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Genetics Unit (R.St-A., A.A., R.T., F.H.G.), Shriners Hospital for Children, Montréal (Québec) Canada H3G 1A6; Departments of Surgery and Human Genetics (R.St-A., F.H.G.), McGill University, Montréal (Québec) Canada H3A 1B1; Department of Biochemistry and Molecular Biology (F.B., M.R.-P., S.C.), New Jersey Medical School, Newark, New Jersey 07103; Endocrine Unit (K.C., M.B.D.), Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114; and Laboratorium voor experimentele geneeskunde en endocrinologie (J.D., C.M.), Catholic University of Leuven, B-3000 Leuven, Belgium
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.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase (1-hydroxylase), the
final metabolic enzyme in the production of the active, hormonal form
of vitamin D, thereby increasing the synthesis of
1,25-dihydroxyvitamin D [1,25(OH)2D]. The
elevated production of 1,25(OH)2D serves to
increase intestinal absorption of calcium (1).
To control mineral homeostasis, both the production and degradation of
1,25(OH)2D need to be tightly regulated. Whereas
PTH increases the transcription of the 1-hydroxylase,
1,25(OH)2D suppresses PTH release and inhibits
1-hydroxylase messenger RNA (mRNA) production in a classic negative
feedback loop (1). To provide even greater control, the
1,25(OH)2D hormone induces the expression of the
gene encoding a key effector of its catabolic breakdown:
25-hydroxyvitamin D-24-hydroxylase (CYP24, herein referred to as
24-OHase) (2).
The 24-OHase enzyme is a cytochrome P450 molecule that catalyzes the addition of an hydroxyl group on carbon 24 of the vitamin D secosteroid backbone. When the substrate is 1,25(OH)2D, this leads to the production of 1,24,25-trihydroxyvitamin D, the initial reactant in the 24-oxidation pathway that leads to metabolite inactivation (3). This pathway comprises five enzymatic steps involving successive hydroxylation/oxidation reactions at carbons 24 and 23 followed by cleavage of the secosteroid at the C-23/C-24 bond and subsequent oxidation of the cleaved product to calcitroic acid (3). The role of the 24-OHase enzyme in the catabolism of 1,25(OH)2D has been examined in cultured cells using cytochrome P450 inhibitors: blocking P450 activity by treatment with ketoconazole inhibits catabolism and results in increased specific accumulation of 1,25(OH)2D (4).
The 25-hydroxyvitamin D [25(OH)D] metabolite can also serve as the substrate for the 24-OHase enzyme, leading to the production of 24,25-dihydroxyvitamin D [24,25(OH)2D]. A prevalent hypothesis is that the synthesis of 24,25(OH)2D provides a mechanism to inactivate circulating 25(OH)D and thus regulate production of 1,25(OH)2D. In this view, 24,25(OH)2D is considered a catabolite of 25(OH)D. The normal intestinal calcium transport and bone histology of rats treated with analogs of vitamin D fluorinated at position 24 (thus preventing further hydroxylation at that position) as the sole source of vitamin D support the notion that the 24,25(OH)2D molecule is a metabolite exhibiting little physiological activity (5, 6).
Evidence gathered in vitro and in vivo seems to contradict this conclusion. The development of vitamin D-deficient chick embryos is impaired, and normal development and egg hatchability requires both 1,25(OH)2D and 24,25(OH)2D (7). Growth plate chondrocytes respond to 24,25(OH)2D in a cell maturation-dependent fashion (8). Treatment with high doses of 24,25(OH)2D increases bone mass in vitamin D-replete rats, rabbits, and dogs (9). Finally, recent results support a role of physiological concentrations of 24,25(OH)2D as an essential vitamin D metabolite for fracture repair (10, 11).
We have inactivated the 24-OHase gene in mice to examine the physiological role of the 24-OHase enzyme and address the putative role of 24,25(OH)2D. Our studies confirm the role of the 24-OHase enzyme in the regulation of vitamin D homeostasis. The survival of some 24-OHase mutant animals to adulthood has also allowed us to breed them and study the effect of an absence of 24,25(OH)2D and a perturbed vitamin D metabolism during development. Bone development is abnormal in homozygous mutants born of homozygous females. Crossing the 24-OHase-deficient animals with mice carrying an inactivating mutation of the vitamin D receptor gene (VDR) (12) rescued the abnormal bone phenotype, providing genetic evidence that expression of the VDR is necessary for the manifestation of the impaired mineralization phenotype of the 24-OHase -/- animals. These results show that elevated 1,25(OH)2D levels during gestation, and not the absence of 24,25(OH)2D, affect mineralization during development.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). ES cells
carrying the disrupted 24-OHase gene were injected into C57BL/6 embryos
at the blastocyst stage, using standard methodology (17).
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Serum biochemistry
Circulating 1,25(OH)2D levels were
measured from serum or plasma samples using a specific RIA
(ImmunoDiagnostic Systems Ltd., Boldon, UK). Serum concentrations of
24,25(OH)2D were assessed using the
25-hydroxyvitamin D 125I RIA kit for the
quantitative determination of vitamin D hydroxylated metabolites
(DiaSorin, Inc., Stillwater, MN) or by competitive
protein-binding assay, as previously described (19). Total calcium was
measured using a Monarch automated analyzer.
Analysis of macrophage function
Macrophages were isolated from the mouse peritoneum in the
following way: mice were anesthetized with 200 mg/kg ketamine + 10
mg/kg xylazine im and then injected ip with 2 ml sterile PBS using a
25G needle. After withdrawal of the needle, mice were replaced in their
cages and rested for 3 min. Cells were then harvested by puncturing the
peritoneum using an 18G needle. Medium containing macrophages was
collected in sterile plastic tubes and kept immediately on ice. By
using this procedure, a purity of more than 90% macrophages was
reached, as assessed by fluorescence-activated cell sorting
(FACS) analysis (FACSsort, Becton Dickinson and Co., Erembodegem, Belgium) using the monoclonal antibody CD11b
(Serotec, Oxford, UK). On average, 2 x
106 macrophages per mouse were harvested. Cells
from two mice were pooled and resuspended in a final vol of 10 ml, in
ice-cold PBS, and counted. Living cells were identified by trypan blue
exclusion. Cells were then centrifuged during 5 min at 930 x
g ( at 4 C) and resuspended in PBS at a final concentration
of 106 cells/ml. For phagocytosis evaluation,
fluorescein-labeled Zymosan particles (Saccharomyces cerevisiae;
Molecular Probes, Inc. Europe, Leiden, The Netherlands)
were added to 1 ml of the cell suspension (1–100 particles
zymosan/cell). After incubation for 2 h at 37 C, the cells were
washed and resuspended in 100 µl trypan blue. One ml PBS was added
after exactly 1 min. The cells were then centrifuged (930 x
g for 5 min at 4 C) and resuspended in 0.5 ml of 2%
paraformaldehyde. Fluorescein staining was analyzed using a FACSsort
(Becton Dickinson and Co.). Phagocytosis was expressed as
percent fluorescein positive cells. Chemotaxis capacity was evaluated
by measuring the ability to bind casein and fnlpntl
(formyl-Nle-Leu-Phe-Nle-Tyr-Lys fluorescein derivative)
(Molecular Probes, Inc. Europe), as described
previously (20, 21). Briefly, 50 µl of a standardized solution of
fluorescein-labeled casein or fnlpntl was added to 100 µl of the cell
suspension. After incubation for 1 h at 4 C, the cells were washed
with PBS, fixed, and analyzed using the FACSort, as described above.
Chemotactic capacity was expressed as percent fluorescein positive
cells. Statistical analysis was performed using an unpaired Student’s
t test.
1,25(OH)2D homeostasis
Sixty-day-old heterozygote controls or 24-OHase -/-
mice received 1,25(OH)2D3
either acutely (45 ng; and death at 3 h or 24 h) or
chronically (10 ng/day for 7 days). Injections were given ip in 0.1 ml
of 10% ethanol-90% propylene glycol. Mice were killed at the
indicated intervals or at the end of the chronic treatment, and blood
was collected by cardiac puncture. Plasma was also collected from
homozygote mutant and heterozygote controls before, and on days 14 and
20 of gestation, by tail bleeding (17). Urine samples were obtained at
the same time. Circulating 1,25(OH)2D levels and
total calcium concentrations in plasma and urine were measured as
described above.
Northern blot assays and probes
Total RNA was isolated from kidneys of heterozygote controls and
24-OHase -/- mice by the guanidinium isothiocyanate-phenol/chloroform
procedure (22), and Northern blots were performed using standard
methodology. The probes used included a 1.2-kb EcoRI
fragment from the mouse calbindin-D28k cDNA (23),
a 170-bp EcoRI fragment from the mouse
calbindin-D9k cDNA (24), a 1.7-kb
EcoRI fragment from the rat VDR cDNA (25), and a 2.1-kb
HindIII fragment from the chicken ßactin cDNA (26). All
probes were labeled using a random primed DNA labeling kit (Roche Molecular Biochemicals, Indianapolis, IN). The intensities of
mRNA bands in autoradiographs of varying exposures were quantified by
densitometry using a dual-wavelength Flying-spot Scanner (Shimadzu
Scientific Instruments Inc., Columbia, MD). The relative optical
density obtained, after probing with VDR,
calbindin-D28k, or
calbindin-D9k cDNAs, was divided by the relative
optical density value obtained after probing with the ßactin
cDNA.
Histology
Kidneys from chronically treated animals were dissected and
fixed overnight in 4% paraformaldehyde before paraffin embedding.
Twelve-micrometer sections were prepared and stained for mineral
deposition using the method of von Kossa (27). Counterstaining was with
toluidine blue. Bones were dissected, fixed overnight in 10% formalin
in PBS, and embedded in methylmethacrylate (27). Sections of 6 µm
were deplastified and stained by von Kossa or Goldner (27). When
required, counterstain was with hematoxilin-eosin.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The engineered
mutation was transmitted to the progeny with the expected Mendelian
ratio (Fig. 1). The mutation effectively removes the Heme binding
domain of the cytochrome P450 molecule (15), generating a null allele.
In mice homozygous for the engineered mutation, no 24-OHase mRNA could
be detected (data not shown). Using an immunoassay with a sensitivity
of 0.5 nmol/liter, the circulating concentration of
24,25(OH)2D in heterozygote animals was measured
as 31.8 ± 2.3 nmol/liter (mean ± SEM,
n = 6). Circulating 24,25(OH)2D was
undetectable in 24-OHase -/- mutants using this immunoassay.
Similarly, no 24,25(OH)2D could be detected in
the serum of 24-OHase -/- mutants using HPLC fractionation of
hydroxylated metabolites and a conventional competitive protein-binding
assay (19). Genotyping of live animals around the time of weaning demonstrated that 50% of homozygous mutant animals died before 3 weeks of age (28). Because the antimicrobial action of macrophages can be modulated by vitamin D metabolites (29), we first examined macrophage function to determine whether impaired responses to infection could be responsible for perinatal death. No abnormalities in macrophage function were detected in 24-OHase -/- mice. Both markers for chemotaxis were present in comparable levels between homozygote mutant and wild-type mice [20 ± 8% vs. 22 ± 9% casein positivity and 65 ± 12% vs. 70 ± 21% fnlpntl positivity, not significant (NS)]. Phagocytosis ability, measured as capacity to ingest Zymosan particles, was also normal in 24-OHase -/- mice (62 ± 7% vs. 64 ± 9% in wild-type mice, NS).
The perinatal lethality is most likely a consequence of hypercalcemia
secondary to hypervitaminosis, because the inactivation of the
24-OHase gene in mice impaired the ability of the animals to maintain
1,25(OH)2D homeostasis (Fig. 2). Somewhat surprisingly, baseline
circulating levels of 1,25(OH)2D were lower in
24-OHase mutant animals that survived past weaning than in wild-type
controls (93 ± 10 vs. 264 ± 23 pmol/liter for
mutant and wild-type, respectively, mean ±
SEM, n = 10). Those animals must use an
alternative pathway of 1,25(OH)2D catabolism to
regulate circulating levels of the hormone (see Discussion).
Whereas heterozygote controls could efficiently clear from their
bloodstream a bolus of 1,25(OH)2D, the
circulating levels of the hormone remained elevated in homozygous
mutant mice (Fig. 2A). Chronic 1,25(OH)2D
administration (10 ng/day for 7 days) also resulted in a marked
elevation in serum 1,25(OH)2D levels in the
mutant animals (5800 pmol/liter, compared with 211 pmol/liter in the
heterozygote controls, P < 0.01) (Fig. 2B). Northern
blot analysis of vitamin D responsive genes in the kidney of mutant
mice treated with high doses of 1,25(OH)2D is
shown in Fig. 2C. In response to acute 1,25(OH)2D
administration, a 2.3-fold induction in VDR mRNA, a 3.6-fold induction
in calbindin-D28k mRNA, and a 2.5-fold induction
in calbindin-D9k mRNA were observed in
heterozygote controls (+D vs. basal, P <
0.01 for all three mRNAs). Mice deficient in 24-OHase had 2- to 4-fold
higher basal levels of renal vitamin D-dependent genes, when compared
with basal levels in heterozygote controls (Fig. 2C, P
< 0.01). Perhaps because of higher basal levels of these renal vitamin
D-dependent genes in the mutant mice, no significant induction in the
expression levels of these genes was observed in the 24-OHase -/-
mice after acute 1,25(OH)2D administration.
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). Cortical tubular dilation, necrotic
debris, and mineralization were evident (Fig. 3B). These changes in the
kidney of the mutant mice are consistent with hypervitaminosis D.
Kidney sections of heterozygote control mice chronically treated with
1,25(OH)2D were histologically normal (Fig. 3A).
The inability to regulate 1,25(OH)2D homeostasis
and ECF [Ca2+] concentration presumably leads
to fatal extremes of hypercalcemia. Indeed, we have measured extremely
high levels of circulating 1,25(OH)2D and
[Ca2+] in runted animals that eventually died
before weaning (4.06 ± 0.44 vs. 1.99 ± 0.11 mmol
[Ca2+]/liter for mutant and wild-type,
respectively). Hypercalcemic animals with elevated
1,25(OH)2D concentrations exhibited undetectable
PTH levels (data not shown).
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). Histological
examination of the bones from these animals revealed an accumulation of
osteoid at sites of intramembranous ossification (Fig. 4, e–h). Of
particular note was the severely reduced amount of bone in the mandible
of the mutant animals (Fig. 4f). Control heterozygote littermates
showed normal bone structure (Fig. 4, a–d). No significant disruption
of growth plate organization was noted (data not shown).
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), leading to hypercalcemia (Fig. 5B)
and hypercalciuria (data not shown). Although
1,25(OH)2D does not readily cross the placenta
(30, 31), it is likely that the very high maternal circulating levels
(Fig. 5A) would lead to increased fetal
1,25(OH)2D concentrations. Alternatively, the
mutant bone phenotype could be caused by the complete absence of
vitamin D metabolites hydroxylated at position 24, supporting a key
role of 24,25(OH)2D in bone formation during
development. Attempts to rescue the phenotype by treating gestating
mutant females with 24,25(OH)2D were not
successful. When mutant females were fed a diet supplemented in
24,25(OH)2D during gestation, the mandible and
clavicle of the mutant progeny always remained severely affected (data
not shown). To examine the possible contribution of the elevated
levels of 1,25(OH)2D, measured during gestation,
to the abnormal bone phenotype, we crossed the 24-OHase -/- animals
with mice carrying an inactivating mutation of the vitamin D receptor
gene (12). Homozygote mutant VDR females maintained on a high calcium,
high phosphorus, high lactose rescue diet, to prevent rickets (18),
were mated with homozygote mutant 24-OHase males. Heterozygotes at both
loci were then mated to obtain compound homozygotes. Gestating females
for this last cross were not maintained on the rescue diet. If elevated
1,25(OH)2D levels, acting through the VDR, were
responsible for the observed phenotype, then mice lacking the VDR and
the 24-OHase gene should not show the aberrant intramembranous bone
development.
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, compare A and B). Double mutant homozygotes (24-OHase -/- and VDR
-/-) showed normal intramembranous bone formation at all sites
examined (Fig. 6, C and D; and data not shown). These results show that
expression of the VDR is necessary for the manifestation of the
impaired mineralization phenotype of the 24-OHase -/- animals and
that elevated 1,25(OH)2D levels during gestation
affect mineralization.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The 24-OHase enzyme initiates the 24-oxidation pathway, a succession of hydroxylation/oxidation reactions at carbons 24 and 23 that lead to 1,25(OH)2D inactivation (3).The recombinant 24-OHase protein, when associated with electron-transport cofactors, has been shown to be able to perform multiple steps in this catalytic pathway, including 23- hydroxylation, dehydrogenation of the 24-hydroxyl group, and side-chain cleavage (32). The role of the 24-OHase enzyme in the catabolism of 1,25(OH)2D had already been demonstrated in tissue culture (4). Our results confirm the function of the 24-OHase protein as an effector of 1,25(OH)2D breakdown in vivo. Because half the mutant progeny seems unaffected by the 24-OHase deficiency, an alternative pathway of 1,25(OH)2D catabolism must exist to regulate circulating levels of the hormone in vivo. The efficacy of this alternate pathway may vary stochastically between individuals and could depend on genetic background. The analysis of the metabolism of 1,25(OH)2D in surviving 24-OHase -/- animals would identify the alternate clearance pathway. A candidate catabolic route would involve C-26 hydroxylation followed by 26,23-lactone formation (33).
The skeletal development of 24-OHase -/- embryos from heterozygote
crosses was normal. This is most likely because of the maintenance of
normal 1,25(OH)2D and calcium homeostasis in
heterozygote pregnant females (Fig. 5). It is interesting to note that
the growth plates from all mutant animals did not show major defects
and that all cell types could readily be identified, with the
expression of differentiation markers such as type II collagen and type
X collagen confirmed (R. St-Arnaud, unpublished results). The
24,25(OH)2D metabolite has been shown to
play an important role in chondrocyte maturation and differentiation
in vitro (8). Although our observations suggest that the
absence of 24-OHase activity does not affect growth plate development
and that 24,25(OH)2D is not required for
chondrocyte maturation in vivo, it is possible that a
redundant endocrine system may be able to compensate for the
24,25(OH)2D function in the animal.
On the contrary, 24-OHase -/- pups born from 24-OHase mutant females
had impaired bone mineralization, showing that the complete absence of
24-OHase enzymatic activity during development leads to abnormal bone
structure. Serum concentrations of 1,25(OH)2D
were lower in 24-OHase -/- dams than in control pregnant mice until
midgestation (Fig. 5a). The impaired mineralization in 24-OHase -/-
fetuses could not be caused by the decreased circulating
1,25(OH)2D levels in the mother during early
pregnancy. Indeed, the feto-placental unit can regulate calcemia and
mineralize the fetal skeleton, even in the presence of marked vitamin D
deficiency in the mother (31). Moreover, despite a lack of VDR
throughout embryogenesis, VDR-null mice are born phenotypically normal
(12, 34).
The bone lesions in 24-OHase -/- pups born from 24-OHase -/- dams were detected at specific sites: the calvaria, mandible, clavicle, and the periosteum of long bones, all sites of intramembranous bone formation. This observation suggests that the bone lesions were not caused by abnormal mineral homeostasis, a mechanism that would also have affected endochondral ossification. Considering the previously reported putative roles of 24,25(OH)2D during development (7), we examined the possibility that the absence of this metabolite could be responsible for the impaired mineralization. This hypothesis was tested by attempting to rescue the bone phenotype with exogenous 24,25(OH)2D administered to the gestating females at midgestation. This mode of administration was chosen because it has been demonstrated that the cord concentrations of 24,25(OH)2D are directly related to the maternal concentrations (35). The 24,25(OH)2D supplementation failed to correct the bone abnormalities (data not shown). These results argue against an essential role of the 24,25(OH)2D metabolite for bone development and support the conclusion drawn after treatment of animals with vitamin D analogs fluorinated at position 24 (5, 6). These observations further suggested that the abnormal mineralization of intramembranous bone in 24-OHase -/- pups born from 24-OHase mutant females was caused by the elevated concentrations of 1,25(OH)2D in late gestation that resulted from the absence of 24-OHase enzymatic activity. We tested this hypothesis using genetic means.
Crossing the 24-OHase-deficient animals to VDR-ablated mice totally rescued the bone phenotype, confirming that the elevated 1,25(OH)2D levels, acting through the VDR, were responsible for the observed accumulation of osteoid. The bone histology of the compound homozygote mutants (24-OHase -/- VDR -/-) seemed identical to the bone histology of the single VDR -/- mutant, which is normal until weaning (12, 34). Formal histomorphometric analysis of the bones of compound homozygote mutants would have to be performed post weaning, but it is anticipated that results similar to those reported for the single VDR-ablated mice would be obtained and that normalization of mineral ion homeostasis by feeding a high calcium, high phosphate, high lactose diet would normalize histomorphometric parameters (36).
Chronic treatment of rats with high doses of
1,25(OH)2D perturbs mineralization, leading to
accumulation of osteoid at the endosteal surface of long bones, as well
as in bone trabeculae (37, 38). These sites are mostly normal in
24-OHase mutant animals (Fig. 4), but the differences may reflect
variations in pre- vs. postnatal responses. At any rate, our
results demonstrate the need for controlled
1,25(OH)2D homeostasis during intramembranous
bone formation and suggest that 1,25(OH)2D may
have a different role in intramembranous vs. endochondral
bone. The specificity of the mineralization defect, because it affects
only intramembranous but not endochondral bone, could be related to the
differences observed between the matrix of endochondral bone and the
matrix of intramembranous bone (39).
The possibility that 1,25(OH)2D regulates bone
development at specific sites through a paracrine mechanism is
supported by recent preliminary findings suggesting that overexpression
of the VDR in bones of transgenic animals can affect bone structure
(40). The recent cloning of the 1- hydroxylase enzyme that is
responsible for the synthesis of 1,25(OH)2D
(41, 42, 43) provides an additional tool to address the putative
autocrine/paracrine roles of vitamin D in bone development.
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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 November 16, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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F. Barletta, L. P. Freedman, and S. Christakos Enhancement of VDR-Mediated Transcription by Phosphorylation: Correlation with Increased Interaction Between the VDR and DRIP205, a Subunit of the VDR-Interacting Protein Coactivator Complex Mol. Endocrinol., February 1, 2002; 16(2): 301 - 314. [Abstract] [Full Text] [PDF]
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M. Yamagata, A. Kimoto, T. Michigami, M. Nakayama, and K. Ozono Hydroxylases Involved in Vitamin D Metabolism Are Differentially Expressed in Murine Embryonic Kidney: Application of Whole Mount in Situ Hybridization Endocrinology, July 1, 2001; 142(7): 3223 - 3230. [Abstract] [Full Text] [PDF]
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W. Yang, S. J. Hyllner, and S. Christakos Interrelationship between signal transduction pathways and 1,25(OH)2D3 in UMR106 osteoblastic cells Am J Physiol Endocrinol Metab, July 1, 2001; 281(1): E162 - E170. [Abstract] [Full Text] [PDF]
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D. K. Panda, D. Miao, M. L. Tremblay, J. Sirois, R. Farookhi, G. N. Hendy, and D. Goltzman Targeted ablation of the 25-hydroxyvitamin D 1alpha -hydroxylase enzyme: Evidence for skeletal, reproductive, and immune dysfunction PNAS, June 19, 2001; 98(13): 7498 - 7503. [Abstract] [Full Text] [PDF]
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M. Raval-Pandya, P. Dhawan, F. Barletta, and S. Christakos YY1 Represses Vitamin D Receptor-Mediated 25-Hydroxyvitamin D3 24-Hydroxylase Transcription: Relief of Repression by CREB-Binding Protein Mol. Endocrinol., June 1, 2001; 15(6): 1035 - 1046. [Abstract] [Full Text] [PDF]
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