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.

We employed a genetic approach to determine whether deficiency of 1,25-dihydroxyvitamin D (1,25(OH) 2 D) and deficiency of the vitamin D receptor (VDR) produce the same alterations in skeletal and calcium homeostasis and whether calcium can subserve the skeletal functions of 1,25(OH) 2 D and the VDR. Mice with targeted deletion of the 25-hydroxyvitamin D 1␣-hydroxylase (1␣(OH)ase ؊/؊ ) gene, the VDR gene, and both genes were exposed to 1) a high calcium intake, which maintained fertility but left mice hypocalcemic; 2) this intake plus three times weekly injections of 1,25(OH) 2 D 3 , which normalized calcium in the 1␣(OH)ase ؊/؊ mice only; or 3) a "rescue" diet, which normalized calcium in all mutants. These regimens induced different phenotypic changes, thereby disclosing selective modulation by calcium and the vitamin D system. Parathyroid gland size and the development of the cartilaginous growth plate were each regulated by calcium and by 1,25(OH) 2 D 3 but independent of the VDR. Parathyroid hormone secretion and mineralization of bone reflected ambient calcium levels rather than the 1,25(OH) 2 D/VDR system. In contrast, increased calcium absorption and optimal osteoblastogenesis and osteoclastogenesis were modulated by the 1,25(OH) 2 D/VDR system. These studies indicate that the calcium ion and the 1,25(OH) 2 D/VDR system exert discrete effects on skeletal and calcium homeostasis, which may occur coordinately or independently.Vitamin D plays a major role in modulating calcium and skeletal homeostasis and exerts a significant influence on the growth and differentiation of a variety of tissues (1-3). Vitamin D is absorbed from the diet and generated in skin by exposure to ultraviolet light. The secosteroid is transported in blood bound to vitamin D-binding protein (4)

To determine whether the cardiovascular effect of 1,25(OH)(2)D is dependent on calcium and/or phosphorus, mice with targeted deletion of the 25(OH)D 1alpha-hydroxylase and their wild-type littermates were fed a normal diet or a diet to rescue the ambient serum calcium and phosphorus levels. Mice on the normal diet were treated daily with vehicle or 1,25(OH)(2)D(3) while mice on the rescue diet received vehicle, captopril or losartan. After four weeks the vehicle-treated knockout mice developed hypertension, cardiac hypertrophy and impaired cardiac function along with an up-regulation of the renin-angiotensin system in both renal and cardiac tissues. Although the serum calcium and phosphorus levels were normalized in knockout mice on the rescue diet, abnormalities in blood pressure, cardiac structure-function and the renin-angiotensin system remained. In contrast, 1,25(OH)(2)D(3) not only normalized serum calcium and phosphorus levels but also normalized blood pressure, cardiac structure-function and the renin-angiotensin system. Treatment of the knockout mice with either captopril or losartan normalized blood pressure and cardiac structure and function although renin expression remained elevated. This study shows that 1,25(OH)2D plays a protective role in the cardiovascular system by repressing the renin-angiotensin system independent of extracellular calcium or phosphorus.

Fibroblast growth factor 23 (FGF23) is a recently characterized protein likely involved in the regulation of serum phosphate homeostasis. Increased circulating levels of FGF23 have been reported in patients with renal phosphate-wasting disorders, but it is unclear whether FGF23 is the direct mediator responsible for the decreased phosphate transport at the proximal renal tubules and the altered vitamin D metabolism associated with these states. To examine this question, we generated transgenic mice expressing and secreting from the liver human FGF23 (R176Q), a mutant form that fails to be degraded by furin proteases. At 1 and 2 months of age, mice carrying the transgene recapitulated the biochemical (decreased urinary phosphate reabsorption, hypophosphatemia, low serum 1,25-dihydroxyvitamin D(3)) and skeletal (rickets and osteomalacia) alterations associated with these disorders. Unexpectantly, marked changes in parameters of calcium homeostasis were also observed, consistent with secondary hyperparathyroidism. Moreover, in the kidney the anticipated alterations in the expression of hydroxylases associated with vitamin D metabolism were not observed despite the profound hypophosphatemia and increased circulating levels of PTH, both major physiological stimuli for 1,25-dihydroxyvitamin D(3) production. Our findings strongly support the novel concept that high circulating levels of FGF23 are associated with profound disturbances in the regulation of phosphate and vitamin D metabolism as well as calcium homeostasis and that elevated PTH levels likely also contribute to the renal phosphate wasting associated with these disorders.

Mice heterozygous for targeted disruption of Pthrp exhibit, by 3 months of age, diminished bone volume and skeletal microarchitectural changes indicative of advanced osteoporosis. Impaired bone formation arising from decreased BM precursor cell recruitment and increased apoptotic death of osteoblastic cells was identified as the underlying mechanism for low bone mass. The osteoporotic phenotype was recapitulated in mice with osteoblast-specific targeted disruption of Pthrp, generated using Cre-LoxP technology, and defective bone formation was reaffirmed as the underlying etiology. Daily administration of the 1-34 amino-terminal fragment of parathyroid hormone (PTH 1-34) to Pthrp +/-mice resulted in profound improvement in all parameters of skeletal microarchitecture, surpassing the improvement observed in treated WT littermates. These findings establish a pivotal role for osteoblast-derived PTH-related protein (PTHrP) as a potent endogenous bone anabolic factor that potentiates bone formation by altering osteoblast recruitment and survival and whose level of expression in the bone microenvironment influences the therapeutic efficacy of exogenous PTH 1-34.

Missense mutations in fibroblast growth factor 23 (FGF23) are the cause of autosomal dominant hypophosphatemic rickets (ADHR). The mutations (R176Q, R179W, and R179Q) replace Arg residues within a subtilisin-like proprotein convertase (SPC) cleavage site (RXXR motif), leading to protease resistance of FGF23. The goals of this study were to examine in vivo the biological potency of the R176Q mutant FGF23 form and to characterize alterations in homeostatic mechanisms that give rise to the phenotypic presentation of this disorder. For this, wild type and R176Q mutant FGF23 were overexpressed in the intact animals using a tumorbearing nude mouse system. At comparable circulating levels, the mutant form was more potent in inducing hypophosphatemia, in decreasing circulating concentrations of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), and in causing rickets and osteomalacia in these animals compared with wild type FGF23. Parameters of calcium homeostasis were also altered, leading to secondary hyperparathyroidism and parathyroid gland hyperplasia. However, the raised circulating levels of parathyroid hormone were ineffective in normalizing the reduced 1,25(OH) 2 D 3 levels by increasing renal expression of 25(OH)D 3 -1␣-hydroxylase (Cyp40) to promote its synthesis and by decreasing that of 25(OH)D 3 -24-hydroxylase (Cyp24) to prevent its catabolism. The findings provide direct in vivo evidence that missense mutations from ADHR kindreds are gain-of-function mutations that retain and increase the protein's biological potency. Moreover, for the first time, they define a potential role for FGF23 in dissociating parathyroid hormone actions on mineral fluxes and on vitamin D metabolism at the level of the kidney.Renal phosphate wasting is associated with a number of hereditary disorders including X-linked (XLH) 1 and autosomal dominant (ADHR) forms of hypophosphatemic rickets. In addition to hypophosphatemia, patients with these two conditions exhibit decreased or inappropriately normal serum levels of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), as well as rickets and osteomalacia. Interestingly, ADHR encompasses not only the classic presentation of hypophosphatemia and rickets, but also it displays variable penetrance with delayed onset of the disease and an even more perplexing feature, spontaneous resolution of the biochemical defect (1, 2).The genes responsible for XLH and ADHR have now been identified. Through positional cloning, a gene that spans the deleted region Xp22.1 in XLH patients or is mutated in nondeletion patients with the disorder has been identified (designated PEX and subsequently PHEX, for phosphate-regulating gene with homologies to endopeptidases on the X chromosome) (3). The predicted human PHEX gene product exhibits structural similarity to a family of neutral endopeptidases involved in either activation or degradation of peptide hormones. Therefore, PHEX probably functions as a protease, and it may act by processing factor(s) involved in bone mineral metabolism. Extensive mutation analysis of XL...

Imperfect maintenance of genome integrity has been postulated to be an important cause of aging. Here we provide support for this hypothesis by demonstrating that the disruption of PASG (lsh), a SNF2-like factor that facilitates DNA methylation, causes global hypomethylation, developmental growth retardation and a premature aging phenotype. PASG mutant mice display signs of growth retardation and premature aging, including low birth weight, failure to thrive, graying and loss of hair, reduced skin fat deposition, osteoporosis, kyphosis, cachexia, and premature death. Fibroblasts derived from PASG mutant embryos show a replicative senescence phenotype. Both PASG mutant mice and fibroblasts demonstrate a markedly increased expression of senescence-associated tumor suppressor genes, such as p16 INK4a, that is independent of promoter methylation, but, instead, is associated with down-regulation of bmi-1, a negative regulator of p16 INK4a. These studies show that PASG is essential for properly maintaining DNA methylation and gene expression patterns that are required for normal growth and longevity. PASG mutant mice provide a useful model for the study of aging as well as the mechanisms regulating epigenetic patterning during development and postnatal life.

IntroductionEndogenous parathyroid hormone (PTH) functions to maintain normal extracellular calcium levels in the adult in part by enhancing osteoclastic bone resorption and liberating calcium from the adult skeleton. In contrast, exogenous PTH has been shown to exert significant skeletal anabolic effects in the adult when administered intermittently as a pharmacologic agent (1)(2)(3)(4). No physiologic role for PTH on increasing bone formation has yet been demonstrated, and its role in fetal skeletal development is unknown. In contrast, PTHrelated peptide (PTHrP) is known to play a critical and nonredundant role in fetal endochondral bone formation. Endochondral bone formation, in which bone is generated within a cartilage primordium, is a critical component of vertebrate skeletal development (5). Cells committed to the chondrogenic lineage progress through stages of proliferation, differentiation, hypertrophy, and apoptosis. Vascular invasion and degradation of calcified cartilage matrix then occurs, followed by secretion of trabecular bone matrix by invading osteoblasts. This complex process requires the coordinated activity of growth factors, hormones, proteases, and matrix molecules. Targeted disruption of the PTHrP gene results in lethal dyschondroplasia, caused mainly by a reduction of chondrocyte proliferation in the epiphyseal growth plate (6) and accelerated maturation of chondrocytes to hypertrophy (7). Both PTH and PTHrP interact at a common G protein-linked receptor termed the type I PTH/PTHrP receptor (PTHR). Ablation of the PTHR has been reported to simulate the effect of PTHrP ablation on chondrocyte differentiation (although more slowly) and to delay cartilage matrix mineral deposition, decrease vascular invasion of cartilage, and reduce trabecular bone formation in the primary spongiosa, alterations not seen after PTHrP ablation (8). These alterations were apparently partially alleviated when both PTHrP and the receptor were deficient. The mechanism for these effects, however, remained unclear.To assess the role of PTH in modulating skeletal development in the fetus and any potential interaction of PTH and PTHrP, we analyzed tissues of newborn mice homozygous for targeted ablation of the genes encoding PTH (PTH -/-), PTHrP (PTHrP -/-), and both PTH and PTHrP (PTH -/-, PTHrP -/-) and compared these to each other and to wild-type mice. MethodsMouse models. Mice carrying a disrupted PTH gene (PTH -/-mice) or a disrupted PTHrP gene (PTHrP -/-mice) were derived by homologous recombination in embryonic stem cells (9, 10). To generate PTH -/-mice, the murine PTH gene (GenBank accession numbers AF066074 and AF066075), which has an organizational structure and exon-intron boundaries identical to the human, bovine, and rat PTH genes (11), was introduced into the pPNT vector (12), replacing the entire coding Parathyroid hormone (PTH) is a potent pharmacologic inducer of new bone formation, but no physiologic anabolic effect of PTH on adult bone has been described. We investigated the role of PTH in fetal ...