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1.
Kidney Int ; 74(10): 1343-53, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18784644

ABSTRACT

To examine any potential role for 1,25-dihydroxyvitamin D (1,25(OH)2D) in inflammation associated with chronic kidney disease we measured vitamin D metabolites, markers of inflammation and gene expression in 174 patients with a variety of kidney diseases. Urinary MCP-1 protein and renal macrophage infiltration were each significantly but inversely correlated with serum 1,25(OH)2D levels. Logistic regression analysis with urinary MCP-1 as binary outcome showed that a 10-unit increase in serum 1,25(OH)2D or 25OHD resulted in lower renal inflammation. Analysis of 111 renal biopsies found that renal injury was not associated with a compensatory increase in mRNA for the vitamin D-activating enzyme 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1), its catabolic counterpart 24-hydroxylase, or the vitamin D receptor. There was, however, a significant association between tissue MCP-1 and CYP27B1. Patients with acute renal inflammation had a significant increase in urinary and tissue MCP-1, macrophage infiltration, and macrophage and renal epithelial CYP27B1 expression but significantly lower levels of serum 1,25(OH)2D in comparison to patients with chronic ischemic disease despite similar levels of renal damage. In vitro, 1,25(OH)2D attenuated TNFalpha-induced MCP-1 expression by human proximal tubule cells. Our study indicates that renal inflammation is associated with decreased serum vitamin D metabolites and involves activation of the paracrine/autocrine vitamin D system.


Subject(s)
Inflammation/etiology , Kidney Diseases/pathology , Vitamin D/blood , 25-Hydroxyvitamin D3 1-alpha-Hydroxylase/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Autocrine Communication , Chemokine CCL2/analysis , Chronic Disease , Female , Humans , Male , Middle Aged , Paracrine Communication , RNA, Messenger/analysis , Vitamin D/analogs & derivatives , Vitamin D/metabolism , Young Adult
2.
Diabetes ; 57(10): 2652-60, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18633104

ABSTRACT

OBJECTIVE: The precise molecular mechanisms contributing to the development of insulin resistance, impaired glucose tolerance (IGT), and type 2 diabetes are largely unknown. Altered endogenous glucocorticoid metabolism, including 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), which generates active cortisol from cortisone, and 5alpha-reductase (5alphaR), which inactivates cortisol, has been implicated. RESEARCH DESIGN AND METHODS: A total of 101 obese patients (mean age 48 +/- 7 years, BMI 34.4 +/- 4.3 kg/m(2), 66 women, 35 men) underwent 75-g oral glucose tolerance testing (OGTT), body composition analysis (dual-energy X-ray absorptiometry), assessment of glucocorticoid metabolism (24-h urine steroid metabolite analysis by gas chromatography/mass spectrometry), and subcutaneous abdominal adipose tissue biopsies. RESULTS: A total of 22.7% of women had IGT compared with 34.2% of men. Two women and five men were diagnosed with type 2 diabetes. In women, adipose 11beta-HSD1 expression was increased in patients with IGT and correlated with glucose levels across the OGTT (R = 0.44, P < 0.001) but was independent of fat mass. Total glucocorticoid secretion was higher in men with and without IGT (normal 13,743 +/- 863 vs. 7,453 +/- 469 microg/24 h, P < 0.001; IGT 16,871 +/- 2,113 vs. 10,133 +/- 1,488 microg/24 h, P < 0.05), and in women, it was higher in those with IGT (7,453 +/- 469 vs. 10,133 +/- 1,488 microg/24 h, P < 0.001). In both sexes, 5alphaR activity correlated with fasting insulin (men R = 0.53, P = 0.003; women R = 0.33, P = 0.02), insulin secretion across an OGTT (men R = 0.46, P = 0.01; women R = 0.40, P = 0.004), and homeostasis model assessment of insulin resistance (men R = 0.52, P = 0.004; women R = 0.33, P = 0.02). CONCLUSIONS: Increased adipose 11beta-HSD1 expression in women may contribute to glucose intolerance. Enhanced 5alphaR activity in both sexes is associated with insulin resistance but not body composition. Augmented glucocorticoid inactivation may serve as a compensatory, protective mechanism to preserve insulin sensitivity.


Subject(s)
11-beta-Hydroxysteroid Dehydrogenase Type 1/metabolism , Cholestenone 5 alpha-Reductase/metabolism , Glucose Intolerance/physiopathology , Insulin Resistance/physiology , Absorptiometry, Photon , Adipose Tissue/metabolism , Adult , Body Mass Index , Cortisone/urine , Female , Gas Chromatography-Mass Spectrometry , Glucocorticoids/metabolism , Glucose Tolerance Test , Humans , Liver/enzymology , Male , Middle Aged , Obesity/enzymology , Obesity/metabolism , Obesity/physiopathology , Regression Analysis , Sex Factors
3.
Diabetes ; 57(6): 1536-43, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18340018

ABSTRACT

OBJECTIVE: The epidemics of obesity, insulin resistance, and type 2 diabetes have heightened the need to understand mechanisms that contribute to their pathogenesis. Increased endogenous glucocorticoid production has been implicated based on parallels with Cushing's syndrome. We have assessed the impact of weight loss on glucocorticoid secretion and metabolism (notably 11beta-hydroxysteroid dehydrogenase type 1 and 5alpha-reductase [5alphaR] activity) and insulin sensitivity. RESEARCH DESIGN AND METHODS: Twenty obese volunteers were investigated before and after weight loss. Patients underwent hyperinsulinemic-euglycemic clamps with simultaneous adipose microdialysis and oral cortisone acetate administration. Changes in glucocorticoid secretion and metabolism were assessed using 24-h urine collections. RESULTS: Before weight loss, fat mass correlated with glucocorticoid secretion rate (total fat, r = 0.46, P < 0.05; trunk fat, r = 0.52, P < 0.05); however, glucocorticoid secretion rate was inversely related to insulin sensitivity (r = -0.51, P < 0.05). Hyperinsulinemia failed to suppress adipose tissue interstitial fluid glycerol release (180 +/- 50 micromol [basal] vs. 153 +/- 10 micromol [steady state], NS). After oral cortisone (25 mg), cortisol concentrations within adipose interstitial fluid increased (4.3 +/- 1.1 vs. 14.2 +/- 2.6 nmol/l, P < 0.01), but glycerol concentrations did not change. After weight loss, insulin sensitivity increased. Consistent with insulin sensitization, adipose tissue interstitial fluid glycerol concentrations fell under hyperinsulinemic conditions (186 +/- 16 vs. 117 +/- 9 micromol, P < 0.05). Glucocorticoid secretion decreased (11,751 +/- 1,520 vs. 7,464 +/- 937 microg/24 h, P < 0.05) as did 5alphaR activity (5alpha-tetrahydrocortisol-to-tetrahydrocortisol ratio 1.41 +/- 0.16 vs. 1.12 +/- 0.17, P < 0.005). CONCLUSIONS: Obesity is associated with insulin resistance within adipose tissue and increased cortisol secretion rates; both are reversed with weight loss. Reduced 5alphaR activity after weight loss may decrease hypothalamo-pituitary-adrenal axis activation and reduce glucocorticoid metabolite production.


Subject(s)
Adipose Tissue/physiology , Cholestenone 5 alpha-Reductase/metabolism , Glucocorticoids/biosynthesis , Insulin/physiology , Weight Loss , Adult , Body Composition , Body Mass Index , Diet, Reducing , Energy Intake , Female , Glucocorticoids/metabolism , Glucose Clamp Technique , Humans , Hyperinsulinism , Kinetics , Male , Middle Aged , Obesity/blood , Obesity/enzymology , Obesity/physiopathology
4.
J Clin Endocrinol Metab ; 92(3): 857-64, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17200165

ABSTRACT

CONTEXT: The pathophysiological importance of glucocorticoids (GCs) is exemplified by patients with Cushing's syndrome who develop hypertension, obesity, and insulin resistance. At a cellular level, availability of GCs to the glucocorticoid and mineralocorticoid receptors is controlled by the isoforms of 11beta-hydroxysteroid dehydrogenase (11beta-HSD). In liver and adipose tissue, 11beta-HSD1 converts endogenous, inactive cortisone to active cortisol but also catalyzes the bioactivation of the synthetic prednisone to prednisolone. OBJECTIVE: The objective of the study was to compare markers of 11beta-HSD1 activity and demonstrate that inhibition of 11beta-HSD1 activity limits glucocorticoid availability to adipose tissue. DESIGN AND SETTING: This was a clinical study. PATIENTS: Seven healthy male volunteers participated in the study. INTERVENTION: Intervention included carbenoxolone (CBX) single dose (100 mg) and 72 hr of continuous treatment (300 mg/d). MAIN OUTCOME MEASURES: Inhibition of 11beta-HSD1 was monitored using five different mechanistic biomarkers (serum cortisol and prednisolone generation, urinary corticosteroid metabolite analysis by gas chromatography/mass spectrometry, and adipose tissue microdialysis examining cortisol generation and glucocorticoid-mediated glycerol release). RESULTS: Each biomarker demonstrated reduced 11beta-HSD1 activity after CBX administration. After both a single dose and 72 hr of treatment with CBX, cortisol and prednisolone generation decreased as did the urinary tetrahydrocortisol+5alpha-tetrahydrocortisol to tetrahydrocortisone ratio. Using adipose tissue microdialysis, we observed decreased interstitial fluid cortisol availability with CBX treatment. Furthermore, a functional consequence of 11beta-HSD1 inhibition was observed, namely decreased prednisone-induced glycerol release into adipose tissue interstitial fluid indicative of inhibition of GC-mediated lipolysis. CONCLUSION: CBX is able to inhibit rapidly the generation of active GC in human adipose tissue. Importantly, limiting GC availability in vivo has functional consequences including decreased glycerol release.


Subject(s)
11-beta-Hydroxysteroid Dehydrogenase Type 1/metabolism , Adipose Tissue/metabolism , Glucocorticoids/metabolism , Lipolysis , Adipose Tissue/drug effects , Adult , Carbenoxolone/administration & dosage , Carbenoxolone/pharmacokinetics , Extracellular Fluid/chemistry , Glucocorticoids/pharmacology , Humans , Hydrocortisone/metabolism , Lipolysis/drug effects , Male , Prednisolone/metabolism , Serum/chemistry
6.
Hypertension ; 46(4): 787-98, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16172422

ABSTRACT

Rodents studies suggest that androgens are involved in sex-specific differences in blood pressure. In humans, there is no difference in blood pressure between boys and girls, but after puberty, blood pressure increases more in men than in women. We investigated androgen-dependent regulation of the alpha-subunit of the epithelial sodium channel (alphaEnaC) in human kidney and in the human renal cell line immortalized human renal proximal tubular cell line (HKC-8). We used microarray technique to analyze androgen-dependent gene regulation and performed quantitative RT-PCR for verification. Promoter constructs for human alphaENaC were used in transfection studies to analyze the regulation by testosterone. We investigated the in vivo effect of testosterone on alphaENaC in a rat model and used the mouse collecting duct cell line M-1 for transepithelial electrophysiological measurements. The androgen receptor (AR) was expressed in male kidney and HKC-8 cells. AlphaENaC mRNA expression increased 2- to 3-fold after treatment with testosterone in HKC-8 cells. The induction by testosterone was completely blocked by adding the AR antagonist flutamide. Analysis of the alphaENaC promoter sequence identified a putative AR response element (ARE) located 140 nucleotides upstream from the transcription start site. HKC-8 cell transfection studies showed that testosterone directly upregulated gene expression via this ARE. In vivo, testosterone treatment of orchiectomized rats resulted in an increased renal alphaENaC mRNA expression. In testosterone-treated mouse M-1 cells, amiloride caused a significant stronger decrease in short circuit current than in control cells. These data show that alphaENaC expression is directly regulated by androgens in vitro and in vivo and highlight a potential mechanism explaining the reported gender differences in blood pressure.


Subject(s)
Kidney/metabolism , Receptors, Androgen/physiology , Sodium Channels/metabolism , Animals , Cells, Cultured , Electrophysiology , Epithelial Sodium Channels , Gene Expression/drug effects , Genes, Reporter/drug effects , Humans , Kidney/cytology , Kidney/enzymology , Kidney/physiology , Kidney Tubules, Collecting/cytology , Kidney Tubules, Proximal/cytology , Male , Mice , Oligonucleotide Array Sequence Analysis , Orchiectomy , Polymerase Chain Reaction , Promoter Regions, Genetic/drug effects , Sodium Channels/genetics , Sodium Channels/physiology , Steroids/pharmacology , Testosterone/pharmacology
7.
Circulation ; 112(10): 1435-43, 2005 Sep 06.
Article in English | MEDLINE | ID: mdl-16145013

ABSTRACT

BACKGROUND: Aldosterone has emerged as a deleterious hormone in the heart, with mineralocorticoid receptor (MR) blockade reducing mortality in patients with severe heart failure. There is also experimental evidence that aldosterone contributes to the development of nephrosclerosis and renal fibrosis in rodent models, but little is known of its role in clinical renal disease. METHODS AND RESULTS: We quantified MR, serum- and glucocorticoid-regulated kinase 1 (sgk1), and mRNA expression of inflammatory mediators such as macrophage chemoattractant protein-1 (MCP-1), transforming growth factor-beta1, and interleukin-6 in 95 human kidney biopsies in patients with renal failure and mild to marked proteinuria of diverse etiologic origins. We measured renal function, serum aldosterone, urinary MCP-1 protein excretion, and the amount of chronic renal damage. Macrophage invasion was quantified by CD68 and vascularization by CD34 immunostaining. Serum aldosterone correlated negatively with creatinine clearance (P<0.01) and positively with renal scarring (P<0.05) but did not correlate with MR mRNA expression or proteinuria. Patients with heavy albuminuria (>2 g/24 h; n=15) had the most renal scarring and the lowest endothelial CD34 staining. This group showed a significant 5-fold increase in MR, a 2.5-fold increase in sgk1 expression and a significant increase in inflammatory mediators (7-fold increase in MCP-1, 3-fold increase in transforming growth factor-beta1, and 2-fold increase in interleukin-6 mRNA). Urinary MCP-1 protein excretion and renal macrophage invasion were significantly increased in patients with heavy albuminuria. CONCLUSIONS: These studies support animal data linking aldosterone/MR activation to renal inflammation and proteinuria. Further studies are urgently required to assess the potential beneficial effects of MR antagonism in patients with renal disease.


Subject(s)
Aldosterone/blood , Kidney/pathology , Kidney/physiology , Proteinuria/pathology , Proteinuria/physiopathology , Receptors, Mineralocorticoid/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Biopsy , Blood Pressure , Chemokine CCL2/genetics , Female , Glomerular Basement Membrane/pathology , Humans , Immediate-Early Proteins/genetics , Interleukin-6/genetics , Ischemia/immunology , Ischemia/pathology , Ischemia/physiopathology , Kidney Function Tests , Male , Middle Aged , Protein Serine-Threonine Kinases/genetics , Proteinuria/immunology , RNA, Messenger/analysis , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta1
8.
Eur J Endocrinol ; 153(2): 291-9, 2005 Aug.
Article in English | MEDLINE | ID: mdl-16061836

ABSTRACT

OBJECTIVE: Renal 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) enables selective access of aldosterone to the mineralocorticoid receptor (MR). Impaired 11beta-HSD2 activity has been suggested in patients with hypertension as well as in patients with renal disease, where it may contribute to sodium retention, oedema and hypertension. To date, these studies have relied upon urinary cortisol (F) metabolite levels as surrogate markers of renal 11beta-HSD2 activity. METHODS: We have directly analysed renal 11beta-HSD2 mRNA expression in 95 patients undergoing kidney biopsy using TaqMan real-time PCR. Serum and 24-h urine samples were used to document underlying renal function and endocrine parameters. Urinary F and cortisone (E) metabolites were analysed using gas chromatography/mass spectrometry. RESULTS: Expression of 11beta-HSD2 did not correlate with blood pressure or urinary Na/K ratio, but a significant positive correlation with creatinine clearance was observed (r = 0.284; P < 0.01). Immunofluorescence and confocal laser microscopy confirmed decreased 11beta-HSD2 expression in patients with impaired renal function. For the first time, we showed that 11beta-HSD2 mRNA expression correlated negatively with the urinary free (UF) F/E (UFF/UFE) ratio (r = 0.276; P < 0.05) as well as with the urinary tetrahydrocortisol + 5alpha-tetrahydrocortisol/tetrahydrocortisone ((THF + alphaTHF)/THE) ratio (r = 0.256; P < 0.05). No difference in 11beta-HSD2 mRNA expression or in the UFF/UFE ratio was found between groups with no proteinuria, microalbuminuria, moderate or severe proteinuria. In contrast, the urinary (THF + alphaTHF)/THE ratio increased significantly (P < 0.05) in patients with severe albuminuria, suggesting increased hepatic 11beta-HSD1 in those patients. CONCLUSIONS: These data suggest that renal 11beta-HSD2 expression may be represented only marginally better, if at all, by the UFF/UFE than by the (THF + alphaTHF)/THE ratio. Reduced renal 11beta-HSD2 expression may lead to occupancy of the MR by glucocorticoids such as cortisol and may contribute to the increased sodium retention seen in patients with impaired renal function.


Subject(s)
11-beta-Hydroxysteroid Dehydrogenase Type 2/genetics , 11-beta-Hydroxysteroid Dehydrogenase Type 2/metabolism , Kidney/enzymology , Renal Insufficiency/metabolism , Adolescent , Adult , Aged , Aged, 80 and over , Albuminuria/metabolism , Albuminuria/pathology , Biopsy , Creatinine/urine , Female , Gene Expression Regulation, Enzymologic , Humans , Hydrocortisone/urine , Kidney/pathology , Male , Middle Aged , RNA, Messenger/analysis , Receptors, Mineralocorticoid/metabolism , Renal Insufficiency/pathology , Sodium/metabolism , Tetrahydrocortisol/urine
9.
Blood ; 106(6): 2042-9, 2005 Sep 15.
Article in English | MEDLINE | ID: mdl-15941907

ABSTRACT

Glucocorticoids (GCs) exert powerful anti-inflammatory effects that may relate in part to their ability to restrict the differentiation and function of dendritic cells (DCs). Although these inhibitory effects are dependent upon GCs binding to nuclear glucocorticoid receptors (GRs), fine-tuning of GR signaling is achieved by prereceptor interconversion of cortisol that binds GRs with high affinity and cortisone that does not. We show for the first time that human monocyte-derived DCs are able to generate cortisol as a consequence of up-regulated expression of the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). Immature DCs demonstrate selective enhancement of 11beta-HSD1 reductase activity, leading to increased conversion of inactive cortisone to active cortisol. Enhancement of GC bioavailability is maintained or increased upon terminal differentiation induced by signals associated with innate immune activation. In marked contrast, maturation induced by CD40 ligation leads to a sharp reduction in cortisol generation by DCs. The differentiation of DCs from monocyte precursors is inhibited at physiologic concentrations of inactive cortisone, an effect that requires activity of the 11beta-HSD1 enzyme. In conclusion, prereceptor regulation of endogenous GCs appears to be an important determinant of DC function and represents a potential target for therapeutic manipulation.


Subject(s)
11-beta-Hydroxysteroid Dehydrogenase Type 1/physiology , Dendritic Cells/metabolism , Glucocorticoids/pharmacokinetics , 11-beta-Hydroxysteroid Dehydrogenase Type 1/genetics , Autocrine Communication/immunology , Biological Availability , Cell Differentiation , Cells, Cultured , Cortisone/metabolism , Cortisone/pharmacology , Dendritic Cells/immunology , Glucocorticoids/pharmacology , Humans , Hydrocortisone/biosynthesis , Immunity , Monocytes/cytology , Signal Transduction/immunology
10.
Cancer Res ; 65(6): 2488-97, 2005 Mar 15.
Article in English | MEDLINE | ID: mdl-15781666

ABSTRACT

Recently, we reported that a novel, noncalcemic vitamin D analogue (19-nor-1,25(OH)2D2; paricalcitol) had anticancer activity. In this study, we explored if paricalcitol enhanced anticancer effects of other clinically useful drugs in vitro against a large variety of cancer cells. Paricalcitol, when combined with As2O3, showed a markedly enhanced antiproliferative effect against acute myeloid leukemia (AML) cells. This combination induced monocytic differentiation of NB-4 acute promyelocytic leukemia (APL) cells and HL-60 AML cells and caused both to undergo apoptosis associated with down-regulation of Bcl-2 and Bcl-x(L). Paricalcitol induced monocytic differentiation of U937 AML cells, which was partially blocked by inducing expression of APL-related PML-retinoic acid receptor alpha (RARalpha) chimeric protein in the U937 cells containing a Zn2+-inducible expression vector coding for this fusion protein (PR9 cells). Exposure to As2O3 decreased levels of PML-RARalpha in PR9 cells, and the combination of paricalcitol and As2O3 enhanced their monocytic differentiation in parallel with the As2O3-mediated decrease of PML-RARalpha. Furthermore, As2O3 increased the transcriptional activity of paricalcitol probably by increasing intracellular levels of paricalcitol by decreasing the function of the mitochondrial enzyme 25-hydroxyvitamin D3-24-hydroxylase, which functions to metabolize the active vitamin D in cells. In summary, the combination of paricalcitol and As2O3 potently decreased growth and induced differentiation and apoptosis of AML cells. This probably occurred by As2O3 decreasing levels of both the repressive PML-RARalpha fusion protein and the vitamin D metabolizing protein, 25-hydroxyvitamin D3-24-hydroxylase, resulting in increased activity of paricalcitol. The combination of both of these Food and Drug Administration-approved drugs should be considered for treatment of all-trans retinoic acid-resistant APL patients as well as those with other types of AML.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Leukemia, Myeloid/drug therapy , Acute Disease , Arsenic Trioxide , Arsenicals/administration & dosage , Cell Differentiation/drug effects , Cell Growth Processes/drug effects , Cell Line, Tumor , Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System/metabolism , Drug Screening Assays, Antitumor , Drug Synergism , Ergocalciferols/administration & dosage , Gene Expression/drug effects , HL-60 Cells , Humans , Leukemia, Myeloid/enzymology , Leukemia, Myeloid/genetics , Leukemia, Myeloid/pathology , Mitochondria/drug effects , Neoplasm Proteins/antagonists & inhibitors , Oncogene Proteins, Fusion/antagonists & inhibitors , Oxides/administration & dosage , Steroid Hydroxylases/antagonists & inhibitors , Steroid Hydroxylases/metabolism , Vitamin D3 24-Hydroxylase
12.
J Steroid Biochem Mol Biol ; 89-90(1-5): 121-5, 2004 May.
Article in English | MEDLINE | ID: mdl-15225758

ABSTRACT

A number of studies have suggested that Vitamin D has a potential role in the development/treatment of diabetes. These effects may be mediated by circulating levels of 1alpha,25(OH)(2)D(3), but local production of 1alpha,25(OH)(2)D(3), catalysed by the enzyme 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-OHase), is also likely to be important. RT-PCR analyses demonstrated that both isolated rat islets and MIN6 cells (mouse insulin-secreting cell line, characteristic of beta cells) expressed 1alpha-OHase mRNA. The transcript in both cell types was similar to that seen in HKC-8 cells (a renal cell line, which expresses 1alpha-OHase). Western blot analysis and immunolocalisation identified 1alpha-OHase protein in MIN6 cells and human pancreatic tissue. In addition, suspensions of rat islets were able to convert [3H]-25-hydroxyvitamin D(3) to [3H]-1alpha,25(OH)(2)D(3), demonstrating 1alpha-OHase activity. Both cell systems expressed the Vitamin D receptor and 1alpha,25(OH)(2)D(3) (50nM) evoked a rapid rise in [Ca(2+)](i) in MIN6 cells. This data clearly demonstrates islets are able to produce 1alpha,25(OH)(2)D(3) and respond rapidly to treatment with 1alpha,25(OH)(2)D(3). Therefore, we would postulate that local production of 1alpha,25(OH)(2)D(3) maybe an important autocrine link between Vitamin D status and pancreatic function.


Subject(s)
25-Hydroxyvitamin D3 1-alpha-Hydroxylase/metabolism , Islets of Langerhans/enzymology , 25-Hydroxyvitamin D3 1-alpha-Hydroxylase/genetics , Animals , Blotting, Western , Cells, Cultured , Islets of Langerhans/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Receptors, Calcitriol/genetics , Receptors, Calcitriol/metabolism , Reverse Transcriptase Polymerase Chain Reaction
13.
J Immunol ; 170(11): 5382-90, 2003 Jun 01.
Article in English | MEDLINE | ID: mdl-12759412

ABSTRACT

The functions of dendritic cells (DCs) are tightly regulated such that protective immune responses are elicited and unwanted immune responses are prevented. 1 alpha 25-dihydroxyvitamin D(3) (1 alpha 25(OH)(2)D(3)) has been identified as a major factor that inhibits the differentiation and maturation of DCs, an effect dependent upon its binding to the nuclear vitamin D receptor (VDR). Physiological control of 1 alpha 25(OH)(2)D(3) levels is critically dependent upon 25-hydroxyvitamin D(3)-1 alpha-hydroxylase (1 alpha OHase), a mitochondrial cytochrome P450 enzyme that catalyzes the conversion of inactive precursor 25-hydroxyvitamin D(3) (25(OH)D(3)) to the active metabolite 1 alpha 25(OH)(2)D(3). Using a human monocyte-derived DC (moDC) model, we have examined the relationship between DC VDR expression and the impact of exposure to its ligand, 1 alpha 25(OH)(2)D(3). We show for the first time that moDCs are able to synthesize 1 alpha 25(OH)(2)D(3) in vitro as a consequence of increased 1 alpha OHase expression. Following terminal differentiation induced by a diverse set of maturation stimuli, there is marked transcriptional up-regulation of 1 alpha OHase leading to increased 1 alpha OHase enzyme activity. Consistent with this finding is the observation that the development and function of moDCs is inhibited at physiological concentrations of the inactive metabolite 25(OH)D(3). In contrast to 1 alpha OHase, VDR expression is down-regulated as monocytes differentiate into immature DCs. Addition of 1 alpha 25(OH)(2)D(3) to moDC cultures at different time points indicates that its inhibitory effects are greater in monocyte precursors than in immature DCs. In conclusion, differential regulation of endogenous 1 alpha 25(OH)(2)D(3) ligand and its nuclear receptor appear to be important regulators of DC biology and represent potential targets for the manipulation of DC function.


Subject(s)
Calcitriol/metabolism , Dendritic Cells/metabolism , Monocytes , Receptors, Calcitriol/metabolism , 25-Hydroxyvitamin D3 1-alpha-Hydroxylase/biosynthesis , 25-Hydroxyvitamin D3 1-alpha-Hydroxylase/genetics , 25-Hydroxyvitamin D3 1-alpha-Hydroxylase/metabolism , Autocrine Communication/drug effects , Autocrine Communication/immunology , Calcitriol/biosynthesis , Calcitriol/pharmacology , Calcitriol/physiology , Cell Differentiation/drug effects , Cell Differentiation/immunology , Cells, Cultured , Dendritic Cells/cytology , Dendritic Cells/drug effects , Dendritic Cells/enzymology , Down-Regulation/drug effects , Down-Regulation/immunology , Enzyme Induction/drug effects , Enzyme Induction/genetics , Gene Expression Regulation/drug effects , Gene Expression Regulation/immunology , Growth Inhibitors/biosynthesis , Growth Inhibitors/metabolism , Growth Inhibitors/pharmacology , Growth Inhibitors/physiology , Humans , Ligands , Monocytes/cytology , Monocytes/drug effects , Monocytes/enzymology , Monocytes/metabolism , Paracrine Communication/drug effects , Paracrine Communication/immunology , Receptors, Calcitriol/antagonists & inhibitors , Receptors, Calcitriol/biosynthesis , Up-Regulation/drug effects , Up-Regulation/immunology
14.
Invest Ophthalmol Vis Sci ; 44(4): 1643-51, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12657604

ABSTRACT

PURPOSE: In peripheral sodium-transporting tissues, the serum- and glucocorticoid-regulated kinase (SGK) isoform-1 is an early corticosteroid target gene in the activation of epithelial sodium channels (ENaCs). Sodium transport across the human ocular nonpigmented and pigmented ciliary epithelial bilayer (NPE-PE) is essential for aqueous humor production, but the expression of SGK1 and ENaC subunits remain to be defined. METHODS: SGK1 and ENaC subunits were evaluated by in situ hybridization and RT-PCR analysis on human NPE-PE sections and an NPE cell line (ODM-2). Northern blot analyses were conducted on ODM-2 cells incubated with dexamethasone (DEX) or aldosterone (ALDO) and RU38486 (a glucocorticoid receptor [GR] antagonist) or RU26752 (a mineralocorticoid receptor [MR] antagonist) or both inhibitors. The affinity of the GRs and MRs for DEX and ALDO was assessed by radioligand-binding assays. RESULTS: Expression of SGK1 and ENaC subunits was confirmed in NPE-PE tissues and ODM-2 cells. Dose-dependent induction of SGK1 mRNA in the ODM-2 cells was demonstrated after incubation with DEX or ALDO. While response to DEX was not inhibited by RU38486 or RU26752, there was a moderate reduction in induction by ALDO in the presence of RU26752 that was completely abolished in the presence of both inhibitors. Specific binding of (3)[H]DEX and (3)[H]ALDO was established, revealing greater expression of GRs than MRs. CONCLUSIONS: The expression of ENaCs within the NPE-PE and corticosteroid regulation of SGK1 through the GR and MR, indicate that this mechanism may be a feature of sodium transport in the human ocular ciliary epithelium.


Subject(s)
Ciliary Body/metabolism , Nuclear Proteins , Pigment Epithelium of Eye/metabolism , Protein Serine-Threonine Kinases/genetics , Sodium Channels/genetics , Spironolactone/analogs & derivatives , Aldosterone/pharmacology , Blotting, Northern , Cell Line , Ciliary Body/drug effects , Dexamethasone/pharmacology , Dose-Response Relationship, Drug , Drug Combinations , Epithelial Sodium Channels , Gene Expression Regulation/drug effects , Glucocorticoids/pharmacology , Hormone Antagonists/pharmacology , Humans , Immediate-Early Proteins , In Situ Hybridization , Isoenzymes , Mifepristone/pharmacology , Mineralocorticoid Receptor Antagonists , Pigment Epithelium of Eye/drug effects , Protein Serine-Threonine Kinases/metabolism , RNA, Messenger/metabolism , Receptors, Glucocorticoid/antagonists & inhibitors , Reverse Transcriptase Polymerase Chain Reaction , Sodium Channels/metabolism , Spironolactone/pharmacology
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