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.

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.

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.

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.

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.

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.

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.

Immunological role of vitamin D at the maternal-fetal interface.

During pregnancy, immune activity is tightly regulated so that antimicrobial protection of the mother and fetus is balanced with the need for immune tolerance to prevent fetal rejection. In this setting, the maternal-fetal interface, in the form of the uterine decidua, provides a heterogeneous immune cell population with the potential to mediate diverse activities throughout pregnancy. Recent studies have suggested that vitamin D may be a key regulator of immune function during pregnancy, with the fetal-maternal interface representing a prominent target. Among its non-classical actions are potent immunomodulatory effects, including induction of antibacterial responses and modulation of T-lymphocytes to suppress inflammation and promote tolerogenesis. Thus, vitamin D may play a pivotal role in normal decidual immune function by promoting innate responses to infection, while simultaneously preventing an over-elaboration of inflammatory adaptive immunity. Research to date has focused upon the potential role of vitamin D in preventing infectious diseases such as tuberculosis, as well as possibly suppressing of autoimmune disease. Nevertheless, vitamin D may also influence facets of immune function not immediately associated with primary innate responses. This review summarises our current understanding of decidual immune function with respect to the vitamin D metabolism and signalling, and as to how this may be affected by variations in maternal vitamin D status. There has recently been much interest in vitamin D supplementation of pregnant women, but our knowledge of how this may influence the function of decidua remains limited. Further insight into the immunomodulatory actions of vitamin D during pregnancy will help shed light upon this.

Vitamin D promotes human extravillous trophoblast invasion in vitro.

INTRODUCTION: Incomplete human extravillous trophoblast (EVT) invasion of the decidua and maternal spiral arteries is characteristic of pre-eclampsia, a condition linked to low maternal vitamin D status. It is hypothesized that dysregulated vitamin D action in uteroplacental tissues disrupts EVT invasion leading to malplacentation. METHODS: This study assessed the effects of the active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25-D3), and its precursor, 25-hydroxyvitamin D3 (25-D3), on primary human EVT isolated from first trimester pregnancies. Expression of EVT markers (cytokeratin-7, HLA-G), the vitamin D-activating enzyme (CYP27B1) and 1,25-D3 receptor (VDR) was assessed by immunocytochemistry. EVT responses following in vitro treatment with 1,25-D3 (0-10 nM) or 25-D3 (0-100 nM) for 48-60 h were assessed using quantitative RT-PCR (qRT-PCR) analysis of key target genes. Effects on EVT invasion through Matrigel(®) were quantified alongside zymographic analysis of secreted matrix metalloproteinases (MMPs). Effects on cell viability were assessed by measurement of MTT. RESULTS: EVT co-expressed mRNA and protein for CYP27B1 and VDR, and demonstrated induction of mRNA encoding vitamin D-responsive genes, 24-hydroxylase (CYP24A1) and cathelicidin following 1,25-D3 treatment. EVT could respond to 1,25-D3 and 25-D3, both of which significantly increased EVT invasion, with maximal effect at 1 nM 1,25-D3 (1.9-fold; p < 0.01) and 100 nM 25-D3 (2.2-fold; p < 0.05) respectively compared with untreated controls. This was accompanied by increased pro-MMP2 and pro-MMP9 secretion. The invasion was independent of cell viability, which remained unchanged. DISCUSSION: These data support a role for vitamin D in EVT invasion during human placentation and suggest that vitamin D-deficiency may contribute to impaired EVT invasion and pre-eclampsia.

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.

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.

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.

Vitamin D induces innate antibacterial responses in human trophoblasts via an intracrine pathway.

The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)(2)D), is a potent inducer of the antimicrobial protein cathelicidin, CAMP (LL37). In macrophages this response is dependent on intracrine synthesis of 1,25(OH)(2)D from precursor 25-hydroxyvitamin D (25OHD), catalyzed by the enzyme 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1). In view of the fact that trophoblastic cells also express abundant CYP27B1, we postulated a similar intracrine pathway for induction of CAMP in the placenta. Analysis of placenta explants, primary cultures of human trophoblast, and the 3A trophoblastic cell line treated with 1,25(OH)(2)D (1-100 nM) revealed dose-dependent induction of CAMP similar to that observed with primary cultures of human macrophages. Also consistent with macrophages, induction of trophoblastic CAMP was enhanced via intracrine conversion of 25OHD to 1,25(OH)(2)D. However, in contrast to macrophages, induction of CAMP by vitamin D in trophoblasts was not enhanced by costimulation with Toll-like receptor ligands, such as lipopolysaccharide. Despite this, exposure to vitamin D metabolites significantly enhanced antibacterial responses in trophoblastic cells: 3A cells infected with Escherichia coli showed decreased numbers of bacterial colony-forming units compared with vehicle-treated controls when treated with 25OHD (49.6% +/- 10.9%) or 1,25(OH)(2)D (45.4% +/- 9.2%), both P < 0.001. Treatment with 25OHD (1-100 nM) or 1,25(OH)(2)D (0.1-10 nM) also protected 3A cells against cell death following infection with E. coli (13.6%-26.9% and 22.3%-40.2% protection, respectively). These observations indicate that 1,25(OH)(2)D can function as an intracrine regulator of CAMP in trophoblasts, and may thus provide a novel mechanism for activation of innate immune responses in the placenta.

Progesterone is extensively metabolized in osteoblasts: implications for progesterone action on bone.

The effect of progestogens on bone is controversial with some studies suggesting an anabolic action while others show no effect. Prereceptor metabolism via localized expression of specific enzymes may have major impact on progesterone action in bone and may explain some of the discrepancies between studies. We therefore investigated the metabolism of progesterone in primary cultures of human osteoblasts and MG-63 osteoblastic cells. Osteoblasts and MG-63 cells were incubated with 4- (14)C-progesterone tracer and 50 nM unlabeled progesterone, and magnitude and pattern of progesterone metabolism were determined by two-dimensional thin-layer chromatography. Conventional and Taqman real-time PCR analysis were used to assess expression of progesterone metabolizing enzymes. In both types of cells the two major metabolic products of progesterone were 20 alpha-dihydroprogesterone and 5 alpha-dihydroprogesterone, but conversion to 3 alpha, 5 alpha- and 3 beta, 5 alpha-tetrahydroprogesterone was also detected. This activity was concomitant with expression of mRNAs for the enzymes AKR1C1, 5 alpha-reductase type 1 and AKR1C2, and 3 beta-HSD type 1 and 3-hydroxysteroid epimerase. In MG-63 cells progesterone metabolism was largely mediated via 5 alpha-reductase. In primary osteoblasts progesterone metabolism was unaffected by treatment with dexamethasone or estradiol, but in MG-63 cells dexamethasone pretreatment increased 5 alpha-reductase activity. Progesterone is subject to extensive intracellular inactivation in human osteoblasts, with potential attenuation of local progesterone receptor responses. Conversely, osteoblasts have the capacity to convert progestogens to metabolites reported to have anabolic actions through the estrogen receptor.

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.

Co-chaperone potentiation of vitamin D receptor-mediated transactivation: a role for Bcl2-associated athanogene-1 as an intracellular-binding protein for 1,25-dihydroxyvitamin D3.

The constitutively expressed member of the heat shock protein-70 family (hsc70) is a chaperone with multiple functions in cellular homeostasis. Previously, we demonstrated the ability of hsc70 to bind 25-hydroxyvitamin D3 (25-OHD3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Hsc70 also recruits and interacts with the co-chaperone Bcl2-associated athanogene (BAG)-1 via the ATP-binding domain that resides on hsc70. Competitive ligand-binding assays showed that, like hsc70, recombinant BAG-1 is able to bind 25-OHD3 (Kd=0.71+/-0.25 nM, Bmax 69.9+/-16.1 fmoles/microg protein) and 1,25(OH)2D3 (Kd=0.16+/-0.07 nM, Bmax = 38.1+/-3.5 fmoles/microg protein; both n=3 separate binding assays, P<0.001 for Kd and Bmax). To investigate the functional significance of this, we transiently overexpressed the S, M, and L variants of BAG-1 into human kidney HKC-8 cells stably transfected with a 1,25(OH)2D3-responsive 24-hydroxylase (CYP24) promoter-reporter construct. As HKC-8 cells also express the enzyme 1alpha-hydroxylase, both 25-OHD3 (200 nM) and 1,25(OH)2D3 (5 nM) were able to induce CYP24 promoter activity. This was further enhanced following overexpression of all the three BAG-1 isoforms. By contrast, BAG-1 isoforms had no effect on metabolism of 25-OHD3 by HKC-8 cells (either via 1alpha- or 24-hydroxylase activities). Further studies showed that a mutant form of BAG-1S exhibited decreased binding of 1,25(OH)2D3 and this resulted in a concomitant loss of potentiation of CYP24 promoter transactivation. Similar effects were not observed for 25-OHD3. These data highlight a novel role for BAG-1 as an intracellular-binding protein for 1,25(OH)2D3 and further suggest that BAG-1 is able to potentiate vitamin D receptor-mediated transactivation by acting as a nuclear chaperone for 1,25(OH)2D3.

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.

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.