Additional pathways of sterol metabolism: Evidence from analysis of Cyp27a1-/- mouse brain and plasma.

Cytochrome P450 (CYP) 27A1 is a key enzyme in both the acidic and neutral pathways of bile acid biosynthesis accepting cholesterol and ring-hydroxylated sterols as substrates introducing a (25R)26-hydroxy and ultimately a (25R)26-acid group to the sterol side-chain. In human, mutations in the CYP27A1 gene are the cause of the autosomal recessive disease cerebrotendinous xanthomatosis (CTX). Surprisingly, Cyp27a1 knockout mice (Cyp27a1-/-) do not present a CTX phenotype despite generating a similar global pattern of sterols. Using liquid chromatography - mass spectrometry and exploiting a charge-tagging approach for oxysterol analysis we identified over 50 cholesterol metabolites and precursors in the brain and circulation of Cyp27a1-/- mice. Notably, we identified (25R)26,7α- and (25S)26,7α-dihydroxy epimers of oxysterols and cholestenoic acids, indicating the presence of an additional sterol 26-hydroxylase in mouse. Importantly, our analysis also revealed elevated levels of 7α-hydroxycholest-4-en-3-one, which we found increased the number of oculomotor neurons in primary mouse brain cultures. 7α-Hydroxycholest-4-en-3-one is a ligand for the pregnane X receptor (PXR), activation of which is known to up-regulate the expression of CYP3A11, which we confirm has sterol 26-hydroxylase activity. This can explain the formation of (25R)26,7α- and (25S)26,7α-dihydroxy epimers of oxysterols and cholestenoic acids; the acid with the former stereochemistry is a liver X receptor (LXR) ligand that increases the number of oculomotor neurons in primary brain cultures. We hereby suggest that a lack of a motor neuron phenotype in some CTX patients and Cyp27a1-/- mice may involve increased levels of 7α-hydroxycholest-4-en-3-one and activation PXR, as well as increased levels of sterol 26-hydroxylase and the production of neuroprotective sterols capable of activating LXR.

Synergistic effect of 1α,25-dihydroxyvitamin D3 and 17ß-estradiol on osteoblast differentiation of pediatric MSCs.

Vitamin D is essential for mineral homeostasis and contributes to bone metabolism by stimulating osteoblast differentiation of marrow stromal cells (MSCs). In this study, we used MSCs from pre-pubertal girls and boys to test the hypothesis that 1α,25(OH)2D and 17ß-estradiol have synergistic effects on these MSCs, and what mechanism is involved. With IRB approval, we isolated MSCs from discarded excess iliac marrow graft from children undergoing alveolar cleft repair. Plasma was available from 8 female (9.3±0.2years) and 8 male (9.6±0.1years) subjects for hormone assays [25(OH)D, total testosterone, 17ß-estradiol, estrone, DHEA-S, Growth Hormone, IGF-I]. RT-PCR was used for gene expression. Alkaline phosphatase (ALP) activity was used to measure osteoblast differentiation at day 7; alizarin red was used to measure matrix mineralization at day 21. All subjects were pre-pubertal based on their hormone levels. Serum 25(OH)D levels ranged from 13.1 to 26.4ng/mL, with 75% below 20ng/mL. Constitutive gene expression of VDR and ERα, ß varied from subject to subject with no association with sex or serum chemistries. In osteoblastogenic medium, 1α,25(OH)2D3 (10nM) increased ALP activity by 36% (p<0.05) in MSCs; 10nM of E2 was not stimulatory but the combination of 1α,25(OH)2D3 and E2 increased ALP 151% (p<0.05 vs. control) and by 84.5% (p<0.05 vs. 1α,25(OH)2D3 alone). The combination of 1α,25(OH)2D3 and E2 significantly increased mineralization 11-fold, compared with either agent alone. Twenty-four hour treatment with 1α,25(OH)2D3 (10nM) or E2 (10nM) upregulated each other's receptor by as much as 5.8-fold for ERα and 2.9-fold for the VDR. In summary, 1α,25(OH)2D3 stimulated osteoblast differentiation and matrix mineralization with MSCs from pre-pubertal subjects, with a synergistic effect of E2, mediated by upregulated receptor levels, at least in part. These studies add new information about the regulation of human osteoblast differentiation, effects of 1α,25(OH)2D3 and E2 on MSCs, and the importance of vitamin D for skeletal health.

27-Hydroxycholesterol impairs neuronal glucose uptake through an IRAP/GLUT4 system dysregulation.

Hypercholesterolemia is associated with cognitively deteriorated states. Here, we show that excess 27-hydroxycholesterol (27-OH), a cholesterol metabolite passing from the circulation into the brain, reduced in vivo brain glucose uptake, GLUT4 expression, and spatial memory. Furthermore, patients exhibiting higher 27-OH levels had reduced 18F-fluorodeoxyglucose uptake. This interplay between 27-OH and glucose uptake revealed the engagement of the insulin-regulated aminopeptidase (IRAP). 27-OH increased the levels and activity of IRAP, countered the IRAP antagonist angiotensin IV (AngIV)-mediated glucose uptake, and enhanced the levels of the AngIV-degrading enzyme aminopeptidase N (AP-N). These effects were mediated by liver X receptors. Our results reveal a molecular link between cholesterol, brain glucose, and the brain renin-angiotensin system, all of which are affected in some neurodegenerative diseases. Thus, reducing 27-OH levels or inhibiting AP-N maybe a useful strategy in the prevention of the altered glucose metabolism and memory decline in these disorders.

Evidence for a role of sterol 27-hydroxylase in glucocorticoid metabolism in vivo.

The intracellular availability of glucocorticoids is regulated by the enzymes 11ß-hydroxysteroid dehydrogenase 1 (HSD11B1) and 11ß-hydroxysteroid dehydrogenase 2 (HSD11B2). The activity of HSD11B1 is measured in the urine based on the (tetrahydrocortisol+5α-tetrahydrocortisol)/tetrahydrocortisone ((THF+5α-THF)/THE) ratio in humans and the (tetrahydrocorticosterone+5α-tetrahydrocorticosterone)/tetrahydrodehydrocorticosterone ((THB+5α-THB)/THA) ratio in mice. The cortisol/cortisone (F/E) ratio in humans and the corticosterone/11-dehydrocorticosterone (B/A) ratio in mice are markers of the activity of HSD11B2. In vitro agonist treatment of liver X receptor (LXR) down-regulates the activity of HSD11B1. Sterol 27-hydroxylase (CYP27A1) catalyses the first step in the alternative pathway of bile acid synthesis by hydroxylating cholesterol to 27-hydroxycholesterol (27-OHC). Since 27-OHC is a natural ligand for LXR, we hypothesised that CYP27A1 deficiency may up-regulate the activity of HSD11B1. In a patient with cerebrotendinous xanthomatosis carrying a loss-of-function mutation in CYP27A1, the plasma concentrations of 27-OHC were dramatically reduced (3.8 vs 90-140 ng/ml in healthy controls) and the urinary ratios of (THF+5α-THF)/THE and F/E were increased, demonstrating enhanced HSD11B1 and diminished HSD11B2 activities. Similarly, in Cyp27a1 knockout (KO) mice, the plasma concentrations of 27-OHC were undetectable (<1 vs 25-120 ng/ml in Cyp27a1 WT mice). The urinary ratio of (THB+5α-THB)/THA was fourfold and that of B/A was twofold higher in KO mice than in their WT littermates. The (THB+5α-THB)/THA ratio was also significantly increased in the plasma, liver and kidney of KO mice. In the liver of these mice, the increase in the concentrations of active glucocorticoids was due to increased liver weight as a consequence of Cyp27a1 deficiency. In vitro, 27-OHC acts as an inhibitor of the activity of HSD11B1. Our studies suggest that the expression of CYP27A1 modulates the concentrations of active glucocorticoids in both humans and mice and in vitro.

Altered bone status in unilateral testicular cancer survivors: Role of CYP2R1 and its luteinizing hormone-dependency.

BACKGROUND: Recent data suggest a potential role of testis in vitamin D activation, where Leydig cells could represent key players in this process since they express the highest amount of CYP2R1, a key enzyme involved in vitamin D 25 hydroxylation. AIM: To evaluate bone status in unilateral orchiectomy and to assess in vivo and in vitro LH-dependency of Vitamin D 25 hydroxylation. SUBJECTS AND METHODS: 125 normotestosteronemic patients with testicular cancer (TC), featured by unilateral orchiectomy and 41 age-matched healthy male controls were studied in the Center for Human Reproduction Pathology at the University of Padova. To evaluate LH-dependency of Vitamin D 25 hydroxylation in vitro, Leydig cell cultures were stimulated with hCG and assessed for CYP2R1 expression, whereas in vivo 10 hypogonadotropic hypogonadal (HH) patients were evaluated before and after treatment with gonadotropins for bone metabolism markers. Hormonal pattern and bone metabolism markers were measured in all subjects, whereas 105 patients and 41 controls underwent bone densitometry by DEXA. RESULTS: In TC patients 25-hydroxyvitamin D levels were significantly lower compared to controls. Furthermore, 23.8% of patients with TC displayed low bone density (Z-score <-2 SD). None of the 41 control subjects showed any significant alteration of BMD. In vitro and in vivo studies revealed that CYP2R1 expression in Leydig cells appeared to be hCG dependent. CONCLUSION: Our data show an association between TC and alteration of the bone status, despite unvaried androgen and estrogen levels, suggesting the evaluation of bone status and possible vitamin D deficiency in TC survivors.

Marked change in the balance between CYP27A1 and CYP46A1 mediated elimination of cholesterol during differentiation of human neuronal cells.

Cholesterol metabolism in the brain is distinct from that in other tissues due to the fact that cholesterol itself is unable to pass across the blood-brain barrier. Elimination of brain cholesterol is mainly dependent on a neuronal-specific cytochrome P450, CYP46A1, catalyzing the conversion of cholesterol into 24(S)-hydroxycholesterol (24OHC), which is able to pass the blood-brain barrier. A suitable model for studying this elimination from human neuronal cells has not been described previously. It is shown here that differentiated Ntera2/clone D1 (NT2) cells express the key genes involved in brain cholesterol homeostasis including CYP46A1, and that the expression profiles of the genes observed during neuronal differentiation are those expected to occur in vivo. Thus there was a decrease in the mRNA levels corresponding to cholesterol synthesis enzymes and a marked increase in the mRNA level of CYP46A1. The latter increase was associated with increased levels of CYP46A1 protein and increased production of 24OHC. The magnitude of the secretion of 24OHC from the differentiated NT2 cells into the medium was similar to that expected to occur under in vivo conditions. An alternative to elimination of cholesterol by the CYP46A1 mechanism is elimination by CYP27A1, and the product of this enzyme, 27-hydroxycholesterol (27OHC), is also known to pass the blood-brain barrier. The CYP27A1 protein level decreased during the differentiation of the NT2 cells in parallel with decreased production of 27OHC. The ratio between 24OHC and 27OHC in the medium from the cultured cells increased, by a factor of 13, during the differentiation process. The results suggest that progenitor cells eliminate cholesterol in the form of 27OHC while neurogenesis induces a change to the CYP46A1 dependent pathway. Furthermore this study demonstrates that differentiated NT2 cells are suitable for studies of cholesterol homeostasis in human neurons.

Bone mineral density and testicular failure: evidence for a role of vitamin D 25-hydroxylase in human testis.

WORKING HYPOTHESIS: Mutations in the CYP2R1 gene, highly expressed in the testis and encoding vitamin D 25-hydroxylase, result in a vitamin D deficiency and a defective calcium homeostasis leading to rickets. OBJECTIVE: Our aim was to investigate CYP2R1 expression in pathological testis samples and relate this to vitamin D metabolism in testiculopathic patients. DESIGN, PATIENTS, SETTING: Testis samples for in vitro study and 98 young men were transversally evaluated at Padova's Center for Male Gamete Cryopreservation. METHODS: CYP2R1 mRNA expression and protein production were evaluated by quantitative RT-PCR, Western blot analysis, and immunofluorescence. Hormonal and bone-marker levels, and bone densitometry by dual-energy x-ray absorptiometry, were determined in patients with Sertoli-cell-only syndrome and severe hypospermatogenesis. RESULTS: We found a lower gene and protein expression of CYP2R1 in samples with hypospermatogenesis and Sertoli-cell-only syndrome (P < 0.05) and a colocalization with INSL-3, a Leydig cell marker, at immunofluorescence. In all testiculopathic patients 25-hydroxyvitamin D levels were significantly lower and PTH levels higher compared to controls (P < 0.05). Furthermore, testiculopathic patients showed osteopenia and osteoporosis despite normal testosterone levels compared with controls both with increased bone-marker levels and altered dual-energy x-ray absorptiometry in the femoral neck and lumbar spine (for all parameters, P < 0.05). CONCLUSIONS: Our data show an association between testiculopathy and alteration of the bone status, despite unvaried androgen and estrogen levels and no other evident cause of vitamin D reduction. Further studies in larger cohorts are needed to confirm our results.