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M Hewison

Publications and source records attributed to M Hewison.

At least 19 recordsLinked to original sources
PubMed2004

11beta-Hydroxysteroid dehydrogenase expression and glucocorticoid synthesis are directed by a molecular switch during osteoblast differentiation.

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) plays an important role in the prereceptor regulation of corticosteroids by locally converting cortisone into active cortisol. To investigate the impact of this mechanism on osteoblast development, we have characterized 11beta-HSD1 activity and regulation in a differentiating human osteoblast cell line (SV-HFO). Continuous treatment with the synthetic glucocorticoid dexamethasone induces differentiation of SV-HFO cells during 21 d of culture. Using this cell system, we showed an inverse relationship between 11beta-HSD1 activity and osteoblast differentiation. 11beta-HSD1 mRNA expression and activity were low and constant in differentiating osteoblasts. However, in the absence of differentiation (no dexamethasone), 11beta-HSD1 mRNA and activity increased strongly from d 12 of culture onward, with a peak around d 19. Promoter reporter studies provided evidence that specific regions of the 11beta-HSD1 gene are involved in this differentiation controlled regulation of the enzyme. Functional implication of these changes in 11beta-HSD1 is shown by the induction of osteoblast differentiation in the presence of cortisone. The current study demonstrates the presence of an intrinsic differentiation-driven molecular switch that controls expression and activity of 11beta-HSD1 and thereby cortisol production by human osteoblasts. This efficient mechanism by which osteoblasts generate cortisol in an autocrine fashion to ensure proper differentiation will help to understand the complex effects of cortisol on bone metabolism.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed2004

Measurement of vitamin D levels in inflammatory bowel disease patients reveals a subset of Crohn's disease patients with elevated 1,25-dihydroxyvitamin D and low bone mineral density.

OBJECTIVES: Many patients with Crohn's disease (CD) have low bone mineral density (BMD) that may not be solely attributable to glucocorticoid use. We hypothesised that low BMD in patients with CD is associated with elevated circulating levels of the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)(2)D). We further hypothesised that this was secondary to increased synthesis of 1,25(OH)(2)D by inflammatory cells in the intestine. The aim of this study was to examine the relationship between 1,25(OH)(2)D levels and BMD in patients with CD. METHODS: An IRB approved retrospective review of medical records from patients with CD (n = 138) or ulcerative colitis (UC, n = 29). Measurements of vitamin D metabolites and immunoreactive parathyroid hormone (iPTH) were carried out. BMD results were available for 88 CD and 20 UC patients. Immunohistochemistry or real time reverse transcription-polymerase chain reaction (RT-PCR) for the enzyme 1alpha-hydroxylase was performed on colonic biopsies from patients with CD (14) or UC (12) and normal colons (4). RESULTS: Inappropriately high levels of serum 1,25(OH)(2)D (>60 pg/ml) were observed in 42% of patients with CD compared with only 7% in UC, despite no differences in mean iPTH. Serum 1,25(OH)(2)D levels were higher in CD (57 pg/ml) versus UC (41 pg/ml) (p = 0.0001). In patients with CD, there was a negative correlation between 1,25(OH)(2)D levels and lumbar BMD (r = -0.301, p = 0.005) independent of therapeutic glucocorticoid use. 1,25(OH)(2)D levels also correlated with CD activity. Lastly, immunohistochemistry and RT-PCR demonstrated increased expression of intestinal 1alpha-hydroxylase in patients with CD. CONCLUSIONS: These data demonstrate that elevated 1,25(OH)(2)D is more common in CD than previously appreciated and is independently associated with low bone mineral density. The source of the active vitamin D may be the inflamed intestine. Treatment of the underlying inflammation may improve metabolic bone disease in this subgroup of patients.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed2003

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 11 beta-hydroxysteroid dehydrogenase (11 beta-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 11 beta-HSD2 in normal pituitaries; however, in a small number of pituitary tumors analysed, 11 beta-HSD2 was readily demonstrable. Here we have used real-time RT-PCR to quantify expression of mRNA for 11 beta-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 11 beta-HSDl mRNA compared with normals (0.2-fold, P<0.05). In contrast, expression of 11 beta-HSD2 mRNA was 9.8-fold greater in tumors than in normals (P<0.001). Enzyme assays showed significant 11 beta-HSD2 activity (71.9+/-22.3 pmol/h/mg protein (mean+/-s.d.)) but no detectable 11 beta-HSDl activity. Proliferation assays showed that addition of glycyrrhetinic acid (an 11 beta-HSD2 inhibitor) resulted in a 30.3+/-7.7% inhibition of cell proliferation. In summary, we describe a switch in expression from 11 beta-HSDl to 11 beta-HSD2 in neoplastic pituitary tissue. We propose that abnormal expression of 11 beta-HSD2 acts as a proproliferative prereceptor determinant of pituitary cell growth, and may provide a novel target for future tumor therapy.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed2003

11beta-hydroxysteroid dehydrogenases, cell proliferation and malignancy.

The enzymes 11beta-hydroxysteroid dehydrogenase type 1 and 2 (11beta-HSD1 and 2) have well-defined roles in the tissue-specific metabolism of glucocorticoids which underpin key endocrine mechanisms such as adipocyte differentiation (11beta-HSD1) and mineralocorticoid action (11beta-HSD2). However, in recent studies we have shown that the effects of 11beta-HSD1 and 2 are not restricted to distinct tissue-specific hormonal functions. Studies of normal fetal and adult tissues, as well as their tumor equivalents, have shown a further dichotomy in 11beta-HSD expression and activity. Specifically, most normal glucocorticoid receptor (GR)-rich tissues such as adipose tissue, bone, and pituitary cells express 11beta-HSD1, whereas their fetal equivalents and tumors express 11beta-HSD2. We have therefore postulated that the ability of 11beta-HSD1 to generate cortisol acts as an autocrine anti-proliferative, pro-differentiation stimulus in normal adult tissues. In contrast, the cortisol-inactivating properties of 11beta-HSD2 lead to pro-proliferative effects, particularly in tumors. This proposal is supported by experiments in vitro which have demonstrated divergent effects of 11beta-HSD1 and 2 on cell proliferation. Current studies are aimed at (1) characterizing the underlying mechanisms for a "switch" in 11beta-HSD isozyme expression in tumors; (2) defining the molecular targets for glucocorticoids as regulators of cell proliferation; (3) evaluating the potential for targeting glucocorticoid metabolism as therapy for some cancers. These and other issues are discussed in the present review.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed2003

Characterization of human trophoblast as a mineralocorticoid target tissue.

In mineralocorticoid target tissues, 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) confers mineralocorticoid receptor selectivity by metabolizing hormonally active cortisol to inactive cortisone, allowing aldosterone access to the receptor. This enzyme is also expressed in high abundance in fetal tissues, particularly in placental trophoblast, where a role has been proposed in regulating fetal growth and development by protecting the fetus from maternal hypercortisolaemia and modulating local glucocorticoid receptor (GR), rather than mineralocorticoid receptor-mediated responses. As such the placenta has not been considered a mineralocorticoid target tissue. We have used conventional RT-PCR and real-time quantitative RT-PCR to demonstrate that primary cultures of term human cytotrophoblast express the mineralocorticoid-responsive genes Na/K-ATPase (alpha1 and beta1 subunits), epithelial sodium channel (ENaC, alpha and gamma subunits) and the serum and glucocorticoid-inducible kinase (SGK). SGK expression was found to be rapidly and strongly induced by corticosteroids (24- and 38-fold by 10(-7) mol/l aldosterone and 10(-7) mol/l dexamethasone respectively after 1 h). Dexamethasone-, but not aldosterone-stimulated SGK induction was inhibited by GR antagonist (RU38486), confirming the presence of a functional mineralocorticoid receptor and suggesting that placental trophoblast expresses a functional mineralocorticoid receptor, which is in part responsible for the corticosteroid regulation of SGK expression. Placental 11beta-HSD2 may protect the MR in a fashion analogous to classical mineralocorticoid tissues to modulate trophoblast sodium transport.

Adult↗
PubMed2002

Presentation of a PTHrP-secreting pancreatic neuroendocrine tumour, with hypercalcaemic crisis, pre-eclampsia, and renal failure.

Severe hypercalcaemia during pregnancy is rare and most cases are secondary to hyperparathyroidism. This is the first report of a parathyroid hormone related protein (PTHrP) secreting neuroendocrine tumour of the pancreas manifesting with severe hypercalcaemia during pregnancy. Measurement of PTHrP was useful in both the diagnosis and follow up of our patient and should be considered in the diagnostic workup of patients with unexplained hypercalcaemia. A raised PTHrP concentration is a strong indicator of malignancy.

Acute Kidney Injury↗
PubMed2002

Association studies between microsatellite markers within the gene encoding human 11beta-hydroxysteroid dehydrogenase type 1 and body mass index, waist to hip ratio, and glucocorticoid metabolism.

Two isozymes of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) interconvert active cortisol (F) and inactive cortisone (E). 11beta-HSD1 is an oxo-reductase (E to F) expressed in several glucocorticoid target tissues, including liver and adipose tissue, where it facilitates glucocorticoid-induced gluconeogenesis and adipocyte differentiation, respectively. We have isolated a full-length HSD11B1 genomic clone; the gene is more than 30 kb in length, not 9 kb in length as previously reported, principally due to a large intron 4. Two polymorphic (CA)(n) repeats have been characterized within intron 4: a CA(19) repeat 2.7 kb 3' of exon 4 and a CA(15) repeat 3 kb 5' of exon 5. The microsatellites, CA(19) and CA(15), were PCR amplified using fluorescent primers and were genotyped on an ABI 377 DNA sequencer from DNA of 413 normal individuals enrolled in the MONICA study of cardiovascular risk factors and 557 Danish men (ADIGEN study), of whom 234 were obese [body mass index (BMI), >/=31 kg/m(2) ] at draft board examination and 323 were randomly selected controls from the draftee population with BMI below 31 kg/m(2) (mean +/- SE, 21.7 +/- 0.41). Genotypic data from the normal MONICA cohort was compared with gender, 5beta-tetrahydrocortisol+5alpha-tetrahydrocortisol/tetrahydrocortisone ratio, and waist to hip (W:H) ratio. When analyzed by allele length (0, 1, or 2 short alleles) for the CA(19) marker, there was a trend toward a higher 5beta-tetrahydrocortisol+5alpha-tetrahydrocortisol/tetrahydrocortisone ratio (P = 0.058) and an increased W:H ratio (2 vs. 0.1 short; P(c) = 0.10) with overrepresentation of short alleles. The opposite was true for the CA(15) locus, with longer alleles at this locus predicting increased 11beta-HSD1 activity, particularly in females. Genotypic data from the ADIGEN case-control population was compared with clinical markers of obesity such as BMI and W:H ratio. There was no significant difference in the distribution of either microsatellite marker between lean and obese groups. Allele distributions were binomial, as seen for the MONICA cohort, and the data were split accordingly (zero, one, or two short alleles). No significant association was seen between grouped alleles and the clinical parameters. No association was observed between HSD11B1 genotype and BMI in either population. These data suggest that 11beta-HSD1 is not a major factor in explaining genetic susceptibility to obesity per se. However, weak associations between HSD11B1 genotype, increased 11beta-HSD1 activity, and W:H ratio suggest that polymorphic variability at the HSD11B1 locus may influence susceptibility to central obesity through enhanced 11beta-HSD1 activity (E to F conversion) in visceral adipose tissue.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗
PubMed2002

Expression of 11beta-hydroxysteroid dehydrogenase type 1 in adipose tissue is not increased in human obesity.

Central obesity is associated with increased morbidity and mortality. Preadipocyte proliferation and differentiation contribute to increases in adipose tissue mass, yet the mechanisms that underlie these processes remain unclear. Patients with glucocorticoid excess develop a reversible form of central obesity, but circulating cortisol levels in idiopathic obesity are invariably normal. We have hypothesized that the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), by converting inactive cortisone to active cortisol in adipose tissue, might be an important autocrine regulator of fat mass. Paired omental and sc fat biopsies were obtained from 32 women (median age, 43 yr; range, 28-65; median body mass index, 27.5 kg/m(2); range, 19.7-39.2) undergoing elective abdominal surgery. 11beta-HSD1 activity and mRNA levels were assessed in whole tissue and in isolated preadipocytes and adipocytes using specific enzyme assays and real-time PCR. Preadipocyte proliferation was measured using tritiated thymidine incorporation. Whole adipose tissue 11beta-HSD1 mRNA levels did not differ between omental and sc samples (P = 0.73). In addition, mRNA levels did not correlate with body mass index (omental: r = 0.1; P = 0.6; sc: r = 0.15; P = 0.4). In keeping with earlier studies, 11beta-HSD1 mRNA levels were higher in omental compared with sc preadipocytes. However, in cultured omental preadipocytes, 11beta-HSD1 activity inversely correlated with body mass index (r = -0.47; P = 0.03). In omental preadipocytes, both cortisol and cortisone decreased proliferation (P < 0.05). Inhibition of 11beta-HSD1 with glycyrrhetinic acid partially reversed the cortisone-induced decrease in preadipocyte proliferation (P < 0.05). Enhanced preadipocyte proliferation within omental adipose tissue as a consequence of decreased 11beta-HSD1 mRNA levels and activity may contribute to increases in visceral adipose tissue mass in obese patients.

11-beta-Hydroxysteroid Dehydrogenase Type 1↗
PubMed2001

Functional expression, characterization, and purification of the catalytic domain of human 11-beta -hydroxysteroid dehydrogenase type 1.

11-beta-hydroxysteroid dehydrogenase type 1 catalyzes the conversion of cortisone to hormonally active cortisol and has been implicated in the pathogenesis of a number of disorders including insulin resistance and obesity. The enzyme is a glycosylated membrane-bound protein that has proved difficult to purify in an active state. Extracted enzyme typically loses the reductase properties seen in intact cells and shows principally dehydrogenase activity. The C-terminal catalytic domain is known to contain a disulfide bond and is located within the lumen of the endoplasmic reticulum, anchored to the membrane by a single N-terminal transmembrane domain. We report here the functional expression of the catalytic domain of the human enzyme, without the transmembrane domain and the extreme N terminus, in Escherichia coli. Moderate levels of soluble active protein were obtained using an N-terminal fusion with thioredoxin and a 6xHis tag. In contrast, the inclusion of a 6xHis tag at the C terminus adversely affected protein solubility and activity. However, the highest levels of active protein were obtained using a construct expressing the untagged catalytic domain. Nonreducing electrophoresis revealed the presence of both monomeric and dimeric disulfide bonded forms; however, mutation of a nonconserved cysteine residue resulted in a recombinant protein with no intermolecular disulfide bonds but full enzymatic activity. Using the optimal combination of plasmid construct and E. coli host strain, the recombinant protein was purified to apparent homogeneity by single step affinity chromatography. The purified protein possessed both dehydrogenase and reductase activities with a K(m) of 1.4 micrometer for cortisol and 9.5 micrometer for cortisone. This study indicates that glycosylation, the N-terminal region including the transmembrane helix, and intermolecular disulfide bonds are not essential for enzyme activity and that expression in bacteria can provide active recombinant protein for future structural and functional studies.

11-beta-Hydroxysteroid Dehydrogenase Type 1↗
PubMed2001

Estrogen metabolism and malignancy: analysis of the expression and function of 17beta-hydroxysteroid dehydrogenases in colonic cancer.

Age and sex differences in the incidence of gastrointestinal cancers suggest the involvement of sex steroids. Post-menopausal loss of estrogen in women appears to be associated with a lower risk of colonic cancer, and studies in vitro have shown that estradiol (E2) stimulates the growth of colonic cancer cell lines. Paradoxically more recent epidemiological data have shown that hormone replacement therapy (HRT) is associated with a lower risk of colonic cancer, although this may reflect differences in the composition and route of administration of HRT regimes. The precise mechanism by which estrogens influence colonic cancer in vivo remains unclear, although E2-induced growth of colonic cancer cells in vitro appears to be dependent on estrogen receptor (ER) expression. We have previously demonstrated differential responses to E2 in pre-malignant and malignant colonic cancer cell lines, without any apparent difference in ER expression. Analogous to well documented studies in breast cancer, we have postulated that local steroid metabolism in the colon may play a key role in modulating the effects of oestrogens by determining the tissue availability of active E2. Using biopsy material we have shown that the normal colonic mucosa has a high level of 17beta-hydroxysteroid dehydrogenase (17beta-HSD)-mediated E2 metabolism. Furthermore, the predominant enzyme activity, inactivation of E2 to estrone (E1), was significantly decreased in paired tumor biopsies. The presence of 17beta-HSD activity in the colon appears to be due to expression of the type 2 and 4 isozymes of 17beta-HSD (17beta-HSD2 and 4), and expression of mRNA for the latter was shown to be significantly decreased in tumours compared to normal mucosa. Further studies have characterised the expression of 17beta-HSD2 and 4 in colonic epithelial cells and in colonic cancer cell lines, and have suggested a link between estrogen metabolism and colonic cell proliferation. Data reviewed here provide evidence for the importance of 17beta-HSD isozymes as attenuators of E2 bioavailability in the colon, and emphasise a possible role for 17beta-HSD2 and 4 in the pathogenesis of colon cancer.

17-Hydroxysteroid Dehydrogenases↗
PubMed2001

Expression of 11 beta-hydroxysteroid dehydrogenase in rat osteoblastic cells: pre-receptor regulation of glucocorticoid responses in bone.

11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) acts as a pre-receptor signaling mechanism for corticosteroids by regulating the access of active glucocorticoids to both glucocorticoid (GR) and mineralocorticoid receptors (MR). To examine the relationship between endogenous glucocorticoid metabolism and osteoblast function, we have characterized the expression of 11 beta-HSD isozymes in rat osteosarcoma cells. Analysis of mRNA from ROS 25/1, UMR 106 and ROS 17/2.8 cells revealed transcripts for both 11 beta-HSD type 1 (11 beta-HSD1) and type 2 (11 beta-HSD2) in all three cell lines. However, enzyme activity studies showed only high affinity dehydrogenase activity (inactivation of corticosterone (B) to 11-dehydrocorticosterone (A)), characteristic of 11 beta-HSD2; conversion of B to A was higher in ROS 25/1> UMR 106 cells>ROS 17/2.8. Although all three cell lines had similar numbers of GR (50,000/cell), glucocorticoid modulation of alkaline phosphatase activity and cell proliferation was only detectable in ROS 17/2.8 cells. Further studies showed that 11 beta-HSD2 activity in each of the cells was potently stimulated by both A and B, but not by synthetic dexamethasone. This effect was blocked by the 11 beta-HSD inhibitor, 18 beta-glycyrrhetinic acid (but not by GR or MR antagonists) suggesting direct, allosteric regulation of 11 beta-HSD2 activity. These data indicate that in osteosarcoma cells 11 beta-HSD2 plays a key role in controlling GR-mediated responses; cells with relatively high levels of 11 beta-HSD2 activity were insensitive to glucocorticoids, whilst cells with low levels showed functional responses to both dexamethasone and B. In addition to the established effects of 11 beta-HSD2 in protecting MR in the kidney and colon, our data suggest that 11 beta-HSD2 in bone represents an important pre-receptor mechanism in determining ligand availability to GR.

11-beta-Hydroxysteroid Dehydrogenase Type 1↗
PubMed2001

Regulation of vitamin D-1alpha-hydroxylase in a human cortical collecting duct cell line.

BACKGROUND: Recent studies have shown that renal expression of 25-hydroxyvitamin D3-1alpha-hydroxylase (1alpha-OHase) is not restricted to proximal tubules. To investigate the significance of this expression, we characterized the regulation of 1alpha-OHase expression and activity in a human cortical collecting duct cell line (HCD). METHODS: Expression of 1alpha-OHase mRNA and protein was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses. Enzyme activity was quantified using 25-hydroxyvitamin D3 as the substrate; conversion to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and 24,25-dihydroxyvitamin D3 was then determined by thin-layer chromatography. RESULTS: HCD cells expressed mRNA and protein for 1alpha-OHase. However, basal 1,25(OH)2D3 production was lower than that observed in proximal tubule HKC-8 cells. In both cell lines, synthesis of 1,25(OH)2D3 was increased by forskolin, parathyroid hormone, and low calcium medium. Conversely, treatment with 1,25(OH)2D3 itself decreased 1alpha-OHase activity. This effect was more pronounced in HCD cells, which also demonstrated significantly higher levels of 24-hydroxylase activity. The most striking induction of 1alpha-OHase activity was observed in the HCD cells following incubation with lipopolysaccharide, which was coincident with the expression of mRNA for both CD14 and Toll-like receptor 4. CONCLUSIONS: These results highlight the capacity for synthesis of 1,25(OH)2D3 in cells from more distal areas of the nephron. However, more sensitive feedback regulation and immune induction of 1alpha-OHase in the HCD cells suggest a more localized role for 1,25(OH)2D3 production in the distal nephron.

Calcitriol↗
PubMed2001

Expression of 11 beta-hydroxysteroid dehydrogenase isozymes and corticosteroid hormone receptors in primary cultures of human trophoblast and placental bed biopsies.

Interconversion of active and inactive glucocorticoids, e.g. cortisol (F) and cortisone (E) is catalysed by 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) which exists as two isoforms. We have used human placental bed biopsies and an in-vitro cytotrophoblast cell culture system to examine the expression and activity of the 11 beta-HSD isoforms along with that of the glucocorticoid and mineralocorticoid receptors (GR and MR). Immunohistochemistry localized 11 beta-HSD1 to decidualized stromal cells and 11 beta-HSD2 to villous cytotrophoblast, syncytiotrophoblasts and trophoblast cells invading the placental bed and maternal vasculature. In primary cultures of human cytotrophoblast, 11 beta-HSD2, GR and MR mRNA were expressed. Low levels of 11 beta-HSD1 mRNA were noted in these cultured cells, but could be explained on the basis of contaminating, vimentin-positive decidual stromal cells (< or =5%). Enzyme activity studies confirmed the presence of a high-affinity, NAD-dependent dehydrogenase activity (K(m) 137 nmol/l and V(max) 128 pmol E/h/mg protein), indicative of the 11 beta-HSD2 isoform. No reductase activity was observed. The presence of functional MR and GR was determined using Scatchard analyses of dexamethasone and aldosterone binding (MR K(d) 1.4 nmol/l B(max) 3.0; GR K(d) 6.6 nmol/l B(max) 16.2 fmol/ng protein). The expression of 11 beta-HSD1 in maternal decidua and 11 beta-HSD2 in adjacent trophoblast suggests an important role for glucocorticoids in determining trophoblast invasion. The presence of the MR within trophoblast indicates that some of the effects of cortisol could be MR- rather than GR-mediated.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗
PubMed2001

Regulation of expression of 11beta-hydroxysteroid dehydrogenase type 1 in adipose tissue: tissue-specific induction by cytokines.

Patients with glucocorticoid excess develop central obesity, yet in simple obesity, circulating glucocorticoid levels are normal. We have suggested that the increased activity and expression of the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) generating active cortisol from cortisone within adipose tissue may be crucial in the pathogenesis of obesity. In this study primary cultures of human hepatocytes and adipose stromal cells (ASC) were used as in vitro models to investigate the tissue-specific regulation of 11betaHSD1 expression and activity. Treatment with tumor necrosis factor-alpha (TNFalpha) caused a dose-dependent increase in 11betaHSD1 activity in primary cultures of both sc [1743.1 +/- 1015.4% (TNFalpha, 10 ng/ml); P < 0.05 vs. control (100%)] and omental [375.8 +/- 57.0% (TNFalpha, 10 ng/ml); P < 0.01 vs. control (100%)] ASC, but had no effect on activity in human hepatocytes [90.2 +/- 2.8% (TNFalpha, 10 ng/ml); P = NS vs. control (100%)]. Insulin-like growth factor I (IGF-I) caused a dose-dependent inhibition of 11betaHSD1 activity in sc [49.7 +/- 15.0% (IGF-I, 100 ng/ml]; P < 0.05 vs. control (100%)] and omental [71.6 +/- 7.5 (IGF-I, 100 ng/ml); P < 0.01 vs. control (100%)] stromal cells, but not in human hepatocytes [101.8 +/- 15.7% (IGF-I, 100 ng/ml); P = NS vs. control (100%)]. Leptin treatment did not alter 11betaHSD1 activity in human hepatocytes, but increased activity in omental ASC [135.8 +/- 14.1% (leptin, 100 ng/ml); P = 0.08 vs. control (100%)]. Treatment with interleukin-1beta induced 11betaHSD1 activity and expression in sc and omental ASC in a time- and dose-dependent manner. 15-Deoxy-12,14-PGJ2, the putative endogenous ligand of the orphan nuclear receptor peroxisome proliferator-gamma, significantly increased 11betaHSD1 activity in omental cells [179.7 +/- 29.6% (1 microM); P < 0.05 vs. control (100%)] and sc [185.3 +/- 12.6% (1 microM); P < 0.01 vs. control (100%)] ASC, and it is possible that expression of this ligand may ensure continued cortisol generation to permit adipocyte differentiation. Protease inhibitors used in the treatment of human immunodeficiency virus infection are known to cause a lipodystrophic syndrome and central obesity, but saquinavir, indinavir, and neflinavir caused a dose-dependent inhibition of 11betaHSD1 activity in primary cultures of human omental ASC. 11betaHSD1 expression is increased in human adipose tissue by TNFalpha, interleukin-1beta, leptin, and orphan nuclear receptor peroxisome proliferator-gamma agonists, but is inhibited by IGF-I. This autocrine and/or paracrine regulation is tissue specific and explains recent clinical data and animal studies evaluating cortisol metabolism in obesity. Tissue-specific 11betaHSD1 regulation offers the potential for selective enzyme inhibition within adipose tissue as a novel therapy for visceral obesity.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed2001

Reduced placental 11beta-hydroxysteroid dehydrogenase type 2 mRNA levels in human pregnancies complicated by intrauterine growth restriction: an analysis of possible mechanisms.

11beta-Hydroxysteroid dehydrogenase type 2 (11beta-HSD2) inactivates cortisol to cortisone. In the placenta 11beta-HSD2 activity is thought to protect the fetus from the deleterious effects of maternal glucocorticoids. Patients with apparent mineralocorticoid excess owing to mutations in the 11beta-HSD2 gene invariably have reduced birth weight, and we have recently shown reduced placental 11beta-HSD2 activity in pregnancies complicated by intrauterine growth restriction. This is reflected in the literature by evidence of hypercortisolemia in the fetal circulation of small babies. In this study we have determined the levels of placental 11beta-HSD2 mRNA expression across normal gestation (n = 86 placentae) and in pregnancies complicated by intrauterine growth restriction (n = 19) and evaluated the underlying mechanism for any aberrant 11beta-HSD2 mRNA expression in intrauterine growth restriction. 11beta-HSD2 mRNA expression increased more than 50-fold across gestation, peaking at term. Placental 11beta-HSD2 mRNA levels were significantly decreased in intrauterine growth restriction pregnancies when compared with gestationally matched, appropriately grown placentae [e.g. at term DeltaCt (11beta-hydroxysteroid dehydrogenase type 2/18S) 12.8 +/- 0.8 (mean +/- SE) vs. 10.2 +/- 0.2, respectively, P < 0.001]. These differences were not attributable to changes in trophoblast mass in intrauterine growth restriction placentae, as assessed by parallel analyses of cytokeratin-8 mRNA expression. No mutations were found in the 11beta-HSD2 gene in the intrauterine growth restriction cohort, and imprinting analysis revealed that the 11beta-HSD2 gene was not imprinted. Although the underlying cause is unknown, 11beta-HSD2 gene expression is reduced in intrauterine growth restriction pregnancies. These data highlight the important role of 11beta-HSD2 in regulating fetal growth, a known factor in determining fetal morbidity but also the subsequent development of cardiovascular disease in adulthood.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed2001

Extrarenal expression of 25-hydroxyvitamin d(3)-1 alpha-hydroxylase.

The mitochondrial enzyme 25-hydroxyvitamin D(3)-1 alpha-hydroxylase (1 alpha-hydroxylase) plays an important role in calcium homeostasis by catalyzing synthesis of the active form of vitamin D, 1,25-dihydroxyvitamin D(3), in the kidney. However, enzyme activity assays indicate that 1 alpha-hydroxylase is also expressed in a variety of extrarenal tissues; recent cloning of cDNAs for 1 alpha-hydroxylase in different species suggests that a similar gene product is found at both renal and extrarenal sites. Using specific complementary ribonucleic acid probes and antisera to 1 alpha-hydroxylase, we have previously reported the distribution of messenger ribonucleic acid and protein for the enzyme along the mouse and human nephron. Here we describe further immunohistochemical and Western blot analyses that detail for the first time the extrarenal distribution of 1 alpha-hydroxylase in both normal and diseased tissues. Specific staining for 1 alpha-hydroxylase was detected in skin (basal keratinocytes, hair follicles), lymph nodes (granulomata), colon (epithelial cells and parasympathetic ganglia), pancreas (islets), adrenal medulla, brain (cerebellum and cerebral cortex), and placenta (decidual and trophoblastic cells). Further studies using psoriatic skin highlighted overexpression of 1 alpha-hydroxylase throughout the dysregulated stratum spinosum. Increased expression of skin 1alpha-hydroxylase was also associated with sarcoidosis. In lymph nodes and skin from these patients 1 alpha-hydroxylase expression was observed in cells positive for the surface antigen CD68 (macrophages). The data presented here confirm the presence of protein for 1 alpha-hydroxylase in several extrarenal tissues, such as skin, placenta, and lymph nodes. The function of this enzyme at novel extrarenal sites, such as adrenal medulla, brain, pancreas, and colon, remains to be determined. However, the discrete patterns of staining in these tissues emphasizes a possible role for 1 alpha-hydroxylase as an intracrine modulator of vitamin D function in peripheral tissues.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed2001

Modulation of 11beta-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 11beta-hydroxysteroid dehydrogenase (11beta-HSD), by interconverting hormonally active cortisol (F) to inactive cortisone (E), regulate the peripheral action of corticosteroids 11beta-HSD1 by converting E to F and 11beta-HSD2 by inactivating F to E. In different in vitro and in vivo systems both 11beta-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 11beta-HSD isozyme expression and activity by cytokines and hormones with established roles in bone physiology. In MG-63 cells, interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) potently inhibited 11beta-HSD2 activity (cortisol-cortisone conversion) and messenger RNA (mRNA) levels in a dose-dependent manner while stimulating reciprocal expression of 11beta-HSD1 mRNA and activity (cortisone-cortisol conversion). A similar rise in 11beta-HSD1 reductase activity also was observed in primary cultures of OBs treated with 10 ng/ml TNF-alpha. Pretreatment of MG-63 cells with 0.1 ng/ml IL-1beta 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-1beta 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 (11beta-HSD2) while inducing glucocorticoid activation (11beta-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.

11-beta-Hydroxysteroid Dehydrogenases↗