Endocrinology Vol. 142, No. 7 2734-2735
Copyright © 2001 by The Endocrine Society
Editorial: Vitamin D 1
-Hydroxylase Knockout Mice as a Hereditary Rickets Animal Model
Shigeaki Kato
University of Tokyo
Institute of Molecular & Cellular Biochemistry
Tokyo 113 0032, Japan
Address all correspondence and requests for reprints to: Shigeaki Kato, Ph.D., University of Tokyo, Institute of Molecular & Cellular Biochemistry, Yayoi, Bunkyo-ku, Tokyo 113 0032, Japan.
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Introduction
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Vitamin D is one of the major regulators of mineral homeostasis in
animals. Thus, any disorder involving vitamin D actions results in the
impairment of bone formation, including rickets with growth
retardation, and osteomalacia in adults, due to impaired bone
mineralization. Most of these biological actions of vitamin D are
thought to be exerted through nuclear vitamin D receptor (VDR)-mediated
transcriptional control of target genes. The VDR is a member of the
nuclear hormone receptor superfamily and acts as a
ligand-inducible transcription factor. The VDR forms heterodimers
with one of three retinoid X receptor isotypes and binds specific DNA
elements referred to as vitamin Dresponsive elements (VDRE) in
target gene promoters. The most biologically active form of vitamin D,
1
,25(OH)2D3, serves as an endogenous ligand
for the VDR, and its biosynthesis is strictly regulated through hepatic
and renal hydroxylation of its precursor, vitamin D3, by
two distinct but related p450 enzymes.
25(OH) vitamin D31-hydroxylase (CYP27B1)
[1(OH)ase] is responsible for the final and key step of
hormonal conversion into 1,25(OH)2D3. Its
clinical importance has been established by the identification of
mutations in the human 1(OH)ase gene in vitamin D-dependent
rickets type I (VDDRI)/pseudo vitamin D deficiency rickets (PDDR)
patients. However, due to lack of an animal model of VDDRI (PDDR), the
physiological significance of 1(OH)ase in intact animals remained to
be verified. The report by Dardenne et al. in this issue
(1) now deciphers the critical role of CYP27B1 in vitamin
D actions by generating KO mice with rachitic abnormalities typically
observed in VDDRI patients.1
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25-Hydroxyvitamin D 1-hydroxylase (CYP27B1) as a key enzyme in
vitamin D biosynthesis
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Cholesterol is metabolized into
7-dehydrocholesterol and is then converted to vitamin
D3 by UV light on the skin. Vitamin D is also
ingested from the diet as vitamin D2
(ergocalciferol) mainly from plants, and vitamin
D3 (cholecalciferol) from animals. The hormonal
form of vitamin D,
1,25(OH)2D3, is
metabolically formed through two sequential hydroxylation steps at the
final stage. First, hepatic hydroxylation of vitamin
D3 by vitamin D3
25-hydroxylase (CYP25) generates 25-dihydroxyvitamin
D3 [25(OH)D3], which is
then hydroxylated into
1,25(OH)2D3 by
25-hydroxyvitamin D3 1 hydroxylase
(CYP27B1) [1(OH)ase] mainly in the kidney (2).
Renal hydroxylation is the critical step in this hormonal conversion,
and hence, expression of 1(OH)ase is under precise transcriptional
control by multiple calciotropic hormones to strictly regulate serum
levels of 1,25(OH)2D3 in
response to calcium requirements. Recently, positively regulated
elements sensitive to PTH and calcitonin, and negative response
elements sensitive to
1,25(OH)2D3 have been
mapped in human and rodent 1(OH)ase gene promoters, although the
transcriptional regulatory factors that bind to these elements remain
to be identified (3, 4). Nevertheless, it is evident that
these regulatory elements probably define multihormonal regulatory
events, most likely primarily at the level of gene expression.
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Genetic mutations in the human 1(OH)ase and VDR genes cause
hereditary rickets
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The complementary DNA (cDNA) cloning of the complete 1(OH)ase
gene was successfully achieved in 1997 by several groups, most of which
used PCR-based strategies reliant on the sequence homology among
related p450 enzymes (2). This advance occurred only 1 yr
after the strategy was first presented at the 1996 ASBMR meeting by
St-Arnaud’s group. Following the successful cloning of the murine
1(OH)ase cDNA, the human cDNA was isolated and examined for the
presence of genetic missense mutations in VDDRI patients. In 1998, we
reported (5) that mutations in Japanese VDDRI patients
were responsible for the loss of 1(OH)ase enzymatic activity and
established that CYP27B1 was the gene responsible for VDDRI. To date,
nearly 30 types of distinct genetic mutations have been reported in
VDDR patients throughout the world (6), though it remains
to be elucidated whether some of the reported mutations lead to
complete or only partial loss of enzymatic activity.
Rachitic abnormalities, particularly alopecia, differ even among VDDRII
patients, depending on the remaining function of the VDR mutants.
Unless complete loss of VDR function occurs due to mutations in
essential VDR domains, such as the DNA and hormone binding domains,
partially impaired vitamin D actions are expected to be responsible for
the range of abnormalities reported for VDDRII patients. The VDR KO
mice exhibited severe rickets with alopecia and are considered to be a
model for VDDRII patients with complete loss of VDR function
(7). In contrast, the relationship between impaired
1(OH)ase activity and the VDDRI phenotype has not been well studied
from both the clinical and basic science viewpoints.
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1(OH) ase KO mice as a VDDRI animal model
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The report by Dardenne et al. in this issue
(1) reports for the first time a null-mutant animal for
1(OH)ase (CYP27B1) by targeted gene disruption in mice.
1(OH)ase-/- mice are
born with no obvious abnormalities but develop rickets by 3 weeks of
age with growth retardation and osteomalacia. Expanded growth plates
with disorganized cartilage layers, as well as osteoid in trabecular
bone due to impaired mineralization, were observed, along with
decreased levels of serum calcium and
1,25(OH)2D3 similar to
rickets patients. Unlike
VDR-/- mice, decreased
levels of 24,25(OH)2D3 with
increased levels of 25(OH)D3 were observed in
8-week-old 1(OH)ase-/-
mice, clearly indicating the lack of 1(OH)ase activity in these
mice.
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What we learn from 1(OH)ase-/-
mice
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These findings for the first time establish that 1(OH)ase is
physiologically critical for vitamin D actions in intact animals. The
alterations in 25(OH)D3 and 24,25(OH)
2D3 serum levels in
1(OH)ase-/- mice were
not consistent with those found in VDDRI patients who still retain
levels of these vitamin D derivatives within normal ranges
(8). The authors raise the possibility that this
inconsistency is due to species differences. This idea may be supported
by previous findings that complete loss of 1(OH)ase activity by
genetic mutation of the 1(OH)ase gene in humans did not cause a
reduction in serum
1,25(OH)2D3 levels
sufficient to cause severe rickets (5, 6). It is therefore
likely that, at least in humans, enzyme(s) other than 1(OH)ase
(CYP27B1) are also specifically or nonspecifically responsible for
1,25(OH)2D3
production.
Consistent with the symptoms of VDDRI patients, no alopecia developed
in 1(OH)ase-/- mice in
contrast with VDR-/-
mice. This phenotypic difference and the response to administered
1,25(OH)2D3 are the
major discriminating factors in distinguishing VDDRI from VDDRII. The
molecular basis of alopecia is of particular interest in terms of
vitamin D actions, as VDR appears to be essential for normal
development of hair follicles in the skin only when it heterodimerizes
with retinoid X receptor- (9).
While the kidney is the major tissue expressing 1(OH)ase, detectable
expression levels are also observed in extra-renal tissues.
Overexpression of the 1(OH)ase gene leading to hypercalcemia has
been found in the tumors of sarcoidosis patients, possibly through
transcriptional activation by cytokines overexpressed in the tumors
through regulatory elements other than those already reported in the
gene promoter. Thus, the physiological roles of 1(OH)ase, including
in extra-renal tissues and tumors, remains largely unknown. These
problems will be hopefully clarified in the near future by the group of
St-Arnaud, as the flox 1(OH)ase mice, as seen in this issue, is now
ready to disrupt this gene in a spatio-temporal way.
Received April 25, 2001.
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Introduction
25-Hydroxyvitamin D 1{alpha}...