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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 B95–8 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. 1Go, left panel) and 630-bp (Fig. 1Go, right panel) cDNAs were then used as probes in Northern blots of total cellular RNA extracted from hormone-resistant New World primate B95–8 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 B95–8, 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.

 
These two cDNAs were then used to screen an unamplified B95–8 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. 2Go). 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. 9Go).

 
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. 3Go). 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 (80–90% 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; Student’s test for unpaired samples).

 
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. 4AGo) and demonstrated maximum stimulation of 24-hydroxylase mRNA levels in primate cells after overnight incubation with 100 nM 1,25-(OH)2D3 (Fig. 4BGo). 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. 4CGo). 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).

 
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 B95–8 New World primate cells transiently transfected with antisense IDBP-1, with antisense IDBP-2, or with both (Fig. 5Go). 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. 6Go. 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. 6AGo). 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. 6BGo).



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Figure 5. Specific 25-OHD3 Binding in Hormone-Resistant B95–8 Cells Transfected with Antisense IDBP cDNA Constructs

Hormone-resistant New World primate B95–8 cells (80–90% 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; Student’s 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.

 
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. 7Go); 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. 8Go). Similar to what was observed with the 24-hydroxylase gene (see left-most panels of Fig. 8Go), 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 66–68 and 386–411, respectively) and osteocalcin (nucleotides 14–39 and 294–320, 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).

 
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. 2Go), 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. 9AGo). Transfection of amino-terminal cDNA constructs spanning nucleotides 1–315 and 1–474 of the open reading frame for IDBP-1 as well as the 1156–1938 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. 9BGo (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. 9BGo).



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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 1–1938 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 1–315, 1–474, and 1156–1938 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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 3Go). 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. 9BGo). 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. 4Go, 6Go, and 8Go) 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. 4Go). Furthermore, transfection of sense and antisense cDNA constructs for IDBP-1 and IDBP-2 cDNA into vitamin D-responsive and -resistant cells (Figs. 3Go and 5Go, 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. 9Go). 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture and Transfection of IDBP cDNAs
B95–8, OMK, MLA-144, and COS-7 primate cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA). B95–8, 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 B95–8 or COS-7 cells by lipofection (LipoTAXI, Stratagene, La Jolla, CA). Briefly, cells were seeded into 100-mm dishes, grown to 80–90% 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 0–100 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 1421–1449 in the IDBP-1 cDNA and 1285–1305 in the IDBP-2 cDNA), while the more 5'-sense strand primers were 5'-ATGAAGGAGACCGCAGAAGCCTACCTTG-3' and 5'-ATGAAAGAGACGGCCGAGG-3' (corresponding to nucleotides 436–463 and 676–694 in the IDBP-1 and IDBP-2 cDNA, respectively). Total RNA extracted from B95–8 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 B95–8, OMK, and MLA-144 cells and for B95–8 cDNA library screening (see below).

cDNA Library Synthesis and Screening
A cDNA library was prepared from 5 µg B95–8 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 406–426 and 660–678, respectively (42); the sense oligomer 5'-CCATGAGAATTGCAGTGATTTGC-3' and antisense oligomer 5'-TCATGTTCATCTACATCATCAGAGTC-3', corresponding to human osteopontin cDNA sequences 66–88 and 386–411, respectively (43); and the sense oligomer 5'-ACACCATGAGAGCCCTCACACTCCTC-3' and antisense oligomer 5'-TAGACCGGGCCGTAGAAGCGCCGATAG-3', corresponding to human osteocalcin cDNA sequences 14–39 and 294–320, 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 B95–8, 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. Back

Received for publication December 31, 1999. Revision received May 19, 2000. Accepted for publication June 5, 2000.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Pilbeam D 1984 The descent of hominoids and hominids. Sci Am 250:84–96[Medline]
  2. Milton K 1993 Diet and primate evolution. Sci Am 269:86–93
  3. Brown GM, Grota LJ, Penney DP, Reichlin S 1970 Pituitary-adrenal function in the squirrel monkey. Endocrinology 86:519–529[Medline]
  4. Lipsett MB, Chrousos GP, Tomita M, Brandon DD, Loriaux DL 1985 The defective glucocorticoid receptor in man and nonhuman primates. Recent Prog Horm Res 41:199–247[Medline]
  5. Brandon DD, Markwick AJ, Chrousos GP, Loriaux DL 1989 Glucocorticoid resistance in humans and nonhuman primates. Cancer Res 49:2203s–2213s
  6. Reynolds PD, Ruan Y, Smith DF, Scammell JG 1999 Glucocorticoid resistance in the squirrel monkey is associated with overexpression of the immunophilin FKBP51. J Clin Endocrinol Metab 84:663–669[Abstract/Free Full Text]
  7. Chrousos GP, Brandon D, Renquist DM, Tomita M, Johnson E, Loriaux DL, Lipsett MB 1984 Uterine estrogen and progesterone receptors in an estrogen- and progesterone- "resistant" primate. J Clin Endocrinol Metab 58:516–520[Abstract]
  8. Chrousos GP, Loriaux DL, Brandon D, Shull J, Renquist D, Hogan W, Tomita M, Lipsett MB 1984 Adaptation of the mineralocorticoid target tissues to the high circulating cortisol and progesterone plasma levels in the squirrel monkey. Endocrinology 115:25–32[Abstract]
  9. Gacad MA, Deseran MW, Adams JS 1992 Influence of ultraviolet B radiation on vitamin D3 metabolism in New World primates. Am J Primatol 28:263–270
  10. Shinki T, Shiina Y, Takahashi N, Tanioka Y, Koizumi H, Suda T 1983 Extremely high circulating levels of 1{alpha},25-dihydroxyvitamin D3 in the marmoset, a New World monkey. Biochem Biophys Res Commun 114:452–457[Medline]
  11. Adams JS, Gacad MA, Baker AJ, Gonzales B, Rude R 1985 Serum concentrations of 1,25-dihydroxyvitamin D3 in Platyrrhini and Catarrhini: a phylogenetic appraisal. Am J Primatol 9:219–224
  12. Gacad MA, Adams JS 1991 Endogenous blockade of 1,25-dihydroxyvitamin D-receptor binding in New World primate cells. J Clin Invest 87:996–1001[Medline]
  13. Arbelle JE, Chen H, Gaead MA, Allegretto EA, Pike JW, Adams JS 1996 Inhibition of vitamin D receptor-retinoid X receptor-vitamin D response element complex formation by nuclear extracts of vitamin D-resistant New World primate cells. Endocrinology 137:786–789[Abstract]
  14. Chen H, Hu B, Gacad MA, Adams JS 1998 Cloning and expression of a novel dominant-negative-acting estrogen response element-binding protein in the heterogeneous nuclear ribonucleoprotein family. J Biol Chem 273:31352–31357[Abstract/Free Full Text]
  15. Hewison M, Rut AR, Kristjansson K, Walker RE, Dillon MJ, Hughes MR, O’Riordan JL 1993 Tissue resistance to 1,25-dihydroxyvitamin D without a mutation of the vitamin D receptor gene. Clin Endocrinol (Oxf) 39:663–670[Medline]
  16. Albertson BD, Brandon DD, Chrousos GP, Loriaux DL 1987 Adrenal 11-hydroxylase activity in a hypercortisolemic New World primate: adaptive intra-adrenal changes. Steroids 49:497–505[CrossRef][Medline]
  17. Tomita M, Brandon DD, Chrousos GP, Vingerhoeds AC, Foster CM, Fowler D, Loriaux DL, Lipsett MB 1986 Glucocorticoid receptors in Epstein-Barr virus-transformed lymphocytes from patients with glucocorticoid resistance and a glucocorticoid-resistant New World primate species. J Clin Endocrinol Metab 62:1145–1154[Abstract]
  18. Jones G, Strugnell SA, DeLuca HF 1998 Current understanding of the molecular actions of vitamin D. Physiol Rev 78:1193–1231[Abstract/Free Full Text]
  19. Gacad MA, Adams JS 1993 Identification of a competitive binding component in vitamin D- resistant New World primate cells with a low affinity but high capacity for 1,25-dihydroxyvitamin D3. J Bone Miner Res 8:27–35[Medline]
  20. Gacad MA, Chen H, Arbelle JE, LeBon T, Adams JS 1997 Functional characterization and purification of an intracellular vitamin D-binding protein in vitamin D-resistant New World primate cells. Amino acid sequence homology with proteins in the hsp-70 family. J Biol Chem 272:8433–8440[Abstract/Free Full Text]
  21. Freedman LP, Lemon DP 1997 Structural and functional determinants of DNA binding and dimerization by the vitamin D receptor. In: Feldman D, Glorieux FH, Pike JW (eds) Vitamin D. Academic Press, San Diego, CA, pp 127–148
  22. Gacad MA, Adams JS 1998 Proteins in the heat shock-70 family specifically bind 25-hydroxyvitamin D3 and 17ß-estradiol. J Clin Endocrinol Metab 83:1264–1267[Abstract/Free Full Text]
  23. Hartl FU 1996 Molecular chaperones in cellular protein folding. Nature 381:571–579[CrossRef][Medline]
  24. Adams JS, Gacad MA 1988 Phenotypic diversity of the cellular 1,25-dihydroxyvitamin D3-receptor interaction among different genera of New World primates. J Clin Endocrinol Metab 66:224–229[Abstract]
  25. Gacad MA, Adams JS 1992 Specificity of steroid binding in New World primate B95–8 cells with a vitamin D-resistant phenotype. Endocrinology 131:2581–2587[Abstract]
  26. Cooke NE, Haddad JG 1989 Vitamin D binding protein (Gc-globulin). Endocr Rev 10:294–307[Medline]
  27. Nykjaer A, Dragun D, Walther D, Vorum H, Jacobsen C, Herz J, Melsen F, Christensen EI, Willnow TE 1999 An endocytic pathway essential for renal uptake and activation of the steroid 25-(OH) vitamin D3. Cell 96:507–515[Medline]
  28. Hussain MM, Strickland DK, Bakillah A 1999 The mammalian low-density lipoprotein receptor family. Ann Rev Nutr 19:141–172[CrossRef][Medline]
  29. Leung TK, Rajendran MY, Monfries C, Hall C, Lim L 1990 The human heat-shock protein family. Expression of a novel heat-inducible HSP70 (HSP70B') and isolation of its cDNA and genomic DNA. Biochem J 267:125–132[Medline]
  30. Bhattacharyya T, Karnezis AN, Murphy SP, Hoang T, Freeman BC, Phillips B, Morimoto RI 1995 Cloning and subcellular localization of human mitochondrial hsp70. J Biol Chem 270:1705–1710[Abstract/Free Full Text]
  31. Kallen CB, Billheimer JT, Summers SA, Stayrook SE, Lewis M, Strauss JF 3rd 1998 Steroidogenic acute regulatory protein (StAR) is a sterol transfer protein. J Biol Chem 273:26285–26288[Abstract/Free Full Text]
  32. Kallen CB, Arakane F, Christenson LK, Watari H, Devoto L, Strauss JF 3rd 1998 Unveiling the mechanism of action and regulation of the steroidogenic acute regulatory protein. Mol Cell Endocrinol 145:39–45[CrossRef][Medline]
  33. Takayama S, Bimston DN, Matsuzawa S, Freeman BC, Aime-Sempe C, Xie Z, Morimoto RI, Reed JC 1997 BAG-1 modulates the chaperone activity of Hsp70/Hsc70. EMBO J 16:4887–4896[Abstract/Free Full Text]
  34. Hohfeld J, Minami Y, Hartl FU 1995 Hip, a novel cochaperone involved in the eukaryotic Hsc70/Hsp40 reaction cycle. Cell 83:589–598[Medline]
  35. Takayama S, Sato T, Krajewski S, Kochel K, Irie S, Millan JA, Reed JC 1995 Cloning and functional analysis of BAG-1: a novel Bcl-2 binding protein with anti-cell death activity. Cell 80:279–284[Medline]
  36. Zeiner M, Gehring U 1995 A protein that interacts with members of the nuclear hormone receptor family: identification and cDNA cloning. Proc Natl Acad Sci USA 92:11456–11469[Abstract]
  37. Takayama S, Xie Z, Reed JC 1999 An evolutionarily conserved family of Hsp70/Hsc70 molecular chaperone regulators. J Biol Chem 274:781–786[Abstract/Free Full Text]
  38. Hohfeld J 1998 Regulation of the heat shock conjugate Hsc70 in the mammalian cell: the characterization of the anti-apoptotic protein BAG-1 provides novel insights. Biol Chem 379:269–274
  39. Craig TA, Lutz WH, Kumar R 1999 Association of prokaryotic and eukaryotic chaperone proteins with the human 1{alpha},25-dihydroxyvitamin D(3) receptor. Biochem Biophys Res Commun 260:446–452[CrossRef][Medline]
  40. Adams JS, Singer FR, Gacad MA, Sharma OP, Hayes MJ, Vouros P, Holick MF 1985 Isolation and structural identification of 1,25-dihydroxyvitamin D3 produced by cultured alveolar macrophages in sarcoidosis. J Clin Endocrinol Metab 60:960–996[Abstract]
  41. St-Arnaud R, Glorieux FH 1998 24,25-dihydroxyvitamin D-active metabolite or inactive catabolite? Endocrinology 139:337–3374
  42. Chen KS, Prahl JM, Deluca HF 1993 Isolation and expression of human 1,25-dihydroxyvitamin D3 24-hydroxylase cDNA. Proc Natl Acad Sci USA 90:4543–4547[Abstract]
  43. Kiefer MC, Bauer DM, Barr PJ 1989 The cDNA and derived amino acid sequence for human osteopontin. Nucleic Acids Res 17:3306[Medline]
  44. Kiefer MC, Saphire AC, Bauer DM, Barr PJ 1990 The cDNA and derived amino acid sequence of human and bovine bone Gla protein. Nucleic Acids Res 18:1909[Medline]