| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
-Hydroxylase and Mutations Causing Vitamin D-Dependent Rickets Type 1
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
Children’s Hospital Research
Institute (C.H.L.S.) Oakland, California 94609
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-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. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-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.
|
RESULTS AND DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-Hydroxylase cDNA 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).
|
mRNA Expression
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).
|
-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 enzyme’s 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).
|
). 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 patient’s
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.
|
gene expressed in keratinocytes is the gene
mutated in VDDR-1, we sought inactivating mutations in P450c1 cDNA
prepared from the patient’s keratinocyte mRNA. Using primers based on
our human P450c1 sequence (Fig. 1), RT-PCR of mRNA from the
patient’s 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 patient’s 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.
|
-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. |
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
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 1226–1248 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 777–797 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 619–638 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 50–60% 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 patient’s 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 patient’s
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
1–797) 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 |
|---|
|
FOOTNOTES |
|---|
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 |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-hydroxylase. J Biol Chem 247:7528–7532-hydroxylase and 24-hydroxylase along the rat
nephron. Proc Natl Acad Sci USA 78:1199–1203- and
24R-hydroxylases. Endocrinology 125:1224–1230-hydroxylase.
J Bone Miner Res 11:S124 (Abstract)
-hydroxylase, P450c1. DNA Cell Biol 16:1499–1507[Medline]
This article has been cited by other articles:
 |
|
 |
|
  H. Liu, J. Guo, L. Wang, N. Chen, A. Karaplis, D. Goltzman, and D. Miao Distinctive anabolic roles of 1,25-dihydroxyvitamin D3 and parathyroid hormone in teeth and mandible versus long bones J. Endocrinol., November 1, 2009; 203(2): 203 - 213. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Bikle Nonclassic Actions of Vitamin D J. Clin. Endocrinol. Metab., January 1, 2009; 94(1): 26 - 34. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. M. Chlon, D. A. Taffany, J. Welsh, and M. J. Rowling Retinoids Modulate Expression of the Endocytic Partners Megalin, Cubilin, and Disabled-2 and Uptake of Vitamin D-Binding Protein in Human Mammary Cells J. Nutr., July 1, 2008; 138(7): 1323 - 1328. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. J. Kim, L. E. Kaplan, F. Perwad, N. Huang, A. Sharma, Y. Choi, W. L. Miller, and A. A. Portale Vitamin D 1{alpha}-Hydroxylase Gene Mutations in Patients with 1{alpha}-Hydroxylase Deficiency J. Clin. Endocrinol. Metab., August 1, 2007; 92(8): 3177 - 3182. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. I. Ellison, R. L. Eckert, and P. N. MacDonald Evidence for 1,25-Dihydroxyvitamin D3-independent Transactivation by the Vitamin D Receptor: UNCOUPLING THE RECEPTOR AND LIGAND IN KERATINOCYTES J. Biol. Chem., April 13, 2007; 282(15): 10953 - 10962. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. D. Bikle For Whom the Bugs Toll; They Toll for D IBMS BoneKEy, April 1, 2006; 3(4): 12 - 15. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. M. Kemmis, S. M. Salvador, K. M. Smith, and J. Welsh Human Mammary Epithelial Cells Express CYP27B1 and Are Growth Inhibited by 25-Hydroxyvitamin D-3, the Major Circulating Form of Vitamin D-3 J. Nutr., April 1, 2006; 136(4): 887 - 892. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Kong, S. A. Grando, and Y. C. Li Regulation of IL-1 Family Cytokines IL-1{alpha}, IL-1 Receptor Antagonist, and IL-18 by 1,25-Dihydroxyvitamin D3 in Primary Keratinocytes J. Immunol., March 15, 2006; 176(6): 3780 - 3787. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. L. Vanhooke, J. M. Prahl, C. Kimmel-Jehan, M. Mendelsohn, E. W. Danielson, K. D. Healy, and H. F. DeLuca CYP27B1 null mice with LacZreporter gene display no 25-hydroxyvitamin D3-1{alpha}-hydroxylase promoter activity in the skin PNAS, January 3, 2006; 103(1): 75 - 80. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Xie, S. Chang, Y. Oda, and D. D. Bikle Hairless Suppresses Vitamin D Receptor Transactivation in Human Keratinocytes Endocrinology, January 1, 2006; 147(1): 314 - 323. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Yamamoto, E. Uchida, N. Urushino, T. Sakaki, N. Kagawa, N. Sawada, M. Kamakura, S. Kato, K. Inouye, and S. Yamada Identification of the Amino Acid Residue of CYP27B1 Responsible for Binding of 25-Hydroxyvitamin D3 Whose Mutation Causes Vitamin D-dependent Rickets Type 1 J. Biol. Chem., August 26, 2005; 280(34): 30511 - 30516. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. S. Dusso, A. J. Brown, and E. Slatopolsky Vitamin D Am J Physiol Renal Physiol, July 1, 2005; 289(1): F8 - F28. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. L. Miller Minireview: Regulation of Steroidogenesis by Electron Transfer Endocrinology, June 1, 2005; 146(6): 2544 - 2550. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Ren, L. Nguyen, S. Wu, C. Encinas, J. S. Adams, and M. Hewison Alternative Splicing of Vitamin D-24-Hydroxylase: A NOVEL MECHANISM FOR THE REGULATION OF EXTRARENAL 1,25-DIHYDROXYVITAMIN D SYNTHESIS J. Biol. Chem., May 27, 2005; 280(21): 20604 - 20611. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Skorija, M. Cox, J. M. Sisk, D. R. Dowd, P. N. MacDonald, C. C. Thompson, and M. B. Demay Ligand-Independent Actions of the Vitamin D Receptor Maintain Hair Follicle Homeostasis Mol. Endocrinol., April 1, 2005; 19(4): 855 - 862. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I Hendrix, P H Anderson, J L Omdahl, B K May, and H A Morris Response of the 5'-flanking region of the human 25-hydroxyvitamin D 1{alpha}-hydroxylase gene to physiological stimuli using a transgenic mouse model J. Mol. Endocrinol., February 1, 2005; 34(1): 237 - 245. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Welsh Vitamin D and breast cancer: insights from animal models Am. J. Clinical Nutrition, December 1, 2004; 80(6): 1721S - 1724S. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. D. Bikle Vitamin D and Skin Cancer J. Nutr., December 1, 2004; 134(12): 3472S - 3478S. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. N. Evans, J. N. Bulmer, M. D. Kilby, and M. Hewison Vitamin D and Placental-Decidual Function Reproductive Sciences, July 1, 2004; 11(5): 263 - 271. [Abstract] [PDF]
|
|
|
|
|
|
D. K. Panda, D. Miao, I. Bolivar, J. Li, R. Huo, G. N. Hendy, and D. Goltzman Inactivation of the 25-Hydroxyvitamin D 1{alpha}-Hydroxylase and Vitamin D Receptor Demonstrates Independent and Interdependent Effects of Calcium and Vitamin D on Skeletal and Mineral Homeostasis J. Biol. Chem., April 16, 2004; 279(16): 16754 - 16766. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Yokomura, T. Suda, S. Sasaki, N. Inui, K. Chida, and H. Nakamura Increased Expression of the 25-Hydroxyvitamin D3-1{alpha}-Hydroxylase Gene in Alveolar Macrophages of Patients with Lung Cancer J. Clin. Endocrinol. Metab., December 1, 2003; 88(12): 5704 - 5709. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-C. Hsieh, J. M. Sisk, P. W. Jurutka, C. A. Haussler, S. A. Slater, M. R. Haussler, and C. C. Thompson Physical and Functional Interaction between the Vitamin D Receptor and Hairless Corepressor, Two Proteins Required for Hair Cycling J. Biol. Chem., October 3, 2003; 278(40): 38665 - 38674. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. P. Guengerich Cytochromes P450, Drugs, and Diseases Mol. Interv., June 1, 2003; 3(4): 194 - 204. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Hewison, L. Freeman, S. V. Hughes, K. N. Evans, R. Bland, A. G. Eliopoulos, M. D. Kilby, P. A. H. Moss, and R. Chakraverty Differential Regulation of Vitamin D Receptor and Its Ligand in Human Monocyte-Derived Dendritic Cells J. Immunol., June 1, 2003; 170(11): 5382 - 5390. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Chen, M. Hewison, B. Hu, and J. S. Adams Heterogeneous nuclear ribonucleoprotein (hnRNP) binding to hormone response elements: A cause of vitamin D resistance PNAS, May 13, 2003; 100(10): 6109 - 6114. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. A. Jelinsky, H. A. Harris, E. L. Brown, K. Flanagan, X. Zhang, C. Tunkey, K. Lai, M. V. Lane, D. K. Simcoe, and M. J. Evans Global Transcription Profiling of Estrogen Activity: Estrogen Receptor {alpha} Regulates Gene Expression in the Kidney Endocrinology, February 1, 2003; 144(2): 701 - 710. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. C. Huang, V. Papavasiliou, J. S. Rhim, R. L. Horst, and R. Kremer Targeted Disruption of the 25-Hydroxyvitamin D3 1{alpha}-Hydroxylase Gene in ras-Transformed Keratinocytes Demonstrates That Locally Produced 1{alpha},25-Dihydroxyvitamin D3 Suppresses Growth and Induces Differentiation in an Autocrine Fashion Mol. Cancer Res., November 1, 2002; 1(1): 56 - 67. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Xie, S. J. Munson, N. Huang, A. A. Portale, W. L. Miller, and D. D. Bikle The Mechanism of 1,25-Dihydroxyvitamin D3 Autoregulation in Keratinocytes J. Biol. Chem., September 27, 2002; 277(40): 36987 - 36990. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. O. Carpenter Variable Degrees of 1-{alpha} Hydroxylase Activity--Fine Tuning the Rachitic Rheostat J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2421 - 2423. [Full Text] [PDF]
|
|
|
|
|
|
X. Wang, M. Y. H. Zhang, W. L. Miller, and A. A. Portale Novel Gene Mutations in Patients with 1{alpha}-Hydroxylase Deficiency That Confer Partial Enzyme Activity in Vitro J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2424 - 2430. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Yoshida, J. Yoshino, M. Hayashi, and T. Saruta Identification of a Renal Proximal Tubular Cell-Specific Enhancer in the Mouse 25-Hydroxyvitamin D 1{alpha}-Hydroxylase Gene J. Am. Soc. Nephrol., June 1, 2002; 13(6): 1455 - 1463. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Zehnder, R. Bland, R. S. Chana, D. C. Wheeler, A. J. Howie, M. C. Williams, P. M. Stewart, and M. Hewison Synthesis of 1,25-Dihydroxyvitamin D3 by Human Endothelial Cells Is Regulated by Inflammatory Cytokines: A Novel Autocrine Determinant of Vascular Cell Adhesion J. Am. Soc. Nephrol., March 1, 2002; 13(3): 621 - 629. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Y. H. Zhang, X. Wang, J. T. Wang, N. A. Compagnone, S. H. Mellon, J. L. Olson, H. S. Tenenhouse, W. L. Miller, and A. A. Portale Dietary Phosphorus Transcriptionally Regulates 25-Hydroxyvitamin D-1{alpha}-Hydroxylase Gene Expression in the Proximal Renal Tubule Endocrinology, February 1, 2002; 143(2): 587 - 595. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Dardenne, J. Prud'homme, A. Arabian, F. H. Glorieux, and R. St-Arnaud Targeted Inactivation of the 25-Hydroxyvitamin D3-1{{alpha}}-Hydroxylase Gene (CYP27B1) Creates an Animal Model of Pseudovitamin D-Deficiency Rickets Endocrinology, July 1, 2001; 142(7): 3135 - 3141. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
T. Yoshida, N. Yoshida, T. Monkawa, M. Hayashi, and T. Saruta Dietary Phosphorus Deprivation Induces 25-Hydroxyvitamin D3 1{{alpha}}-Hydroxylase Gene Expression Endocrinology, May 1, 2001; 142(5): 1720 - 1726. [Abstract] [Full Text]
|
|
|
|
 |
|
 J.-Y. Hsu, D. Feldman, J. E. McNeal, and D. M. Peehl Reduced 1{{alpha}}-hydroxylase Activity in Human Prostate Cancer Cells Correlates with Decreased Susceptibility to 25-Hydroxyvitamin D3-induced Growth Inhibition Cancer Res., April 1, 2001; 61(7): 2852 - 2856. [Abstract] [Full Text]
|
|
|
|
 |
|
 R. M. Maas, K. Reus, B. Diesel, W.-I. Steudel, W. Feiden, U. Fischer, and E. Meese Amplification and Expression of Splice Variants of the Gene Encoding the P450 Cytochrome 25-Hydroxyvitamin D3 1,{{alpha}}-Hydroxylase (CYP 27B1) in Human Malignant Glioma Clin. Cancer Res., April 1, 2001; 7(4): 868 - 875. [Abstract] [Full Text]
|
|
|
|
|
|
H. S. Tenenhouse, J. Martel, C. Gauthier, M. Y. H. Zhang, and A. A. Portale Renal Expression of the Sodium/Phosphate Cotransporter Gene, Npt2, Is Not Required for Regulation of Renal 1{{alpha}}-Hydroxylase by Phosphate Endocrinology, March 1, 2001; 142(3): 1124 - 1129. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. St-Arnaud, A. Arabian, R. Travers, F. Barletta, M. Raval-Pandya, K. Chapin, J. Depovere, C. Mathieu, S. Christakos, M. B. Demay, et al. Deficient Mineralization of Intramembranous Bone in Vitamin D-24-Hydroxylase-Ablated Mice Is Due to Elevated 1,25-Dihydroxyvitamin D and Not to the Absence of 24,25-Dihydroxyvitamin D Endocrinology, July 1, 2000; 141(7): 2658 - 2666. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. ZEHNDER, R. BLAND, E. A. WALKER, A. R. BRADWELL, A. J. HOWIE, M. HEWISON, and P. M. STEWART Expression of 25-Hydroxyvitamin D3-1{alpha}-Hydroxylase in the Human Kidney J. Am. Soc. Nephrol., December 1, 1999; 10(12): 2465 - 2473. [Abstract] [Full Text]
|
|
|
|
|
|
S. Kitanaka, A. Murayama, T. Sakaki, K. Inouye, Y. Seino, S. Fukumoto, M. Shima, S. Yukizane, M. Takayanagi, H. Niimi, et al. No Enzyme Activity of 25-Hydroxyvitamin D3 1{alpha}-Hydroxylase Gene Product in Pseudovitamin D Deficiency Rickets, Including That with Mild Clinical Manifestation J. Clin. Endocrinol. Metab., November 1, 1999; 84(11): 4111 - 4117. [Abstract] [Full Text]
|
|
|
|
|
|
A. J. Brown, A. Dusso, and E. Slatopolsky Vitamin D Am J Physiol Renal Physiol, August 1, 1999; 277(2): F157 - F175. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Shinki, Y. Ueno, H. F. DeLuca, and T. Suda Calcitonin is a major regulator for the expression of renal 25-hydroxyvitamin D3-1alpha -hydroxylase gene in normocalcemic rats PNAS, July 6, 1999; 96(14): 8253 - 8258. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Jones, H. Ramshaw, A. Zhang, R. Cook, V. Byford, J. White, and M. Petkovich Expression and Activity of Vitamin D-Metabolizing Cytochrome P450s (CYP1{alpha} and CYP24) in Human Nonsmall Cell Lung Carcinomas Endocrinology, July 1, 1999; 140(7): 3303 - 3310. [Abstract] [Full Text]
|
|
|
|
|
|
R. Bland, E. A. Walker, S. V. Hughes, P. M. Stewart, and M. Hewison Constitutive Expression of 25-Hydroxyvitamin D3-1{alpha}-Hydroxylase in a Transformed Human Proximal Tubule Cell Line: Evidence for Direct Regulation of Vitamin D Metabolism by Calcium Endocrinology, May 1, 1999; 140(5): 2027 - 2034. [Abstract] [Full Text]
|
|
|
|
|
|
A. Murayama, K.-i. Takeyama, S. Kitanaka, Y. Kodera, Y. Kawaguchi, T. Hosoya, and S. Kato Positive and Negative Regulations of the Renal 25-Hydroxyvitamin D3 1{alpha}-Hydroxylase Gene by Parathyroid Hormone, Calcitonin, and 1{alpha},25(OH)2D3 in Intact Animals Endocrinology, May 1, 1999; 140(5): 2224 - 2231. [Abstract] [Full Text]
|
|
|
|
 |
|
 P. J. Malloy, J. W. Pike, and D. Feldman The Vitamin D Receptor and the Syndrome of Hereditary 1,25-Dihydroxyvitamin D-Resistant Rickets Endocr. Rev., April 1, 1999; 20(2): 156 - 188. [Abstract] [Full Text]
|
|
|
|
|
|
Y. C. Li, M. Amling, A. E. Pirro, M. Priemel, J. Meuse, R. Baron, G. Delling, and M. B. Demay Normalization of Mineral Ion Homeostasis by Dietary Means Prevents Hyperparathyroidism, Rickets, and Osteomalacia, But Not Alopecia in Vitamin D Receptor-Ablated Mice Endocrinology, October 1, 1998; 139(10): 4391 - 4396. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. JONES, S. A. STRUGNELL, and H. F. DeLUCA Current Understanding of the Molecular Actions of Vitamin D Physiol Rev, October 1, 1998; 78(4): 1193 - 1231. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. St-Arnaud and F. H. Glorieux Editorial: 24, 25-Dihydroxyvitamin D--Active Metabolite or Inactive Catabolite? Endocrinology, August 1, 1998; 139(8): 3371 - 3374. [Full Text] [PDF]
|
|
|
|
 |
|
 R. Bouillon The Many Faces of Rickets N. Engl. J. Med., March 5, 1998; 338(10): 681 - 682. [Full Text]
|
|
|
|
|
|
H. Chen, B. Hu, E. A. Allegretto, and J. S. Adams The Vitamin D Response Element-binding Protein. A NOVEL DOMINANT-NEGATIVE REGULATOR OF VITAMIN D-DIRECTED TRANSACTIVATION J. Biol. Chem., November 3, 2000; 275(45): 35557 - 35564. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
