-HSD1 expression, highlighting the contribution of cross talk between GC target tissues in determining metabolic phenotype.

A Switch in Hepatic Cortisol Metabolism across the Spectrum of Non Alcoholic Fatty Liver Disease

Context Non alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome. NAFLD represents a spectrum of liver disease ranging from reversible hepatic steatosis, to non alcoholic steato-hepatitis (NASH) and cirrhosis. The potential role of glucocorticoids (GC) in the pathogenesis of NAFLD is highlighted in patients with GC excess, Cushings syndrome, who develop central adiposity, insulin resistance and in 20% of cases, NAFLD. Although in most cases of NAFLD, circulating cortisol levels are normal, hepatic cortisol availability is controlled by enzymes that regenerate cortisol (F) from inactive cortisone (E) (11β-hydroxysteroid dehydrogenase type 1, 11β-HSD1), or inactivate cortisol through A-ring metabolism (5α- and 5β-reductase, 5αR and 5βR). Objective and Methods In vitro studies defined 11β-HSD1 expression in normal and NASH liver samples. We then characterised hepatic cortisol metabolism in 16 patients with histologically proven NAFLD compared to 32 obese controls using gas chromatographic analysis of 24 hour urine collection and plasma cortisol generation profile following oral cortisone. Results In patients with steatosis 5αR activity was increased, with a decrease in hepatic 11β-HSD1 activity. Total cortisol metabolites were increased in this group consistent with increased GC production rate. In contrast, in patients with NASH, 11β-HSD1 activity was increased both in comparison to patients with steatosis, and controls. Endorsing these findings, 11β-HSD1 mRNA and immunostaining was markedly increased in NASH patients in peri septal hepatocytes and within CD68 positive macrophages within inflamed cirrhotic septa. Conclusion Patients with hepatic steatosis have increased clearance and decreased hepatic regeneration of cortisol and we propose that this may represent a protective mechanism to decrease local GC availability to preserve hepatic metabolic phenotype. With progression to NASH, increased 11β-HSD1 activity and consequent cortisol regeneration may serve to limit hepatic inflammation.