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Original Studies |
Department of Pediatrics, Rambam Medical Center, Haifa 31096, Israel; and Bone Disease Unit, Tel-Aviv Medical Center (Y.W.), Tel-Aviv 64230, Israel
Address all correspondence and requests for reprints to: Dr. Dov Tiosano, Rambam Medical Center, POB 9602, Haifa 31096, Israel. E-mail: d_tiosano{at}rambam.health.gov.il
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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-Hydroxylase
(1-OHase) and 24-OHase activities were estimated by the
product/substrate ratio. In the PTH-N group, 1-OHase activity was
higher and 24-OHase activity was lower than in controls. In the PTH-H
group, 1-OHase activity was even higher, probably due to an additive
effect of PTH. Thus, 1,25-(OH)2D-liganded VDR is a major
control mechanism for vitamin D metabolism, and PTH exerts an additive
effect. Assessment of the influence of 1,25-(OH)2D shows
reciprocal control of enzyme activity in man, suppressing 1-OHase and
stimulating 24-OHase activity. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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,25-dihydroxyvitamin D [1,25-(OH)2D] and
the mostly inactive 24,25-(OH)2D
(1). The biosynthesis of these two metabolites seems to be
reciprocally controlled by 1,25-(OH)2D itself, as
the expression of the former is diminished and that of the latter
enhanced upon administration of vitamin D (2, 3). This
control mechanism is mediated in part by the vitamin D receptor (VDR)
(4). Binding of
1,25-(OH)2D3 to the nuclear
VDR activates the receptor and induces specific transcription activity
at target genes (4). Among such genes,
25OHD-24-hydroxylase (25OHD-24-OHase) possesses a VDR response element
and is activated by
1,25-(OH)2D3. Inhibition by
vitamin D of the other renal enzyme 25OHD-1-OHase could result from a
direct or an indirect effect of the VDR, down-regulation of the VDR, or
a VDR-suppressing effect on 1-OHase transcription. In a study of VDR
knockout mice, 1,25-(OH)2D was shown to inhibit
1-OHase transcription and expression in wild-type
VDR+/+ and heterozygote
VDR+/-, but not in
VDR-/-, animals
(5). To investigate the direct or indirect role of VDR in controlling vitamin D metabolism in humans, we studied patients with a defective VDR. Vitamin D-dependent rickets type II (VDDR-II) (6, 7, 8) is an autosomal recessive disease caused by loss of function mutations of the VDR. All patients in the present study had a truncation mutation with complete loss of function (7, 8). Findings in such patients are equivalent to a knockout experiment and have contributed to our understanding of vitamin D physiology (9, 10, 11). In the absence of any VDR-mediated biological activity in VDDR-II patients and after normalizing their serum calcium and PTH through exogenous delivery of calcium, it becomes possible to make a more precise assessment of the influence of the VDR by comparing these patients to normal controls with the same circulating levels of PTH, calcium, and phosphorus. The importance of PTH can also be assessed by comparing VDDR-II patients with normal PTH and normal controls to VDDR-II patients with high PTH.
The present study was designed to investigate the metabolism of vitamin D in VDDR-II patients. Special attention was paid to prepare the patients by normalizing the levels of serum calcium, phosphorus, and PTH. Under these conditions a load of vitamin D was followed by reassessment of vitamin D metabolites. The results show that although PTH enhances both 1-OHase and 24-OHase independently of the VDR, the latter plays a critical role in the regulation of vitamin D metabolism. The magnitude of this effect surpasses that of PTH. After 1,25-(OH)2D binding to VDR the production of 1,25-(OH)2D is down-regulated and that of 24,25-(OH)2D is up-regulated.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Ten patients with hereditary vitamin D-dependent rickets type 2 (five males and five females) and with an age range from 4–16 yr were the subjects of this study. They had been described previously (10, 11). Six of them belong to an extended pedigree with a nonsense mutation in exon 7 resulting in a stop codon (6) and consequently in the expression of a truncated receptor unable to bind 1,25-(OH)2D and devoid of any biological function (7). Three siblings have a missense mutation in exon 3, expressing a modified zinc finger of the DNA-binding domain (12). One patient with VDDR-II was also reported previously (13). The absence of any biological function of 1,25-(OH)2D and the VDR characterized these patients as having VDDR-II.
They had undergone treatment for several years, first with iv and then
with oral calcium therapy, and their rickets had healed
(14). In retrospect, at the outset of the present study
seven of the patients had normal serum calcium and plasma PTH levels,
and three showed increased plasma PTH due, apparently, to insufficient
compliance with therapy (Fig. 1
). Their
serum calcium, phosphorus, and urinary calcium/creatinine ratio tended
to be lower, and their serum alkaline phosphatase activity higher,
although this did not become statistical significant. Seven healthy
siblings of these patients, of similar age, served as normal controls.
Informed consent was obtained from the parents of all children.
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The VDDR-II patients have for several years been receiving oral calcium at a dose of 5 g/m2 and 2 weeks before the study they were instructed to increase the dose to 7.5 g/m2. Basal serum calcium, phosphorous, creatinine, 25OHD, 1,25-(OH)2D, 24,25-(OH)2D, plasma PTH, and urinary calcium, phosphorus, and creatinine were determined, and the threshold concentration of serum phosphate, the renal phosphate threshold concentration (TmP/GFR), was calculated from the tubular reabsorption of phosphate and the serum phosphate level (15). Cholecalciferol was then given as a single oral load of 50,000 U/m2. Two weeks later the same blood and urine tests were repeated.
Materials and methods
Serum 25OHD and 24,25-(OH)2D3 concentrations were measured by competitive binding radioassay (DiaSorin, Inc., Stillwater, MN) after separation of vitamin D metabolites in serum extracted by Sephadex LH-20 chromatography. Serum 1,25-(OH)2D was measured by RRA kit (DiaSorin, Inc.). Plasma PTH was measured by an immunoradiometric assay (Nichols Institute Diagnostics, San Juan Capistrano, CA).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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: 1)
the control group (n = 7); 2) patients with good compliance to
calcium therapy and normal serum calcium, phosphorus, PTH, and alkaline
phosphatase (PTH-N; n = 7; Fig. 1, A–D); and 3) patients with
poor compliance to calcium therapy, high PTH, and a tendency for low
serum calcium and high alkaline phosphate levels (PTH-H; n = 3).
Serum phosphorus (Fig. 1C) and TmP/GFR (Fig. 1E) were similar in all
three groups, whereas urinary calcium was significantly higher in the
calcium-compliant PTH-N group (P = 0.01; Fig. 1F). Two
weeks after cholecalciferol loading, serum calcium increased slightly
and insignificantly in all three groups. Serum phosphorus tended to
decrease in the PTH-H group, as did TmP/GFR. Plasma PTH remained higher
in the PTH-H group, compared with that in the PTH-N group
(P = 0.06). Vitamin D metabolites
25OHD levels were similar in the three groups before
cholecalciferol loading (Fig. 2A).
Whereas it increased in the control group by 85 ± 50%
(P = 0.01) and in the PTH-N group by 50 ± 41%
(P = 0.09), it did not change significantly in the
PTH-H group (17.8 ± 1.5%; P = 0.71).
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).
The control group had the highest basal levels of
24,25-(OH)2D. These levels were significantly
lower in the PTH-N group (P < 0.003) and the PTH-H
group (P < 0.002). After cholecalciferol loading they
rose to a similar extent in all three group (Fig. 2C).
1-OHase activity
1-OHase activity was estimated by the ratio of the
product/substrate [1,25-(OH)2D/25OHD]. In the
control group 1-OHase activity basally (0.30 ± 0.15%) and
that after loading (0.19 ± 0.05%) were similar. In the PTH-N
group, 1-OHase activity was significantly higher than in the control
group (P < 0.005; Fig. 2D); basal 1-OHase activity
(1.0 ± 0.5%) and that after loading (1.2 ± 1.0%) were
similar. In the PTH-H group, basal 1-OHase activity was even higher
than that in the control group (1.4 ± 0.5%; P <
0.05); after loading it increased further to 2.5 ± 0.5%. At that
point it was higher than the levels in the control group
(P < 0.01) and the PTH-N group (P <
0.02).
24-OHase activity
24-OHase activity was estimated by the product/substrate ratio
[24,25-(OH)2D/25OHD]. 24-OHase activity in the
control group was similar before (11.9 ± 2.8%) and after
(8.7 ± 0.5%) cholecalciferol loading (Fig. 2E). In the control
group, 24-OHase activity was 56 ± 28- and 48.9 ± 10.8-fold
the 1-OHase activity before and after loading, respectively (Fig. 2F). In the PTH-N group, basally (7.2 ± 1.5%) and after loading
(6.0 ± 1.8%), 24-OHase activity was lower than in the control
group, and 24-OHase activity was only 10.3 ± 7.9- and 8.6 ±
6.8-fold the 1-OHase activity before and after loading,
respectively. In the PTH-H group, basal 24-OHase activity (8.7 ±
4.5%) and that after loading (9.6 ± 2.5%) were intermediate
between PTH-N and control group activities, and only 6.0 ± 0.9-
and 3.9 ± 1.6-fold the 1-OHase activity. 24-OHase activity was
approximately 40% higher in the PTH-H group than in the PTH-N
group.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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To single out the role of the VDR, the protocol of this study was designed with special care to ensure that the calcium milieu would be as close as possible to normal. Toward that end, patients were prescribed enough oral calcium to normalize its levels, and normalization of PTH and phosphorus levels followed. We further identified patients who did not normalize PTH (PTH-H), and they underscored the additive role of PTH in vitamin D metabolism. The control group consisted of siblings of VDDR-II patients who might be heterozygote carriers of the disease. Such heterozygotes were previously shown to express normal VDR (6), to have in vitro VDR activity, and to be indistinguishable from normal subjects both clinically and biochemically (7).
VDR is expressed in the proximal convoluted tubules (16)
where 25OHD is hydroxylated to 1,25-(OH)2D and
24,25-(OH)2D. It is also the site of PTH
induction of tubular phosphate transport and 1-OHase expression. We
have previously shown that these effects of PTH are not mediated by the
VDR (11). We now show that the paradigm developed from VDR
knockout mice (5) and in vitro experiments
(4) also applies to humans. Trans-activation
function of 1,25-(OH)2D-liganded VDR regulates
reciprocally 1-OHase and 24-OHase activity.
As the most active metabolite of vitamin D, the levels of
1,25-(OH)2D are tightly regulated. It depends on
its synthesis by 1-OHase as well as on its conversion to
1,24,25-(OH)3 D by 24-OHase. Indirectly, it is
also influenced by 24-hydroxylation of 25OHD into
24,25-(OH)2D, and the latter may be
1-hydroxylated into 1,24,25-(OH)3D. The
capacity of these two enzymes is rather low. As shown by the control
group, only about 10% of 25OHD is 24-hydroxylated, and only about
0.3% is 1-hydroxylized. Thus, enzymatic activity can be estimated
with good approximation from the products/substrate ratios. Differences
between PTH-N and PTH-H groups allowed us to estimate the relative
contributions of VDR and PTH to vitamin D metabolism. It is interesting
that 25OHD levels increased more in the PTH-N group than in the PTH-H
group. This could be related to the increased metabolism of 25OHD to
1,25-(OH)2D due to secondary hyperparathyroidism
in the PTH-H group or through a novel effect of PTH on 25-OHase
activity. 1-OHase activity increased by 3- to 6-fold in the
VDR-defective subjects compared with the controls. A similar increase
in 1,25-(OH)2D levels was reported for the VDR
knockout mouse (5). 1-OHase activity increased by an
additional 40–100% in the PTH-H group, and at the same time, 24-OHase
activity was approximately 40% higher in the PTH-H group than in the
PTH-N group, apparently due to hyperparathyroidism. These results
exclude a role for the VDR in PTH-stimulated 1-hydroxylation
(11) and 24-hydroxylation. This clinical experiment
supports the working hypothesis that vitamin D metabolism in the human
is controlled in a similar fashion. Based on this analogy and the
present results, it is reasonable to assume that regulation by vitamin
D of 1-OHase and 24-OHase gene expression in humans occurs at the
transcriptional level.
Mouse experiments showed that regulations of 1-OHase by PTH and
calcitonin are also transcriptional events. Indeed, cloned 1-OHase
gene promoter confers responses to PTH, calcitonin and
1,25-(OH)2D (17) and cloned 24-OHase
gene promoter confers responses to 1,25-(OH)2D
(3). The present quantitative estimation suggests that
1,25-(OH)2D, through the VDR, has a direct effect
on enzymatic activity and is the major control mechanism for vitamin D
metabolism and that PTH exerts an additive effect.
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Footnotes |
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Received October 9, 2000.
Revised January 10, 2001.
Accepted January 14, 2001.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-hydroxilase in a transformed human
proximal tubule cell line: evidence of direct regulation of vitamin D
metabolism by calcium. Endocrinology. 140:2027–2034.,25-dihydroxvitamin
D3. Endocrinology. 138:2233–2240.-hydroxylase and vitamin D synthesis. Science. 277:1827–1830.,hydroxylase gene by
parathyroid hormone, calcitonin, and
1,25(OH)2D3 in intact
animals. Endocinology. 140:2224–2231.
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