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-Hydroxylase Gene (CYP27B1) Creates an Animal Model of Pseudovitamin D-Deficiency Rickets1
Genetics Unit (O.D., J.P., A.A., F.H.G., R.St.-A.), Shriners Hospital for Children, Montréal (Quebéc) Canada H3G 1A6; and Departments of Surgery and Human Genetics (F.H.G., R.St.-A.), McGill University, Montréal (Québec) Canada H3A 1B1
Address all correspondence and requests for reprints to: René St-Arnaud, Genetics Unit, Shriners Hospital for Children, 1529 Cedar Avenue, Montréal (Québec), Canada H3G 1A6. E-mail: rst-arnaud{at}shriners.mcgill.ca
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase (1-OHase). Patients with the disease
exhibit growth retardation, rickets, and osteomalacia. Serum
biochemistry is characterized by hypocalcemia, secondary
hyperparathyroidism, and undetectable levels of
1,25-dihydroxyvitamin D3. We have inactivated the
1-OHase gene in mice after homologous recombination in embryonic
stem cells. Serum analysis of homozygous mutant animals confirmed that
they were hypocalcemic, hypophosphatemic, hyperparathyroidic, and that
they had undetectable 1,25-dihydroxyvitamin D3.
Histological analysis of the bones from 3-week-old mutant animals
confirmed the evidence of rickets. At the age of 8 weeks, femurs from
1-OHase-ablated mice present a severe disorganization in the
architecture of the growth plate and marked osteomalacia. These results
show that we have successfully inactivated the 1-OHase gene in mice
and established a valid animal model of pseudovitamin D-deficiency
rickets. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-dihydroxyvitamin
D3
[1,25(OH)2D3], by two
successive hydroxylation reactions. The first of these takes place in
the liver, giving rise to 25-hydroxyvitamin D3
[25(OH)D3]. This inactive metabolite is
converted to 1,25(OH)2D3 by
the renal cytochrome P450 enzyme, 25-hydroxyvitamin
D3-1-hydroxylase (CYP27B1, hereafter referred
to as 1-OHase). The kidney is the major site of 1-OHase activity,
but expression of the gene has been detected in other cell types such
as keratinocytes, osteoblasts, chondrocytes, and macrophages (1, 2).
Once synthesized,
1,25(OH)2D3 plays a key
role in the regulation of calcium homeostasis by promoting calcium
absorption from the intestinal lumen. The homeostatic feedback loop
involves both 1,25(OH)2D3
and PTH. Decreases in blood calcium stimulate synthesis and secretion
of PTH, which, in turn, leads to increased expression of the 1-OHase
gene, 1,25(OH)2D3
synthesis, and intestinal calcium absorption. To prevent sustained
production of 1,25(OH)2D3
that would lead to hypercalcemia, the vitamin D hormone, in turn,
inhibits PTH and 1-OHase gene expression (3).
Pseudovitamin D-deficiency rickets (PDDR) is a rare autosomal recessive
disease characterized by growth retardation, failure to thrive,
rickets, and osteomalacia (4, 5). Serum biochemistry
reveals hypocalcemia, secondary hyperparathyroidism, and undetectable
levels of 1,25(OH)2D3.
Measurements of circulating vitamin D metabolite levels and 1-OHase
enzymatic activity have long suggested that the disease is caused by
inactivating mutations in the cytochrome P450 gene responsible for the
synthesis of 1,25(OH)2D3
(4, 5). The cloning of the 1-OHase complementary DNA
(cDNA) and gene (6, 7, 8, 9, 10) has confirmed this hypothesis,
first by mapping of the gene to the disease locus (7, 8, 11, 12, 13, 14), then by the identification of mutations in affected
patients (4, 6, 11, 12, 15, 16, 17).
We have generated an animal model of PDDR by targeted inactivation of
the 1-OHase gene in mice. Homozygous mutant animals were
phenotypically normal at birth but progressively developed the full
range of PDDR symptoms. Hypocalcemia, hyperparathyroidism, and rickets
were evident as soon as weaning, whereas osteomalacia was detected in
young adult mutant animals.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-OHase cDNA was amplified by PCR, using
primers based on the rat cDNA sequence (8), and used as
probe to clone a 12-kb fragment from a 129-SV genomic library
(Stratagene, La Jolla, CA). A 927-bp KpnI
genomic fragment containing a portion of exon 7, intron 7, and exon 8
was subcloned in pGEM3Zf(-). This template was used to introduce a
first loxP site, 35-bp into intron 7, by inverse PCR (primer
information available on demand). The loxP-neo-loxP selection cassette
was engineered by subcloning the 1.6-kb Mst II fragment (containing the
SV40 promoter, neo resistance gene, and thymidine kinase
polyadenylation signal) of pBK-CMV (Stratagene) into the
HindIII site of pBS246 (Life Technologies, Inc., Burlington, Ontario, Canada). The cassette was excised as
a NotI fragment and subcloned into the Msc I site downstream
from exon 8 in the pGEM3Zf(-) subclone. The engineered KpnI
subfragment containing exons 7 and 8, the neo cassette, and the three
loxP sites was then reintroduced into the 12-kb 1-OHase genomic
fragment at the corresponding site. A linear 8.8-kb ApaLI
fragment from this targeting vector (Fig. 1a
) was electroporated into R1 embryonic
stem (ES) cells (18) using conventional protocols
(19). Targeted ES cell clones were identified by Southern
blotting of BamHI-digested DNA using a 450-bp PCR-amplified
probe from upstream of the region of homology (upstream primer:
5'-ACACACACACACACACCAATATG-3'; downstream primer:
5'-TGCACCACCACGCCCGGC-3') (Fig. 1a). We then excised exon 8 and the neo
selection cassette by transfecting targeted ES cells with the Cre
expression vector, pBS185 (Life Technologies, Inc.). We
confirmed the in vitro excision using Southern blotting of
BamHI genomic digests with the previously described probe.
For routine genotyping of tail DNA samples (19), we used
an internal PCR-amplified 374-bp probe (upstream primer:
5'-ACCATTTTAGACCCACCCACAGT-3'; downstream primer:
5'-GAGGAATGATCAGGAGAGGCAC-3'), yielding a wild-type fragment of 5428 bp
and a diagnostic deleted allele fragment of 4797 bp.
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-OHase allele
into C57BL/6 embryos at the blastocyst stage used standard methodology
(19). The resulting chimeras and their progeny were housed
in a virus- and parasite-free barrier facility. They were exposed to a
12-h light, 12-h dark cycle and were fed tap water and regular chow
at libitum. All procedures involving animals were previously
approved by the Institutional Animal Care and Use Committee.
RT-PCR analysis of 1-OHase transcripts
The RT-PCR protocol used total RNA prepared with the
Trizol reagent (Life Technologies, Inc.) from kidney of
+/+, +/-, and -/- animals. The RT reaction contained oligo-dT and
Superscript II reverse transcriptase (Life Technologies, Inc.). PCR amplification required two pairs of primers. The
first pair (5'-CTGCGAGGAGGGGTAAGGTGTT-3' and
5'-GGAAACGGGGGAGGGGA-3') allowed detection of exon 8-containing
transcripts. The second pair (5'-TCTATGAGCTTTCCCGGCACCCC-3' and
5'-TCAGGTAGCTCTTCAAAATGGGTCAA-3') permitted detection of truncated
transcripts. Control ß-actin amplification reactions used the
following primer pair: 5'-GCTGCGTGTGGCCCCTAGG-3' and
5'-CAAGAAGGAAGGCTGGAAAAGAG-3'. Amplimers were detected by ethidium
bromide staining of agarose electrophoresis gels.
Serum biochemistry
Circulating
1,25(OH)2D3 levels were
measured from serum samples using a specific RIA (ImmunoDiagnostic
Systems Ltd., Boldon, UK). Serum concentrations of
25(OH)D3 and 24,25(OH)2 D
were assessed using the 25-hydroxyvitamin D (125) I RIA
kit for the quantitative determination of vitamin D hydroxylated
metabolites (DiaSorin, Inc., Stillwater, MN). Total
calcium and phosphate were measured using a Monarch automated analyzer.
We measured serum PTH levels using the mouse intact PTH
enzyme-linked immunosorbent assay kit (Immunotropics, San
Clemente, CA). All serological data are shown as mean ±
SEM.
Northern blot assays
We used the Trizol Reagent (Life Technologies, Inc.) to isolate total RNA from the intestine, kidney, and tibia
of 8-week-old +/- and -/- animals, and we performed Northern blots
using standard methodology. The probes used were the 170-bp
EcoRI fragment from the mouse calbindin
D9k cDNA (20), the 1.1-kb
BamHI-EcoRI fragment from the mouse osteopontin
cDNA (21), the 470-bp EcoRI-PstI
fragment from the mouse osteocalcin cDNA (22), the 263-bp
HincII-KpnI fragment from the rat CYP24 cDNA
(23), the PstI fragment of the rat
glyceraldehyde-3-phosphate-dehydrogenase cDNA (24), and a
420-bp PCR fragment from the mouse vitamin D receptor (VDR) cDNA
(upstream primer: 5'-AGGGTTTCTTCAGGCGGAGCAT-3'; downstream primer:
5'-CATGTCCAGTGAGGGGGTGTAC-3').
Histology
The thyroid, parathyroids, trachea, and heart were dissected
en bloc from 8-week-old littermates to facilitate
orientation of the specimens and sectioning in the same plane. Samples
were fixed overnight in 4% paraformaldehyde before paraffin embedding.
Six-micrometer sections were prepared and stained with
hematoxylin-eosin. Bones were dissected, fixed overnight in 4%
paraformaldehyde, and embedded in methylmethacrylate. Sections of 6
µm were deplastified and stained by von Kossa or Goldner
(25).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-OHase gene-OHase gene from a 129-SV mouse genomic library
to construct a targeting vector in which exon 8, encoding the heme
binding domain (26), was flanked by a 5'-loxP recognition
site and by a 3'-loxP-neo-loxP selection cassette (Fig. 1a
). Homologous
recombination at the 1-OHase locus, followed by transient
transfection of the ES cells with the Cre recombinase, generated a
targeted allele in which exon 8 was deleted (Fig. 1A). The engineered
mutation was transmitted to the progeny (Fig. 1A), with the expected
Mendelian ratio (not shown). Heterozygous animals had no discernible
phenotype and were fertile. Homozygous mutant animals were
phenotypically normal at birth but exhibited retarded growth, as
measured by weight gain from 3–8 weeks of age and femur length at 8
weeks (data not shown).
We did not detect 1-OHase transcripts that contained exon 8 in
homozygous -/- animals (Fig. 1b, left panel); however,
mutant animals expressed a truncated 1-OHase messenger RNA (mRNA)
(Fig. 1b, right panel). The product of this aberrant
transcript was inactive, as evidenced by undetectable
1,25(OH)2D3 levels in the
blood of -/- animals (Fig. 1c), confirming that we have engineered a
true null allele. In adult animals, circulating concentrations of
24,25-dihydroxyvitamin D3
[24,25(OH)2D3] were
negligible (1.4 ± 0.2 vs. 11.1 ± 0.4 ng/ml for
mutants and heterozygotes, respectively, P < 0.001)
(Fig. 1d), whereas the levels of 25(OH)D3 were
elevated (21.9 ± 5.6 vs. 12.0 ± 0.4 ng/ml for
mutants and heterozygotes, respectively, P < 0.001)
(Fig. 1e).
Serum biochemistry
A slight, but statistically significant (P <
0.01), hypocalcemia was detected in -/- animals as early as weaning
(3 weeks, Fig. 2a). The hypocalcemic
status worsened in the following week, remained constant for a while,
before worsening again when the animals were killed at 8 weeks
(1.30 ± 0.04 vs. 2.33 ± 0.02
mM for -/- and +/- animals, respectively,
P < 0.001) (Fig. 2a). In homozygous mutant pups,
marked hypophosphatemia was detected at 3 weeks of age (Fig. 2b). The
difference in serum phosphate levels was less pronounced in adult -/-
animals, compared with wild-type and heterozygous littermate controls,
because of the decrease in circulating phosphate levels in control
animals (Fig. 2b). Nonetheless, the serum phosphate levels measured in
-/- animals at 8 weeks remained significantly lower than in +/-
littermates (2.5 ± 0.3 vs. 3.2 ± 0.2
mM, respectively, P < 0.05)
(Fig. 2b). When compared with control littermates (+/+ or +/-), serum
alkaline phosphatase activity was significantly elevated in -/-
animals at all times (data not shown).
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-OHaseablated pups showed increased PTH
blood levels as early as weaning, and the secondary hyperparathyroidism
was even more pronounced when the animals were killed at 8 weeks
(1444 ± 123 vs. 20 ± 2 pg/ml for mutant and
wild-type, respectively, P < 0.001) (Fig. 2c). The
parathyroid glands also increased in size in 8-week-old homozygous
mutant mice (Fig. d; the arrows point to the
parathyroids).
Vitamin D-dependent gene expression
We measured the expression levels of vitamin D-regulated genes
(27) in several target tissues, using Northern blot
assays. Expression of the calbindin D9k gene in
the duodenum was completely inhibited in 1-OHase -/- animals (Fig. 3, left panel), as was the
expression of CYP24 (25-hydroxyvitamin D-24-hydroxylase) in kidney
(data not shown). On the contrary, the expression levels of the
osteocalcin and osteopontin genes in tibia were unaffected in mutant
animals (Fig. 3, center and right panels). Expression of the
vitamin D receptor gene in kidney from homozygous mutants was reduced
(not shown).
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, a and b). The
femurs presented evidence of rickets, as demonstrated by the impaired
calcification of the maturing cartilage, disorganization of the
columnar alignment of hypertrophic chondrocytes, increased width of the
growth plate, and accumulation of osteoid (unmineralized matrix) in
trabecular bone (Fig. 4, c and d). At 8 weeks of age, the defect in
mineralization was more profound (Fig. 4, e and f), and the -/-
animals showed a more severe disorganization in the architecture of the
growth plate (Fig. 4, g and h). The chondrocytes completely lost
their columnar organization. Marked osteomalacia (accumulation of
osteoid) was evident when we compared the femoral cortex from -/- and
+/- littermates (Fig. 4, i and j).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-OHase gene. The engineered
mouse strain represents a valid animal model for PDDR. The homozygous
mutant animals presented with the same clinical phenotype as patients
with the disease: retarded growth, failure to thrive, undetectable
1,25(OH)2D3, hypocalcemia,
secondary hyperparathyroidism, and bone anomalies that included rickets
and osteomalacia.
Other aspects of the phenotype of 1-OHase -/- animals also matched
the clinical manifestations of PDDR. These included x-ray features,
hypophosphatemia, and elevated serum alkaline phosphatase. Contact
radiography of femurs from mutant animals revealed diffuse osteopenia
(hypomineralization) and rachitic metaphyseal changes (data not shown).
Serum phosphate concentrations may be normal or low in PDDR patients
(4, 5). Similarly, we have measured a marked
hypophosphatemia in young -/- animals, which tended toward normal at
later stages, an observation reminiscent of the human pathology.
Individuals affected by PDDR consistently have elevated serum alkaline
phosphatase activity (4, 5), and we observed the same
feature in homozygous mutant animals (not shown).
Patients with PDDR have normal serum levels of
25(OH)D3 (28, 29, 30), and circulating
levels of 24,25(OH)2D3 also
remain within the normal range (31). This observation
contrasts with our findings, which show elevated levels of
25(OH)D3 and very low levels of
24,25(OH)2D3 in 1-OHase
mutant animals. The
1,25(OH)2D3 hormone is the
main in vivo regulator of the expression of the CYP24 enzyme
that catalyzes the synthesis of
24,25(OH)2D3
(32). It was thus not surprising to measure low
circulating 24,25(OH)2D3
levels and undetectable CYP24 expression in 1-OHase mutant animals.
The discrepancy with the human disease remains to be explained but
could result from species differences. This observed inhibition of
CYP24 expression in mice, combined with the targeted ablation of the
1-OHase enzyme, leads to a metabolic block in mutant animals and an
accumulation of the unprocessed 25(OH)D3
substrate.
The osteocalcin gene promoter is modularly organized and contains
several positive and negative regulatory elements (33).
Similarly, osteopontin gene expression is regulated by many agents
acting via diverse signaling pathways in specific cell types
(34). Though
1,25(OH)2D3 has been shown
to regulate the expression of these genes both in vitro and
in vivo (33, 34), the unaltered mRNA levels for
both genes measured in 1-OHase mutant animals suggest that the
vitamin D hormone is not the main modulator of the steady-state
expression of these genes in murine bone tissue.
Two independent laboratories engineered strains of animals deficient
for the vitamin D receptor (35, 36). The mutant mice are
valid animal models for another hereditary type of rickets: hereditary
vitamin D-resistant rickets (4, 37). Some aspects of the
phenotype of these mice are identical to the phenotype of the mutant
1-OHase mice that we engineered: retarded growth, hypocalcemia,
secondary hyperparathyroidism, and rickets. The VDR-null mice also
develop alopecia, a clinical feature shared with some hereditary
vitamin D-resistant rickets patients but not exhibited by PDDR patients
or the 1-OHase-ablated mice. It seems that the hypocalcemia measured
in 1-OHase homozygous -/- pups, statistically significant as early
as weaning, manifests itself earlier than in the VDR-ablated mice
(36). This is difficult to precisely evaluate from the
literature, however, and may simply reflect different diets and housing
conditions in various laboratories around the world.
The 1-OHase -/- animals developed clear histological evidence of
rickets and osteomalacia. The severe disorganization of the growth
plate of the adult -/- mice exceeded what is normally observed in
PDDR patients, because those are usually treated before the symptoms
reach their full manifestation. The treatment of choice for PDDR is
long-term replacement therapy with
1,25(OH)2D3 (4, 5, 30). Vitamin D and 25(OH)D3, at high
doses, have been used, in the past, with some success
(38). In this case, it is likely that massive
concentrations of 25(OH)D3 can bind to the
vitamin D receptor and induce some response of the target organs.
Physicians in some countries also treat PDDR patients with the
monohydroxylated analog, 1(OH)D3, which is
activated to the hormonal form by hydroxylation at position 25 in the
liver (39). It will be interesting to compare the various
therapeutic interventions using the animal model of PDDR that we have
engineered. Such rescue experiments are currently underway.
Although the kidney is the main site of expression of the 1-OHase
gene, its expression has been documented in other cell types, including
osteoblasts, chondrocytes, keratinocytes, and macrophages (6, 40, 41, 42, 43, 44). It is thought that local production of
1,25(OH)2D3 could play an
important autocrine or paracrine role in the differentiation or
function of these tissues (45). The conditional 1-OHase
allele that we have engineered will provide an invaluable genetic tool
to test these hypotheses in the context of normocalcemic animals. Mice
carrying the loxP-bearing (floxed) allele are now being bred to
transgenic animals expressing the Cre recombinase in relevant target
tissues to address these questions.
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Acknowledgments |
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Footnotes |
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2 A Chercheur-Boursier from the Fonds de la Recherche en Santé
du Québec.
Received February 15, 2001.
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References |
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-hydroxylase cDNA. Proc Natl Acad Sci USA 94:12920–12925-hydroxylase and vitamin D
synthesis. Science 277:1827–1830-hydroxylase mutations in French-Canadians with vitamin D
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gene in patients with pseudovitamin D-deficiency rickets. N Engl
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deficiency in 17 families. Am J Hum Genet 63:1694–1702[CrossRef][Medline]
-hydroxylase (P450c1) gene in three
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functional enzyme activity in blood-derived macrophages. J Bone
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-hydroxylase gene product in pseudovitamin D deficiency rickets,
including that with mild clinical manifestation. J Clin Endocrinol
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isolated calvarial cells. Proc Natl Acad Sci USA 77:5720–5724This article has been cited by other articles:
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K. Skorija, M. Cox, J. M. Sisk, D. R. Dowd, P. N. MacDonald, C. C. Thompson, and M. B. Demay Ligand-Independent Actions of the Vitamin D Receptor Maintain Hair Follicle Homeostasis Mol. Endocrinol., April 1, 2005; 19(4): 855 - 862. [Abstract] [Full Text] [PDF]
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K. D. Healy, J. L. Vanhooke, J. M. Prahl, and H. F. DeLuca Parathyroid hormone decreases renal vitamin D receptor expression in vivo PNAS, March 29, 2005; 102(13): 4724 - 4728. [Abstract] [Full Text] [PDF]
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S. Masuda, V. Byford, A. Arabian, Y. Sakai, M. B. Demay, R. St-Arnaud, and G. Jones Altered Pharmacokinetics of 1{alpha},25-Dihydroxyvitamin D3 and 25-Hydroxyvitamin D3 in the Blood and Tissues of the 25-Hydroxyvitamin D-24-Hydroxylase (Cyp24a1) Null Mouse Endocrinology, February 1, 2005; 146(2): 825 - 834. [Abstract] [Full Text] [PDF]
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 P. Capuano, T. Radanovic, C. A. Wagner, D. Bacic, S. Kato, Y. Uchiyama, R. St.-Arnoud, H. Murer, and J. Biber Intestinal and renal adaptation to a low-Pi diet of type II NaPi cotransporters in vitamin D receptor- and 1{alpha}OHase-deficient mice Am J Physiol Cell Physiol, February 1, 2005; 288(2): C429 - C434. [Abstract] [Full Text] [PDF]
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 J. G. J. Hoenderop, B. Nilius, and R. J. M. Bindels Calcium Absorption Across Epithelia Physiol Rev, January 1, 2005; 85(1): 373 - 422. [Abstract] [Full Text] [PDF]
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 G. J. Downing and J. F. Battey Jr. Technical Assessment of the First 20 Years of Research Using Mouse Embryonic Stem Cell Lines Stem Cells, December 1, 2004; 22(7): 1168 - 1180. [Abstract] [Full Text] [PDF]
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