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Burns and Allen Research Institute and Division of Endocrinology and Metabolism, Cedars-Sinai Medical Center, University of California School of Medicine, Los Angeles, California 90048
Address all correspondence and requests for reprints to: John S. Adams, M.D., University of California School of Medicine, Division of Endocrinology and Metabolism, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Room B 131, Los Angeles, California 90048.
Abstract
Most New World primates evolved to express a form of compensated
resistance to steroid hormones from the gonads and adrenal glands as
well as to the hydroxylated vitamin D3 prohormone,
25-hydroxyvitamin D3 (25OHD3), and the vitamin
D hormone 1,25-dihydroxyvitamin D3
[1,25-(OH)2D3] originating from the liver and
kidney, respectively. We recently demonstrated that this form of
resistance is associated with the overexpression of a novel member of
the 70-kDa heat shock protein (hsp-70) molecular chaperone family,
which we have termed the intracellular vitamin D binding protein
(IDBP). In the current report we more closely examine the
ligand-binding capability of purified IDBP and two other mammalian
hsp-70 family members, heat-inducible (hsp-70) and constitutively
expressed (hsc-70) hsp-70 proteins. Purified IDBP, hsp-70, and hsc-70
all bound 25OHD3 with relatively high affinity; the mean
Kd for 25OHD3 ranged from 0.5–2.2 nmol/L (rank
order: IDBP 
hsp-70 hsc-70). By Scatchard analysis, high
affinity, specific binding of 1,25-(OH)2D3 was
not reproducibly observed for any of the three members of the hsp-70
family. Unlike purified IDBP, hsc-70 and hsp-70 were also competent
binders of the gonadal steroid 17ß-estradiol (mean Kd for
25OHD3, 2.5 and 6.6 nmol/L by hsc-70 and hsp-70,
respectively), but not of two other gonadal hormones, progesterone and
testosterone. These data suggest that IDBP is relatively specific for
25OHD3 and that additional hsp-70-like binding proteins are
present in unpurified New World primate cell extracts that are specific
for 1-hydroxylated vitamin D metabolites as well as other gonadal
steroid hormones.
MOLECULAR chaperones of the 70-kDa
heat shock protein (hsp-70) family are known to reside in all major
compartments of eukaryotic cells (1). Although hsps undergo selective
expression during conditions of metabolic stress (i.e. heat
exposure), they are also expressed constitutively and participate in a
variety of normal cellular processes, including protein folding,
multimeric protein assembly, protein degradation, and the translocation
of proteins across membranes (1, 2, 3, 4, 5). hsp-70 proteins are examples of
energy-supplying chaperone proteins. The NH2-terminal
portion of hsp-70 molecules (
44 kDa) binds ATP avidly, possesses ATP
hydrolytic (adenosine triphosphatase) activity (6), and influences the
binding and release of polypeptide substrates in the COOH-terminal
(27 kDa) part of the protein (7, 8).
We recently identified a novel member of the hsp-70 protein family, the intracellular vitamin D-binding protein (IDBP), and demonstrated that it interacts specifically with sterol and steroid ligands (9). IDBP is selectively overexpressed in New World primate (NWP) cells (10) and is associated with a vitamin D-, gonadal steroid-, and adrenal steroid-resistant state that is characteristic of most primates in the suborder Platyrrhini (11, 12, 13, 14, 15). Because of its extraordinarily high capacity for ligand (16), IDBP is proposed to act as an intracellular repository for hormones, disallowing access of these small lipid-soluble molecules to their cognate receptor proteins and thus contributing to the state of relative hormone resistance (9). Preliminary data (9) indicated that 25OHD3 binding may be shared by other hsp-70 proteins. The purpose of the current report is to confirm that observation and more clearly define the range of sterol/steroid hormone-binding capability of prototypical members of the hsp-70 family, including purified IDBP.
Materials and Methods
Compounds
[3H]25-hydroxyvitamin D3 ([3H]25OHD3; SA, 181 Ci/mmol) was purchased from Amersham (Arlington Heights, IL). Crystalline vitamin D3 metabolites were provided by Dr. Milan Uskokovic (Hoffmann-La Roche, Nutley, NJ). Steroids and buffer reagents were obtained from Sigma Chemical Co. (St. Louis, MO). Stress-inducible recombinant human hsp-70 and purified bovine constitutive hsp-70 (hsc-70) were obtained from StressGen (Victoria, Canada).
Cell culture
The B95–8 cell line (American Type Culture Collection, Rockville, MD) is a B lymphoblastoid cell line established by Epstein-Barr virus transformation of blood leukocytes from the vitamin D-resistant NWP Callithrix jacchus (common marmoset). The cell line was maintained in RPMI 1640 medium (Irvine Scientific, Santa Ana, CA) supplemented with 10% FCS (Gemini BioProducts, Calabasas, CA), 100 U/mL penicillin, 100 µg/mL streptomycin, and 4 mmol/L glutamine (all from Life Technologies, Grand Island, NY) within an atmosphere of 95% air-5% CO2.
IDBP extraction and purification
Extraction and purification of IDBP were accomplished as previously described by us (9). In summary, B95–8 cells were harvested, washed twice in ice-cold phosphate-buffered saline (20 mmol/L Na2HPO4 and 150 mmol/L NaCl, pH 7.2), resuspended in ETD buffer [1 mmol/L ethylenediamine tetraacetate, 10 mmol/L Tris-HCl (pH 7.4), and 5 mmol/L dithiothreitol] containing 1 mmol/L phenylmethylsulfonylfluoride and homogenized on ice in five 15-s bursts. Nuclei, with associated sterol/steroid receptor proteins, were pelleted at 4,000 x g for 30 min at 4 C. The supernatant was centrifuged at 100,000 x g for 1 h at 4 C and then either used in competitive ligand binding analyses or subjected to the following fast protein liquid chromatography (FPLC) sequence for purification of IDBP: 1) anion- exchange chromatography over bps-diethylamine cellulose (MetaChem Technologies, Torrance, CA) through an inclining NaCl gradient, 2) hydrophobic interaction chromatography over phenyl-Sepharose (Pharmacia Biotech, Alameda, CA) through a declining NaCl gradient, and 3) affinity chromatography over HTP-hydroxyapatite (Bio-Rad, Hercules, CA) through a declining Na2HPO4 gradient. The peak of specific [3H]25OHD3 binding was pooled, microconcentrated, and desalted through a Centricon concentrator with a nominal molecular mass cut-off of 30 kDa (Amicon, Beverly, MA).
Ligand binding analyses
Saturable, specific [3H]ligand binding was measured in crude B95–8 extract, in chromatographically purified protein preparations, and in solutions of hsp-70 and hsc-70 by competitive protein binding assay, and data were transformed by the method of Scatchard as previously described by us (16). Protein samples were adjusted to 0.5 mol/L with NaCl-containing ETD buffer (pH 8.0) and incubated overnight at 4 C with 4 nmol/L radioligand in the presence or absence of 1–100 nmol/L unlabeled competitive ligand. Protein-bound hormone was separated from unbound ligand with dextran-coated charcoal buffer.
Statistical analysis
When appropriate, experimental values for ligand binding under varying conditions were compared using Student’s t test for unpaired samples.
Results
hsp-70 and hsc-70 as vitamin D-binding proteins
Among the most well investigated members of the 70-kDa stress
protein family in mammals are heat-inducible hsp-70 and constitutive
hsc-70. Preparations of recombinant human hsp-70 and purified bovine
hsc-70 were used as a source of potential binding protein. Five hundred
nanograms per mL of either protein in 0.5 mol/L NaCl-ETD buffer
rendered the highest yields of specific 25OHD3
binding and were optimal for 25OHD3 binding to
IDBP in crude and purified cell extracts (9). The ability of increasing
concentrations of 25OHD3 and
1,25-(OH)2D3 to displace 5
nmol/L [3H]25OHD3 from hsp-70 and
hsc-70, respectively, is shown in the upper panels of Fig. 1
. Maximal displacement of
[3H]25OHD3 from hsp-70 was achieved
with 100 nmol/L unlabeled 25OHD3. Scatchard
transformation of the saturable binding data disclosed a mean
Kd for 25OHD3 in the range
of 2 nmol/L (Table 1). Similar
observations were made with hsc-70 (Table 1 and Fig. 1B). At least a
100-fold greater concentration of
1,25-(OH)2D3 was required
to achieve the same 50% decrement in
[3H]25OHD3 binding to hsp-70 and
hsc-70 (Table 1 and Fig. 1). The preference of hsp-70 and hsc-70 for
25OHD3 was confirmed using 5 nmol/L
[3H]1,25-(OH)2D3
as the displaceable ligand (Fig. 1, C and D). A greater than 20%
displacement of labeled hormone from either protein was not achieved
until the competitive ligand concentration reached 100 nmol/L.
Reminiscent of our observations in crude B95–8 cell extracts (9),
internal alterations to the 25OHD metabolite side-chain
(i.e. 25OHD2) and the presence of additional hydroxyl groups
at the C-24 and C-26 positions did little to alter displaceable
[3H]25OHD3 binding.
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We previously showed (9) that crude NWP cell extracts were not
specific for vitamin D metabolite binding; 17ß-estradiol, a
steroid hormone of gonadal origin, was also shown to specifically bind
in heterologous as well as homologous binding assays (17). As depicted
in Fig. 2 and Table 1
, both hsp-70 and
hsc-70 exhibited competitive displacement of
[3H]25OHD3 when exposed to
increasing concentrations of 17ß-estradiol. There was some
apparent displacement of [3H]25OHD3
from hsp-70 and hsc-70 by high concentrations of testosterone
and progesterone; however, 17ß-estradiol was at least 2 orders of
magnitude more effective (Table 1 and Fig. 2) and similar to
1,25-(OH)2D3 in hsp-70 and
hsc-70 binding potential (Fig. 1, C and D). By contrast, estrone, the
major circulating product of the adrenal androgen androstenedione, and
its estrogen receptor-binding metabolites, 16
-hydroxyestrone and
2-hydroxyestrone (18), did not specifically bind to any of the hsps
(data not shown).
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), suggesting the presence of other binding
proteins in crude NWP cell extracts (9) that share biochemical
characteristics with IDBP, but interact specifically with other gonadal
steroid hormones. To resolve this issue, we examined the
displacement of 5 nmol/L [3H]25OHD3
by 100 nmol/L/25OHD3 and 17ß-estradiol after
each chromatographic step used to purify IDBP (Fig. 3).
[3H]25OHD3 displaceable binding by
25OHD3 and 17ß-estradiol copurified through
extraction and diethylamine-anion exchange chromatography. However,
there was preferential retention of 25OHD3
binding and loss of 17ß-estradiol binding to IDBP during the course
of hydrophobic interaction chromatography. Further enrichment of
25OHD3 binding was observed after hydroxyapatite
chromatography and was confirmed in homologous binding assays with
either [3H]25OHD3 or
[3H]17ß-estradiol and their respective radioinert
competitive ligands (9). These results suggest that IDBP, unlike hsp-70
and hsc-70, is specific only for 25-hydroxylated vitamin D ligands.
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Primates inhabiting the South American subcontinent (NWPs) have evolved independently of primates inhabiting the African subcontinent [Old World primates (OWP)] (19). The only OWP over the last 30–50 million yr to successfully inhabit the New World is Homo sapiens, and that event occurred only in the last few thousand years (20). As a consequence of their independent evolution, platyrrhines (NWP) and catarrhines (OWP) have developed divergent phenotypes. One phenotypic difference is the presence of high circulating concentrations of sterol/steroid prohormones and hormones originating from the liver (25OHD3), kidney 1,25-(OH)2D3, adrenal cortex, and gonads in the presence of apparently normally functioning cellular receptor proteins for these hormones (10, 21). The proximal cause of hormone resistance remains uncertain, but may be due at least in part to the constitutive overexpression of two functionally distinct sets of intracellular proteins in NWP cells. One family of proteins exerts a dominant negative effect on hormone receptor-directed transcription (22, 23). A second set of proteins binds sterol/steroid hormones in the cytoplasmic and nuclear compartments (10); the first identified member of this second set, IDBP, effectively alters access of the vitamin D hormone to its cognate receptor (9).
We recently showed that IDBP was structurally related to hsp-70
proteins and that its preferred ligands were 25-hydroxylated
vitamin D metabolites (9). This study also suggested that recombinant
hsp-70 may be capable of specific 25OHD3 binding,
albeit with lower affinity than purified IDBP. The purpose of the
current experiments was to confirm this observation and extend it to
another stress protein member, hsc-70, which is recognized as an
important component of the cellular chaperone system in primate cells
(24). Binding studies using radiolabeled 25OHD3
as displaceable ligand indicated that both recombinant hsp-70 and
purified hsc-70 as well as purified IDBP were specific binding proteins
for 25OHD3. The affinity of all three hsp
proteins for 25OHD3 ranged from 0.5–2.2 nmol/L,
with the rank order of binding potency being purified IDBP
hsp-70 hsc-70.
An unexpected finding was the ability of hsp-70 and hsc-70, but not purified IDBP, to bind 17ß-estradiol in a competitive manner. These data suggest that hormone binding specificities may vary among the hsp-70 proteins, with some hsps being more versatile than others; hsp-70 and hsc-70 are apparently capable of incorporating a closed B ring steroid (17ß-estradiol) into the ligand-binding pocket, which also accepts a sterol with an open B ring (25OHD3), whereas purified IDBP cannot. This is supported by the fact that 25OHD3 and 17ß-estradiol specific binding activities were separated during IDBP purification and confirms our preliminary observations (9) that there is more than one hsp-70-related protein in NWP cell extracts capable of preferentially binding different gonadal steroids.
Before our recent observations (9), hsp-70s were not recognized as specific sterol/steroid ligand-binding proteins. It is possible that binding of small lipid-soluble ligands to hsp-70, like that of cyclosporine-like molecules binding to hsps in the immunophillin family (25), alters the protein ligand-binding potential of hsp-70-related molecules. It is also possible that hormone ligands somehow modify the cytoplasmic aporeceptor complexes for those steroid hormone receptors, including the estrogen, androgen, and glucocorticoid receptors, which are known to be associated with a number of heat shock-like proteins in the inactivated state (26); in this context, the hsp-70s may function as "approximator" proteins, bringing hormone in close proximity to receptor and promoting the hormone-receptor interaction. A potential caveat in this hypothesis is that neither hsp-70 nor hsp-90 is known to associate with the vitamin D receptor, and a hsp-70-related intracellular glucocorticoid-binding protein has not yet been discovered (9). Considering that IDBP appears to be more specific for 25OHD than for gonadal steroids, it is also possible that IDBP overexpression in NWP cells is essential for amplified translocation of substrate 25OHD3 to the inner mitochondrial membrane vitamin D 1- and 24-hydroxylase enzymes that are crucial for insuring that the demand for high circulating levels of the vitamin D hormone are met in NWP (27). In this regard, we have not yet fully characterized the NH2-terminal region of IDBP to determine whether it harbors a mitochondria-targeting sequence found in other hsp-70 proteins (1).
Acknowledgments
This work is dedicated to the memory of Dr. Bayard Catherwood (1948–1995), whose advice and encouragement were greatly appreciated. We are also grateful for the administrative assistance provided by Ms. R. Martinez.
Footnotes
1 This work was supported by NIH Grant AR-37399. 
Received August 19, 1997.
Revised January 5, 1998.
Accepted January 8, 1998.
References
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0.02) reduction in total binding.
