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Find video protocols related to scientific articles indexed in Pubmed.
The retinoic acid-metabolizing enzyme Cyp26b1 regulates CD4 T cell differentiation and function.
PLoS ONE
PUBLISHED: 01-01-2013
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The vitamin A metabolite retinoic acid (RA) has potent immunomodulatory properties that affect T cell differentiation, migration and function. However, the precise role of RA metabolism in T cells remains unclear. Catabolism of RA is mediated by the Cyp26 family of cytochrome P450 oxidases. We examined the role of Cyp26b1, the T cell-specific family member, in CD4(+) T cells. Mice with a conditional knockout of Cyp26b1 in T cells (Cyp26b1 (-/-) mice) displayed normal lymphoid development but showed an increased sensitivity to serum retinoids, which led to increased differentiation under both inducible regulatory T (iTreg) cell- and TH17 cell-polarizing conditions in vitro. Further, Cyp26b1 expression was differentially regulated in iTreg and TH17 cells. Transfer of naïve Cyp26b1 (-/-) CD4(+) T cells into Rag1 (-/-) mice resulted in significantly reduced disease in a model of T cell-dependent colitis. Our results show that T cell-specific expression of Cyp26b1 is required for the development of T cell-mediated colitis and may be applicable to the development of therapeutics that target Cyp26b1 for the treatment of inflammatory bowel disease.
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Genetic deletion of Cyp26b1 negatively impacts limb skeletogenesis by inhibiting chondrogenesis.
J. Cell. Sci.
PUBLISHED: 08-03-2011
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Cyp26b1, a retinoic acid (RA)-metabolising enzyme, is expressed in the developing limb bud, and Cyp26b1(-/-) mice present with severe limb defects. These malformations might be attributable to an RA-induced patterning defect; however, recent reports suggest that RA is dispensable for limb patterning. In this study, we examined the role of endogenous retinoid signalling in skeletogenesis using Cyp26b1(-/-) mice and transgenic mice in which Cyp26b1 is conditionally deleted under control of the Prrx1 promoter beginning at ~E9.5 (Prrx1Cre(+)/Cyp26b1(fl/fl)). We found that the limb phenotype in Prrx1Cre(+)/Cyp26b1(fl/fl) mice was less severe than that observed in Cyp26b1(-/-) animals and that a change in retinoid signalling contributed to the difference in phenotypes. We systematically examined the role of endogenous RA signalling in chondrogenesis and found that Cyp26b1(-/-) cells and limb mesenchymal cells treated with a CYP inhibitor, are maintained in a pre-chondrogenic state, exhibit reduced chondroblast differentiation and have modestly accelerated chondrocyte hypertrophy. Furthermore, Cyp26b1(-/-) mesenchyme exhibited an increase in expression of genes in a closely related tendogenic lineage, indicating that retinoid signals in the limb interfere with differentiation and maintain progenitor status. Together, these findings support an important function for RA in regulating the behaviour of mesenchymal progenitors, and their subsequent differentiation and maturation.
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CYP24A1 and kidney disease.
Curr. Opin. Nephrol. Hypertens.
PUBLISHED: 05-26-2011
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Patients with chronic renal disease have elevated serum phosphate levels, elevated fibroblast-like growth factor 23 (FGF-23), and declining vitamin D status. These changes are related and may be responsible for elevated 25-hydroxyvitamin D-24-hydroxylase (CYP24A1) and dysfunctional vitamin D metabolism. This review focuses on the biochemistry and pathophysiology of CYP24A1 and the utility of blocking this enzyme with CYP24A1 inhibitors in chronic kidney disease (CKD) patients.
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Functional properties and substrate characterization of human CYP26A1, CYP26B1, and CYP26C1 expressed by recombinant baculovirus in insect cells.
J Pharmacol Toxicol Methods
PUBLISHED: 04-29-2011
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The cytochrome P450 CYP26 family of retinoic acid (RA) metabolizing enzymes, comprising CYP26A1, CYP26B1, and CYP26C1 is critical for establishing patterns of RA distribution during embryonic development and retinoid homeostasis in the adult. All three members of this family can metabolize all trans-RA. CYP26C1 has also been shown to efficiently metabolize the 9-cis isomer of RA.
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The role of CYP26 enzymes in defining appropriate retinoic acid exposure during embryogenesis.
Birth Defects Res. Part A Clin. Mol. Teratol.
PUBLISHED: 09-16-2010
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Retinoic acid (RA) is a pleiotropic derivative of vitamin A, or retinol, which is responsible for all of the bioactivity associated with this vitamin. The teratogenic influences of vitamin A deficiency and excess RA in rodents were first observed more than 50 years ago. Efforts over the last 15-20 years have refined these observations by defining the molecular mechanisms that control RA availability and signaling during murine embryonic development. This review will discuss our current understanding of the role of RA in teratogenesis, with specific emphasis on the essential function of the RA catabolic CYP26 enzymes in preventing teratogenic consequences caused by uncontrolled distribution of RA. Particular focus will be paid to the RA-sensitive tissues of the caudal and cranial regions, the limb, and the testis, and how genetic mutation of factors controlling RA distribution have revealed important roles for RA during embryogenesis.
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Dysregulation of renal vitamin D metabolism in the uremic rat.
Kidney Int.
PUBLISHED: 06-09-2010
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The progressive decline in kidney function and concomitant loss of renal 1alpha-hydroxylase (CYP27B1) in chronic kidney disease (CKD) are associated with a gradual loss of circulating 25-hydroxyvitamin D(3) (25(OH)D(3)) and 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)). However, only the decrease in 1alpha,25(OH)(2)D(3) can be explained by the decline of CYP27B1, suggesting that insufficiency of both metabolites may reflect their accelerated degradation by the key catabolic enzyme 24-hydroxylase (CYP24). To determine whether CYP24 is involved in causing vitamin D insufficiency and/or resistance to vitamin D therapy in CKD, we determined the regulation of CYP24 and CYP27B1 in normal rats and rats treated with adenine to induce CKD. As expected, CYP24 decreased whereas CYP27B1 increased when normal animals were rendered vitamin D deficient. Unexpectedly, renal CYP24 mRNA and protein expression were markedly elevated, irrespective of the vitamin D status of the rats. A significant decrease in serum 1alpha,25(OH)(2)D(3) levels was found in uremic rats; however, we did not find a coincident decline in CYP27B1. Analysis in human kidney biopsies confirmed the association of elevated CYP24 with kidney disease. Thus, our findings suggest that dysregulation of CYP24 may be a significant mechanism contributing to vitamin D insufficiency and resistance to vitamin D therapy in CKD.
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Vitamin D analogues targeting CYP24 in chronic kidney disease.
J. Steroid Biochem. Mol. Biol.
PUBLISHED: 01-19-2010
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The cytochrome P450 enzyme 24-hydroxylase (CYP24) plays a critical role in regulating levels of vitamin D hormone. Aberrant expression of CYP24 has been implicated in vitamin D insufficiency and resistance to vitamin D therapy. We have demonstrated amplified CYP24 expression in uremic rats, suggesting that CYP24 has an etiological role in vitamin D insufficiency commonly associated with chronic kidney disease (CKD). We have designed two new analogues of 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3), namely CTA091 and CTA018/MT2832, which are potent inhibitors of CYP24. In vitro studies with CTA091 show that it enhances the potency of 1alpha,25(OH)2D3. In vivo studies demonstrate that CTA091 decreases serum intact parathyroid hormone (iPTH) levels and increases circulating 1alpha,25(OH)2D3. CTA091 increases both Cmax and AUC of co-administered 1alpha,25(OH)2D3. These studies indicate that CYP24 inhibition can increase cellular responsiveness to vitamin D hormone and potentiate vitamin D therapy. CTA018/MT2832 differs from CTA091 in that it also has the ability to activate vitamin D receptor-mediated transcription. CTA018/MT2832 effectively suppresses elevated iPTH secretion at doses which do not affect serum calcium or phosphorus levels in a rodent model of CKD. Studies with both new analogues underscore the potential utility of CYP24 inhibition in the treatment of secondary hyperparathyroidism in CKD.
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Tulp3 is a critical repressor of mouse hedgehog signaling.
Dev. Dyn.
PUBLISHED: 04-01-2009
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Precise regulation of the morphogen sonic hedgehog (Shh) and modulation of the Shh signaling pathway is required for proper specification of cell fate within the developing limbs and neural tube, and resultant tissue morphogenesis. Tulp3 (tubby-like protein 3) is a protein of unknown function which has been implicated in nervous system development through gene knockout studies. We demonstrate here that mice lacking the Tulp3 gene develop abnormalities of both the neural tube and limbs consistent with improper regulation of Shh signaling. Tulp3(-/-) embryos show expansion of Shh target gene expression and display a ventralization of neural progenitor cells in the caudal neural tube. We further show that Tulp3(-/-)/Shh(-/-) compound mutant embryos resemble Tulp3 mutants, and express Shh target genes in the neural tube and limbs which are not expressed in Shh(-/-) embryos. This work uncovers a novel role for Tulp3 as a negative regulatory factor in the Hh pathway.
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Cyp26b1 expression in murine Sertoli cells is required to maintain male germ cells in an undifferentiated state during embryogenesis.
PLoS ONE
PUBLISHED: 03-16-2009
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In mammals, germ cells within the developing gonad follow a sexually dimorphic pathway. Germ cells in the murine ovary enter meiotic prophase during embryogenesis, whereas germ cells in the embryonic testis arrest in G0 of mitotic cell cycle and do not enter meiosis until after birth. In mice, retinoic acid (RA) signaling has been implicated in controlling entry into meiosis in germ cells, as meiosis in male embryonic germ cells is blocked by the activity of a RA-catabolizing enzyme, CYP26B1. However, the mechanisms regulating mitotic arrest in male germ cells are not well understood. Cyp26b1 expression in the testes begins in somatic cells at embryonic day (E) 11.5, prior to mitotic arrest, and persists throughout fetal development. Here, we show that Sertoli cell-specific loss of CYP26B1 activity between E15.5 and E16.5, several days after germ cell sex determination, causes male germ cells to exit from G0, re-enter the mitotic cell cycle and initiate meiotic prophase. These results suggest that male germ cells retain the developmental potential to differentiate in meiosis until at least at E15.5. CYP26B1 in Sertoli cells acts as a masculinizing factor to arrest male germ cells in the G0 phase of the cell cycle and prevents them from entering meiosis, and thus is essential for the maintenance of the undifferentiated state of male germ cells during embryonic development.
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Genetic disruption of CYP26B1 severely affects development of neural crest derived head structures, but does not compromise hindbrain patterning.
Dev. Dyn.
PUBLISHED: 02-25-2009
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Cyp26b1 encodes a cytochrome-P450 enzyme that catabolizes retinoic acid (RA), a vitamin A derived signaling molecule. We have examined Cyp26b1(-/-) mice and report that mutants exhibit numerous abnormalities in cranial neural crest cell derived tissues. At embryonic day (E) 18.5 Cyp26b1(-/-) animals exhibit a truncated mandible, abnormal tooth buds, reduced ossification of calvaria, and are missing structures of the maxilla and nasal process. Some of these abnormalities may be due to defects in formation of Meckels cartilage, which is truncated with an unfused distal region at E14.5 in mutant animals. Despite the severe malformations, we did not detect any abnormalities in rhombomere segmentation, or in patterning and migration of anterior hindbrain derived neural crest cells. Abnormal migration of neural crest cells toward the posterior branchial arches was observed, which may underlie defects in larynx and hyoid development. These data suggest different periods of sensitivity of anterior and posterior hindbrain neural crest derivatives to elevated levels of RA in the absence of CYP26B1.
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Focal facial dermal dysplasia, type IV, is caused by mutations in CYP26C1.
Hum. Mol. Genet.
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Focal facial dermal dysplasia (FFDD) Type IV is a rare syndrome characterized by facial lesions resembling aplasia cutis in a preauricular distribution along the line of fusion of the maxillary and mandibular prominences. To identify the causative gene(s), exome sequencing was performed in a family with two affected siblings. Assuming autosomal recessive inheritance, two novel sequence variants were identified in both siblings in CYP26C1-a duplication of seven base pairs, which was maternally inherited, c.844_851dupCCATGCA, predicting p.Glu284fsX128 and a missense mutation, c.1433G>A, predicting p.Arg478His, that was paternally inherited. The duplication predicted a frameshift mutation that led to a premature stop codon and premature chain termination, whereas the missense mutation was not functional based on its in vitro expression in mammalian cells. The FFDD skin lesions arise along the sites of fusion of the maxillary and mandibular prominences early in facial development, and Cyp26c1 was expressed exactly along the fusion line for these facial prominences in the first branchial arch in mice. Sequencing of four additional, unrelated Type IV FFDD patients and eight Type II or III TWIST2-negative FFDD patients revealed that three of the Type IV patients were homozygous for the duplication, whereas none of the Type II or III patients had CYP26C1 mutations. The seven base pairs duplication was present in 0.3% of healthy controls and 0.3% of patients with other birth defects. These findings suggest that the phenotypic manifestations of FFDD Type IV can be non-penetrant or underascertained. Thus, FFDD Type IV results from the loss of function mutations in CYP26C1.
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Sevelamer hydrochloride binds phosphate released from phytate in chicks fed 1?-hydroxy cholecalciferol.
J Ren Nutr
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Hyperphosphatemia in animal models of human renal disease has been linked to increased risk of death. Phosphate binders (e.g., sevelamer hydrochloride) and plant-based, low phosphate diets are used to reduce dietary phosphate load; however, animal models show that treatment with active forms of vitamin D(3) (e.g., calcitriol, a renal disease therapy) renders plant phytate phosphate available for absorption. Using an established chick model, the effectiveness of sevelamer in preventing the apparent absorption of liberated phytate phosphate during active vitamin D use was investigated in two separate experiments.
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CYP24 inhibition preserves 1?,25-dihydroxyvitamin D(3) anti-proliferative signaling in lung cancer cells.
Mol. Cell. Endocrinol.
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Human lung tumors aberrantly express the 1?,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3))-catabolizing enzyme, CYP24. We hypothesized that CYP24 reduces 1,25(OH)(2)D(3)-mediated transcription and allows lung cancer cells to escape its growth-inhibitory action. To test this, H292 lung cancer cells and the CYP24-selective inhibitor CTA091 were utilized. In H292 cells, CTA091 reduces 1,25(OH)(2)D(3) catabolism, significantly increases 1,25(OH)(2)D(3)-mediated growth inhibition, and increases 1,25(OH)(2)D(3) effects on induced and repressed genes in gene expression profiling studies. Pathway mapping of repressed genes uncovered cell cycle as a predominant 1,25(OH)(2)D(3) target. In H292 cells, 1,25(OH)(2)D(3) significantly decreases cyclin E2 levels and induces G(0)/G(1) arrest. A broader set of cyclins is down-regulated when 1,25(OH)(2)D(3) is combined with CTA091, and cell cycle arrest further increases. Effects of CTA091 on 1,25(OH)(2)D(3) signaling are vitamin D receptor-dependent. These data provide evidence that CYP24 limits 1,25(OH)(2)D(3) anti-proliferative signaling in cancer cells, and suggest that CTA091 may be beneficial in preserving 1,25(OH)(2)D(3) action in lung cancer.
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A local effect of CYP24 inhibition on lung tumor xenograft exposure to 1,25-dihydroxyvitamin D(3) is revealed using a novel LC-MS/MS assay.
Steroids
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The vitamin D(3) catabolizing enzyme, CYP24, is frequently over-expressed in tumors, where it may support proliferation by eliminating the growth suppressive effects of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). However, the impact of CYP24 expression in tumors or consequence of CYP24 inhibition on tumor levels of 1,25(OH)(2)D(3)in vivo has not been studied due to the lack of a suitable quantitative method. To address this need, an LC-MS/MS assay that permits absolute quantitation of 1,25(OH)(2)D(3) in plasma and tumor was developed. We applied this assay to the H292 lung tumor xenograft model: H292 cells eliminate 1,25(OH)(2)D(3) by a CYP24-dependent process in vitro, and 1,25(OH)(2)D(3) rapidly induces CYP24 expression in H292 cells in vivo. In tumor-bearing mice, plasma and tumor concentrations of 1,25(OH)(2)D(3) reached a maximum of 21.6 and 1.70ng/mL, respectively, following intraperitoneal dosing (20?g/kg 1,25(OH)(2)D(3)). When co-administered with the CYP24 selective inhibitor CTA091 (250?g/kg), 1,25(OH)(2)D(3) plasma levels increased 1.6-fold, and tumor levels increased 2.6-fold. The tumor/plasma ratio of 1,25(OH)(2)D(3) AUC was increased 1.7-fold by CTA091, suggesting that the inhibitor increased the tumor concentrations of 1,25(OH)(2)D(3) independent of its effects on plasma disposition. Compartmental modeling of 1,25(OH)(2)D(3) concentration versus time data confirmed that: 1,25(OH)(2)D(3) was eliminated from plasma and tumor; CTA091 reduced the elimination from both compartments; and that the effect of CTA091 on tumor exposure was greater than its effect on plasma. These results provide evidence that CYP24-expressing lung tumors eliminate 1,25(OH)(2)D(3) by a CYP24-dependent process in vivo and that CTA091 administration represents a feasible approach to increase tumor exposure to 1,25(OH)(2)D(3).
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