Rescue of the phenotype of CYP27B1 (1alpha-hydroxylase)-deficient mice

The treatment of choice for pseudo Vitamin D deficiency rickets (PDDR), caused by mutations in the 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1; 1alpha-OHase) gene, is replacement therapy with 1,25(OH)(2)D(3). We have previously engineered an animal model of PDDR by targeted inactivation of the 1alpha-OHase gene in mice (Endocrinology 142 (2001) 3135). Replacement therapy was performed in this model, and compared to feeding with a high calcium diet containing 2% calcium, 1.25% phosphorus, 20% lactose (rescue diet). Blood biochemistry analysis revealed that both rescue treatments corrected the hypocalcemia and secondary hyperparathyroidism. Bone histology and histomorphometry confirmed that the rickets and osteomalacia were cured by both rescue protocols. However, despite the restoration of normocalcemia, the rescue diet did not entirely correct bone growth as femur size remained significantly smaller than control in 1alpha-OHase(-/-) mice fed the rescue diet. These results demonstrate that correction of the abnormal mineral ion homeostasis by feeding with a high calcium rescue diet is effective to rescue the PDDR phenotype of 1alpha-OHase mutant mice. This treatment, however, does not appear as effective as 1,25(OH)(2)D(3) replacement therapy since bone growth remained impaired.     

Molecular basis for pseudo vitamin D-deficiency rickets in the Hannover pig

The molecular basis for pseudo vitamin D deficiency rickets (PDDR) in the Hannover pig model was determined in the current study. Consistent with the inability of Hannover PDDR pigs to maintain ambient levels of 1,25-dihydroxyvitamin D (i.e., 1,25D), the bioactivation enzyme cytochrome P450C1 (or CYP27B1) was determined to contain coding-region deletions that rendered the enzyme ineffective due to frame-shift mutations and expression of a premature termination codon. Expression levels of P450C1mRNA were up-regulated in response to the low-1,25D high-parathyroid hormone state of the PDDR animals. In a complementary manner, cytochrome P450C24 mRNA was not detectable in PDDR pigs. Two different deletions were detected within the Hannover pig strain in which the P450C1 coding region contained either 173 bp or 329 bp deletions that resulted in the expression of non-sense products beginning within the I-helix region and extending through the truncated C-terminal domains. The boundaries for the deletion segments aligned with derived mRNA processing sites. This observation was consistent with an mRNA processing error as the causative factor for the coding-region deletions. Based upon the expression of a non-functional P450C1 enzyme, the Hannover pig model for PDDR was determined to be identical to the human disease in which enzyme-inhibitory mutations are the molecular basis for the calcium disorder.     

Homology modeling of human 25-hydroxyvitamin D3 1alpha-hydroxylase (CYP27B1) based on the crystal structure of rabbit CYP2C5

Seventeen missense mutations of 25-hydroxyvitamin D(3) 1alpha-hydroxylase (CYP27B1) that cause Vitamin D-dependent rickets type I (VDDR-I) have been identified. To understand the mechanism by which each mutation disrupts 1alpha-hydroxylase activity and to visualize the substrate-binding site, we performed the homology modeling of CYP27B1. The three-dimensional (3D) structure of CYP27B1 was modeled on the basis of the crystal structure of rabbit CYP2C5, the first solved X-ray structure of a eukaryotic CYP. The 3D structure of CYP27B1 contains 17 helices and 6 beta-strands, and the overall structural folding is similar to the available structures of soluble CYPs as well as to the template CYP2C5. Mapping of the residues responsible for VDDR-I has provided much information concerning the function of each mutant. We have previously reported site-directed mutagenesis studies on several mutants of CYP27B1 causing VDDR-1, and suggested the role of each residue. All these suggestions are in good agreement with our 3D-model of CYP27B1. Furthermore, this model enabled us to predict the function of the other mutation residues responsible for VDDR-I.     

Identification of the amino acid residue of CYP27B1 responsible for binding of 25-hydroxyvitamin D3 whose mutation causes vitamin D-dependent rickets type 1

We previously reported the three-dimensional structure of human CYP27B1 (25-hydroxyvitamin D3 1alpha-hydroxylase) constructed by homology modeling. Using the three-dimensional model we studied the docking of the substrate, 25-hydroxyvitamin D3, into the substrate binding pocket of CYP27B1. In this study, we focused on the amino acid residues whose point mutations cause vitamin D-dependent rickets type 1, especially unconserved residues among mitochondrial CYPs such as Gln65 and Thr409. Recently, we successfully overexpressed mouse CYP27B1 by using a GroEL/ES co-expression system. In a mutation study of mouse CYP27B1 that included spectroscopic analysis, we concluded that in a 1alpha-hydroxylation process, Ser408 of mouse CYP27B1 corresponding to Thr409 of human CYP27B1 forms a hydrogen bond with the 25-hydroxyl group of 25-hydroxyvitamin D3. This is the first report that shows a critical amino acid residue recognizing the 25-hydroxyl group of the vitamin D3.     

Critical role of vitamin D in sulfate homeostasis: regulation of the sodium-sulfate cotransporter by 1,25-dihydroxyvitamin D3

As the fourth most abundant anion in the body, sulfate plays an essential role in numerous physiological processes. One key protein involved in transcellular transport of sulfate is the sodium-sulfate cotransporter NaSi-1, and previous studies suggest that vitamin D modulates sulfate homeostasis by regulating NaSi-1 expression. In the present study, we found that, in mice lacking the vitamin D receptor (VDR), NaSi-1 expression in the kidney was reduced by 72% but intestinal NaSi-1 levels remained unchanged. In connection with these findings, urinary sulfate excretion was increased by 42% whereas serum sulfate concentration was reduced by 50% in VDR knockout mice. Moreover, levels of hepatic glutathione and skeletal sulfated proteoglycans were also reduced by 18 and 45%, respectively, in the mutant mice. Similar results were observed in VDR knockout mice after their blood ionized calcium levels and rachitic bone phenotype were normalized by dietary means, indicating that vitamin D regulation of NaSi-1 expression and sulfate metabolism is independent of its role in calcium metabolism. Treatment of wild-type mice with 1,25-dihydroxyvitamin D3 or vitamin D analog markedly stimulated renal NaSi-1 mRNA expression. These data provide strong in vivo evidence that vitamin D plays a critical role in sulfate homeostasis. However, the observation that serum sulfate and skeletal proteoglycan levels in normocalcemic VDR knockout mice remained low in the absence of rickets and osteomalacia suggests that the contribution of sulfate deficiency to development of rickets and osteomalacia is minimal.     

Structure-function analysis of CYP27B1 and CYP27A1. Studies on mutants from patients with vitamin D-dependent rickets type I (VDDR-I) and cerebrotendinous xanthomatosis (CTX).

We have determined eight types of missense mutants of CYP27B1 from Japanese vitamin D-dependent rickets type I (VDDR-I) patients [Kitanaka, S., Takeyama, K., Murayama, A., Sato, T., Okumura, K., Nogami, M., Hasegawa, Y., Niimi, H., Yanagisawa, J., Tanaka, T. & Kato, S. (1998) New England J. Med., 338, 653-661 and Kitanaka, S., Murayama, A., Sakaki, T., Inouye, K., Seino, Y., Fukumoto, S., Shima, M., Yukizane, S., Takayanagi, M., Niimi, H., Takeyama, K. & Kato, S. (1999) J. Clin. Endocrine Metab., 84, 4111-4117]. None of the CYP27B1 mutants showed 1alpha-hydroxylase activity towards 25-hydroxyvitamin D3. Thus, it was assumed that the mutated amino-acid residues play important roles in the 1alpha-hydroxylase activity, such as substrate binding, activation of molecular oxygen, interaction with adrenodoxin, and folding of the cytochrome P450 structure. To examine our hypothesis, we generated various mutants of CYP27B1 and studied their enzymatic properties. In addition, the corresponding mutations were introduced to CYP27A1, which belongs to the same family as CYP27B1. As CYP27A1 showed much higher expression level than CYP27B1 in Escherichia coli, further analysis including heme-binding and substrate-binding was performed with CYP27A1 in place of CYP27B1. Western blot analysis, spectral analysis including reduced CO-difference spectra and substrate-induced difference spectra, and enzymatic analysis of the mutant CYP27A1 gave information on the structure-function relationships of both CYP27A1 and CYP27B1. Although the sequence alignment suggested that Arg107, Gly125, and Pro497 of CYP27B1 might be involved in substrate binding, the experimental data strongly suggested that mutations of these amino-acid residues destroyed the tertiary structure of the substrate-heme pocket. It was also suggested that Arg389 and Arg453 of CYP27B1 were involved in heme-propionate binding, and Asp164 stabilized the four-helix bundle consisting of D, E, I and J helices, possibly by forming a salt bridge. Thr321 was found to be responsible for the activation of molecular oxygen.     

RNAi-mediated silencing of CYP27B1 abolishes 1,25(OH)2D3 synthesis and reduces osteocalcin and CYP24 mRNA expression in human osteosarcoma (HOS) cells

Although local synthesis of 1,25D has been postulated to regulate parameters of cell growth and differentiation in non-renal cells, the physiological role of 1,25D production in bone cells remains unclear. We used the technique of RNA interference to inhibit the mRNA encoding the enzyme responsible for 1,25D synthesis, 25-hydroxyvitamin D 1alpha-hydroxylase (CYP27B1). Human osteosarcoma (HOS) cells were transfected with siRNA for CYP27B1 or non-silencing RNA before being treated with 25D for 48h under normal growth conditions. De novo synthesis of 1,25D was measured in the media as well as mRNA levels for CYP27B1, osteocalcin (OCN) and 25-hydroxyvitamin D 24-hydroxylase (CYP24). We demonstrated that HOS cells express CYP27B1 mRNA, metabolize 25D and secrete detectable levels of de novo synthesized 1,25D. CYP27B1 mRNA silencing by RNAi, resulted in the suppression of 1,25D production and subsequent reduction of OCN and CYP24 mRNA expression. Our findings suggest that local 1,25D synthesis has paracrine effects in the bone microenvironment implying that vitamin D metabolism in human osteoblasts represents a physiologically important pathway, possibly regulating the maturation of osteoblasts.     

RNAi-mediated silencing of CYP27B1 abolishes 1,25(OH)2D3 synthesis and reduces osteocalcin and CYP24 mRNA expression in human osteosarcoma (HOS) cells

Although local synthesis of 1,25D has been postulated to regulate parameters of cell growth and differentiation in non-renal cells, the physiological role of 1,25D production in bone cells remains unclear. We used the technique of RNA interference to inhibit the mRNA encoding the enzyme responsible for 1,25D synthesis, 25-hydroxyvitamin D 1α-hydroxylase (CYP27B1). Human osteosarcoma (HOS) cells were transfected with siRNA for CYP27B1 or non-silencing RNA before being treated with 25D for 48 h under normal growth conditions. De novo synthesis of 1,25D was measured in the media as well as mRNA levels for CYP27B1, osteocalcin (OCN) and 25-hydroxyvitamin D 24-hydroxylase (CYP24). We demonstrated that HOS cells express CYP27B1 mRNA, metabolize 25D and secrete detectable levels of de novo synthesized 1,25D. CYP27B1 mRNA silencing by RNAi, resulted in the suppression of 1,25D production and subsequent reduction of OCN and CYP24 mRNA expression. Our findings suggest that local 1,25D synthesis has paracrine effects in the bone microenvironment implying that vitamin D metabolism in human osteoblasts represents a physiologically important pathway, possibly regulating the maturation of osteoblasts.     

Reduced chondrocyte proliferation and chondrodysplasia in mice lacking the integrin-linked kinase in chondrocytes

Chondrocyte proliferation and differentiation requires their attachment to the collagen type II-rich matrix of developing bone. This interaction is mediated by integrins and their cytoplasmic effectors, such as the integrin-linked kinase (ILK). To elucidate the molecular mechanisms whereby integrins control these processes, we have specifically inactivated the ILK gene in growth plate chondrocytes using the Cre-lox methodology. Mice carrying an ILK allele flanked by loxP sites (ILK-fl) were crossed to transgenic mice expressing the Cre recombinase under the control of the collagen type II promoter. Inactivation of both copies of the ILK-fl allele lead to a chondrodysplasia characterized by a disorganized growth plate and to dwarfism. Expression of chondrocyte differentiation markers such as collagen type II, collagen type X, Indian hedgehog and the PTH-PTHrP receptor was normal in ILK-deficient growth plates. In contrast, chondrocyte proliferation, assessed by BrdU or proliferating cell nuclear antigen labeling, was markedly reduced in the mutant growth plates. Cell-based assays showed that integrin-mediated adhesion of primary cultures of chondrocytes from mutant animals to collagen type II was impaired. ILK inactivation in chondrocytes resulted in reduced cyclin D1 expression, and this most likely explains the defect in chondrocyte proliferation observed when ILK is inactivated in growth plate cells.     

CYP2R1-, CYP27B1- and CYP24-mRNA expression in German type 1 diabetes patients

1,25(OH)(2)D(3) and 25(OH)D(3) have been associated with type 1 diabetes. Diverse enzymes are involved in the synthesis of these metabolites: the 25-Vitamin-D-hydroxylase (CYP2R1), the 25-hydroxyvitamin-D(3)-1-alpha-hydroxylase (CYP27B1) and the 25(OH)D(3)-24-hydroxylase (CYP24) among others. Serum levels of 25(OH)D(3) and 1,25(OH)(2)D(3) were investigated in type 1 diabetes patients (n=173) and the mRNA expression of the CYP2R1, CYP27B1 and CYP24 genes in type 1 diabetes patients (n=33) and healthy controls (n=23). These parameters were correlated with the -1260 (C/A) polymorphism in the CYP27B1 gene. Lower expression of CYP27B1 mRNA in comparison with healthy controls (1.7165 versus 1.7815, P=0.0268) was found. Additionally, patients carrying the genotype CC possessed a reduced amount of CYP27B1 mRNA compared to healthy controls (1.6855 versus 1.8107, respectively, P=0.0220). The heterozygosity rate of the -1260 C/A polymorphism was more frequent in patients with normal levels of 1,25(OH)(2)D(3) (> or =19.9 pmol/ml) than in whose with a level of less than 19.9 pmol/ml (46.7% versus 22.2%, P=0.0134). No correlation with serum levels of 25(OH)D(3) was found. Thus, CYP27B1 gene could play a functional role in the pathogenesis of type 1 diabetes through modulation of its mRNA expression and influence serum levels of 1,25(OH)(2)D(3) via the -1260 C/A polymorphism.     

Cooperation between p27 and p107 during endochondral ossification suggests a genetic pathway controlled by p27 and p130

Pocket proteins and cyclin-dependent kinase (CDK) inhibitors negatively regulate cell proliferation and can promote differentiation. However, which members of these gene families, which cell type they interact in, and what they do to promote differentiation in that cell type during mouse development are largely unknown. To identify the cell types in which p107 and p27 interact, we generated compound mutant mice. These mice were null for p107 and had a deletion in p27 that prevented its binding to cyclin-CDK complexes. Although a fraction of these animals survived into adulthood and looked similar to single p27 mutant mice, a larger number of animals died at birth or within a few weeks thereafter. These animals displayed defects in chondrocyte maturation and endochondral bone formation. Proliferation of chondrocytes was increased, and ectopic ossification was observed. Uncommitted mouse embryo fibroblasts could be induced into the chondrocytic lineage ex vivo, but these cells failed to mature normally. These results demonstrate that p27 carries out overlapping functions with p107 in controlling cell cycle exit during chondrocyte maturation. The phenotypic similarities between p107(-/-) p27(D51/D51) and p107(-/-) p130(-/-) mice and the cells derived from them suggest that p27 and p130 act in an analogous pathway during chondrocyte maturation.     

Enzymatic properties of human 25-hydroxyvitamin D3 1alpha-hydroxylase coexpression with adrenodoxin and NADPH-adrenodoxin reductase in Escherichia coli.

We have cloned human 25-hydroxyvitamin D3 1alpha-hydroxylase cDNAs from normal subjects and patients with pseudovitamin D-deficient rickets (PDDR), and expressed the cDNAs in Escherichia coli JM109 cells. Kinetic analysis of normal 1alpha-hydroxylase in the reconstituted system revealed that Km values for 25(OH)D3 and (24R), 25(OH)2D3 were 2.7 and 1.1 microM, respectively. The lower Km value and higher Vmax/Km value for (24R),25(OH)2D3 indicated that it is a better substrate than 25(OH)D3 for 1alpha-hydroxylase. These results are quite similar to those of mouse 1alpha-hydroxylase. To establish a highly sensitive in vivo system, 1alpha-hydroxylase, adrenodoxin and NADPH-adrenodoxin reductase were coexpressed in E. coli cells. The recombinant E. coli cells showed remarkably high 1alpha-hydroxylase activity, suggesting that the electrons were efficiently transferred from NADPH-adrenodoxin reductase through adrenodoxin to 1alpha-hydroxylase in E. coli cells. Using this system, the activities of four mutants of 1alpha-hydroxylase, R107H, G125E, R335P and P382S, derived from patients with PDDR were examined. Although no significant reduction in expression of these mutants was observed, none showed detectable activity. These results strongly suggest that the mutations found in the patients with PDDR completely abolished 1alpha-hydroxylase activity by replacement of one amino acid residue.     

Igf1 promotes longitudinal bone growth by insulin-like actions augmenting chondrocyte hypertrophy.

Longitudinal bone growth, and hence stature, are functions of growth plate chondrocyte proliferation and hypertrophy. Insulin-like growth factor 1 (Igf1) is reputed to augment longitudinal bone growth by stimulating growth plate chondrocyte proliferation. In this study, however, we demonstrate that chondrocyte numbers and proliferation are normal in Igf1 null mice despite a 35% reduction in the rate of long bone growth. Igf1 null hypertrophic chondrocytes differentiate normally in terms of expressing specialized proteins such as collagen X and alkaline phosphatase, but are smaller than wild-type at all levels of the hypertrophic zone. The terminal hypertrophic chondrocytes, which form the scaffold on which long bone growth extends, are reduced in linear dimension by 30% in Igf1 null mice, accounting for most of their decreased longitudinal growth. The expression of the insulin-sensitive glucose transporter, GLUT4, is significantly decreased and the insulin-regulated enzyme glycogen synthase kinase 3beta (GSK3) is hypo-phosphorylated in Igf1 null chondrocytes. Glycogen levels were significantly decreased and ribosomal RNA levels were reduced by almost 75% in Igf1 null chondrocytes. These data suggest that Igf1 promotes longitudinal bone growth by 'insulin-like' anabolic actions which augment chondrocyte hypertrophy.     

Rat growth plate chondrocytes express low levels of 25-hydroxy-1alpha-hydroxylase

Long standing disturbances of Vitamin D-metabolism as well as null-mutant animals for 25-hydroxy-1alpha-hydroxylase results in disorganised growth plates. Cultured chondrocytes were shown to be target for the hydroxylated Vitamin D-metabolites 1alpha,25(OH)(2)D(3) and 24,25(OH)(2)D(3). Because studies on production of these metabolites were inconclusive in in vitro systems, the expression of the Vitamin D-system was examined in rat growth plate chondrocytes in vitro as well as ex vivo. Gene expression for 25-hydroxy-1alpha-hydroxylase, 25-hydroxy-24-hydroxylase as well as Vitamin D-receptor and collagen II and X were analysed on mRNA level by RT-PCR and quantitative real-time PCR, on protein level by western blotting and by immunohistochemistry in isolated growth plate chondrocytes or intact growth plates. Compared to UMR or CaCo(2) cells and renal homogenates cultured growth plate chondrocytes expressed low levels of 25-hydroxy-1alpha-hydroxylase mRNA and 25-hydroxy-24-hydroxylase mRNA. The expression of both was modulated by 25(OH)D(3), but 1alpha,25(OH)(2)D(3) affected only 25-hydroxy-24-hydroxylase. These data were confirmed by Western blotting. Immunohistochemistry demonstrated predominant staining for 25-hydroxy-1alpha-hydroxylase in chondrocyte nodules and cells embedded in matrix in vitro. Ex vivo, 25-hydroxy-1alpha-hydroxylase was detected predominantly in late proliferative and hypertrophic zone of the growth plate. In conclusion, growth plate chondrocytes express the key components for a paracrine/autocrine Vitamin D-system.     

Regulation of intracrine production of 1,25-dihydroxyvitamin D and its role in innate immune defense against infection

Vitamin D was discovered as the cure for nutritional rickets. Classically, hormonal 1,25-dihydroxyvitamin D (1,25D), produced in the kidney by CYP27B1-catalyzed 1α-hydroxylation from its circulating 25-hydroxy precursor, has been considered to function as a critical endocrine regulator of calcium homeostasis. However, our appreciation of vitamin D metabolism and physiological function has evolved dramatically in recent years. First, vitamin D is now recognized as a pleiotropic regulator of human physiology, with emerging roles in cancer chemoprevention, cardio-protection, and, in particular, regulation of immune system functions. Moreover, CYP27B1 is very widely expressed, and evidence is rapidly accumulating that local CYP27B1-catalyzed production of 1,25D, controlled by tissue-specific signals, is critical for its physiological actions. Nowhere is this more apparent than in the innate immune system, where recent studies have shown that CYP27B1 expression is under control of several immune signaling pathways, and that signaling by 1,25D in macrophages and dendritic cells is critical for innate immune responses to infection. This review will describe our current knowledge of the signaling pathways that lead to 1,25D production in the immune system and the downstream signaling events it controls in response to pathogen recognition.