Cloning and expression of rat 25-hydroxyvitamin D 3 -1α-hydroxylase cDNA

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242

138

The DNA f lanking the 5 sequence of the mouse 1␣-hydroxylase gene has been cloned and sequenced. A TATA box has been located at ؊30 bp and aCCAAT box has been located at ؊79 bp. The gene's promoter activity has been demonstrated by using a luciferase reporter gene construct transfected into a modified pig kidney cell line, AOK-B50. Parathyroid hormone stimulates this promoter-directed synthesis of luciferase by 17-fold, whereas forskolin stimulates it by 3-fold. The action of parathyroid hormone is concentration-dependent. 1,25-Dihydroxyvitamin D 3 does not suppress basal promoter activity and marginally suppresses parathyroid hormone-driven luciferase reporter activity. The promoter has three potential cAMP-responsive element sites, and two perfect and one imperfect AP-1 sites, while no DR-3 was detected. These results indicate that parathyroid hormone stimulates 25-hydroxyvitamin D 3 -1␣-hydroxylase by acting on the promoter of the 1␣-hydroxylase gene.Vitamin D is a major actor in calcium homeostasis of higher animals (1). To carry out these functions, vitamin D must be metabolized to its biologically active hormonal form, 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ). This is a two-step process requiring 25-hydroxylation in the liver and 1␣-hydroxylation in the kidney. The resulting hormone, 1,25-(OH) 2 D 3 , then binds to a nuclear vitamin D receptor (VDR) in target tissues, and the liganded receptor acts as a transcription factor to modulate the expression of specific genes encoding proteins that bring about the actions of vitamin D (2).The importance of the 1␣-hydroxylase enzyme is emphasized by the occurrence of a genetic disorder of vitamin D metabolism, vitamin D dependency rickets type I (VDDRI) (3). This disorder is characterized by very low serum 1,25-(OH) 2 D 3 levels despite normal vitamin D intakes (4) and is thought to be the result of a defect in the 1␣-hydroxylase gene (5). This link has recently been reinforced with the identification of the gene for human 1␣-hydroxylase (6). The gene was mapped to chromosomal region 12q13.1-q13.3, which contains the VDDRI disease locus.The 1␣-hydroxylation step is the most tightly regulated step in vitamin D metabolism. Several physiological factors interact to regulate 1␣-hydroxylase activity that, in turn, determines serum and tissue levels of 1,25-(OH) 2 D 3 . These regulators include parathyroid hormone (PTH) and hypophosphatemia, which stimulate 1␣-hydroxylase activity, and 1,25-(OH) 2 D 3 , which suppresses it (7-9).

Section: Methodsmentioning

confidence: 99%

“…Recently, a major advance in technology of the 1␣-hydroxylase has been achieved by the cloning of the mouse (18), rat (6,19), and human (6,29) cDNAs encoding the 1␣-hydroxylase, an accomplishment first described by St. Arnaud et al (20). This provided an opportunity for our group to clone the mouse gene including its promoter.…”

mentioning

confidence: 99%

Parathyroid hormone activation of the 25-hydroxyvitamin D ₃ -1α-hydroxylase gene promoter

Brenza

Kimmel-Jehan

Jehan

et al. 1998

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242

138

“…1␣,25-dihydroxyvitamin D [1␣,25(OH) 2 D] is the most potent metabolite of vitamin D (5)(6)(7) and is believed to exert most of its actions via the 1␣,25(OH) 2 D receptor (VDR) (8,9), a member of the nuclear hormone receptor superfamily. The synthesis of 1␣,25(OH) 2 (10), rat (11,12), and human (13,14) enzyme have been cloned, and the structures of the human (11,14,15) and mouse (16,17) gene have been reported.…”

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confidence: 99%

Targeted ablation of the 25-hydroxyvitamin D 1α-hydroxylase enzyme: Evidence for skeletal, reproductive, and immune dysfunction

Panda

Miao²,

Tremblay³

et al. 2001

591

407

Vitamin D-dependent rickets type I (VDDR-I), also known as pseudovitamin D deficiency rickets (PDDR), is an autosomal recessive disorder characterized by low or undetectable levels of 1␣,25(OH) 2 D, secondary hyperparathyroidism, hypocalcemia, hypophosphatemia, and severe rachitic lesions (18 -21). VDDR-I is assumed to result from impaired synthesis of 1␣,25(OH) 2 D, and, indeed, a number of 1␣(OH)ase gene mutations have been reported in this disorder that result in diminished or absent 1␣(OH)ase activity (13,(22)(23)(24)(25)(26).To further investigate the functional role of the 1␣(OH)ase enzyme, we generated mice deficient in 1␣(OH)ase by gene targeting. Materials and MethodsMethods including construction of the 1␣(OH)ase targeting vector; transfection of embryonic stem (ES) cells and generation of 1␣(OH)ase-deficient mice; Southern blot and PCR analysis of ES cell and mouse tail DNA; Northern blot analysis; biochemical and hormonal analyses; histological analysis; computer-assisted image analysis; immunohistochemistry; and f luorescenceactivated cell sorter (FACS) lymphocyte phenotyping are presented in the supplemental data (which is published on the PNAS web site, www.pnas.org). ResultsThe targeting vector shown in Fig. 1A was used to inactivate one allele of the 1␣(OH)ase gene in ES cells. The inactivated allele lacked both the hormone-binding domain and the heme-binding domain of the enzyme. Two independent ES cell clones were used to generate two lines of mice heterozygous for the mutation, which were then interbred to generate 1␣(OH)ase null (Ϫ͞Ϫ) mice (Fig. 1B). Litter sizes were no different from normal, and the mutated allele was transmitted to the progeny with the expected Mendelian frequency. Thus, haploinsufficiency of the 1␣(OH)ase did not affect embryonic survival. By reverse transcription (RT)-PCR, renal expression of the kidney 1␣(OH)ase mRNA in (ϩ͞Ϫ) mice was reduced relative to that in (ϩ͞ϩ) mice, and, in (Ϫ͞Ϫ) mice, it was undetectable (Fig. 1C).Circulating concentrations of 1,25(OH) 2 D were undetectable in the homozygous null mice and were somewhat lower (although not significantly so) in the heterozygotes relative to normals at 7 weeks of age (Table 1). Serum 25(OH)D concentrations were elevated in (Ϫ͞Ϫ) mice relative to the heterozygotes and normals. Both serum calcium and phosphate concentrations were reduced in (Ϫ͞Ϫ) mice relative to the (ϩ͞Ϫ) mice that were normal, and urinary phosphate was increased in the homozygous null mice. Serum parathyroid hormone concentrations were markedly elevated, the alkaline phosphatase concentrations were twice normal, and the body weight was substantially reduced in the homozygous null mice at this time (Table 1). The null mutant mice appeared grossly normal from birth until This paper was submitted directly (Track II) to the PNAS office.Abbreviations: 1␣(OH)ase, 25(OH)D-1␣-hydroxylase; VDDR-I, vitamin D dependent rickets type I; VDR, vitamin D receptor.

“…Subsequent 1␣-hydroxylation of 25-OH-D 3 in the kidney produces the physiologically active metabolite, 1␣,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ]. The second hydroxylation is catalyzed by the 25-hydroxyvitamin D 3 -1␣-hydroxylase (1␣-hydroxylase), a mitochondrial cytochrome P450 enzyme that is the product of the CYP27B1 gene (2)(3)(4)(5)(6). Activity of 1␣-hydroxylase is tightly regulated through complex mechanisms that depend on the circulating levels of calcium, phosphorus, parathyroid hormone, and 1,25(OH) 2 D 3 .…”

mentioning

confidence: 99%

CYP27B1 null mice with LacZ reporter gene display no 25-hydroxyvitamin D ₃ -1α-hydroxylase promoter activity in the skin

Vanhooke

Prahl

Kimmel-Jehan

et al. 2005

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. The second hydroxylation is catalyzed by the 25-hydroxyvitamin D 3 -1␣-hydroxylase (1␣-hydroxylase), a mitochondrial cytochrome P450 enzyme that is the product of the CYP27B1 gene (2-6). Activity of 1␣-hydroxylase is tightly regulated through complex mechanisms that depend on the circulating levels of calcium, phosphorus, parathyroid hormone, and 1,25(OH) 2 D 3 .Mutations in the 1␣-hydroxylase gene are known to cause vitamin D-dependency rickets type I (VDDR-I) (2, 7-9). Patients afflicted with this disease are unable to maintain normal serum calcium and suffer from secondary hyperparathyroidism, rickets, and osteomalacia (10). VDDR-I is cured by administration of physiological doses of 1,25(OH) 2 D 3 (11). Physiological doses of 25-OH-D 3 are noncurative, but high dose administration can be effective (11), presumably due to the ability of 25-OH-D 3 to bind and activate the vitamin D receptor when present in vast excess.The experiments reported by Fraser and Kodicek in 1970 (12) were the first to demonstrate the kidney as the major, if not the only, tissue in which 1,25(OH) 2 D 3 is produced under normal physiological conditions. Over the next several years, extrarenal production of 1,25(OH) 2 D 3 was convincingly demonstrated in pregnant nephrectomized rats and in an anephric patient suffering from sarcoidosis (13-15). In these cases, synthesis was localized to the placenta and the sarcoid macrophages (14,16,17). Production of 1,25(OH) 2 D 3 at other sites has remained a subject of much investigation. A number of research groups have reported 1␣-hydroxylase activity in cultured cells, including those of the skin, bone, cartilage, intestine, prostate, and vascular epithelium (18 -25). Bikle et al. (26) have also reported 1,25(OH) 2 D 3 production in perfused flaps of porcine skin. Local production of 1,25(OH) 2 D 3 has been proposed to regulate cellular function and͞or differentiation in an autocrine or paracrine fashion (18,19,24,27,28), and it has been suggested that keratinocytes could supply 1,25(OH) 2 D 3 to the systemic circulation when renal production of the hormone is impaired (26,29). Production of extrarenal 1,25(OH) 2 D 3 in these experiments is not supported, however, by in vivo metabolic studies in nephrectomized nonpregnant rats. In these studies, two independent research groups were unable to detect 3 H-1,25(OH) 2 D 3 in the tissue or plasma after administering a dose of 3 H-25-OH-D 3 of high specific radioactivity (30,31). These conflicting results demonstrate a need for further investigation of the in vivo expression of the 1␣-hydroxylase. We have approached such an investigation by using gene targeting to replace the 1␣-hydroxylase coding sequence with a bacterial lacZ gene controlled by the 1␣-hydroxylase promoter. The lacZ gene codes for ␤-galactosidase, whose activity is readily detected in situ through histochemical staining with X-Gal (32). Herein we report the successful production of 1␣-hydroxylase null mice harboring the lacZ gene and present our analysis of in vivo 1␣-hydroxylase...