Intracellular Vitamin D Binding Proteins: Novel Facilitators of Vitamin D-Directed Transactivation
Shaoxing Wu,
Songyang Ren,
Hong Chen,
Rene F. Chun,
Mercedes A. Gacad and
John S. Adams
Burns and Allen Research Institute and Division of
Endocrinology, Diabetes and Metabolism Cedars-Sinai Medical
Center University of California Los Angeles School of Medicine
Los Angeles, California 90048
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ABSTRACT
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Previously recognized intracellular proteins with
an affinity for vitamin D metabolites include the vitamin D receptor
and the cytochrome P-450-based vitamin D metabolizing mixed-function
oxidases. We recently characterized a third set of high-capacity,
intracellular vitamin D binding proteins (IDBPs) in the inducible heat
shock protein-70 (hsp-70) family. Here we report the cloning and
expression of cDNAs coding for two IDBPs. The full-length cDNAs for
IDBP-1 and IDBP-2 demonstrated 95% and 94% nucleotide homology,
respectively, with the cDNAs for human constitutively expressed heat
shock protein 70 (hsc-70) and hsp-70. Transient expression of the IDBP
cDNAs in a vitamin D-responsive primate cell line increased extractable
25-hydroxylated vitamin D metabolite-IDBP-binding 25-fold. Transfection
experiments also demonstrated that the majority of the constitutively
expressed 25-hydroxylated vitamin D metabolite binding activity
was attributable to expression of the hsc-70-related IDBP-1 and that
metabolite binding activity sublocalized to the highly conserved
ATP-binding/ATPase domain of hsp-70s. Stable overexpression of IDBP-1
in wild-type cells enhanced vitamin D-directed responsiveness of
endogenous vitamin D-24-hydroxylase, osteopontin, and osteocalcin genes
by several-fold over that observed in cells transfected with an empty
vector. These results suggest that IDBP-1 facilitates the intracellular
localization of active vitamin D metabolites and vitamin D
receptor-mediated transactivation.
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INTRODUCTION
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During the Eocene, the major continental land masses separated
from one another and led to the independent evolution of primates in
the Platyrrhini, Catarrhini, and Lemuridae infra-orders. These primates
inhabit the regions of South and Central America, Africa and Asia, and
the islands of Madagascar, respectively (1). Many million years of
independent evolution resulted in major phenotypic differences among
the New World primates (platyrrhines), Old World primates
(catarrhines), and lemurs (2). One distinctive phenotype,
characteristic of almost all genera of platyrrhines, is resistance to
steroid hormones that originate from the adrenal cortex (3, 4, 5, 6) and
gonads (7, 8), as well as resistance to the vitamin D prohormone,
25-hydroxyvitamin D (25-OHD) (9) and its active hormone,
1,25-dihydroxyvitamin D [1,25-(OH)2D]
(10, 11, 12), produced in the liver and kidney, respectively.
Glucocorticoid resistance is associated with the overexpression of the
immunophilin FKBP51 (6), while the estrogen- and vitamin D-resistant
phenotype is associated with constitutive overexpression of a subfamily
of heterogeneous nuclear ribonucleoproteins (hnRNPs) that compete with
steroid hormone receptor dimers for binding to their cognate hormone
response elements (13, 14). In fact, there is preliminary evidence (H.
Chen, M. Hewison, and J. S. Adams, unpublished) of the existence of a
human counterpart to the hnRNP-overexpressing vitamin D-resistant New
World primate, a patient with inherited vitamin D resistance but
possessing a fully functional vitamin D receptor (15).
To preserve normal target tissue responsiveness to the hormone,
platyrrhines maintain high circulating concentrations of these hormones
and hormone substrates. With respect to the adrenal and gonadal
steroids, high serum levels are achieved, at least in part, by an
increase in the enzymatic synthesis of steroids from endogenous
cholesterol stores (3, 16). By contrast, compensation for the vitamin
D-resistant state does not involve cholesterol, but an ultraviolet B
(UVB)-derived photo product of 7-dehydrocholesterol, previtamin
D3. As a result, platyrrhines must ingest and/or
produce endogenously more vitamin D to compensate for the increased
demand for 25-OHD and 1,25-(OH)2D. Hence, by comparison
with catarrhines, endogenous production of vitamin
D3 in platyrrhines is limited by both the
quantity of 7-dehydrocholesterol present in their skin as well as the
rate of nonenzymatic photoconversion by sunlight (UVB photons) of that
7-dehydrocholesterol to previtamin D3 (17).
Platyrrhines in all diurnal genera maintain high cutaneous vitamin
D3 production rates (9) by inhabiting the
sunlight (UVB)-rich canopy of the equatorial rain forests of South and
Central America. Although postulated (11), it is not known whether
platyrrhines, obligate herbivores (2), also ingest significant
quantities of plants containing ergocalciferol or vitamin
D2, which is comparable to vitamin
D3 as a substrate for vitamin D-25-hydroxylase
(18). Also unknown is the existence of additional pathways in
platyrrhine target cells that would maximize the effectiveness of
25-OHD and 1,25-(OH)2D to which the cells are exposed.
During our investigation of vitamin D resistance in platyrrhines, we
uncovered the presence of a high-capacity intracellular vitamin D
binding protein (IDBP) that was constitutively overexpressed in vitamin
D-resistant platyrrhine cells and virtually absent in vitamin
D-responsive, catarrhine (Old World primate) cells (12, 19). We
recently determined this protein to possess a high degree of homology
with proteins in the inducible heat shock protein-70 (hsp-70) family
(20). In this report we describe the molecular cloning, expression, and
function of two IDBP gene products.1
We demonstrate the ability of IDBP cDNAs, when transiently and stably
transfected and expressed in catarrhine cells, to increase specific
vitamin D metabolite binding activity and enhance
1,25-(OH)2D-directed transactivation. Our results suggest
that the constitutive overexpression of these hsp-70-related proteins
may serve to direct 25-hydroxylated vitamin D metabolites to
critical sites of hormone action and metabolism. Additionally, it may
provide a means of antagonizing the dominant-negative, hormone
resistance-causing actions of the hnRN-Prelated vitamin D response
element binding protein (13, 14).
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RESULTS
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IDBP cDNA Sequence
A panel of degenerate oligonucleotide primers (see Materials
and Methods) was designed on the basis of the amino acid sequence
of two tryptic peptides from the amino-terminal half of the New World
primate IDBP molecule (20). These primers were used with total RNA
extracted from B958 New World primate cells to amplify by RT-PCR two
major, overlapping cDNA fragments, 1013 and 630 bp in length. These
cDNAs demonstrated, respectively, 95% and 94% sequence identity with
cDNA sequences for human constitutively expressed heat shock protein-70
(hsc-70) and inducible heat shock protein-70 (hsp-70). The 1013-bp
(Fig. 1
, left panel) and
630-bp (Fig. 1
, right panel) cDNAs were then used as probes
in Northern blots of total cellular RNA extracted from
hormone-resistant New World primate B958 cells, from New World
primate OMK cells with partial hormone resistance, and from
hormone-responsive Old World primate MLA-144 cells. Both cDNA probes
revealed a 2.2-kb message that was most abundant in New World primate
cells and the least plentiful in Old World primate cells; specific mRNA
levels in the OMK cell line were intermediate.

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Figure 1. Northern Blot Detection of an Approximately 2.2-kb
Message (IDBP) in Total Cellular RNA of Different Primate Cells
Total cellular RNA was extracted from the hormone-resistant New World
primate cell line B958, partially hormone-resistant New World primate
cell line OMK, and hormone-responsive Old World primate MLA cell line.
Thirty micrograms each were fractionated by size through a formaldehyde
gel, transferred onto nylon membrane, and then hybridized with a
1013-bp (probe 1, left panel ) or a 630-bp (probe 2,
right panel ) IDBP probe. The 28S RNA bands are shown
below.
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These two cDNAs were then used to screen an unamplified B958 cDNA
library. After secondary and tertiary screening, two distinct clones,
termed IDBP-1 and IDBP-2, containing a 2260-bp and 2573-bp insert,
respectively, were isolated and sequenced. The cDNAs for IDBP-1 and
IDBP-2 predicted proteins with an open reading frame of 646 and 643
amino acid residues, respectively (Fig. 2
). When the deduced amino acid sequence
for IDBP-1 was aligned with that of IDBP-2 using the GAP program of the
Genetics Computer Group (GCG, Madison, WI), 77%
amino acid sequence identity was calculated. The full-length cDNA for
IDBP-1 showed 95% sequence identity with human hsc-70. The full-length
cDNA sequence for IDBP-2 showed 94% sequence identity with human
hsp-70.

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Figure 2. Comparison of the Amino Acid Sequences for the
IDBP-1 and IDBP-2
The sequence of 646 amino acids deduced for IDBP-1 and the sequence of
643 amino acids deduced for IDBP-2 were aligned with the use of the GAP
program from the Genetics Computer Group (GCG). The
lightly shadowed region corresponds to the ATP-binding
domain of hsp-70s. The more darkly shaded area
corresponds to the vitamin D sterol binding domain of IDBP-1 and IDBP-2
(see Fig. 9 ).
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Transient Expression of IDBP-1 and IDBP-2
If IDBP-1 and IDBP-2 are among the principal intracellular
proteins responsible for enhanced expression of 25-OHD binding in New
World primate cells, then transient expression of IDBP-1 and IDBP-2
cDNA constructs in wild-type, Old World primate cells harboring small
amounts of IDBP should dramatically enhance metabolite binding. As
expected, transient transfection of hormone-responsive Old World
primate COS-7 cells with sense IDBP-1 or IDBP-2 cDNAs increased
extractable 25-OHD3 binding significantly (13-fold and
8-fold, respectively), compared with cells transfected with vector
alone (Fig. 3
). Cotransfection of the
IDBP-1 and IDBP-2 constructs was additive, increasing
25-OHD3 binding nearly 25-fold over that observed in
hormone-responsive cells transfected with the empty vector.
Collectively, these data suggest that relatively more of the
constitutively expressed vitamin D metabolite binding activity in
hormone-resistant New World primate cells can be attributed to
IDBP-1-like than to IDBP-2-like proteins.

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Figure 3. Specific 25-OHD3 Binding in
Hormone-Responsive COS-7 Cells Transfected with Sense IDBP cDNA
Constructs
COS-7 cells (8090% confluent) were transfected with 5 µg sense
p3.1-IDBP-1 and/or p3.1-IDBP-2 constructs. After 48 h the cells
were harvested and protein was extracted for measurement of
25-OHD3 binding. Each value is the mean ±
SD of nine assessments of specific 25-OHD3
binding. A significant increase over basal binding is marked by
bullets (P < 0.001; Students test
for unpaired samples).
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Effects of IDBP Expression on Transactivation of the Vitamin
D-Target Genes
The above transient transfection experiments established that
coexpression of the two IDBP molecules resulted in enhanced ability of
hormone-responsive primate cells to specifically bind a 25-hydroxylated
vitamin D metabolite. We next questioned whether the enhanced vitamin D
binding capacity had a functional consequence in a cell relatively
devoid of the dominant-negative-acting vitamin D response element
binding protein (VDRE-BP). Because it possesses a highly inducible VDRE
in its promoter (21), analysis of expression of the endogenous
25-hydroxyvitamin D-24-hydroxylase gene was selected as our initial
bioassay for the transactivating potential of
1,25-(OH)2D3. Using primate RNA as template, we
cloned by RT-PCR a 275-bp primate-specific 24-hydroxylase cDNA (Fig. 4A
) and demonstrated maximum stimulation
of 24-hydroxylase mRNA levels in primate cells after overnight
incubation with 100 nM 1,25-(OH)2D3
(Fig. 4B
). Vitamin D-responsive Old World primate COS-7 cells
cotransfected with the sense IDBP-1 and IDBP-2 constructs exhibited
1,25-(OH)2D3-induced increase in
24-hydroxylase mRNA expression 9-fold above that observed in cells
harboring only the empty vector (Fig. 4C
). By comparison, transient
coexpression of the antisense IDBP constructs reduced
1,25-(OH)2D3-stimulated 24-hydroxylase mRNA
expression by 50%, to the levels below that observed for cells
transfected with vector alone.

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Figure 4. Cloning and Expression of Primate-Specific
24-Hydroxylase
A primate-specific 275-bp cDNA fragment with high homology to the human
24-hydroxylase cDNA sequence (panel A) was obtained by RT-PCR (see
Materials and Methods). This cDNA was used in Northern
blot analysis to detect a
1,25-(OH)2D3-dependent increase in endogenous
24-hydroxylase gene expression in wild-type, hormone-responsive COS-7
cells (panel B). Expression of 24-hydroxylase was also detected in
COS-7 cells before and after cotransfection with sense or antisense
IDBP-1 and IDBP-2 constructs (panel C).
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These transient transfection experiments did not distinguish whether
the effect on the 24-hydroxylase mRNA levels was due to IDBP-1
expression, IDBP-2 expression, or a combination of the two. To address
this issue, we assessed specific 25-OHD3 binding activity
in B958 New World primate cells transiently transfected with
antisense IDBP-1, with antisense IDBP-2, or with both (Fig. 5
). Antisense IDBP-1 significantly
reduced metabolite binding, while antisense IDBP-2 did not. These data
suggested that it was the hsc-70-like, non-heat-inducible IDBP-1 that
was largely responsible for the protransactivating potential of the
IDBPs constitutively overexpressed in New World primate cells.
Confirmation was sought in two clonal COS-7 cell lines stably
transfected with IDBP-1. Representative experiments are shown in
Fig. 6
. 24-Hydroxylase gene
expression exhibited a substantial 1,25(OH)2D3
concentration-dependent enhancement that was higher in IDBP-1.4 clone
than in wild-type cells. Maximum enhancement were observed in both
cells after exposure to 100 nM
1,25(OH)2D3 (Fig. 6A
). A comparable
1,25(OH)2D3-dependent increase in
24-hydroxylase gene expression was observed in a second clonal cell
line, IDBP-1.8, stably transfected with IDBP-1 (Fig. 6B
).

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Figure 5. Specific 25-OHD3 Binding in
Hormone-Resistant B958 Cells Transfected with Antisense IDBP cDNA
Constructs
Hormone-resistant New World primate B958 cells (8090% confluent)
were transfected with 5 µg antisense p3.1-IDBP-1 and/or antisense
p3.1-IDBP-2 constructs. After 48 h the cells were harvested and
protein was extracted for measurement of 25-OHD3 binding.
Each value is the mean ± SD of nine assessments of
specific 25-OHD3 binding. A significant increase over basal
binding is marked by bullets (P <
0.001; Students test for unpaired samples).
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Figure 6. Comparison of 24-Hydroxylase (24-OHase) Expression
between Wild-Type and Stably Transfected COS-7 Cells after
1,25-Dihydroxyvitamin D3 (1,25-D3) Stimulation
Northern blot analyses of total RNA extracts from wild-type COS-7 cells
and two COS-7 clones, IDBP-1.4 and IDBP-1.8, both stably transfected
with sense p3.1-IDBP-1. Wild-type COS-7 and clone IDBP-1.4 cells were
incubated overnight in the presence of increasing concentrations of
1,25-(OH)2D3, and fractionated RNA extracts
were probed for primate-specific 24-hydroxylase (panel A). Panel B
demonstrates 24-hydroxylase mRNA expression after clone IDBP-1.8 and
wild-type COS-7 cells were incubated overnight in the presence or
absence of 100 nM 1,25-(OH)2D3.
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Furthermore, the IDBP-1-mediated protransactivating activity was not
limited to the 24-hydroxylase gene. Primate-specific cDNAs for
osteocalcin and osteopontin were cloned (Fig. 7
); both genes possess stimulatory VDREs
in their promoters (21). These cDNAs were then used as probes to
detect steady-state mRNA levels in COS-7 IDBP-1-overexpressing clones,
IDBP-1.4 and IDBP-1.8, that were incubated with increasing
concentrations of 1,25-(OH)2D3 (Fig. 8
). Similar to what was observed with the
24-hydroxylase gene (see left-most panels of Fig. 8
), IDBP-1
boosted the 1,25-(OH)2D3 responsiveness for
both the osteopontin and osteocalcin genes.

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Figure 7. Cloning of Primate-Specific Osteopontin and
Osteocalcin cDNAs
Sense and antisense primers corresponding to human osteopontin
(nucleotides 6668 and 386411, respectively) and osteocalcin
(nucleotides 1439 and 294320, respectively) cDNA sequences were
used in RT-PCR with 1 µg RNA extracted from OMK cells as template.
The cDNA sequences of the RT-PCR unique 344-bp and 291-bp bands were
aligned with the sequences of the human osteopontin (panel A) and
osteocalcin genes (panel B), respectively, with the FASTA
program in GCG.
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Figure 8. Comparison of mRNA Expression of Different Vitamin
D-Directed Genes in Wild-Type and Stably Transfected COS-7 Cells
Stimulated with 1,25-(OH)2D3
Total RNA were extracted from wild-type COS-7 cells and stably
transfected (with sense IDBP-1) COS-7 cell clones stimulated by
1,25-(OH)2D3 overnight. Ten micrograms each of
the above RNAs were analyzed by Northern blot with primate-specific
24-hydroxylase, osteopontin, and osteocalcin cDNA probes, respectively.
The corresponding gene expression signals were measured by Mean Density
in NIH Image 1.62. 1,25-(OH)2D3
dose-dependent increases in gene expression in clone IDBP-1.4
(closed squares, panel A) and clone IDBP-1.8
(closed squares, panel B) were compared with those in
wild-type COS-7 cells (open squares, panels A and B).
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Mapping the Vitamin D Metabolite Binding Domain in New World
Primate Cells
Human hsc-70 and hsp-70 are now recognized as high-capacity
25-OHD3 binding proteins (22) and most likely share a
structurally conserved ligand binding domain for the vitamin D
metabolite. Because the percent identity between these two molecules is
greatest within their amino-terminal and midportions (Fig. 2
), we
predicted that the vitamin D ligand binding domain would reside in the
highly conserved ATP-binding/ATPase binding segments of these proteins.
We prepared by RT-PCR a panel of "in-frame" sense IDBP-1 cDNA
constructs of varying length for transient transfection into wild-type
Old World primate COS-7 cells (Fig. 9A
).
Transfection of amino-terminal cDNA constructs spanning nucleotides
1315 and 1474 of the open reading frame for IDBP-1 as well as the
11561938 construct comprising the carboxy-terminal third of the
molecule resulted in little or no specific 25-OHD3 binding
capability in transfected cells. By comparison, transfection of
constructs spanning nucleotides 605-1155 showed substantial, but not
maximum, metabolite binding activity in transfected cells. For
illustrative purposes, the general domain structure of proteins in the
hsp-70/IDBP family is shown in Fig. 9B
(23). These results suggested
that the amino-terminal and carboxy-terminal extent of the sterol
binding domain, respectively, resided between nucleotides 474 (amino
acid 158) and 1155 (amino acid 385) within the ATP-binding domain of
the protein (Fig. 9B
).

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Figure 9. Mapping of the Sterol Binding Domain (Panel A) and
Functional Domain Structure (Panel B) of the hsp-70-Related IDBPs
Specific binding of 25-OHD3 in hormone-responsive Old
World primate COS-7 cells after transient transfection of IDBP-1 cDNA
constructs that span different regions of the open reading frame of
IDBP-1 were compared (panel A). Data are expressed as the percent of
specific 25-OHD3 binding obtained with transfection of the
11938 holo IDBP-1 construct (*). Each value is the mean ±
SE of seven to nine replicates in three separate
experiments. Binding values for vector alone and constructs
encompassing nucleotides 1315, 1474, and 11561938 were all
significantly reduced (P < 0.005) below maximal
binding. The mean binding value for the 605-1155 construct was also
significantly reduced (P < 0.02) below maximal
binding. Panel B depicts the domain structure of the hsp-70 family of
proteins, including the sterol binding domain determined from
experiments presented in panel A.
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DISCUSSION
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Activation of vitamin D to its hormonal form requires that vitamin
D and its metabolites be enzymatically modified in a serial manner
(18). In avian and mammalian species, this process of enzymatic
modification is catalyzed by cytochrome P450-mixed function oxidases in
various tissues. In liver, the vitamin D molecule is hydroxylated at
the C-25 position to 25-OHD. In kidney, 25-OHD is hydroxylated
at the C-1 position to form 1,25-(OH)2D. Vitamin D
and its metabolites are shuttled through the circulation from tissue to
tissue bound to serum vitamin D binding protein (DBP), a member of the
albumin superfamily of proteins (26). The mechanism by which vitamin D
metabolites, in general, and 1,25-(OH)2D, in particular,
enter these different tissues and how they reach a specific destination
within target cells (i.e. nucleus, mitochondria, etc.) is
just beginning to be understood. It has been the generally accepted
tenet that it is the lipophilic nature of vitamin D molecules that
drives their movement into target cells. Once inside the cell, the laws
of mass action drive the association between vitamin D molecules and
their high affinity binding proteins, such as cytochrome
P450-linked-hydroxylases or the vitamin D receptor (VDR). However, it
is unlikely that the trafficking of vitamin D molecules is solely
random. It is more likely that vitamin D traffic, particularly with
respect to 25-hydroxylated vitamin D metabolites, is finely directed by
a set of intervening molecular shuttle proteins. For example, it was
recently discovered that entrance of 25-hydroxylated vitamin D
metabolites into the proximal tubular epithelial cell of the kidney,
the central site of 25-OHD-1-hydroxylation, occurs at the brush border
(i.e. at the membrane abutting the urinary space) of the
epithelial cell (27). Vitamin D and its metabolites, bound to DBP, are
largely filtered through the glomerulus into the tubular lumen and the
DBP-bound vitamin D molecules reclaimed from the urine by megalin (27).
Megalin, a member of the LDL (low density lipoprotein) receptor
family of proteins (28), is concentrated in coated pits along the
tubular cell membrane and acts as a specific binding protein for DBP.
The megalin-DBP-25-hydroxylated vitamin D metabolite complexes are
internalized by invagination of the coated pits.
The mechanism by which vitamin D metabolites dissociate from the
internalized complex and travel to specific intracellular destinations
is not known. We propose that the hsp-70-related family of IDBPs play a
central role in this transport process. However, if this is the
case, then four criteria must be fulfilled by the IDBPs. First, the
IDBPs must possess a capacity and affinity for vitamin D metabolites
that are sufficiently greater than those expressed by the megalin-DBP
complex. The dissociation constant (Kd) of
IDBPenriched cell extracts of primate cells for 25-OHD is 0.5
nM, 100-fold less than that of 25-OHD for the serum DBP,
indicating the existence of a substantial 25-OHD3
affinity gradient from DBP TO IDBP (22). In the present study, we
demonstrate that transient expression of IDBP-1 and IDBP-2 in
wild-type, Old World primate cells results in a 13-fold and 8-fold
increase in specific 25-OHD3 binding capacity, respectively
(Fig. 3
). This is particularly significant when one considers the
innate inefficiency of transient transfections. Second, once bound to
IDBP in the cytoplasm, 25-hydroxylated vitamin D metabolites must have
the opportunity to move with a favorable gradient from IDBP to other
intracellular proteins that have an affinity for vitamin
D3. An affinity gradient clearly exists for
1,25-(OH)2D that favors its movement from IDBP to
the VDR. The VDR has a Kd for
1,25-(OH)2D in the range of 0.1 nM
(18), 3 orders of magnitude lower (i.e. higher affinity)
than IDBP for 1,25-(OH)2D. Moreover, we
previously demonstrated that cells from New World primate genera
possess a capacity for specific, intracellular 1,25-(OH)2D
binding that exceeds by 1.5 to 2.0 orders of magnitude the capacity of
the VDR to bind the vitamin D hormone (19). The other 25-OHD metabolite
binding proteins of recognized significance in the cell are the
mitochondrial-based 25-OHD-hydroxylases. The true affinity of these
enzymes for vitamin D substrates is not known. Presumably, an affinity
gradient exists that favors the movement of 25-hydroxylated vitamin D
metabolites from IDBP to both 1-hydroxylase and the multicatalytic
24-hydroxylase.
The third criterion that should be fulfilled if IDBPs are to be
considered as intracellular transit proteins for vitamin D metabolites
is that they must be in association with, or in close proximity to,
other vitamin D binding proteins in the cell. These binding proteins
include megalin-bound DBP, the mitochondrial vitamin D hydroxylases,
and the VDR. The mechanism by which cytoplasmic IDBP gains access to
intravesicular DBP-bound vitamin D molecules is not understood;
nevertheless, once in the cytoplasm, 25-hydroxylated vitamin D
metabolites are known to be associated with IDBPs (20). In fact, it was
this specific binding interaction that provided the critical tool for
purification of the first IDBP family members (20). Intracellular
distribution studies demonstrate that the same IDBPs recovered from the
cytoplasm are also found in the nucleus (19), suggesting
intercompartmental transit involving one or more members of the IDBP
family. Although neither of the IDBPs characterized here possesses an
amino-terminal targeting sequence for a specific intracellular
compartment, ample evidence shows that other members of the hsp-70
superfamily are specifically targeted to specific intracellular sites
(i.e. to mitochondrial, nucleus, etc.). We have preliminary
data (R. F. Chun and J. S. Adams, unpublished)
demonstrating that the chimeric proteins, gst-vitamin D-1-hydroxylase
and gst-megalin, specifically interact with IDBP/hsp-70 family members.
Hence, it is tempting to speculate that the IDBPs have the same role
for vitamin D substrates that the steroid acute regulatory (StAR)
proteins have for cholesterol substrates (31, 32), moving cholesterol
metabolites to specific intracellular destinations.
It is also possible that organelle targeting is directed by a companion
protein, independent of IDBP. For example, two recently discovered
proteins, BAG-1 (33) and Hip (34), have been shown to bind to the
ATP-binding/ATPase domain of hsp-70 proteins (see Fig. 9B
). BAG-1 is a
multifunctional protein first recognized for its ability to bind the
antiapoptotic protein Bcl-2 and promote cell survival (35). Included
among its other functions are an ability to bind to numerous members of
the steroid/retinoid/T3 superfamily (36) and act
as a corepressor of hormone-directed transcription (33). While BAG-1
promotes the release of ADP and target substrates (37), Hip acts to
stabilize the hsp-70-ADP chaperone function by promoting the
interaction of target peptides with hsp-70 (34, 38). Moreover, BAG-1
and Hip may be direct competitors for hsp-70 binding; the association
of Hip with hsp-70 prevents an interaction between BAG-1 and hsp-70.
Considering that the hsp-70/IDBP family of proteins can interact with
small lipophilic signaling molecules other than vitamin Ds (20), it is
possible that BAG-1 or Hip acts as a molecular bridge between two sets
of hormone binding proteins in the cell, the hsp-70/IDBP family of
proteins and sterol/steroid/retinoid/T3 receptor
superfamily of proteins. In fact, Craig, et al. (39) have
recently described the association of a gst-VDR construct with human
hsp-70.
If IDBPs have a role in the intracellular vitamin D transit system,
then a fourth criterion requires that alterations in the expression of
these proteins be linked to a relevant functional consequence. The
transfection experiments presented here (Figs. 4
, 6
, and 8
) permit us
for the first time to determine whether IDBPs act 1) to amplify hormone
resistance in New World primate cells by acting as an intracellular
repository for steroid/sterol hormones and their intermediate
substrates (40) (i.e. sequestering them from cognate
receptors or activation enzymes); or 2) to antagonize the
hormone-resistant state that is maintained by the hnRNP-related VDRE-BP
(13) by facilitating the movement of ligand to its cognate receptor in
the nucleus. The latter situation appears to be most likely. Wild-type
Old World primate cells express little IDBP and VDRE-BP; however, when
transfected with IDBP-1 and IDBP-2, a marked increase in the ability of
vitamin D hormone to enhance expression of the vitamin D-24-hydroxylase
gene was measured (Fig. 4
). Furthermore, transfection of sense and
antisense cDNA constructs for IDBP-1 and IDBP-2 cDNA into vitamin
D-responsive and -resistant cells (Figs. 3
and 5
, respectively)
indicated that most, if not all, of the 25-hydroxyvitamin D3 binding
potential in IDBPs resides within the general domain of the molecule
also responsible for ATP, BAG-1, and Hip binding (Fig. 9
). When only
IDBP-1 is stably overexpressed in wild-type cells, up-regulation of
CYP24, osteopontin, and osteocalcin gene expression, three well known
1,25-(OH)2D-VDR-directed events (41), was clearly measured.
These results support the hypothesis that hsc-70-related IDBP-1 may act
as an intracellular chaperone for 1,25-(OH)2D movement to
the VDR.
We have not yet investigated whether binding of 25-hydroxylated vitamin
D metabolites or other steroid ligands have any influence on BAG-1 or
Hip binding, or on ATP binding and ATPase activity of the parent
hsp-70. However, we suspect that vitamin D binding is associated with
the displacement of BAG-1 and Hip from IDBPs. Purification of IDBPs was
accomplished on the ability of 25-OHD3 to specifically bind
these proteins in nondenaturing conditions (14), and neither BAG-1 nor
Hip-like proteins copurified with the IDBPs. Finally, we have no
evidence yet that ATP binding or hydrolysis has any influence on the
binding of sterol ligands by these proteins. It is tempting to
speculate that the binding of certain hydrophilic ligands in this
region of the molecule may influence the ATP/ATPase-regulated process
of protein-protein interaction, characteristic of the amino-terminal
and midregions of hsp-70 molecules.
 |
MATERIALS AND METHODS
|
---|
Cell Culture and Transfection of IDBP cDNAs
B958, OMK, MLA-144, and COS-7 primate cell lines were obtained
from the American Type Culture Collection
(ATCC, Manassas, VA). B958, an Epstein-Barr virus
(EBV)-transformed lymphoblastoid cell line from a vitamin D-resistant,
New World primate marmoset and MLA-144, a gibbon lymphoma cell line
harvested from a vitamin D-responsive Old World primate, were both
cultured in RPMI 1640. The OMK cell line, derived from the kidney of
the partially vitamin D-responsive Owl monkey, was cultured in MEM.
COS-7 kidney cells, from a vitamin D-responsive African green monkey,
were grown in DMEM. All media were supplemented with 10% FCS, 100 U/ml
penicillin, 100 µg/ml streptomycin, 2 mM
L-glutamine.
A panel of in-frame sense and antisense IDBP-1 and IDBP-2
constructs were subcloned into the eukaryotic expression vector,
pcDNA3.1 (Invitrogen, Carlsbad, CA). The plasmids
p3.1-IDBP-1 sense, p3.1-IDBP-1 antisense, p3.1-IDBP-2 sense, and
p3.1-IDBP-2 antisense were transiently transfected into B958 or COS-7
cells by lipofection (LipoTAXI, Stratagene, La Jolla, CA).
Briefly, cells were seeded into 100-mm dishes, grown to 8090%
confluence, and washed in serum-free DMEM. Plasmid DNA (5 µg) in
media was suspended with 100 µl LipoTAXI reagent, added to each dish,
and incubated for 5 h. FCS (20%)-supplemented DMEM was added and
after 12 h replaced with culture media for 48 h. For analysis
of IDBP expression, cells were extracted as described below. For
24-hydroxylase, osteopontin, and osteocalcin expression experiments,
transfected COS-7 cells were treated with 0100 nM
1,25-(OH)2D3 and, after 16 h, extracted
for total RNA with Trizol reagent (Life Technologies, Inc., Gaithersburg, MD). COS-7 clones stably transfected with
IDBP-1 were created by G418 selecting. Cells transfected with
p3.1-IDBP-1 sense constructs were cultured and maintained in DMEM
containing 500 µg/ml G418 (Omega, Tarzana, CA), and the media were
changed every 3 days. Four weeks later, G418-resistant clones were
picked, grown, and selected for the highest expression of IDBP-1.
Subclones IDBP-1.4 and IDBP-1.8 were identified and tested for
24-hydroxylase, osteopontin, and osteocalcin gene expression after
1,25-(OH)2D3 stimulation.
Generation by RT-PCR of IDBP cDNAs
Degenerate oligonucleotides were deduced from the amino acid
sequence of two tryptic fragments from the amino-terminal half of
purified IDBP (20); the antisense strand primers were
5'-AGGACAAAAGTGCCAAGCAGGTGGTTATC-3' and 5'-CAGGACGACGTCATGAATCTG-3'
(corresponding to nucleotides 14211449 in the IDBP-1 cDNA and
12851305 in the IDBP-2 cDNA), while the more 5'-sense strand primers
were 5'-ATGAAGGAGACCGCAGAAGCCTACCTTG-3' and 5'-ATGAAAGAGACGGCCGAGG-3'
(corresponding to nucleotides 436463 and 676694 in the IDBP-1 and
IDBP-2 cDNA, respectively). Total RNA extracted from B958 cells was
used as template. Forty cycles of RT-PCR were performed using the
reverse transcription-PCR kit from Perkin Elmer Corp.
(Branchburg, NJ) at a denaturing, reannealing and extension temperature
of 95 C, 60 C and 72 C, respectively. Two major PCR products (a 1013-bp
and 630-bp fragment) were subcloned into a pCR2.1 vector
(Invitrogen), sequenced, and used as probes for Northern
blot analysis of total RNA extracts from B958, OMK, and MLA-144 cells
and for B958 cDNA library screening (see below).
cDNA Library Synthesis and Screening
A cDNA library was prepared from 5 µg B958 cell poly A+ RNA
using the ZAP-cDNA synthesis kit and constructed in pBluescript.SK
phage (Stratagene). Phages were plated on
Escherichia coli strain XL-1 and transferred onto nylon
membranes. The membranes were hybridized in 20 mM
PIPES, 50% deionized formamide, 0.5% SDS, 100 µg/ml denatured
salmon sperm DNA with the two different
[32P]CTP-labeled IDBP cDNA probes at a specific
activity of approximately 1 million cpm per ml by random priming. After
incubation for 20 h at 42 C, the membranes were washed three times
for 10 min each in 0.1x SSC and 0.1% SDS at 60 C, and then exposed to
x-ray film for 16 h. Positive clones were successively spread and
rescreened with either the 1013-bp or 630-bp cDNA probes until pure
clones were obtained. Clones containing the largest inserts (
2.2 to
2.6 kb) were rescued in the E. coli strain SOLR after
in vivo excision of the pBluescript phagemid from Uni-ZAP XR
vector (Stratagene); two distinct cDNAs, IDBP-1 and
IDBP-2, corresponding to the 2.2-kb and 2.6-kb inserts, respectively,
were obtained and subjected to manual sequencing using Sequenase
Version 2.0 kit (United States Biochemical Corp.,
Cleveland, OH). The sense strand and opposite strand sequences were
verified using fluorescent dye terminator chemistry on a PE 377XL
automated DNA sequencer (PE Applied Biosystems, Foster
City, CA).
Protein Extraction and 25-Hydroxyvitamin
D3 Binding Analysis
Confluent cultures of cells were harvested, pelleted, and washed
twice in ice-cold PBS (PBS; 20 mM
Na2HPO4 and 150 mM NaCl, pH 7.2).
Cell pellets were resuspended in ETD buffer (1 mM EDTA, 10
mM Tris-HCl [pH 7.4], 5 mM dithiothreitol,
and 1 mM phenylmethylsulfonylfluoride) and homogenized by
Polytron on ice in five 15-sec bursts. Nuclei, with associated
proteins, were pelleted at 4,000 x g, and the
supernatant was subjected to further centrifugation at 100,000 x
g for 1 h at 4 C. 25-OHD3 binding in the
supernatants of cell extracts was determined by competitive protein
binding assay as previously described (20, 21); extracts were adjusted
with NaCl-containing ETD buffer (pH 8.0) to achieve a final salt
concentration of 0.5 M NaCl for assay.
Protein-bound [3H]25-hydroxyvitamin
D3 (specific activity, 181 Ci/mmol,
Amersham Pharmacia Biotech, Arlington Heights, IL) was
separated from unbound sterol by incubation with dextran-coated
charcoal. Specifically bound 25-OHD3 was
determined by subtracting the mean of duplicate determinations of
binding in the presence of 100 nM radioinert
competitive ligand (nonspecific binding) from the mean of triplicate
determinations of binding in the absence of added competitive ligand
(total binding).
Molecular Cloning of the Primate Vitamin D-24-Hydroxylase,
Osteopontin, and Osteocalcin cDNAs
To obtain a primate-specific 24-hydroxylase, osteopontin, and
osteocalcin cDNA as probe, the following sense and antisense oligomers
were synthesized: the sense oligomer 5'-ATCAGCAAGAGCCGCTCGCTT-3' and
antisense oligomer 5'-CTTGCCATACTTCTTGTGG-3', corresponding to human
24-hydroxylase cDNA sequences 406426 and 660678, respectively (42);
the sense oligomer 5'-CCATGAGAATTGCAGTGATTTGC-3' and antisense oligomer
5'-TCATGTTCATCTACATCATCAGAGTC-3', corresponding to human osteopontin
cDNA sequences 6688 and 386411, respectively (43); and the sense
oligomer 5'-ACACCATGAGAGCCCTCACACTCCTC-3' and antisense oligomer
5'-TAGACCGGGCCGTAGAAGCGCCGATAG-3', corresponding
to human osteocalcin cDNA sequences 1439 and 294320,
respectively (44). These synthetic oligomers were used in RT-PCR with 1
µg total RNA extracted from OMK cells as template and amplified in 40
thermal cycles. Unique 275-bp, 346-bp, and 307-bp RT-PCR products from
each pair of primers were subcloned into the pCR2.1 vector
(Invitrogen) and sequenced. These cDNAs, confirmed to
possess 91%, 95%, and 94% sequence homology with human
24-hydroxylase, osteopontin, and osteocalcin cDNA sequences,
respectively, were used as probes in Northern Blot analysis (see
below).
Northern Blot Analysis
Total cellular RNA was extracted as previously described from
B958, OMK, and MLA 144 cells and subjected to Northern blot analysis
with probes for IDBP-1 and IDBP-2. Wild-type COS-7 and COS-7 cells
transfected with IDBP were incubated in the presence or absence of
1,25-(OH)2D3 were also
extracted for total cellular RNA and subjected to Northern blot
analysis with primate-specific 24-hydroxylase, osteopontin, and
osteocalcin cDNA probes. RNA was electrophoresed through a 1%
agarose-10% formaldehyde gel and then transferred onto nylon membranes
in 0.45 M NaCl/0.045 M sodium citrate, pH 7.
Membranes were hybridized with [32P]-labeled
primate IDBP-1, IDBP-2, vitamin D-24-hydroxylase, osteopontin, or
osteocalcin cDNA probes overnight, washed, and exposed to x-ray film
for 16 h.
 |
ACKNOWLEDGMENTS
|
---|
This work is dedicated to the memory of Bayard D. Catherwood. We
thank B. Sharifi for technical advice and L. Pei and G. Brendt for
helpful discussions and comments on the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. John S. Adams, Burns and Allen Research Institute, Division of Endocrinology, Diabetes and Metabolism, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Room B 131, Los Angeles, California 90048.
This work was supported by NIH Grants DK-07682 and AR-37399.
1 IDBP-1 and IDBP-2 sequence data have been
submitted to GenBank databases under accession numbers AF142571 and
AF142572, respectively. 
Received for publication December 31, 1999.
Revision received May 19, 2000.
Accepted for publication June 5, 2000.
 |
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