Cloning of Human 25-Hydroxyvitamin D-1
-Hydroxylase and Mutations Causing Vitamin D-Dependent Rickets Type 1
Glenn K. Fu,
Dong Lin,
Martin Y. H. Zhang,
Daniel D. Bikle,
Cedric H. L. Shackleton,
Walter L. Miller and
Anthony A. Portale
Departments of Pediatrics (G.K.F., D.L., M.Y.H.Z., W.L.M., A.A.P),
Medicine (D.D.B., A.A.P.), and Dermatology (D.D.B.) Child Health
Research Center Veterans Affairs Medical Center (D.D.B.) San
Francisco, California 94121
Childrens Hospital Research
Institute (C.H.L.S.) Oakland, California 94609
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ABSTRACT
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The secosteroid hormone, 1,25-dihydroxyvitamin D
[1,25(OH)2D], plays a crucial role in normal
bone growth, calcium metabolism, and tissue differentiation. The key
step in the biosynthesis of 1,25-(OH)2D is its
1
-hydroxylation from 25-hydroxyvitamin D (25-OHD) in the kidney.
Because its expression in the kidney is very low, we cloned and
sequenced cDNA for 25-OHD-1
-hydroxylase (P450c1
) from human
keratinocytes, in which 1
-hydroxylase activity and mRNA expression
can be induced to be much greater. P450c1
mRNA was expressed at much
lower levels in human kidney, brain, and testis. Mammalian cells
transfected with the cloned P450c1
cDNA exhibit robust
1
-hydroxylase activity. The identity of the
1,25(OH)2D3 product
synthesized in transfected cells was confirmed by HPLC and gas
chromatography-mass spectrometry. The gene encoding P450c1
was
localized to chromosome 12, where the 1
-hydroxylase deficiency
syndrome, vitamin D-dependent rickets type 1 (VDDR-1), has been
localized. Primary cultures of human adult and neonatal keratinocytes
exhibit abundant 1
-hydroxylase activity, whereas those from a
patient with VDDR-1 lacked detectable activity. Keratinocyte P450c1
cDNA from the patient with VDDR-1 contained deletion/frameshift
mutations either at codon 211 or at codon 231, indicating that the
patient was a compound heterozygote for two null mutations. These
findings establish the molecular genetic basis of VDDR-1, establish a
novel means for its study in keratinocytes, and provide the sequence of
the key enzyme in the biological activation of vitamin D.
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INTRODUCTION
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The hormone 1,25-dihydroxyvitamin D [1,25(OH)2D]
plays an essential role in calcium metabolism, bone growth, and
cellular differentiation. Synthesis of 1,25(OH)2D from
its endogenous precursor, 25-hy-droxyvitamin D (25-OHD), is
catalyzed by 25-OHD-1
-hydroxylase (1
-hydroxylase), a
mitochondrial cytochrome P450 enzyme (1, 2, 3) that is subject to complex
regulation by PTH, calcium, 1,25(OH)2D, and phosphorus
(4, 5, 6, 7). The principal site of 1,25(OH)2D synthesis is the
proximal renal tubule (1, 8, 9, 10, 11). Serum concentrations of
1,25(OH)2D are decreased in patients with chronic renal
insufficiency and are greatly reduced yet detectable in anephric
patients (12, 13, 14, 15, 16). 25-OHD also is converted to 24,25-dihydroxyvitamin
D, a hormone whose physiological role is uncertain, by
25-OHD-24-hydroxylase (24-hydroxylase). The cDNAs for both the renal
24-hydroxylase (P450c24) and hepatic 25-hydroxylase (P450c25) have been
cloned, demonstrating that each is a unique mitochondrial cytochrome
P450 enzyme (17, 18, 19, 20).
Synthesis of 1,25(OH)2D is impaired in numerous disorders
including chronic renal insufficiency, renal tubular diseases, and
autosomal recessive vitamin D-dependent rickets type 1 (VDDR-1).
VDDR-1 (or pseudo-vitamin D-deficiency rickets) is characterized by
failure to thrive, muscle weakness, skeletal deformities, hypocalcemia,
secondary hyperparathyroidism, and greatly reduced serum concentrations
of 1,25(OH)2D despite normal concentrations of 25-OHD
(21, 22). These abnormalities are reversed by administration of
physiological amounts of 1,25-(OH)2D3. The
genetic defect is unknown but is presumed to result in defective renal
1
-hydroxylation of 25-OHD (23). Although such a defect in renal
tissue has not been demonstrated directly, cells isolated from human
placental decidua of patients with VDDR-1 fail to convert 25-OHD to
1,25(OH)2D, which suggests that 1
-hydroxylase in decidua
and kidney, or a regulator of its activity, are encoded by the same
gene (24). The mutation causing VDDR-1 has been mapped to chromosome
12q14 by linkage analysis (25, 26).
Because the activity and presumably mRNA content of renal
1
-hydroxylase are very low, we sought an alternative source of RNA
for its cloning. Keratinocytes, which synthesize vitamin D3
from endogenous 7-dehydrocholesterol upon exposure to UV light, also
synthesize 1,25(OH)2D from exogenous 25-OHD with high
activity in vitro (27, 28, 29). Such 1
-hydroxylase activity
is tightly regulated and coupled to the differentiation of these cells
(27, 28, 29). We now report the cloning of the cDNA for
25-OHD-1
-hydroxylase, designated P450c1
, from human
keratinocytes, its expression in transfected mammalian cells, and that
mutations in its gene cause VDDR-1.
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RESULTS AND DISCUSSION
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Cloning of the Human 1
-Hydroxylase cDNA
Our initial efforts to clone the P450c1
cDNA from vitamin
D-deficient rat kidney by cross-hybridization with an oligonucleotide,
based on the presumably conserved heme-binding site of mitochondrial
P450c24, were unsuccessful. As an alternative source of RNA, we used
human keratinocytes, which exhibit high 1
-hydroxylase activity
in vitro (27, 28). Efforts to screen a human keratinocyte
cDNA library, by probe hybridization and by expression cloning, were
unsuccessful. We then performed RT-PCR using multiple sets of
degenerate-sequence primers corresponding to the region of the
ferredoxin-binding and heme-binding sites of P450c24 and P450c25. One
set of primers (see Materials and Methods for details)
yielded a predominant cDNA fragment of
300 bp with a unique DNA
sequence homologous to both P450c24 and P450c25 (17, 18, 19, 20). This PCR
product was used to screen a human keratinocyte cDNA library, yielding
a partial-length, 1.9-kb clone, whose complete sequence was then
obtained by rapid amplification of cDNA ends (5'-RACE). The
full-length, 2.4-kb 1
-hydroxylase cDNA, P450c1
, encodes a protein
of 508 amino acids with predicted molecular mass of 56 kDa (Fig. 1A
). The predicted
topology is similar to that of known mitochondrial cytochrome P450
enzymes, with the NH2 terminus having characteristics of a
mitochondrial signal sequence (30). Within the heme-binding region, its
amino acid sequence identity is 73% and 65% to that of human P450c24
and P450c25, respectively (17, 18, 19, 20). However, the overall sequence
identity of P450c1
to that of other mitochondrial P450 enzymes is
limited: P450c25 (39%), P450c24 (30%), P450scc (32%), and P450c11ß
(33%) (Fig. 1B
).

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Figure 1. Human P450c1 cDNA
A, The full-length nucleotide and predicted amino acid sequences are
shown; the ferredoxin-binding site (beginning at base 1116) and
heme-binding site (beginning at base 1374) are underlined.
Human keratinocyte poly(A)+ RNA was reverse transcribed
into cDNA, which was PCR-amplified using degenerate sequence primers,
yielding a predominant 300-bp fragment. This PCR product was cloned,
sequenced, and used to screen a human keratinocyte cDNA library,
yielding a partial-length 1.9-kb clone containing the 3'-sequence
shown, 5'-RACE was then performed to yield a 650-bp nested PCR product,
which was cloned, sequenced, and ligated to the 5'-end of clone pDL1.9
containing the 3'-end of the cDNA. The full-length, 2.4-kb
1 -hydroxylase cDNA (Genbank accession number AF020192) encodes a
protein of 508 amino acids with predicted molecular mass of 56 kDa. B,
Sequence comparison of P450c1 with other human mitochondrial P450
sequences. The alignments were performed with the ClustalW program
(52). The top line depicts the sequence of P450c1 ,
followed by P450c25 (53), P450c24 (20), P450scc (54), and P450c11ß
(55). The 62 residues identical in all five sequences are designated by
an asterisk (*), the 68 highly conservative substitutions by
a colon (:), and the 32 moderately conservative
substitutions by a period (.). The conserved
ferredoxin-binding and heme-binding sites are underlined, as
in panel A.
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Tissue Distribution of P450c1
mRNA Expression
Northern blotting of keratinocyte RNA revealed a single P450c1
band of
2.5 kb (Fig. 2A
), but
ribonuclease protection assays did not detect the mRNA in other
tissues. Therefore, we used RT-PCR to amplify a specific 553-bp
fragment of the cDNA, which was then detected by Southern blotting.
This approach demonstrated expression of P450c1
mRNA in human adult
kidney (not shown) and fetal kidney, brain, and testis, all at less
than 1/25 of the level of its expression in cultured neonatal
keratinocytes (Fig. 2B
). Expression was not detectable in human
placenta or fetal liver, heart, adrenal, or lung, whereas the ß-actin
RT-PCR product was obtained from all tissues examined. The absence of
detectable P450c1
mRNA in placental tissue is surprising, since
1
-hydroxylase activity has been observed in human placental decidual
cells (24). However, it has been suggested that 1
-hydroxylase
activity in placental trophoblastic tissue is nonenzymatic, since it
can be abolished by antioxidants and is unaffected by ketoconazole,
unlike that in kidney (31). Southern blotting of human genomic DNA
probed with a small fragment of cDNA under low-stringency conditions
revealed a single band in each restriction digest, which suggests that
there is only one copy of the gene for P450c1
(not shown).
While the mRNA abundance in the cultured keratinocytes is relatively
great, it should be noted that these cells were grown in serum-free
calcium-deficient medium, which maximizes their 1
-hydroxylase
activity (27) and presumably their P450c1
mRNA. Similarly, the
relatively low abundance of P450c1
mRNA in kidney tissue presumably
reflects the small contribution of P450c1
mRNA from proximal tubule
cells, the site of 1
-hydroxylase activity, relative to that of
remaining renal tissue, where 1
-hydroxylase activity is absent.
Expression of P450c1
cDNA in Mammalian Cells
To determine whether the cloned P450c1
indeed catalyzes
1
-hydroxylase activity, we expressed the cDNA in mammalian cells.
The enzymatic activity of all mitochondrial P450 enzymes requires the
expression of the electron transport proteins, ferredoxin and
ferredoxin reductase (32). Therefore, we transfected a plasmid
expressing the full-length P450c1
cDNA into cultured mouse Leydig
MA-10 cells (33), which contain both of these electron transfer
proteins and which exhibit marked enzymatic activity when expressing
P450c11AS, another mitochondrial P450 found in low abundance (34).
Minimal 1
-hydroxylase activity was detected both in untransfected
MA-10 cells and in those transfected with empty vector. MA-10 cells
expressing the P450c1
cDNA converted 25-OHD3 to putative
1,25(OH)2D3 at levels
200 times above
control transfections (Fig. 3A
). The
1
-hydroxylated product coeluted with authentic
1,25(OH)2D3 on HPLC using two solvent systems
that distinguish it from other metabolites of vitamin D (35). Thus, the
cloned cDNA encodes an enzyme, P450c1
, with robust 1
-hydroxylase
activity. Lineweaver-Burke analysis of the cloned enzymes kinetics
yielded an apparent Michaelis-Menten constant (Km) of
2.6 x 10-7 M (Fig. 3B
). This value,
obtained in whole cells, may not represent the true kinetic constant,
as substrate access to the inner mitochondrial membrane may be rate
limiting (36, 37). However, this value is comparable to that obtained
previously in chick and pig kidney and human placenta and skin (27, 38, 39, 40).

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Figure 3. Characterization of 1 -Hydroxylase Activity
A, 1 -hydroxylase activity, expressed as picograms of
1,25(OH)2D3 produced per 10-cm plate of
cultured cells. The y-axis (logarithmic scale) begins at
the detection limit of 1,25(OH)2D per plate. The three
left bars depict MA-10 cells transfected with:
1 , vector expressing 1 -hydroxylase cDNA; V, empty vector; or
Un, untransfected cells. The three right bars depict
keratinocytes from: N, neonatal foreskin; Ad, healthy adult subject; or
Pt, patient with VDDR-1. MA-10 cells expressing P450c1 converted
25-OHD3 to 1,25(OH)2D3 at levels
200 times above control transfections. Minimal 1 -hydroxylase
activity was detected in untransfected MA-10 cells, but no activity was
detected (ND) in keratinocytes from the patient with VDDR-1. MA-10
cells were transfected at 50% confluence with 2 µg/ml DNA using
adenovirus-mediated transfection. Keratinocytes were cultured in KGM
containing 0.07 mM calcium. Cells were incubated with 0.1
µM 25-OHD3 for 1 h in serum-free medium,
and the 1 -hydroxylated product was determined by RRA after
extraction and C-18 and silica SepPak chromatography (49). Each
bar represents the mean and range of activity determined
in two separate transfections of MA-10 cells or two separate cultures
of keratinocytes, each assayed in duplicate. Identity and
quantification of the assayed product as authentic
1,25(OH)2D3 was confirmed by HPLC. B,
Lineweaver-Burke plot of 1 -hydroxylase activity in MA-10 cells
expressing the cloned P450c1 . C, GC/MS selected-ion recording of
five ions diagnostic for the trimethylsilyl ether of
1,25(OH)2D3 (41). The left panel
represents authentic 1,25(OH)2D3, and the
right panel represents that biosynthesized in MA-10
cells expressing P450c1 . To conserve space, only the central
portions of the recordings showing the major pyro- form at 19.5 min is
depicted. The ions found and their intensities and retention times are
identical.
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To prove the identity of the assayed product as authentic
1,25(OH)2D3, a
1,25(OH)2D3 reference standard and the material
biosynthesized (230 ng) in transfected MA-10 cells were
trimethylsilylated and analyzed by gas chromatography/mass spectrometry
(GC/MS). Due to limited amount of material, both samples were analyzed
by selected ion monitoring of five representative ions of
1,25(OH)2D3 (see Ref. (41) for published
spectrum): m/z 632 (molecular ion), m/z 542 (M -90, loss of
trimethylsilanol), m/z 501 (M -131, loss of terminal three carbons of
side chain with trimethylsilylated 25-hydroxyl), m/z 452 (loss of
two trimethylsilanols), and m/z 131 (base peak, the fragment
representing the terminal part of the side chain). Analysis of both
authentic and biosynthesized 1,25-(OH)2D3 gave
rise to two chromatographic peaks of retention time 19.5 min (major)
and 21.3 min (minor), respectively, which represent the pyro- and
isopyro- forms of the ring B recyclized hormone (41). Proof of identify
of biosynthesized 1,25(OH)2D3 was established
by the presence of the appropriate ions, with intensities and retention
time identical to those of authentic hormone (Fig. 3C
). Further
confirmation of identity was achieved by obtaining a full mass spectrum
of the hormone.
Since the surface area of skin is large and its in vitro
1
-hydroxylase activity high relative to that in kidney, the question
arises as to whether 1,25(OH)2D produced in skin
contributes to the maintenance of its serum concentration (27). The
1,25(OH)2D produced in skin is rapidly degraded, and its
export from the cell is very inefficient (27). Furthermore,
1
-hydroxylase activity in skin is much more sensitive to suppression
by exogenous 1,25(OH)2D3 than that in kidney
(27), which suggests that at physiological concentrations of
1,25-(OH)2D, its production by skin is inhibited. However,
the skin can contribute significant amounts of 1,25-(OH)2D
to the circulation when renal function is absent or greatly decreased,
provided that sufficient amounts of 25-OHD are provided (42, 43).
Mutations in P450c1
Causing VDDR-1
Keratinocytes from patients with VDDR-1 have not been studied
previously. Primary cultures of keratinocytes from a patient with
clinical and laboratory features characteristic of VDDR-1 (see
Materials and Methods and Table 1
), failed to convert 25-OHD3
to 1,25-(OH)2D3 in amounts above assay
background, whereas keratinocytes from adult and neonatal tissue,
examined in an identical fashion, had 1
-hydroxylase activity
1000
times and
3000 times, respectively, above assay background (Fig. 3A
). Since VDDR-1 is presumed to result from a defect in renal
1
-hydroxylase, the failure to detect its activity in the patients
keratinocytes strongly suggests that renal and keratinocyte
1
-hydroxylase activities are encoded by the same gene, as suggested
by our finding of one size of mRNA and expression of the same cDNA in
kidney, keratinocytes, and other tissues.
To prove that the P450c1
gene expressed in keratinocytes is the gene
mutated in VDDR-1, we sought inactivating mutations in P450c1
cDNA
prepared from the patients keratinocyte mRNA. Using primers based on
our human P450c1
sequence (Fig. 1
), RT-PCR of mRNA from the
patients keratinocytes yielded cDNA of
2.4 kb, demonstrating that
the mutation was not a gene deletion or a severe promoter mutation.
Sequencing of multiple cDNA clones revealed that all clones had a
deletion/frameshift mutation either at codon 211 or at codon 231 (Fig. 4
), but no clones had both mutations.
Each of these mutations leads to the creation of a premature TGA
transitional stop codon after reading 233 amino acids. Although the
RT-PCR of the patients keratinocyte cDNA was performed in two
fragments, both mutations lie on the same fragment. Each frameshift
created a novel restriction endonuclease cleavage site; digestion of
multiple cDNA clones confirmed that all clones carried either one
mutation or the other, but no clones carried both, indicating that the
patient was a compound heterozygote, having inherited a different
frame-shift mutation from each parent (Fig. 4
). Unfortunately, neither
parent was available for study. Thus, the gene encoding the cloned
keratinocyte vitamin D 1
-hydroxylase cDNA is homozygously disrupted
in VDDR-1, giving rise to a severely truncated protein that cannot bind
heme and thus cannot have P450 catalytic activity. Screening of a panel
of rodent/human somatic cell hybrid cell lines demonstrated that the
gene for P450c1
lies on chromosome 12, where the mutation causing
VDDR-1 has previously been mapped (25, 26). Thus, our findings that
cloned keratinocyte P450c1
confers authentic 1
-hydroxylase
activity, and that its disruption causes VDDR-1, demonstrate that the
keratinocyte and renal 1
-hydroxylase enzymes are encoded by the
same gene and establish the molecular genetic basis of VDDR-1.
Furthermore, our studies demonstrate that P450c1
can be
characterized both enzymatically and genetically in cultured
keratinocytes.

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Figure 4. Mutations Causing VDDR-1
A, Sequence of cDNA from normal (left) and patient
(right) keratinocytes in the region of codon 211.
Deletion of a G (arrow) changes the normal sequence
TGCCTGGAGGCT to TGCCTGAGGCT, destroying an EcoRII/Bst NI
site (CCNGG) and creating the Bsu 36I recognition sequence CCTNAGG. B,
Normal (left) and patient (right)
sequence in the region of codon 231. Deletion of a C
(arrow) changes the normal sequence GTGTCCACG to
GTGTCACG, creating the Tsp 45I site GTNAC. C, Confirmation of compound
heterozygosity. A 796-bp fragment containing both codon 211 and codon
231 was amplified from patient keratinocyte RNA using primers GF41 and
GF21. The four left hand lanes show digestion with Bsu
36I (top panel) and Tsp 45I (bottom
panel); the right hand lanes are the same
samples undigested and DNA size markers. Two independently prepared
samples of patient keratinocyte DNA could be half-digested by extensive
treatment with each enzyme, but keratinocyte cDNA from both a normal
control and the cDNA clone remained uncut.
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VDDR-1 is a rare disorder; however, defective 1
-hydroxylation of
vitamin D is characteristic of chronic renal insufficiency and other
renal tubular disorders. The cloning of the cDNA for 1
-hydroxylase,
the key enzyme in the metabolic activation of vitamin D, will permit
the detailed study of the molecular genetic basis of its regulation in
health and disease.
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MATERIALS AND METHODS
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Cell Culture
Keratinocytes were isolated from human neonatal foreskins by
incubation with 25 U/ml Dispase (Collaborative Biomedical Products,
Bedford, MA) in Hanks basal salt solution, calcium- and magnesium
free, containing 0.05 mg/ml gentamycin, at 4 C overnight. The epidermis
was separated from the remaining tissue and incubated in 0.05% trypsin
for 15 min. Cells were centrifuged, suspended in keratinocyte growth
medium (KGM; Clonetics, San Diego, CA) containing 0.07 mM
calcium, plated into T-185 plastic flasks, and grown at 37 C, under 5%
CO2/95% air. Keratinocytes were isolated from human skin
obtained by biopsy by incubation with Dispase and trypsin as above.
Cells were suspended in keratinocyte serum-free medium (GIBCO-BRL,
Grand Island, NY) containing 5 ng/ml epidermal growth factor
(GIBCO-BRL) and 0.09 mM calcium, plated into T-25 plastic
flasks, and grown as above. Keratinocytes were passaged at
80%
confluence, and preconfluent cultures between the second to fourth
passage used for subsequent procedures. Mouse Leydig MA-10 cells (33)
(American Type Culture Collection, Rockville, MD) were maintained in
Waymouth medium containing 2.24 g/liter NaHCO3, 0.35
g/liter glutamine, and 10% fortified bovine calf serum (Hyclone,
Logan, UT) as previously described (44). Cells between the fourth to
tenth passage were plated at a density of 1.5 x 106
per 10-cm plate and incubated for 36 h before transfection.
Construction and Screening of cDNA Library
Using poly(A)+ RNA obtained from preconfluent
neonatal keratinocytes, a unidirectional, oligo(dT)-primed,
size-selected (>1000 bp) cDNA library was prepared in the mammalian
expression vector pcDNA3 (Invitrogen, Carlsbad, CA). The unamplified
library size was 1.2 x 106 recombinants and was
amplified for use in subsequent procedures.
Poly(A)+ RNA (0.5 µg) isolated from neonatal
keratinocytes was reverse transcribed into cDNA using 20 µl 10
mM Tris-HCl (pH 8.3), 50 mM KCl, 5
mM MgCl2, 1 mM each of
deoxynucleotide triphosphate, 2.5 U of Moloney murine leukemia virus
(MMLV) reverse transcriptase (Perkin-Elmer, Foster City, CA), and 2.5
mM of random primers. PCR amplification of the
single-stranded cDNA product was performed using Taq
polymerase (Perkin-Elmer) and multiple sets of degenerate-sequence
oligonucleotide primers based on the relatively conserved regions of
ferredoxin-binding and heme-binding regions of P450c24 and P450c25
(17, 18, 19, 20). The primers, 5'-CTSCTSAARGCYGTSATYAARGA-3' and
5'-TGYMTBGGYCGCCGCMTGGCYGAR-3' (where R = G or A, Y = C or T,
S = G or C, M = A or C, and B = C, T, or G), yielded a
predominant PCR product of
300 bp. Degeneracy of these primers was
decreased slightly based on a partial amino acid sequence of rat
1
-hydroxylase reported in preliminary form (45). PCR was performed
in a Perkin-Elmer DNA Thermal Cycler under the following program: 94 C,
45 sec; 60 C, 45 sec; 72 C, 30 sec for 30 cycles. The PCR product was
extracted from gels, purified through a Centricon-100 filter (Amicon,
Beverly, MA), subcloned into vector pCR2.1 (Invitrogen), and sequenced
by the dideoxy chain-termination technique using Sequenase (United
States Biochemical, Cleveland, OH) (46). This clone was used to screen
the human keratinocyte cDNA library, yielding 18 positive colonies.
After rescreening, representative clones were subcloned into pcDNA3 and
sequenced.
5'-RACE
Poly(A)+ mRNA (1 µg) isolated from neonatal
keratinocytes was reverse transcribed using 100 ng of primer DR2,
5'-GTGACACAGAGTGACCAGCATAT- 3' (bases 12261248 in Fig. 1
), and
Superscript reverse transcriptase (GIBCO-BRL), Escherichia
coli DNA ligase (20 U), E. coli DNA polymerase (80 U),
and E. coli RNase H (4 U). The resulting double-stranded
cDNA was ligated to 10 µg of the double-stranded adaptor GF6, 5'-
CCTCACGCTGCAGAAATTCCAGACTGAACCTTGAT-3', corresponding to bases
-160/-126 of the human P450scc promoter (47), using 10 U of T4 DNA
ligase (Boeringer-Mannheim, Indianapolis, IN) at 4 C for 24 h.
This material was PCR amplified using primers GF6 and GF21,
5'-GCAAACATCTGGTCCCAGTCT- 3' (bases 777797 in Fig. 1
), with the
program: 94 C, 40 sec; 60 C 40 sec; 72 C, 50 sec; for 30 cycles. A
second nested PCR amplification was performed using GF6 and GF29,
5'-CAGCCCAAGCGCGAGCCGAG-3' (bases 619638 in Fig. 1
) under the same
conditions. The resulting 650-bp PCR product was subcloned into pCR2.1
and sequenced. The full-length cDNA was assembled by ligating the RACE
clone containing the 5'-end of the cDNA into clone pDL1.9 containing
the 3'-end of the cDNA, through the EcoRI and
SacII sites.
Characterization of the P450c1
mRNA
For Northern blotting, 30 µg of total RNA from keratinocytes
was size-fractionated on a 0.7% formaldehyde-agarose gel, blotted to a
nylon membrane (Amersham, Arlington Heights, IL), and probed with a
340-bp KpnI fragment of the P450c1
cDNA labeled with
[32P]dCTP by random priming. Hybridization was performed
for 18 h at 60 C. For analysis of tissue distribution, 1 µg
total RNA from various human tissues was used to synthesize cDNA using
MMLV-RT and random primers. PCR amplification of the single-stranded
cDNA product was performed using primers DF2, 5'-ACGCTGTTGACCATGGC-3',
and DR2 for 30 cycles at 94 C, 45 sec; 60 C, 45 sec; 72 C, 30 sec; to
yield a 553-bp 1
-hydroxylase cDNA fragment. PCR also was performed
using ß-actin primers to yield a 439-bp actin cDNA fragment as
control. The amplified products were separated on 1% agarose gel,
blotted to nylon membrane, and probed with [32P]P450c1
or actin cDNA probes.
Expression of Human P450c1
cDNA and Characterization of the
1,25(OH)2D3 Product
The full-length P450c1
cDNA was subcloned into pcDNA3, which
contains the cytomegalovirus promoter for expression of DNA in
mammalian cells. MA-10 cells at 5060% confluence were transfected
with 2 µg/ml plasmid DNA using adenovirus-mediated transfection
modified from that described (48). Seventy-two hours after
transfection, cells were transferred to serum-free medium and incubated
with 0.1 µM chromatographically purified
25-OHD3 for 1 h. Cells and medium were extracted
with acetonitrile, and the 1
-hydroxylated product was determined in
duplicate by RRA after C-18 and silica SepPak chromatography (49). To
confirm quantification of the assayed product, after SepPak
chromatography a fraction of selected samples was subjected to
sequential HPLC using a Zorbax Sil column (0.46 x 25 cm) (Dupont
Instruments, Wilmington, DE) at a flow rate of 2.0 ml/min, equilibrated
first in isopropanol-hexane (11:89) and then in
isopropanol-dichloromethane (5:95) (35). The paired values for each
sample, quantitated by RRA, did not differ significantly from one
another. The identity of the assayed product as authentic
1,25(OH)2D3 was determined by two methods:
MA-10 cells expressing P450c1
were incubated with 200,000 dpm of
chromatographically purified [3H]25-OHD for 1 h.
After extraction and SepPak chromatography, the radiolabeled
1
-hydroxylated product was subjected to sequential HPLC in the
solvent systems described. GC/MS of the assayed product also was
performed using a Hewlett-Packard 5970 instrument housing a DB 17 fused
silica capillary column as described (41).
Patient with VDDR-1
A caucasian female first came to medical attention at the age of
9 months because of respiratory distress. Radiographic examinations
revealed osteopenia and metaphyseal abnormalities consistent with
rickets, and treatment was initiated with Vitamin D2, 4000
U/day. At age 13 months, the patients weight and height were below
the fifth percentile for age. The skin was normal but she had developed
no deciduous teeth; she had enlargement of the costochondral junctions
of the ribs, moderate genu varus, enlargement of the wrists and ankles,
and inability to sit, crawl, or stand. These clinical findings and the
data in Table 1
were characteristic of VDDR-1 (21, 22). At age 14
months, therapy with 1,25-(OH)2D3 and calcium
carbonate was initiated with doses adjusted to maintain normocalcemia.
The patient was able to stand at age 18 months and to walk at 23
months. At 54 months of age, genu varus is minimal, but the patients
height and weight remain below the 5th percentile for age although
growth velocity has been normal.
Characterization of P450c1
cDNA in VDDR-1
Total RNA was isolated from keratinocytes from the patient with
VDDR-1 and from a healthy adult as control. After RT with random
primers, cDNA from the patient with VDDR-1 was PCR amplified in two
overlapping pieces with primers GF41,
5'-CTGACCCAGACCATGACCCAGACCCTCAA-3', and GF21 for the 5'-half (bases
1797) and DF2 and GF42, 5'-GGTCAGATAGGCATTAGGGGAAGATGT-3', for the
3'-half (bases 705-1619). The PCR products were cloned into pCR2.1 and
several clones were picked for sequencing. Complementary cDNA from the
healthy control was PCR amplified using primers GF41 and GF42, the PCR
product was cloned into pCR2.1, and two clones were picked for
sequencing. To confirm the mutations detected by DNA sequencing, the
RT-PCR products from the patient and normal control were digested with
either Tsp 45I (New England Biolabs, Beverly, MA) or Bsu 36I (Promega,
Madison, WI), and the products were separated on a 1% agarose gel. The
chromosomal location of the P450c1
gene was determined by PCR
amplification of genomic DNA samples in NIGMS rodent/human somatic cell
hybrid mapping panel 2 (50, 51) (Coriell Institute, Camden, NJ). PCR,
performed with primers GF41 and GF21, yielded a 2-kb genomic
fragment.
 |
ACKNOWLEDGMENTS
|
---|
We thank John Forsayeth for help with adenovirus-mediated
transfection and Sally Pennypacker for help with keratinocyte
culture.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Anthony A. Portale, M.D., University of California San Francisco, 533 Parnassus Avenue, Room U-585, San Francisco, California 94143.
Dr. Portale was supported by the Department of Pediatrics, grants from
the University of California, San Francisco Academic Senate and
Research Evaluation and Allocation Committee, and generous gifts from
the Carmel David Trust. Dr. Miller was supported by NIH Grants DK-37922
and DK-42154 and a grant from the March of Dimes. Dr. Fu was supported
by Pediatric Endocrinology Training Grant DK-07161 and Dr. Lin by the
UCSF Child Health Research Center Grant HD-28825. Dr. Bikle was
supported by NIH Grants RO1 AR-38386 and PO1 AR-39448.
Received for publication August 20, 1997.
Revision received September 16, 1997.
Accepted for publication September 17, 1997.
 |
REFERENCES
|
---|
-
Fraser DR, Kodicek E 1970 Unique biosynthesis by kidney
of a biologically active vitamin D metabolite. Nature 228:764766[Medline]
-
Ghazarian JG, Jefcoate CR, Knutson JC, Orme-Johnson WH,
DeLuca HF 1974 Mitochondrial cytochrome P450. A component of chick
kidney 25-hydroxycholecalciferol-1alpha-hydroxylase. J Biol Chem 249:30263033[Abstract/Free Full Text]
-
Pedersen JI, Ghazarian JG, Orme-Johnson NR, DeLuca HF 1976 Isolation of chick renal mitochondrial ferredoxin active in the
25-hydroxyvitamin D3-1-hydroxylase system. J Biol Chem 251:39333941[Abstract]
-
Fraser DR 1980 Regulation of the metabolism of vitamin D.
Physiol Rev 60:551613[Free Full Text]
-
Reichel H, Koeffler HP, Norman AW 1989 The role of the
vitamin D endocrine system in health and disease. N Engl J
Med 320:980991[Medline]
-
Portale AA, Halloran BP, Morris Jr RC 1989 Physiologic
regulation of the serum concentration of 1,25-dihydroxyvitamin D by
phosphorus in normal men. J Clin Invest 83:14941499[Medline]
-
Feldman D, Malloy PJ, Gross C 1996 Vitamin D: metabolism and
action. In: Marcus R, Feldman D, Kelsey J (eds) Osteoporosis. Academic
Press, San Diego, pp 205235
-
Gray R, Boyle I, DeLuca HF 1971 Vitamin D metabolism: the
role of kidney tissue. Science 172:12321234[Medline]
-
Gray RW, Omdahl JL, Ghazarian JG, DeLuca HF 1972 25-hydroxycholecalciferol-1
-hydroxylase. J Biol Chem 247:75287532[Abstract/Free Full Text]
-
Brunette MG, Chan M, Ferriere C, Roberts KD 1978 Site of
1,25(OH)2 vitamin D3 synthesis in the kidney.
Nature 276:287289[Medline]
-
Kawashima H, Torikai S, Kurokawa K 1981 Localization of
25-hydroxyvitamin D 1
-hydroxylase and 24-hydroxylase along the rat
nephron. Proc Natl Acad Sci USA 78:11991203[Abstract]
-
Brumbaugh PF, Haussler DH, Bressler R, Haussler MR 1974 Radioreceptor assay for 1,25-dihydroxyvitamin D3. Science 183:10891091[Medline]
-
Eisman JA, Hamstra AJ, Kream BE, DeLuca HF 1976 1,25-dihydroxyvitamin D in biological fluids: a simplified and
sensitive assay. Science 193:10211023[Medline]
-
Portale AA, Booth BE, Tsai HC, Morris Jr RC 1982 Reduced
plasma concentration of 1,25-dihydroxyvitamin D in children with
moderate renal insufficiency. Kidney Int 21:627632[Medline]
-
Lambert PW, Stern PH, Avioli RC, Brackett NC, Turner RT,
Greene A, Fu IY, Bell NH 1982 Evidence for extrarenal production of
1,25-dihydroxyvitamin D in man. J Clin Invest 69:722725[Medline]
-
Pitts TO, Piraino BH, Mitro R, Chen TC, Segre GV,
Greenberg A, Puschett JB 1988 Hyperparathyroidism and
1,25-dihydroxyvitamin D deficiency in mild, moderate, and severe renal
failure. J Clin Endocrinol Metab 67:876881[Abstract]
-
Usui E, Noshiro M, Okuda K 1990 Molecular cloning of cDNA for
vitamin D3 25-hydroxylase from rat liver mitochondria. FEBS
Lett 262:135138[CrossRef][Medline]
-
Su P, Rennert H, Shayiq RM, Zheng YM, Addya S, Strauss JF3,
Avadhani NG 1990 A cDNA encoding a rat mitochondrial cytochrome P450
catalyzing both the 26-hydroxylation of cholesterol and
25-hydroxylation of vitamin D3: gonadotropic regulation of
the cognate RNA in ovaries. DNA Cell Biol 9:657667[Medline]
-
Ohyama Y, Noshiro M, Okuda K 1991 Cloning and expression of
cDNA encoding 25-hydroxyvitamin D3 24-hydroxylase. FEBS
Lett 278:195198[CrossRef][Medline]
-
Chen KS, Prahl JM, DeLuca HF 1993 Isolation and expression of
human 1,25-dihydroxyvitamin D3 24-hydroxylase cDNA. Proc
Natl Acad Sci USA 90:45434547[Abstract]
-
Scriver CR, Reade TM, DeLuca HF, Hamstra AJ 1978 Serum
1,25-dihydroxyvitamin D levels in normal subjects and in patients with
hereditary rickets or bone disease. N Engl J Med 299:976979[Abstract]
-
Delvin EE, Glorieux FH, Marie PJ, Pettifor JM 1981 Vitamin D
dependency: replacement therapy with calcitriol. J Pediatr 99:2634[Medline]
-
Fraser D, Kooh SW, Kind HP, Holick MF, Tanaka Y, DeLuca HF 1973 Pathogenesis of hereditary vitamin-D-dependent rickets. An inborn
error of vitamin D metabolism involving defective conversion of
25-hydroxyvitamin D to 1 alpha,25-dihydroxyvitamin D. N Engl
J Med 289:817822[Medline]
-
Glorieux FH, Arabian A, Delvin EE 1995 Pseudo-vitamin D
deficiency: absence of 25-hydroxyvitamin D-1 alpha-hydroxylase activity
in human placenta decidual cells. J Clin Endocrinol Metab 80:22552258[Abstract]
-
Labuda M, Morgan K, Glorieux FH 1990 Mapping autosomal
recessive vitamin D dependency type I to chromosomal 12q14 by linkage
analysis. Am J Hum Genet 47:2836[Medline]
-
Labuda M, Fujiwara TM, Ross MV, Morgan K, Garcia-Heras J,
Ledbetter DH, Hughes MR, Glorieux FH 1992 Two hereditary defects
related to vitamin D metabolism map to the same region of human
chromosome 12q1314. J Bone Miner Res 7:14471453[Medline]
-
Bikle DD, Nemanic MK, Gee E, Elias P 1986 1,25-dihydroxyvitamin D3 production by human keratinocytes.
J Clin Invest 78:557566[Medline]
-
Bikle DD, Nemanic MK, Whitney JO, Elias PW 1986 Neonatal human
foreskin keratinocytes produce 1,25-dihydroxyvitamin D3.
Biochemistry 25:15451548[Medline]
-
Bikle DD, Pillai S 1993 Vitamin D, calcium, and epidermal
differentiation. Endocr Rev 14:319[Medline]
-
Douglas MG, McCammon MT, Vassarotti A 1986 Targeting proteins
into mitochondria. Microbiol Rev 50:166178
-
Hollis BW, Iskersky VN, Chang MK 1989 In vitro
metabolism of 25-hydroxyvitamin D3 by human trophoblastic
homogenates, mitochondria, and microsomes: lack of evidence for the
presence of 25-hydroxyvitamin D3-1
- and
24R-hydroxylases. Endocrinology 125:12241230[Abstract]
-
Miller WL 1988 Molecular biology of steroid hormone synthesis.
Endocr Rev 9:295318[Medline]
-
Ascoli M 1981 Characterization of several clonal lines of
cultured leydig tumor cells: gonadotropin receptors and steroidogenic
responses. Endocrinology 108:8895[Abstract]
-
Fardella CE, Hum DW, Rodriguez H, Zhang G, Barry FL, Ilicki A,
Bloch CA, Miller WL 1996 Gene conversion in the CYP11B2 gene encoding
P450c11AS is associated with, but does not cause, the syndrome of
corticosterone methyloxidase II deficiency. J Clin Endocrinol
Metab 81:321326[Abstract]
-
Napoli JL, Koszewski NJ, Horst RL 1986 Isolation and
identification of vitamin D metabolites. Methods Enzymol 123:127140[Medline]
-
Lin D, Sugawara T, Strauss JF, Clark BJ, Stocco DM, Saenger P,
Rogoi A, Miller WL 1995 Role of steroidogenic acute regulatory protein
in adrenal and gonadal steroidogenesis. Science 267:18281831[Medline]
-
Bose HS, Sugawara T, Strauss JF, Miller WL 1996 The
pathophysiology and genetics of congenital lipoid adrenal hyperplasia.
N Engl J Med 335:18701878[Abstract/Free Full Text]
-
Bikle DD, Rasmussen H 1974 A comparison of the metabolism of
25-hydroxyvitamin D3 by chick renal tubules, homogenates, and
mitochondria. Biochim Biophys Acta 362:439447[Medline]
-
Delvin EE, Dussault M 1985 Kinetics of kidney mitochondrial
25-hydroxycholecalciferol 1-hydroxylase in vitamin D-repleted weanling
guinea pigs. Arch Biochem Biophys 240:337344[Medline]
-
Delvin EE, Arabian A 1987 Kinetics and regulation of
25-hydroxycholecalciferol 1 alpha-hydroxylase from cells isolated from
human term decidua. Eur J Biochem 163:659662[Abstract]
-
Shackleton CHL, Roitman E, Whitney J 1980 Urinary metabolites
of vitamin D3. J Steroid Biochem 11:523529[CrossRef]
-
Littledike ET, Horst RL 1982 Metabolism of vitamin
D3 in nephrectomized pigs given pharmacological amounts of
vitamin D3. Endocrinology 111:20082013[Abstract]
-
Halloran BP, Schaefer P, Lifschitz M, Levens M, Goldsmith
RS 1984 Plasma vitamin D metabolite concentrations in chronic renal
failure: effect of oral administration of 25-hydroxyvitamin
D3. J Clin Endocrinol Metab 59:10631069[Abstract]
-
Hum DW, Staels B, Black SM, Miller WL 1993 Basal
transcriptional activity and cyclic adenosine 3',5'-monophosphate
responsiveness of the human cytochrome P450scc promoter transfected
into MA-10 Leydig cells. Endocrinology 132:546552[Abstract]
-
St-Arnaud R, Moir M, Messerlian S, Glorieux FH 1996 Molecular
cloning and characterization of a cDNA for vitamin D 1
-hydroxylase.
J Bone Miner Res 11:S124 (Abstract)
-
Sanger F, Nicklen S, Coulson AR 1977 DNA sequencing with
chain-terminating inhibitors. Proc Natl Acad Sci USA 74:54635467[Abstract]
-
Moore CCD, Brentano ST, Miller WL 1990 Human P450scc gene
transcription is induced by cyclic AMP and repressed by
12-O-tetradecanoylphorbol-13-acetate and A23187 through
independent cis elements. Mol Cell Biol 10:60136023[Medline]
-
Forsayeth JR, Garcia PD 1994 Adenovirus-mediated transfection
of cultured cells. BioTechniques 17:354359[Medline]
-
Reinhardt TA, Horst RL, Orf JW, Hollis BW 1984 A microassay
for 1,25-dihydroxyvitamin D not requiring high performance liquid
chromatography: application to clinical studies. J Clin Endocrinol
Metab 58:9198[Abstract]
-
Drwinga HL, Toji LH, Kim CH, Greene AE, Mulivor RA 1993 NIGMS
human/rodent somatic cell hybrid mapping panels 1 and 2. Genomics 16:311314[CrossRef][Medline]
-
Dubois BL, Naylor SL 1993 Characterization of NIGMS
human/rodent somatic cell hybrid mapping panel 2 by PCR. Genomics 16:315319[CrossRef][Medline]
-
Thompson JD, Higgins DG, Gibson TJ 1994 CLUSTAL W: improving
the sensitivity of progressive multiple sequence alignment through
sequence weighting, position-specific gap penalties and weight matrix
choice. Nucleic Acids Res 22:46734680[Abstract]
-
Cali JJ, Russell DW 1991 Characterization of human sterol
27-hydroxylase. A mitochondrial cytochrome P-450 that catalyzes
multiple oxidation reaction in bile acid biosynthesis. J Biol Chem 266:77747778[Abstract/Free Full Text]
-
Chung BC, Matteson KJ, Voutilainen R, Mohandas TK, Miller WL 1986 Human cholesterol side-chain cleavage enzyme, P450scc: cDNA
cloning, assignment of the gene to chromosome 15, and expression in the
placenta. Proc Natl Acad Sci USA 83:89628966[Abstract]
-
Mornet E, Dupont J, Vitek A, White PC 1989 Characterization of two genes encoding human steroid 11
beta-hydroxylase (P-450(11) beta). J Biol Chem 264:2096120967[Abstract/Free Full Text]
-
Fu GK, Portale AA, Miller WL 1997 Complete structure of the
human gene for the vitamin D 1
-hydroxylase, P450c1
. DNA Cell Biol 16:14991507[Medline]