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Genetics Unit Shriners Hospital for Children, and Departments of Surgery and Human Genetics McGill University Montréal H3G 1A6, Québec, Canada
Address all correspondence and requests for reprints to: René St-Arnaud, Ph.D., Genetics Unit, Shriners Hospital for Children, 1529 Cedar Avenue, Montréal H3G 1A6, Québec, Canada.
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Introduction |
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 Top Introduction References |
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,25-dihydroxyvitamin D
[1,25(OH)2D]. This active metabolite is responsible
for intestinal calcium and phosphorus absorption, mobilization of
calcium from bone, and renal reabsorption of calcium and phosphorus
(1).
To maintain mineral homeostasis, the production and degradation of
1,25(OH)2D needs to be tightly regulated. Hypocalcemia
results in increased production of 1,25(OH)2D (2). This
induction is secondary to increased PTH (2). The production of
1,25(OH)2D is caused by the action of the cytochrome
P450 enzyme, 25-hydroxyvitamin D-1-hydroxylase (1-OHase). The
results of classic experiments demonstrating the role of the
parathyroid gland in the regulation of the production of
1,25(OH)2D in animals were recently confirmed at the
molecular level through the cloning of the 1-OHase gene (3, 4, 5, 6) and
promoter (7), showing direct effects of PTH on the expression of the
1-OHase messenger RNA (mRNA) (7).
The expression and activity of the 1-OHase enzyme is controlled in a
classic feedback loop (2) to prevent a sustained production of
1,25(OH)2D that would lead to hypercalcemia. To provide
for an even faster "shut-off" mechanism, 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 (24-OHase).
The 24-OHase enzyme is also a mixed-function oxydase cytochrome P450
molecule. It resides in the mitochondrial membrane and 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-oxydation pathway that leads to metabolite inactivation (8).
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 (8). Interestingly,
the recombinant 24-OHase protein, when associated with its
electron-transport cofactors, NADPH-ferredoxin reductase and
ferredoxin, has been shown to be able to perform multiple steps in this
catalytic pathway, including 23-hydroxylation (9, 10), dehydrogenation
of the 24-hydroxyl group (9, 10), and side-chain cleavage (10). In cell
culture systems, the role of the 24-OHase enzyme in the catabolism of
1,25(OH)2D has been examined using cytochrome P450
inhibitors: blocking P450 activity by treatment with ketoconazole
inhibits catabolism and results in increased specific accumulation of
1,25(OH)2D (11). The function of the 24-OHase protein as
an effector of 1,25(OH)2D breakdown has also been
confirmed in vivo through targeted inactivation of the
24-OHase gene in mice (12). Animals homozygous for the 24-OHase
mutation cannot effectively clear 1,25(OH)2D from their
circulation (our unpublished observations). This leads to perinatal
lethality, most probably secondary to hypercalcemia caused by sustained
elevated levels of 1,25(OH)2D. It is interesting to note
that the perinatal lethality phenotype shows only partial
penetrance, suggesting that alternative pathways of
1,25(OH)2D catabolism can effectively regulate
circulating levels of the hormone in vivo. The efficiency of
this alternate catabolic pathway may vary depending on the genetic
background of the animals (our unpublished observations). We have begun
to study the metabolism of 1,25(OH)2D in
24-OHase-deficient animals to identify the alternate clearance pathway
(possibly C-26 hydroxylation/26,23-lactone formation; Ref. 13). The
24-OHase mutation is also being bred on different genetic backgrounds
to investigate the possibility that modifier loci participate in
vitamin D homeostasis in vivo.
The mechanism through which 1,25(OH)2D affects its own
fate is a classic genomic response: several groups have studied the
promoter region of the 24-OHase gene from various species and shown
that it contains functional vitamin D-response elements (see Ref. 14
for review). Thus, in target cells that express the vitamin D receptor,
the hormone can induce the expression of the enzyme that leads to its
inactivation. Expression of the 24-OHase gene has been detected in
kidney, intestine, bone, placenta, skin, and macrophages (14).
The effect of PTH on vitamin D catabolism varies depending on the
target cells. In the proximal convoluted tubule cells of the kidney,
the major site of 1,25(OH)2D production, PTH inhibits
the expression of the 24-OHase mRNA (15). This presumably allows for
1,25(OH)2D production with minimal concomitant
catabolism. The expression of the 24-OHase gene in the intestine is not
affected by PTH as gut cells lack PTH receptors. However, PTH has been
shown to synergize with 1,25(OH)2D to stimulate 24-OHase
expression in established lines of osteoblastic cells (16). The
molecular mechanisms underlying this effect are dissected in a paper by
Armbrecht et al. (17) in this issue of
Endocrinology.
Their results show that PTH potentiates from 3- to 10-fold the
stimulation induced by 1,25(OH)2D, whereas it has no
effect on its own. The PTH effect was shown to be rapid (peaks at
1.5 h), dose dependent, and mediated through cAMP, as it can be
mimicked by the cAMP analog, 8-bromo-cAMP. The data presented shows
that PTH acts at the transcriptional level and that the increased
24-OHase mRNA levels are translated into protein. It should be
mentioned that other investigators had reported no effect of PTH on the
1,25(OH)2D-dependent expression of 24-OHase in bone
cells (18). This discrepancy is most likely due to cell line
dependency, as Armbrecht et al. (17) demonstrate the
synergistic effect of PTH in primary cultures of osteoblasts.
The physiological rationale for the synergistic action of PTH and
1,25(OH)2D on the expression of the 24-OHase gene seems
perplexing when compared with the actions of the peptide hormone in
kidney cells. This could reflect tissue-specific requirements for
1,25(OH)2D catabolism between various target tissues.
Alternatively, increased bone cell expression of 24-OHase due to the
synergism between PTH and 1,25(OH)2D could be meant to
increase synthesis of 24,25-dihydroxyvitamin D
[24,25(OH)2D], the metabolite produced by the action of
the 24-OHase enzyme on the 25-hydroxyvitamin D substrate. This leads us
to a discussion of the controversial physiological role of
24,25(OH)2D.
One of the most abundant circulating metabolite of vitamin D is
24,25(OH)2D. Some authors have proposed that
24,25(OH)2D production represents a mean to inactivate
circulating 25-hydroxyvitamin D and thus regulate production of
1,25(OH)2D. In this view, 24,25(OH)2D is
considered a catabolite of 25-hydroxyvitamin D. Support for the notion
that the 24,25(OH)2D molecule serves no physiological
function came primarily from experiments using analogs of vitamin D
fluorinated at position 24 (thus preventing further hydroxylation at
that position). When these analogs were used as the sole source of
vitamin D, they produced the same biological responses as those
resulting from 25-hydroxyvitamin D regarding intestinal calcium
transport, mobilization of calcium from bone, and mineralization of
vitamin D-deficient bone (19). These results have been cited repeatedly
to substantiate the hypothesis that 24-hydroxylation does not play a
significant role in vitamin D function and bone growth and development
in the rat.
Results from in vitro studies and experiments in other
species contradict this conclusion. A large body of literature
demonstrates that the 24-OHase gene is expressed in growth plate
chondrocytes and that cells from the growth plate respond to
24,25(OH)2D in a cell maturation-dependent fashion
(reviewed in Ref. 20). Most of these studies have been performed
in vitro using primary cultures of rat costochondral
chondrocytes. Dissection of the tissue allows to culture cells from
different regions of the growth plate that represent different
maturation stages along the chondrocytic differentiation pathway. In
this system, the less differentiated cells of the resting zone,
also called the reserve zone, appear to respond to
24,25(OH)2D, whereas the more mature cells of the
growth zone, comprising the prehypertrophic and hypertrophic
compartments, respond primarily to 1,25(OH)2D. Moreover,
treatment of resting zone chondrocytes with 24,25(OH)2D
causes a change in maturation state (21), supporting the hypothesis
that 24,25(OH)2D plays a role in cartilage development. The
maturation-stage specific responses of chondrocytes to the vitamin D
metabolites include both genomic and nongenomic effects such as plasma
membrane and matrix vesicle enzyme activity and fluidity, cell
proliferation and protein synthesis, calcium signaling, phospholipid
metabolism, and production of vitamin D metabolites (reviewed in detail
in Ref. 20).
Evidence gathered in vivo also supports a physiological role
for 24,25(OH)2D during embryogenesis and in processes
regulating bone growth, development, and repair. Experiments dating
back 20 yr revealed that the development of vitamin D-deficient chick
embryos is impaired (22), and that a large proportion of the eggs from
the vitamin D-depleted hens fail to hatch (23). Whereas treatment of
the vitamin D-deficient eggs with vitamin D itself leads to normal
development, treatment with 1,25(OH)2D alone can rescue
some but not all of the observed defects (22), suggesting that vitamin
D metabolites other than 1,25(OH)2D are necessary for
normal chick embryo development. Similarly, normal egg hatchability
requires both 1,25(OH)2D and 24,25(OH)2D
(23), and the unnatural epimer 24S,25(OH)2D3
cannot substitute for 24R,25(OH)2D3 in this
system (24), supporting a unique role for the natural
24R,25(OH)2D metabolite.
Several in vivo studies have also examined a putative role for 24,25(OH)2D in bone biology. Treatment with high doses of 24,25(OH)2D increases bone mass in vitamin D-replete rats, rabbits, and dogs (25 and references therein). In hypophosphatemic mice, a model for the human disease familial X-linked hypophosphatemic rickets, treatment with 24,25(OH)2D showed dose-dependent effects in increasing bone formation without inducing excessive bone resorption (25). These experiments support a clear pharmacological activity for 24,25(OH)2D, although they do not establish a clear physiological role for the metabolite.
Another aspect of bone biology in which investigators have sought to
identify a role for 24,25(OH)2D concerns fracture repair.
The circulating levels of 24,25(OH)2D increase during
fracture repair due to an increase in renal 24-OHase activity (26).
These results suggest that 24,25(OH)2D may be involved in
the early phases of the healing process. Whether the increased
production of the metabolite relates to its putative role in the
maturation of chondrocytes (20, 21) or to other aspects of bone biology
remains to be established; the level of expression and activity of the
24-OHase at the fracture site was not assessed and the cells that could
produce 24,25(OH)2D within the fracture callus have not
been identified. Nevertheless, when the effect of various vitamin D
metabolites on the mechanical properties of healed bones was tested,
treatment with 1,25(OH)2D3 alone resulted in
poor healing (27). On the contrary, the strength of healed bones in
animals fed 24,25(OH)2D3 in combination with
1,25(OH)2D3 was equivalent to that measured
in a control population fed 25-hydroxyvitamin D3 (27).
These results support a role of physiological concentrations of
24,25(OH)2D as an essential vitamin D metabolite for
fracture repair.
Our own laboratory has taken a different approach to examine this issue. We have used the powerful technique of homologous recombination in embryonic stem cells to engineer a strain of mice deficient for the 24-OHase enzyme and address the putative physiological role of 24,25(OH)2D. Analysis of the phenotype of these mutant animals has revealed previously unrecognized roles for vitamin D metabolites hydroxylated at position 24, most presumably 24,25(OH)2D. The survival of some 24-OHase mutant animals to adulthood has allowed us to breed them and address the role of 24,25(OH)2D during development. When fertile mutant homozygous females are mated to heterozygous males, litters comprise an equal proportion of mutant homozygotes and control heterozygous littermates. Because of the impaired 24-OHase activity of the female, homozygous embryos are completely deprived of vitamin D metabolites hydroxylated at position 24 during development. Heterozygous littermates can synthesize those metabolites because they carry one functional allele of the 24-OHase gene. We have observed a mineralization defect in homozygous mutants born of homozygous females (our unpublished observations). Histological examination of the bones from these animals revealed an accumulation of unmineralized osteoid matrix at sites of intramembranous ossification, such as the calvaria, mandible, clavicle, and the exocortical (periosteal) surface of long bones (28). Control heterozygote littermates show normal bone structure. These results show that a complete absence of vitamin D metabolites hydroxylated at position 24 during embryogenesis leads to abnormal bone structure and suggest a key role for 24,25(OH)2D in the developmental regulation of intramembranous bone formation. We surmise that homozygous mutant animals born from heterozygous females do not exhibit the bone phenotype because the heterozygous female can supply 24,25(OH)2D across the feto-placental barrier.
At first glance, the growth plates from these mutant animals did not show major defects, and all cell types could readily be identified, suggesting that 24,25(OH)2D is not a major regulator of chondrocyte maturation in vivo. Further examination of the precise architecture of each zone of the growth plate and of the gene expression patterns of the chondrocytes at different stages of maturation within the growth plate may reveal subtle differences not apparent at this stage of the analysis.
Several hypotheses can be formulated to account for the phenotype of
the 24-OHase-deficient embryos from 24-OHase mutant mice. A potential
role of the 24-OHase enzyme affecting 1,25(OH)2D action,
rather than a role for 24,25(OH)2D, remains possible. In
collaboration with Dr. Marie Demay (29), we have initiated a cross
between the 24-OHase-deficient animals and mice carrying an
inactivating mutation of the vitamin D receptor gene. If
1,25(OH)2D, acting through the vitamin D receptor, is
responsible for the observed phenotype, then mice lacking the receptor
and the 24-OHase gene should not show the aberrant intramembranous bone
development. It seems unlikely, however, that the observed
mineralization defect could be due to the loss of the C-24 oxidation
pathway and subsequent hypervitaminosis D. There appears to be
tremendous species variations to the effects of elevated levels of
1,25(OH)2D. While treatment with high doses of the
hormonal form of vitamin D perturbs mineralization in rats (30), it has
little or no consequence on bone structure in mice (31). Moreover, the
defects observed in rats treated with high doses of
1,25(OH)2D were localized at the endosteal surface of
long bones as well as in bone trabeculae (30), sites that are mostly
normal in 24-OHase mutant animals (28).
It is also very unlikely that the observed phenotype is secondary to a perturbation in mineral homeostasis because the growth plate is normal in homozygotes. Biochemical analysis reveals equivalent blood calcium levels in homozygotes compared with heterozygotes. The most likely hypothesis remains that 24,25(OH)2D is required for normal intramembranous ossification. To test this hypothesis, we have attempted to rescue the bone phenotype by treating gestating homozygote mutant females with 24,25(OH)2D3. Histological examination of the calvaria from mutant homozygote pups born of homozygote mothers treated with 24,25(OH)2D3 show that treatment with the metabolite can rescue the calvarial phenotype. Interestingly, treatment with the unnatural epimer 24S,25(OH)2D3 is less effective at correcting the development of the calvaria (our unpublished observations). Our results show that a complete absence of vitamin D metabolites hydroxylated at position 24 during embryogenesis leads to abnormal bone structure and support a key role for 24,25(OH)2D in the developmental regulation of intramembranous bone formation.
What is the mechanism of action of the 24,25(OH)2D
metabolite in its target cells? Receptor-mediated signaling remains a
logical possibility. Autoradiographic and binding saturation analyses
raised the possibility of a hypothetical 24,25(OH)2D
receptor in bone tissue from several species (reviewed in Ref. 28).
These initial studies, more than a decade old, have not been pursued
with any measure of success. By analogy with the vitamin D receptor
that binds 1,25(OH)2D, it can be hypothesized that if a
24,25(OH)2D receptor exists, it would be a member of the
nuclear hormone receptor superfamily. Considering that more than 150
members of this superfamily have now been identified, it can be
envisaged that a nuclear receptor binding 24,25(OH)2D may
be expressed in target tissues. Alternatively, it should be mentioned
that nongenomic effects of 24,25(OH)2D have been described,
for example in resting zone chondrocytes (20), raising the possibility
that some type of membrane receptor could mediate the effects of the
metabolite. Interestingly, a putative transmembrane receptor kinase
molecule involved in mediating steroid hormone signaling in plants has
recently been described (32). Perhaps such a receptor, entirely
different from the classic vitamin D receptor, is responsible for the
actions of 24,25(OH)2D in target tissues. That would
certainly explain why it has remained elusive until now. We surmise
that it is only when the effector molecules of 24,25(OH)2D
function have been cloned and characterized that all the vitamin D
field will be converted to accept the physiological function of
the 24,25(OH)2D metabolite. In the meantime, believers in
several laboratories are hard at work trying to identify the components
of the signaling cascade that would prove their beliefs beyond the
shadow of a doubt.
Received April 2, 1998.
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References |
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TopIntroductionReferences |
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-hydroxylase and
vitamin D synthesis. Science 277:1827–1830-hydroxylase cDNA. Proc Natl Acad
Sci USA 94:12920–12925-hydroxylase and mutations causing vitamin D-dependent rickets
type 1. Mol Endocrinol 11:1961–1970-hydroxylase gene promoter.
Proc Natl Acad Sci USA 95:1387–1391,25-dihydroxyvitamin D3-24-hydroxylase in
rat osteoblasts. Endocrinology 134:1794–1799[Abstract]
,25-dihydroxycholecalciferol and 24R,25-dihydroxycholecalciferol. J
Nutr 113:2505–2515
This article has been cited by other articles:
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  G. R. Mundy and T. A. Guise Hormonal Control of Calcium Homeostasis Clin. Chem., August 1, 1999; 45(8): 1347 - 1352. [Abstract] [Full Text] [PDF]
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