Inflammation dynamically regulates steroid hormone metabolism and action within macrophages in rheumatoid arthritis.

RATIONALE: In inflammatory diseases such as rheumatoid arthritis (RA), steroid metabolism is a central component mediating the actions of immuno-modulatory glucocorticoids and sex steroids. However, the regulation and function of cellular steroid metabolism within key leukocyte populations such as macrophages remain poorly defined. In this study, the inflammatory regulation of global steroid metabolism was assessed in RA macrophages. METHODS: Bulk RNA-seq data from RA synovial macrophages was used to assess transcripts encoding key enzymes in steroid metabolism and signalling. Changes in metabolism were assessed in synovial fluids, correlated to measures of disease activity and functionally validated in primary macrophage cultures. RESULTS: RNA-seq revealed a unique pattern of differentially expressed genes, including changes in genes encoding the enzymes 11ß-HSD1, SRD5A1, AKR1C2 and AKR1C3. These correlated with disease activity, favouring increased glucocorticoid and androgen levels. Synovial fluid 11ß-HSD1 activity correlated with local inflammatory mediators (TNFα, IL-6, IL-17), whilst 11ß-HSD1, SRD5A1 and AKR1C3 activity correlated with systemic measures of disease and patient pain (ESR, DAS28 ESR, global disease activity). Changes in enzyme activity were evident in inflammatory activated macrophages in vitro and revealed a novel androgen activating role for 11ß-HSD1. Together, increased glucocorticoids and androgens were able to suppress inflammation in macrophages and fibroblast-like-synoviocytes. CONCLUSIONS: This study underscores the significant increase in androgen and glucocorticoid activation within inflammatory polarized macrophages of the synovium, contributing to local suppression of inflammation. The diminished profile of inactive steroid precursors in postmenopausal women may contribute to disturbances in this process, leading to increased disease incidence and severity.

Transcriptomic analysis of vitamin D responses in uterine and peripheral NK cells.

Vitamin D deficiency is prevalent in pregnant women and is associated with adverse pregnancy outcomes, in particular disorders of malplacentation. The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), is a potent regulator of innate and adaptive immunity, but its immune effects during pregnancy remain poorly understood. During early gestation, the predominant immune cells in maternal decidua are uterine natural killer cells (uNK), but the responsivity of these cells to 1,25(OH)2D3 is unknown despite high levels of 1,25(OH)2D3 in decidua. Transcriptomic responses to 1,25(OH)2D3 were characterised in paired donor uNK and peripheral natural killer cells (pNK) following cytokine (CK) stimulation. RNA-seq analyses indicated 911 genes were differentially expressed in CK-stimulated uNK versus CK-stimulated pNK in the absence of 1,25(OH)2D3, with predominant differentially expressed pathways being associated with glycolysis and transforming growth factor ß (TGFß). RNA-seq also showed that the vitamin D receptor (VDR) and its heterodimer partner retinoid X receptor were differentially expressed in CK-stimulated uNK vs CK-stimulated pNK. Further analyses confirmed increased expression of VDR mRNA and protein, as well as VDR-RXR target in CK-stimulated uNK. RNA-seq analysis showed that in CK-stimulated pNK, 1,25(OH)2D3 induced 38 and suppressed 33 transcripts, whilst in CK-stimulated uNK 1,25(OH)2D3 induced 46 and suppressed 19 genes. However, multiple comparison analysis of transcriptomic data indicated that 1,25(OH)2D3 had no significant overall effect on gene expression in either CK-stimulated pNK or uNK. These data indicate that CK-stimulated uNK are transcriptionally distinct from pNK and, despite expressing abundant VDR, neither pNK nor uNK are sensitive targets for vitamin D.

Associations of total and free 25OHD and 1,25(OH)2D with serum markers of inflammation in older men.

UNLABELLED: Vitamin D is hypothesized to suppress inflammation. We tested total and free vitamin D metabolites and their association with inflammatory markers. Interleukin-6 levels were lower with higher 25-hydroxyvitamin D. 1,25-dihydroxyvitamin D and free 25OHD associations mirrored those of 25OHD. However, associations for the two metabolites diverged for tumor necrosis factor alpha (TNF-α) soluble receptors. INTRODUCTION: Vitamin D is hypothesized to suppress inflammation, and circulating 25-hydroxyvitamin D (25OHD) and inflammatory markers are inversely correlated. However, total serum 25OHD may not be the best indicator of biologically active vitamin D. METHODS: We tested serum total 25OHD, total 1,25(OH)2D, vitamin D binding protein (DBP), and estimated free 25OHD and free 1,25(OH)2D associations with inflammatory markers serum interleukin-6 (IL-6), TNF-α and their soluble receptors, interleukin-10 (IL-10), and C-reactive protein (CRP) as continuous outcomes and the presence of ≥2 inflammatory markers in the highest quartile as a dichotomous outcome, in a random subcohort of 679 men in the Osteoporotic Fractures in Men (MrOS) study. RESULTS: IL-6 was lower in men with higher 25OHD (-0.23 µg/mL per standard deviation (SD) increase in 25OHD, 95 % confidence intervals (CI) -0.07 to -0.38 µg/mL) and with higher 1,25(OH)2D (-0.20 µg/mL, 95 % CI -0.0004 to -0.39 µg/mL); free D associations were slightly stronger. 25OHD and DBP, but not 1,25(OH)2D, were independently associated with IL-6. TNF-α soluble receptors were inversely associated with 1,25(OH)2D but positively associated with 25OHD, and each had independent effects. The strongest association with ≥2 inflammatory markers in the highest quartile was for free 1,25(OH)2D (odds ratios (OR) 0.70, 95 % CI 0.54 to 0.89 per SD increase in free 1,25(OH)2D). CONCLUSIONS: Associations of 1,25(OH)2D and free 25OHD with IL-6 mirrored those of 25OHD, suggesting that 1,25(OH)2D and free D do not improve upon 25OHD in population-based IL-6 studies. However, associations for the two metabolites diverged for TNF-α soluble receptor, warranting examination of both metabolites in studies of TNF-α and its antagonists.

Modelling the influence of vitamin D and probiotic supplementation on the microbiome and immune response.

The intestinal microbiota play a critical role in human health and disease, maintaining metabolic and immune/inflammatory health, synthesising essential vitamins and amino acids and maintaining intestinal barrier integrity. The aim of this paper is to develop a mathematical model to describe the complex interactions between the microbiota, vitamin D/vitamin D receptor (VDR) pathway, epithelial barrier and immune response in order to understand better the effects of supplementation with probiotics and vitamin D. This is motivated by emerging data indicating the beneficial effects of vitamin D and probiotics individually and when combined. We propose a system of ordinary differential equations determining the time evolution of intestinal bacterial populations, concentration of the VDR:1,25(OH)2D complex in epithelial and immune cells, the epithelial barrier and the immune response. The model shows that administration of probiotics and/or vitamin D upregulates the VDR complex, which enhances barrier function and protects against intestinal inflammation. The model also suggests co-supplementation to be superior to individual supplements. We explore the effects of inflammation on the populations of commensal and pathogenic bacteria and the vitamin D/VDR pathway and discuss the value of gathering additional experimental data motivated by the modelling insights.

Vitamin D, the placenta and pregnancy.

Impaired vitamin D status is common to many populations around the world. However, data suggest that this is a particular problem for specific groups such as pregnant women. This has raised important questions concerning the physiological and clinical impact of low vitamin D levels during pregnancy, with implications for classical skeletal functions of vitamin D, as well as its diverse non-classical actions. The current review will discuss this with specific emphasis on the classical calciotropic effects of vitamin D as well as the less well established immunological functions of vitamin D that may influence pregnancy outcome. The review also describes the pathways that are required for metabolism and function of vitamin D, and the various clinical complications that have been linked to impaired vitamin D status during pregnancy.

Extrarenal expression of the 25-hydroxyvitamin D-1-hydroxylase.

Like the vitamin D receptor (VDR), the CYP27B1-hydroxylase is expressed widely in human tissues. This expression profile establishes the potential for interaction of the VDR with the product of the CYP27B1, 1,25-dihydroxyvitamin D (1,25-(OH)(2)D), in either an intracrine or paracrine mode. This expansive expression profile also suggests that the local production and action of 1,25-(OH)(2)D to regulate VDR-directed gene expression may be similarly wide-ranging and distinct from what occurs in the kidney; the proximal renal tubular epithelial cell is the richest source of the CYP27B1 and the site for production of 1,25-(OH)(2)D destined to function as a hormone. Existence of the CYP27B1 at extrarenal sites has been widely documented, although the functional impact of the enzyme in these tissues has yet to be fully demonstrated. Two notable exceptions are the disease-activated macrophage (e.g., in sarcoidosis or tuberculosis) and the placenta. These two tissues are capable of generating enough 1,25-(OH)(2)D so as to be detectable in the general circulation. As such, this review will focus on CYP27B1 expression only at these two sites, theorizing that 1,25-(OH)(2)D production at these sites is for the purpose of local immunoregulatory function, not for controlling calcium balance in the host or the fetus.

Development of a LC-MS/MS method to measure serum 3-sulfate and 3-glucuronide 25-hydroxyvitamin D3 metabolites; comparisons to unconjugated 25OHD in pregnancy and polycystic ovary syndrome.

Vitamin D status is routinely assessed by measuring circulating concentrations of 25-hydroxyvitamin D (25OHD2 or 25OHD3). However as deconjugation is not routinely incorporated into sample treatment prior to analysis, conjugated forms of 25OHD (particularly the more abundant 25OHD3) are often not considered in determining serum concentrations of total 25OHD. Two major circulating conjugated forms of 25OHD3 are 25-hydroxyvitamin D3-3-sulfate (25OHD3-S) and 25-hydroxyvitamin D3-3-glucuronide (25OHD3-G). Incorporating these two conjugated metabolites into the measurement of vitamin D status could improve our understanding of vitamin D status in health, particularly if there are changes in sulfation and glucuronidation activities. The aim of this study was to develop a liquid chromatography tandem-mass spectrometry (LC-MS/MS) targeted method for measurement of 25OHD3-S and 25OHD3-G in serum to enable comparisons with circulating levels of the free 25OHD3 form. We developed and validated a new LC-MS/MS method that measured both 25OHD3-S and 25OHD3-G following a solid phase extraction sample preparation method. Partial separation of analytes by LC, and the separation of analytes by the optimized multiple reaction monitoring transitions enabled the quantitation of both 25OHD3-S and 25OHD3-G in the single method. Serum concentrations of 25OHD3-S (24.7 ± 11.8 ng/mL) and 25OHD3-G (2.4 ± 1.2 ng/mL) were shown to be a significant proportion of circulating vitamin D metabolites in healthy donor serums. These levels of 25OHD3-S and 25OHD3-G closely associated with 25OHD3 concentrations, r = 0.728, p = 0.001 and r = 0.632, p = 0.006 respectively. However in serum from pregnant women and non-pregnant women with polycystic ovary syndrome (PCOS) significant differences in the ratios between conjugated and free 25OHD3 were observed between pregnancy groups (25OHD3/25OHD3-S and 25OHD3/25OHD3-G p < 0.001), and between healthy and PCOS subjects (25OHD3/25OHD3-G p < 0.050). Development of this novel high-throughput LC-MS/MS method indicates that 25OHD3-S and 25OHD3-G are substantial components of circulating vitamin D metabolites. The concentrations of these metabolites relative to conventional 25OHD3 may vary in different physiological and pathophysiological settings, and may therefore play an unrecognized but important role in the actions of vitamin D.

Synergistic induction of local glucocorticoid generation by inflammatory cytokines and glucocorticoids: implications for inflammation associated bone loss.

OBJECTIVES: Synovial fibroblasts and osteoblasts generate active glucocorticoids by means of the 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) enzyme. This activity increases in response to proinflammatory cytokines or glucocorticoids. During inflammatory arthritis synovium and bone are exposed to both these factors. This study hypothesised that glucocorticoids magnify the effects of inflammatory cytokines on local glucocorticoid production in both synovium and bone. METHODS: The effects of inflammatory cytokines (IL-1beta/tumour necrosis factor alpha; TNFalpha) and glucocorticoids, alone or combined, were assessed on the expression and activity of 11beta-HSD1 in primary synovial fibroblasts, primary human osteoblasts and MG-63 osteosarcoma cells. A range of other target genes and cell types were used to examine the specificity of effects. Functional consequences were assessed using IL-6 ELISA. RESULTS: In synovial fibroblasts and osteoblasts, treatment with cytokines or glucocorticoids in isolation induced 11beta-HSD1 expression and activity. However, in combination, 11beta-HSD1 expression, activity and functional consequences were induced synergistically to a level not seen with isolated treatments. This effect was seen in normal skin fibroblasts but not foreskin fibroblasts or adipocytes and was only seen for the 11beta-HSD1 gene. Synergistic induction had functional consequences on IL-6 production. CONCLUSIONS: Combined treatment with inflammatory cytokines and glucocorticoids synergistically induces 11beta-HSD1 expression and activity in synovial fibroblasts and osteoblasts, providing a mechanism by which synovium and bone can interact to enhance anti-inflammatory responses by increasing localised glucocorticoid levels. However, the synergistic induction of 11beta-HSD1 might also cause detrimental glucocorticoid accumulation in bone or surrounding tissues.

Expression of 25-hydroxyvitamin D3-1alpha-hydroxylase in subcutaneous fat necrosis.

BACKGROUND: The most serious complication of subcutaneous fat necrosis (SCFN), a rare condition of the newborn characterized by indurated purple nodules, is hypercalcaemia. However, the mechanism for this hypercalcaemia remains unclear. OBJECTIVES: To determine whether the hypercalcaemia associated with SCFN involves expression of the vitamin D-activating enzyme 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-hydroxylase) in affected tissue. METHODS: Skin biopsies from two male patients with SCFN and hypercalcaemia were taken. The histological specimens were assessed using a polyclonal antibody against 1alpha-hydroxylase. RESULTS: Histology in both cases showed strong expression of 1alpha-hydroxylase protein (brown staining) within the inflammatory infiltrate associated with SCFN. This was consistent with similar experiments in other granulomatous conditions. CONCLUSIONS: Hypercalcaemia in SCFN appears to be due to abundant levels of 1alpha-hydroxylase in immune infiltrates associated with tissue lesions. This is consistent with previous observations of extrarenal 1alpha-hydroxylase in skin from other granulomatous conditions such as sarcoidosis and slack skin disease.

Free versus total serum 25-hydroxyvitamin D in a murine model of colitis.

Inflammatory bowel diseases (IBD) such as ulcerative colitis and Crohn's disease have been linked to vitamin D-deficiency. Using a dextran sodium sulphate (DSS)-induced model of IBD we have shown previously that mice raised on vitamin D-deficient diets from weaning have lower serum 25-hydroxyvitamin D (25OHD) levels and develop more severe colitis compared to vitamin D-sufficient counterparts. We have also shown in vitro that immune responses to 25OHD may depend on 'free' rather than total serum concentrations of 25OHD. To investigate the possible effects of free versus total 25OHD on anti-inflammatory immune responses in vivo we have studied DSS-induced colitis in wild type C57BL/6 mice raised from weaning on diets containing vitamin D2 (D2) or vitamin D3 (D3) only (both 1000 IU/kg feed). 25OHD2 has lower binding affinity for the vitamin D binding protein than 25OHD3 which results in higher levels of free 25OHD2 relative to free 25OHD3 in mice raised on a D2-only diet. Total serum 25OHD concentrations, measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS), showed that D2 mice had significantly lower levels of 25OHD than D3 mice (6.85 ± 2.61 nmol/L vs. 49.16 ± 13.8 nmol/L for D2 and D3 respectively). Despite this, direct ELISA measurement showed no difference in free serum 25OHD levels between D2 and D3 mice (13.62 ± 2.26 pmol/L vs. 14.11 ± 2.24 pmol/L for D2 and D3 respectively). Analysis of DSS-induced colitis also showed no difference in weight loss or disease progression between D2 and D3 mice. These data indicate that despite D2-fed mice being vitamin D-deficient based on serum total 25OHD concentrations, these mice showed no evidence of increased inflammatory colitis disease relative to vitamin D-sufficient D3 mice. We therefore propose that free, rather than total serum 25OHD, may be a better marker of immune responses to vitamin D in vivo.

Mobilising vitamin D from adipose tissue: The potential impact of exercise.

Vitamin D is lipophilic and accumulates substantially in adipose tissue. Even without supplementation, the amount of vitamin D in the adipose of a typical adult is equivalent to several months of the daily reference nutrient intake (RNI). Paradoxically, despite the large amounts of vitamin D located in adipose tissue, individuals with obesity are often vitamin D deficient according to consensus measures of vitamin D status (serum 25-hydroxyvitamin D concentrations). Thus, it appears that vitamin D can become 'trapped' in adipose tissue, potentially due to insufficient lipolytic stimulation and/or due to tissue dysfunction/adaptation resulting from adipose expansion. Emerging evidence suggests that exercise may mobilise vitamin D from adipose (even in the absence of weight loss). If exercise helps to mobilise vitamin D from adipose tissue, then this could have important ramifications for practitioners and policymakers regarding the management of low circulating levels of vitamin D, as well as chronically low levels of physical activity, obesity and associated health conditions. This perspective led us to design a study to examine the impact of exercise on vitamin D status, vitamin D turnover and adipose tissue vitamin D content (the VitaDEx project). The VitaDEx project will determine whether increasing physical activity (via exercise) represents a potentially useful strategy to mobilise vitamin D from adipose tissue.

Defining vitamin D status using multi-metabolite mathematical modelling: A pregnancy perspective.

Vitamin D deficiency is linked to adverse pregnancy outcomes such as pre-eclampsia (PET) but remains defined by serum measurement of 25-hydroxyvitamin D3 (25(OH)D3) alone. To identify broader changes in vitamin D metabolism during normal and PET pregnancies we developed a relatively simple but fully parametrised mathematical model of the vitamin D metabolic pathway. The data used for parametrisation were serum vitamin D metabolites analysed for a cross-sectional group of women (n = 88); including normal pregnant women at 1 st (NP1, n = 25) and 3rd trimester (NP3, n = 21) and pregnant women with PET (n = 22), as well as non-pregnant female controls (n = 20). To account for the effects various metabolites have upon each other, data were analysed using an ordinary differential equation model of the vitamin D reaction network. Information obtained from the model was then also applied to serum vitamin D metabolome data (n = 50) obtained from a 2nd trimester pregnancy cohort, of which 25 prospectively developed PET. Statistical analysis of the data alone showed no significant difference between NP3 and PET for serum 25(OH)D3 and 24,25(OH)2D3 concentrations. Conversely, a statistical analysis informed by the reaction network model revealed that a better indicator of PET is the ratios of vitamin D metabolites in late pregnancy. Assessing the potential predicative value, no significant difference between NP3 and PET cases at 15 weeks gestation was found. Mathematical modelling offers a novel strategy for defining the impact of vitamin D metabolism on human health. This is particularly relevant within the context of pregnancy, where major changes in vitamin D metabolism occur across gestation, and dysregulated metabolism is evidenced in women with established PET.

Local and systemic glucocorticoid metabolism in inflammatory arthritis.

BACKGROUND: Isolated, primary synovial fibroblasts generate active glucocorticoids through expression of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-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 11beta-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 11beta-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 11beta-HSD2 expression in synovial macrophages, whereas 11beta-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 11beta-HSD1 activity in untreated patients with RA compared with controls and a significant correlation between total body 11beta-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.

Serum and urine vitamin D metabolite analysis in early preeclampsia.

Vitamin D deficiency is common in pregnant women and may contribute to adverse events in pregnancy such as preeclampsia (PET). To date, studies of vitamin D and PET have focused primarily on serum concentrations vitamin D, 25-hydroxyvitamin D3 (25(OH)D3) later in pregnancy. The aim here was to determine whether a more comprehensive analysis of vitamin D metabolites earlier in pregnancy could provide predictors of PET. Using samples from the SCOPE pregnancy cohort, multiple vitamin D metabolites were quantified by liquid chromatography-tandem mass spectrometry in paired serum and urine prior to the onset of PET symptoms. Samples from 50 women at pregnancy week 15 were analysed, with 25 (50%) developing PET by the end of the pregnancy and 25 continuing with uncomplicated pregnancy. Paired serum and urine from non-pregnant women (n = 9) of reproductive age were also used as a control. Serum concentrations of 25(OH)D3, 25(OH)D2, 1,25(OH)2D3, 24,25(OH)2D3 and 3-epi-25(OH)D3 were measured and showed no significant difference between women with uncomplicated pregnancies and those developing PET. As previously reported, serum 1,25(OH)2D3 was higher in all pregnant women (in the second trimester), but serum 25(OH)D2 was also higher compared to non-pregnant women. In urine, 25(OH)D3 and 24,25(OH)2D3 were quantifiable, with both metabolites demonstrating significantly lower (P < 0.05) concentrations of both of these metabolites in those destined to develop PET. These data indicate that analysis of urinary metabolites provides an additional insight into vitamin D and the kidney, with lower urinary 25(OH)D3 and 24,25(OH)2D3 excretion being an early indicator of a predisposition towards developing PET.

Splice variants of the CYP27b1 gene and the regulation of 1,25-dihydroxyvitamin D3 production.

The cytochrome P450 25-hydroxyvitamin D3-1alpha-hydroxylase (CYP27b1) plays a pivotal role in vitamin D physiology by catalyzing synthesis of active 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. In common with other P450s, CYP27b1 is known to exhibit alternative splicing. Here we have cloned and sequenced several novel intron 2-containing, noncoding splice variant mRNAs for CYP27b1 in 1,25(OH)2D3-producing HKC-8 human proximal tubule and THP-1 monocytic cells. Regulation of 1,25(OH)2D3 synthesis in these cell lines by calciotropic and noncalciotropic factors was associated with altered expression of the CYP27b1 splice variants. To assess the functional significance of this, HKC-8 cells were transfected with short hairpin RNA (shRNA) to inhibit mRNAs containing sequences from intron 2. This resulted in a significant increase in the expression of CYP27b1 protein and synthesis of 1,25(OH)2D3 by HKC-8 cells compared with control cells for two different intron 2-containing shRNAs (both P<0.001). shRNA to intron 2 had no significant effect on the levels of wild-type CYP27b1 mRNA, suggesting a posttranscriptional mechanism of action. By contrast, shRNA to wild-type CYP27b1 suppressed transcription and activity of the enzyme by 70 and 31%, respectively (both P<0.01). These data indicate that noncoding splice variants of CYP27b1 are functionally active and may play a significant role in the regulation of 1,25(OH)2D3 synthesis during normal physiology.

Two mutations in the hormone binding domain of the vitamin D receptor cause tissue resistance to 1,25 dihydroxyvitamin D3.

We have identified and characterized two mutations in the hormone binding domain of the vitamin D receptor (VDR) in patients with hereditary vitamin D-resistant rickets. One patient was found to have a premature stop mutation (CAG to TAG) in the hinge region affecting amino acid 149 (Q149X) and the other demonstrated a missense mutation (CGC to CTC) resulting in the substitution of arginine 271 by leucine (R271L) in the steroid binding domain. Eukaryotic expression analyses in CV-1 cells showed the inability of both patients' VDR to induce transcription from the osteocalcin hormone gene response element at 10(-7) M 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Normal transcription levels could, however, be elicited by the missense mutated VDR (R271L) in the presence of 1,000-fold higher 1,25-(OH)2D3 concentrations than needed for the wild-type receptor. This shows that Arg 271 directly affects the affinity of the VDR for its ligand and its conversion to leucine decreases its affinity for 1,25(OH)2D3 by a factor of 1,000. Arg 271 is located immediately 3-prime to a 30 amino acid segment (VDR amino acids 241-270) that is conserved among members of the steroid/thyroid/retinoid hormone receptor superfamily. These results represent the first missense mutation identified in the hormone binding domain of VDR and further define the structure-function relationship of 1,25(OH)2D3 ligand binding to its nuclear receptor.

Evidence for auto/paracrine actions of vitamin D in bone: 1alpha-hydroxylase expression and activity in human bone cells.

Vitamin D is an important regulator of mineral homeostasis and bone metabolism. 1Alpha-hydroxylation of 25-(OH)D3 to form the bioactive vitamin D hormone, 1alpha,25-(OH)2D3, is classically considered to take place in the kidney. However, 1alpha-hydroxylase has been reported at extrarenal sites. Whether bone is a 1alpha,25-(OH)2D3 synthesizing tissue is not univocal. The aim of this study was to investigate an autocrine/paracrine function for 1alpha,25-(OH)2D3 in bone. We show that 1alpha-hydroxylase is expressed in human osteoblasts, as well as the vitamin D binding protein receptors megalin and cubilin. Functional analyses demonstrate that after incubation with the 1alpha-hydroxylase substrate 25-(OH)D3, the osteoblasts can produce sufficient 1alpha,25-(OH)2D3 to modulate osteoblast activity, resulting in induced alkaline phosphatase (ALP) activity, osteocalcin (OC) and CYP24 mRNA expression, and mineralization. The classical renal regulators of 1alpha-hydroxylase, parathyroid hormone, and ambient calcium do not regulate 1alpha-hydroxylase in osteoblasts. In contrast, interleukin (IL)-1beta strongly induces 1alpha-hydroxylase. Besides the bone-forming cells, we demonstrate 1alpha-hydroxylase activity in the bone resorbing cells, the osteoclasts. This is strongly dependent on osteoclast inducer RANKL. This study showing expression, activity, and functionality of 1alpha-hydroxylase unequivocally demonstrates that vitamin D can act in an auto/paracrine manner in bone.

Dysregulation of maternal and placental vitamin D metabolism in preeclampsia.

INTRODUCTION: Epidemiology has linked preeclampsia (PET) to decreased maternal serum 25-hydroxyvitamin D3 (25(OH)D3). However, alterations in systemic and placental/decidual transport and metabolism of 25(OH)D3 during pregnancy suggest that other forms of vitamin D may also contribute to the pathophysiology of PET. METHODS: In a cross sectional analysis of normal pregnant women at 1st (n = 25) and 3rd trimester (n = 21), pregnant women with PET (n = 22), and non-pregnant female controls (n = 20) vitamin D metabolites were quantified in paired maternal serum, placental, and decidual tissue. RESULTS: Serum 25(OH)D3 was not significantly different in sera across all four groups. In normal 3rd trimester pregnant women serum active 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) was significantly higher than non-pregnant, normal 1st trimester pregnant, and PET women. Conversely, PET sera showed highest levels of the catabolites 3-epi-25(OH)D3 and 24,25-dihydroxyvitamin D3 (24,25(OH)2D3). Serum albumin was significantly lower in normal 3rd trimester pregnant women and PET relative to normal 1st trimester pregnant women, but there was no change in free/bioavailable 25(OH)D3. In PET placental tissue, 25(OH)D3 and 3-epi-25(OH)D3 were lower than normal 3rd trimester tissue, whilst placental 24,25(OH)2D3 was highest in PET. Tissue 1,25(OH)2D3 was detectable in 1st trimester decidua, which also showed 10-fold higher 25(OH)D3 relative to paired placentae. 3-epi-25(OH)D3 and 24,25(OH)2D3 were not different for decidua and placenta. In normal 3rd trimester pregnant women, total, free and bioavailable maternal 25(OH)D3 correlated with placental 25(OH)D3, but this was not conserved for PET. DISCUSSION: These data indicate that PET is associated with decreased activation, increased catabolism, and impaired placental uptake of 25(OH)D3.

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.

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.