Division of Biomedical Sciences (A.W.N.), Departments of
Biochemistry (A.W.N., J.E.B., M.C.D., M.C.F-C.) and Chemistry (W.H.O.,
M.W.H.), University of California, Riverside, California
92521,
Laboratorium voor Experimentele Geneeskunde en
Endocrinologie (R.B., H.v.B.), Katholieke Universiteit Leuven,
Leuven B-3000, Belgium,
Department of Basic Sciences (A.L.R.,
E.D., R.K., M.C.F-C.), University of Texas Dental Branch,
Houston, Texas 77030
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ABSTRACT |
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INTRODUCTION |
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It is well established that 1,25(OH)2D3 can
stimulate biological responses via signal transduction pathways that
utilize nuclear receptors for 1
,25(OH)2D3
(VDRnuc) to regulate gene transcription (3, 4, 5). Indeed the
VDRnuc for 1
,25(OH)2D3 belongs
to a superfamily of receptors for the steroid hormones, retinoic acid,
and T4 (5, 6).
Also there is considerable evidence that
1,25(OH)2D3 can utilize different signal
transduction pathways to generate rapid, nongenomic biological
responses. Rapid actions of 1
,25(OH)2D3 have
been reported in a variety of systems including: 1) stimulation of
intestinal Ca2+ transport in the perfused chick intestine
(termed transcaltachia) (7, 8, 9), which involves the associated rapid
opening of Ca2+ channels (10, 11); 2) a prolonged opening
of both voltage-gated Ca2+ channels (12, 13) and chloride
channels (14) in rat osteosarcoma cells; 3) rapid effects on
45Ca2+uptake in ROS 17/2.8 cells (15), which is
independent of the VDRnuc effects on gene transcription of
osteopontin (OPN) and osteocalcin (16); 4) rapid changes in
intracellular Ca2+ concentrations in a pancreatic ß-cell
line (17); 5) rapid effects on phospholipid metabolism in the intestine
(18), liver (19), parathyroid cells (20), and kidney (21); 6) rapid
changes in membrane fluidity and protein kinase C (PKC) activity in
chondrocytes (22); 7) rapid effects on the cellular redistribution of
PKC (23, 24, 25, 26, 27); and 8) the direct activation of PKC in phospholipid
bilayers (28).
The A ring, triene, and side chain of vitamin D3 and all
its metabolites are, in comparison to other steroid hormones (29, 30, 31),
unusually conformationally mobile (32, 33). It is pertinent in a
structure-function context to discern whether conformers of
1,25(OH)2D3 differ in their ability to
mediate biological responses, i.e. differ in their ability
to interact with the VDRnuc and the signal transduction
process responsible for rapid responses. In this study, we focus on the
conjugated triene system characteristic of vitamin D. Vitamin D seco
steroids can undergo rotation about the 6,7 carbon-carbon single bond,
which permits generation of a continuum of potential ligand shapes
extending from the 6-s-cis (steroid-like conformation) to the the
6-s-trans (extended steroid conformation); see Fig. 1
. We have previously presented a
detailed study of the biological properties of a 6-s-cis
locked analog, 1,25-(OH)2-d5-pre-D3
(34) and reported that two rapid response/nongenomic systems respond as
effectively to 1,25-(OH)2-d5-pre-D3
as 1,25-(OH)2D3, while all tested genomic
systems discriminated markedly against the 6-s-cis locked
1,25-(OH)2-d5-pre-D3 species. This
suggested that the ligand-binding domain of the VDRnuc
for 1,25-(OH)2D3 may be fundamentally different
from the ligand-binding domain of the transducer, which is associated
with rapid responses to 1
,25(OH)2D3.
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RESULTS |
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The four 6-s-cis locked analogs (JM, JN,
JO, JP), are not seco steroids; thus they are
permanently locked in the 6-s-cis conformation. They
are also diastereomers with respect to the orientation of the hydrogen
on C-9 and the methyl group on C-10. In JM and
JN, the hydrogen atom at carbon 9 and the angular methyl
group at carbon 10 are oriented on opposite faces (anti) of the pseudo
plane defined by the steroid ABCD nucleus. More specifically, JM
possesses the 9,10ß (natural) configuration characteristic of
steroids such as cholesterol; JN by contrast is doubly
epimeric (9ß, 10
) at these positions. In JO and
JP, the corresponding hydrogen and methyl have a syn (on the
same face) relationship to one another (9
, 10
, and 9ß10ß,
respectively) with respect to the pseudo plane of the steroid.
Analogs JB, JC, and JD are all seco
steroids that possess a 6,7 double bond, in contrast to
1,25(OH)2D3, and thus are locked in either
the 6-s-trans (JB, JD) or the 6-s-cis
(JC) conformation. Consequently, although JB and
JD are not able to achieve interconversion to the
6-s-cis conformation like
1
,25(OH)2D3, they are able to display
conformational mobility around their two 5,6 and 7,8 single-carbon
bonds, which permits them to generate a population of conformations not
available to 1
,25(OH)2D3. Similarly,
JC can not achieve interconversion to the
6-s-trans conformation, but is also able to rotate around
its 5,6 and 7,8 single carbon bonds so as to generate a different
population of conformations not available to
1
,25(OH)2D3.
Biological Profile of New Analogs in Classic Vitamin D Assays
Table 1 summarizes the
biological profile of the seven new analogs of
1
,25(OH)2D3 in four assays that document the
relative ability of the various analogs to bind in vitro to
the chick intestinal 1
,25(OH)2D3 nuclear
receptor (VDRnuc) and the plasma transport protein, vitamin
D binding protein (DBP), and to stimulate, under in vivo
conditions in a vitamin D-deficient chick, the classic vitamin D
responses of intestinal 45Ca2+ absorption (ICA)
and bone Ca2+ mobilization (BCM).
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None of the six analogs were able to compete effectively with
[3H]1,25-(OH)2D3 in the steroid
competition assay for binding to the VDRnuc; all RCI values
were less than 2%, with four RCI values less than 0.5%. These results
dramatically emphasize that analogs that are not seco steroids, as is
the case for the four 1,25(OH)2D3
6-s-cis locked provitamins, are not effective ligands for
the VDRnuc. In addition, analog JB, which is a
seco steroid, but which is a 6-s-cis locked analog by virtue
of a double bond between carbons 67 and has a RCI of only 0.01%,
also emphasizes that the 6-s-trans conformation is not
preferred by the VDRnuc. A similar conclusion can be
proposed for the 6-s-trans locked analog JD,
which has a RCI of only 1.0%. Apparently the VDRnuc
ligand-binding domain can not accommodate a 6-s-trans locked
analog.
The RCI results for the DBP (Table 1) also emphasize that this
proteins ligand-binding domain does not favor ligands with either a
6-s-trans or 6-s-cis conformation. With the
exception of analog JB, for the other five analogs studied,
all RCI values were <
2%, with four analogs (JD,
JM, JN, JP) consistently reporting
negative RCI values, suggesting a possible allosteric effect upon the
binding of the [3H]1,25-(OH)2D3
employed in the steroid competition assay.
Stimulation of ICA and BCM Activity Responses
The relative ability of the four 6-s-cis locked
diastereomers to generate within 14 h the traditional vitamin D
biological responses of ICA and BCM in the vitamin D-deficient chick,
in vivo, was determined. It has been previously shown that
both the ICA and BCM responses are largely genomic responses; both
responses can be inhibited by administration of actinomycin D, an
inhibitor of DNA-directed RNA synthesis (38). A portion of Table 1
summarizes the ICA and BCM results for the seven new analogs. The most
potent stimulator of ICA and BCM, as expected, was the reference
compound 1
,25(OH)2D3; the activity produced
by 100 pmol of 1
,25(OH)2D3 was set to 100%
for both ICA and BCM. The dose of the comparison analogs required to
achieve a biological response of either ICA or BCM equivalent to the
100 pmol dose of 1
,25(OH)2D3 was then
calculated and converted to a percentage. None of the analogs were
potent agonists for either assay. The only analog that had a barely
detectable agonist activity was analog JN, which had
5%
of the activity of 1
,25(OH)2D3 for both ICA
and BCM.
Rapid (Nongenomic) Actions
Figures 2 and 3
and Table 2
present an evaluation of the relative
ability of the 6-s-cis and 6-s-trans locked
analogs, in comparison to the conformationally flexible
1
,25(OH)2D3, to stimulate rapid nongenomic
responses. Figure 2
focuses on the
biological response of transcaltachia. Vascular perfusion at 650
pM with each of the four 6-s-cis locked analogs
(Fig. 2A
) resulted in a prompt stimulation within 25 min of
transcaltachia and with a 1.8- to 4-fold increase in
45Ca2+ over control levels at 40 min.
Dose-response studies (data not shown) established that the
maximum effective concentration for each analog was
650
pM. Previous studies with
1
,25(OH)2D3 have established that the
dose-response profile for transcaltachia is biphasic; thus, after
the dose that generates a maximum response has been attained, higher
concentrations of agonist result in lower responses (7, 9). Analog
JN was found to be equipotent with
1
,25(OH)2D3, while the other three related
diastereomers generated maximum transcaltachic responses that were
diminished in relation to that of
1
,25(OH)2D3.
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Figure 3 presents an evaluation of
the ability of the 6-s-cis analogs, in comparison to
1
,25(OH)2D3, to stimulate
45Ca2+ uptake into ROS 17/2.8 cells within 1
min. As originally described by Caffrey and Farach-Carson (13), this
response occurs as a consequence of the ability of
1
,25(OH)2D3 or its analogs to prolong the
open time of dihydropyridine-sensitive Ca2+ channels via a
rapid/nongenomic mechanism (see also Refs. 34 and 39). As shown in Fig. 3
, analogs JM and JN stimulated
45Ca2+ uptake in the concentration range of
0.01100 nM. Analog JN consistently (over five
experiments) displayed a biphasic ability to stimulate
45Ca2+ influx, while analogs JM,
JO, and JP (detailed data not shown) displayed
only one concentration for maximum stimulation. The basis for the
biphasic response of JN is not yet known.
Table 2 presents a summary of the
relative activity of the seven new analogs in the two rapid response
assays (transcaltachia and 45Ca2+ uptake into
ROS 17/2.8 cells). With regard to transcaltachia, the relative order of
agonist effectiveness of the four 6-s-cis diastereomers is
JN >JM >JO >JP; neither
of the two 6-s-trans analogs (JB or
JD) displayed significant transcaltachia activity. For the
45Ca2+uptake, both the concentration of analog
that achieved a maximum stimulation of
45Ca2+uptake and the relative calcium index
(RCX) are reported for all analogs that were biologically active. The
RCX is the relative 45Ca2+ influx, normalized
to the level of stimulation produced by the reference
1
,25(OH)2D3 at a concentration of 1
nM, which was set to 100%. The 6-s-trans locked
analog (JB) was unable to act as an agonist, suggesting that
the response element (receptor) that is coupled to the signal
transduction process stimulating 45Ca2+ uptake
is not responsive to 6-s-trans analogs like
1
,25(OH)2-tachysterol3. In contrast, all
five of the 6-s-cis locked analogs (JC,
JM, JN, JO, JP) were
effective at stimulating a rapid uptake of
45Ca2+, although there was some variability in
the reproducibility of the RCX values over a series of five separate
experiments. The two analogs that were most consistently equivalent in
activity to 1
,25(OH)2D3 were analogs
JN and JP (RCX range
60100%), while analogs
JM and JO were somewhat less active (RCX range
4080%).
Genomic Actions
Figures 46 and Table 3
report an evaluation in three cultured
cell lines of the ability of the various analogs to initiate biological
responses via signal transduction pathways believed to require the
participation of the 1
,25(OH)2D3
VDRnuc. 1
,25(OH)2D3 and analogs
were evaluated in MG-63 cells for their relative ability to induce
human osteocalcin (Fig. 4
). Three of the
four 6-s-cis locked 1
,25(OH)2-provitamins
(JM, JO, JP), were found to be 800- to
30,000-fold less effective than 1
,25(OH)2D3
in inducing osteocalcin; the fourth analog, JN, was the most
effective of the diastereomers; however, it was still only 250-fold
less effective than 1
,25(OH)2D3 (see Table 3
). These results are consistent with the interpretation that the
6-s-cis conformation is not able to efficiently interact
with the VDRnuc present in the MG-63 cells.
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Figure 6 reports the agonist activity of
the five 6-s-cis 1
,25(OH)2D3
analogs (JC, JM, JN, JO,
JP) and the two 6-s-trans locked analogs
(JB, JD) on induction of luciferase activity in
ROS 17/2.8 cells after transient transfection of the VDRE containing
the 1.7RI-Luc fusion construct. Several reports have clearly documented
the ability of 1
,25(OH)2D3 to induce OPN at
the level of transcription in ROS 17/2.8 cells, which are known to
express high levels of the VDRnuc. With the exception of
analog JN, all other analogs from both the
6-s-cis and 6-s-trans families displayed a
potency ranging from only 6% to 0.2% of the activity of
1
,25(OH)2D3, for which the measured
luciferase activity was set to 100%. The results suggest that neither
family of conformationally locked analogs can interact productively
with the VDRE present in the osteopontin promoter so as to result in an
activation of the luciferase reporter. In contrast, analog
JN was consistently found to have 2856% of the activity
of 1
,25(OH)2D3; this result is addressed in
Discussion.
Table 3 presents a summary of the
relative ability of 1
,25(OH)2D3 and its
B-ring analogs to inhibit cell proliferation or modulate gene
expression. The data describing the induction of osteocalcin in MG-63
cells and the induction of hGH in COS-7 cells were presented in Figs. 4
and 5
, respectively, while the original data describing the results
from induction of nitroblue tetrazolium (NBT) activity in HL-60 cells
(evaluated by the appearance of NBT reduction) and inhibition of
proliferation of MCF-7 cells (quantitated by inhibition of
[3H]thymidine incorporation) are not presented.
Collectively, the results obtained for both the HL-60 cells and MCF-7
cells suggest that neither 6-s-cis nor 6-s-trans
locked analogs are able to interact effectively with the
VDRnuc for 1
,25(OH)2D3, which is
known to be present [HL-60 cells (40, 41), MCF-7 cells (42, 43)] and
associated with the process of cell differentiation.
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DISCUSSION |
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For 1,25(OH)2D3 the interconversion between
the 6-s-trans and 6-s-cis forms occurs in
solution millions of times per second at room temperature and generates
a continuum of conformers. As yet, the true equilibrium ratio of the
6-s-trans and 6-s-cis conformers of any vitamin D
seco steroid, including 1
,25(OH)2D3, has not
been rigorously determined. It has been estimated by computational
methods that 8899% of 1
,25(OH)2D3 exists
as the 6-s-trans conformation, with only 112%
existing in the 6-s-cis form. This topic has been reviewed
(33), and it seem more reasonable on steric grounds that
1
,25(OH)2D3 is primarily (closer to 99%
rather than 88%) in the 6-s-trans form. However, a more
significant point is that due to the facile interconversion of the
6-s-trans and 6-s-cis conformers of
1
,25(OH)2D3, there exist kinetically
competent amounts of all conformers available to interact with any
receptors which may, in turn, be linked to the generation of biological
responses. Accordingly, in the absence of direct structural information
on protein-bound ligands, insight as to the relative importance of the
steroid or extended conformation in the biological actions(s) of
1
,25(OH)2D3 can more practically be obtained
through studies of locked analogs.
This present study employed two classes of analogs of
1,25(OH)2D3 which are locked in a defined
conformation. One class consists of four diastereomers of the
provitamin D form of 1
,25(OH)2D3
(JM, JN, JO, JP). Because
these analogs are not seco steroids, i.e. their 9,10
carbon-carbon bond is not broken, they are permanently locked in the
6-s-cis conformation (see Fig. 1C
) and as ligands for
receptors there is only one conformation present in rings A, B, C, and
D. The two relevant asymmetric centers, which are at carbon-9 and
carbon-10, result in four analogs (diastereomers) each with an
or
ß orientation of the hydrogen on C-9 and the methyl group on
C-10.
The second class of analogs employed in our studies consists of three
seco steroids with a double bond between carbons 6 and 7 (see Fig. 1B);
accordingly, there can be no rotation around the 6,7 single bond as in
1
,25(OH)2D3. Thus, analogs JB and
JD are 6-s-trans locked analogs, while analog
JC is a 6-s-cis locked analog. However it should
be appreciated that JB, JC, and JD do
display conformational mobility around their two 5,6 and 7,8 single
carbon bonds, which permits them to generate a population of
conformations not available to
1
,25(OH)2D3.
The uniformity of the inability of both the 6-s-cis and
6-s-trans locked analogs to act as an agonist for the six
genomic assays is impressive. None of the analogs had a significant
affinity for the VDRnuc (Table 1). In addition, the lack of
genomic responses was not limited to one cell type or system; there was
no detectable genomic effect at physiological concentrations of the
analogs in vivo in the vitamin D-deficient chick (Table 1
),
in HL-60, or MCF-7 cells (Table 3
), or in COS-7 (Fig. 5
and Table 3
) or
ROS 17/2.8 cells (Fig. 6
), which had been transfected with a promoter
containing the VDREs for 1
,25(OH)2D3. These
results suggest that the VDRnuc utilizes a
1
,25(OH)2D3 ligand conformation that is not
provided by the conformation of either the 6-s-cis or
6-s-trans locked analogs employed in these studies.
Our results demonstrate that two rapid nongenomic biological systems
are fully responsive to the group of 6-s-cis analogs. Both
the process of transcaltachia, as studied in the isolated perfused
chick duodenum (Fig. 2) (7, 8, 9), and the process of Ca2+
channel opening in the rat osteogenic sarcoma cell line (12, 13, 14) and
45Ca2+ uptake in ROS 17/2.8 cells (Fig. 3
and
Table 2
) (15) respond with approximately equivalent potency to the
6-s-cis analogs. Interestingly though, not all four
1
,25(OH)2-provitamin D3 diastereomers were
100% as active as 1
,25(OH)2D3, which no
doubt reflects the fact that there are subtle differences in the shape
of this molecule resulting from different
or ß orientations of
the hydrogen on C-9 and the methyl group on C-10 (see Fig. 1
). The
relative rank order of potency for transcaltachia (Fig. 2
) was
JN > JM
JP >
JO, while the rank order of potency for
45Ca2+in the ROS 17/2.8 cells (Fig. 3
and Table 2
) was JN
JP > JM
> JO. These results are also noteworthy in that this
represents the first clear demonstration that 6-s-cis locked
provitamins D are capable of being potent mediators of selected vitamin
D responses. Before this observation it was generally believed that
only vitamin D seco steroids could generate biological responses (1, 44).
For one 6-s-cis locked analog (JN) there was some
evidence in ROS 17/2.8 cells of its ability to activate over 24 h
a luciferase reporter linked to the OPN hormone-response element for
VDRnuc (Fig. 6). In light of the inability of JN
to consistently activate four other genomic assays (Table 3
) and its
very weak ability to bind to the VDRnuc under in
vitro conditions (RCI = 1.8%; see Table 1
), the mechanism by
which the nuclear activation of the OPN promoter occurs is open to
speculation. Bhatia et al (27) have recently reported that
NB4 promyelocytic leukemic cells could be stimulated to
differentiate into macrophages by combination treatment with a
6-s-cis locked analog,
1
,25(OH)2-d5-pre-D3
(HF), and phorbol ester; these authors proposed that analog
HF was able to interact with a putative membrane receptor
that engaged in cross-talk in collaboration with phorbol ester to
effect the onset of the nuclear response of cell differentiation. Our
previous studies have clearly established that HF is not
able to bind effectively to the VDRnuc or to initiate
genomic responses (34). Thus it is possible that in the ROS 17/2.8
cells, where there is evidence for both a VDRnuc and
putative membrane receptor (16, 39, 45), analog JN may
interact with the putative membrane receptor, which via signal
transduction cross-talk pathways, then results in the activation of the
nuclear response of induction of the mRNA for OPN by VDRnuc
independent pathways. Consistent with this suggestion are recent
results from this laboratory that indicate that 6-s-cis
locked, but not 6-s-trans locked, analogs (JN
vs. JB) can stimulate mitogen-activated protein
kinases in both chick intestinal cells (46) and NB4 cells (47).
Mitogen-activated protein kinases are known to be able to integrate
multiple intracellular signals transmitted by various second messengers
so as to regulate many cellular functions by phosphorylation of numbers
of cytoplasm kinases and nuclear transcription factors including the
epidermal growth factor receptor, c-Myc, and c-Jun (48). Another
alternative explanation might be that analog JN or
JO is metabolized into seco steroids that have a more
favorable interaction with the VDRnuc; however, this seems
unlikely as there are no known lyase enzymes that can break the 9,10
carbon bond of 1
,25(OH)2-7-dehydrocholesterol.
The responsiveness of the signal transduction process for the two rapid
systems occurs in two species, the rat and chick, and in two different
vitamin D target organs, the intestine and bone. In both systems there
is evidence that the biological response involves the opening of
voltage-sensitive Ca2+ channels that are located in the
outer cell membrane (12, 13, 14). It has been postulated for both systems
that the signal transduction process that results in the opening of the
Ca2+ channel may involve a putative membrane receptor for
1,25(OH)2D3. Biochemical evidence has been
presented for the existence of a binding protein specific for
1
,25(OH)2D3 present in the chick intestinal
basal lateral membrane that is correlated with the process of
transcaltachia; this protein has been purified 4500 fold [dissociation
constant (KD) = 0.72 x 10-9
M for 1
,25(OH)2D3] (49).
In both the ROS 17/2.8 cell system and in the perfused intestinal
transcaltachic system, an evaluation has been made of a series of
analogs with differing structural modifications of the reference
compound, 1,25(OH)2D3. Evidence was obtained
for two classes of analogs, those which bind effectively to the
1
,25(OH)2D3 nuclear receptor but which are
ineffective at opening Ca2+ channels and those analogs
which are effective in stimulating the opening of Ca2+
channels but which bind poorly to the
1
,25(OH)2D3 nuclear receptor (9, 15, 39, 45, 50). Collectively, these results have been interpreted as suggesting
the existence of two forms of the
1
,25(OH)2D3 receptor, one present in the
nucleus/cytosol concerned with genomic responses and a second species
present in the plasma membranes of some cells, which are involved in
some fashion with rapid nongenomic biological responses related to
1
,25(OH)2D3. Further support for two
receptors for 1,25-(OH)2D3 has come from
studies with 1ß,25(OH)2D3. This analog binds
very poorly to the nuclear 1
,25(OH)2D3
receptor (RCI = 0.01), and although it is devoid of agonist
activity in transcaltachia, it has been found that
1ß,25(OH)2D3 is a potent antagonist of both
1
,25(OH)2D3-stimulated transcaltachia (51, 52) and the opening of L-type Ca2+ channels (14).
The principal carrier of vitamin D seco steroids throughout the body is
the plasma vitamin D-binding protein (DBP). This protein, which has a
ligand binding domain for vitamin D seco steroids, transports vitamin
D3, 25(OH)D3,
1,25-(OH)2D3, and
24R,25(OH)2D3 throughout the plasma compartment
(53, 54). It is also of importance to learn whether the ligand-binding
domain of DBP prefers the steroid-like conformation (6-s-cis
conformer) or the extended steroid conformation (6-s-trans
conformer). When the data of Table 1 are evaluated, it is clear that
the four 1
,25(OH)2-provitamins D3
(6-s-cis locked) and analogs JB and JD
(6-s-trans locked) all had very low RCI values in comparison
to the reference 1,25-(OH)2D3, whose RCI is
100%. Thus we tentatively conclude that the ligand-binding domain of
DBP prefers neither the 6-s-cis steroid-like conformation nor that of
the 6-s-trans extended steroid conformation of
1,25-(OH)2D3.
On the basis of the data presented in this communication, we conclude that the generation of rapid biological responses prefers ligands that are 6-s-cis locked rather than ligands that are 6-s-trans locked. In contrast to our positive conclusion relative to the rapid responses, we have gained indirect insight only into the preferred ligand configurations for the VDRnuc. Based on the seven analogs studied to date, we can only conclude that the VDRnuc does not prefer analogs that are either 6-s-trans or 6-s-cis locked. It remains to the future to synthesize conformationally locked analogs that are restrained in intermediate conformations relative to the extreme limits defined by the 6-s-cis and 6-s-trans locked analogs. Also it is possible that the VDRnuc requires that its optimal ligands retain conformational flexibility about the 6,7 single carbon bond in yet some unknown fashion.
We are currently synthesizing additional analogs of
1,25(OH)2D3 and conducting further studies
to explore these various possibilities. Collectively these studies
demonstrate the complexity of the structure-function relationships
in the vitamin D endocrine system and the ligand-binding domain
of the 1
,25(OH)2D3 receptors and
binding proteins.
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MATERIALS and METHODS |
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Chemical Synthesis of Four 6-s-cis
1,25(OH)2-Provitamins D (Analogs
JM, JM, JO, and JP) and the Two
6-s-trans Analogs,
1
,25(OH)-Dihydrotachysterol3
(JB) and
1
,25-(OH)2-trans-Isotachysterol3
(JD)
Figure 1 presents the structure of all the analogs of
1
,25(OH)2D3 used in this study. Presented
below is a description of the chemical synthesis of the four
6-s-cis locked diastereomers.
1,25-Dihydroxytachysterol (Analog JB)
A sample of 1,25-dihydroxyprevitamin D3 (15 mg,
0.0360 mmol) in ether (2 ml) was isomerized with iodine in ether (0.49
mM, 300 µl) using a standard procedure (55). HPLC (Rainin
Microsorb, Rainin Instrument Co., Woburn, MA; 5 µm silica, 10 mm
x 25 cm, 11% isopropanol/hexanes) afforded tachysterol analog
JB (8 mg, 53%). 1H-NMR:
(CDCl3)
0.69 (3H, C18-CH3, s),
0.98 (3H, C21-CH3, d, J
6.4 Hz), 1.22 (6H,
C26,27-CH3, s), 1.91 (3H,
C19-CH3, s, 4.14 (1H, H3, dddd,
J
9.5 Hz, 9.5 Hz, 4.7 Hz, 4.7 Hz), 4.24 (1H, H1, br s),
5.75 (1H, H9, br s), 6.15 (1H, H6 or 7, d,
J
16.2 Hz), 6.65 (1H, H7 or 6, d, J
16.2 Hz).
UV: (100% EtOH)
max 282 nm (
20, 300);
sh 290 nm (
18, 100), 272 nm (
16, 800).
HRMS: m/z 416.3283 (calculated for
C27H44O3, 416.3290).
1,25-Dihydroxy-cis-isotachysterol (Analog JC) and
1
,25-Dihydroxyisotachysterol (Analog JD)
Analog JC was prepared as previously reported by VanAlstyne
et al.
(56). A sample of JC (7.5 mg, 0.0180 mmol)
in ether (1 ml) was isomerized in ether (0.49 mM, 150 µl)
using the standard procedure above (55). HPLC (as above for
JB) afforded in order of elution starting JC (2.1
mg, 28%) and analog JD (3.9 mg, 52%). Data for
JD: 1H-NMR: (CDCl3)
0.91 (3H, C18-CH3, s), 0.97 (3H,
C21-CH3, d, J
6.6 Hz), 1.22 (6H,
C26,27-CH3, s), 1.92 (3H,
C19-CH3, s), 1.02.8, 4.16 (1H,
H3, m), 4.25 (1H, H1, br s), 6.47 (1H, H7
or 6, d, J
16.0 Hz), 6.54 (1H, H7 or 6, d,
J
16.0 Hz). UV: (100% EtOH)
max 292 nm
(
30, 800);
sh 302 nm (
23, 100), 282 nm (
25,
600). HRMS: (FAB, Et2O/NBA) m/z 416.3302
(calculated for C27H44O3,
417.3292).
1,25-Dihydroxy-7-dehydrocholesterol (Analog JM,
9
, 10ß-Isomer) and 1
,25-Dihydroxylumisterol (Analog
JN, 9ß, 10
-Isomer)
After 1,25-dihydroxyprevitamin D3 (120 mg) (55) in
methanol was photolyzed (Hanovia 450 watt medium pressure mercury lamp
(Engelhard Hanovia Inc., Newark, NJ), pyrex filter,
> 300 nm) for
3 h at room temperature the residue after concentration was
subjected to HPLC to afford in order of elution JM (9.1 mg,
7.6%), JN (15.0 mg, 12.5%), and the starting previtamin
(10.6 mg, 8.8%). Analysis of the crude mixture by 1H-NMR
spectroscopy showed the ratio of JN:JM to be 3:1. Data for
JM: 1H-NMR: (CDCl3)
0.63 (3H, C18-CH3, s), 0.95 (3H,
C19-CH3, s), 0.96 (3H,
C21-CH3, d, J
5.6 Hz), 1.22 (6H,
C26,27-CH3, s), 2.70 (1H, m), 3.77 (1H,
H1, br s), 4.07 (1H, H3, m), 5.38 (1H, H6
or 7, ddd, J
5.5 Hz, 2.8 Hz, 2.8 Hz), 5.73 (1H, H7 or
6, dd, J
5.5 Hz, 2.2 Hz). UV: (100% EtOH)
max 294 nm (
8, 400), 282 nm (
13, 400), 272 nm
(
12, 800);
min 290 nm (
7, 800), 278 nm (
11,
500); lsh 264 nm (
9, 600). HRMS: (CI,
CH4) m/z 417.3365 (calcd. for
C27H44O3 plus H, 417.3370 Data for
JN: 1H-NMR: (CDCl3)
0.61 (3H, C18-CH3, s), 0.78 (3H,
C19-CH3, s), 0.91 (3H,
C21-CH3, d, J
5.2 Hz), 1.21 (6H,
C26,27-CH3, s), 4.10 (1H, H1, dd,
J
9.2 Hz, 4.8 Hz), 4.14 (1H, H3, dd, J
3.0 Hz, 3.0 Hz),
5.45 (1H, H6 or 7, m), 5.75 (1H, H7 or 6, dd,
J
5.1 Hz, 1.7 Hz). UV: (100% EtOH)
max 282
nm (
6, 900), 274 nm (
7, 300);
sh 294 nm (
3,
900), 264 nm (
5, 900). HRMS: m/z (CI, CH4)
417.3365 (calculated for C27H44O3
plus H, 417.3370).
1,25-Dihydroxypyrocholecalciferol (Analog JO,
9
, 10
-Isomer) and 1
,25-Dihydroxyisopyrocholecalciferol (Analog
JP, 9ß, 10ß-Isomer)
An argon flushed solution of 1,25-dihydroxyprevitamin
D3 (54.2 mg) dissolved in DMF (15 ml) containing one
drop of 2,4,6-trimethylpyridine was heated in a screw cap vial (156 C)
for 18 h. After concentration the crude residue was purified by
HPLC (as above) to afford in order of elution analog JP (7.3
mg, 13.5%), analog JO (20.1 mg, 37.1%), and
1
,25-dihydroxyvitamin D3 (2.1 mg, 3.9%). Analysis of
the crude mixture by 1H-NMR spectroscopy showed the ratio
of JO to JP to be 3:1. Data for JO:
1H-NMR: (CDCl3)
0.53 (3H,
C18-CH3, s), 0.90 (3H,
C21-CH3, d, J
6.0 Hz), 1.02 (3H,
C19-CH3, s), 1.21 (6H,
C26,27-CH3, s), 0.802.05, 2.15 (1H, dd,
J
12.6 Hz, 7.6 Hz), 2.26 (1H, d with fine structure, J
6.1 Hz),
2.54 (1H, br, d, J
6.1 Hz), 4.16 (1H, H3, dddd, J
2.8
Hz, 2.8 Hz, 2.8 Hz, 2.8 Hz), 4.31 (1H, H1, dd, J
12.0 Hz,
4.6 Hz), 5.34 (1H, H6 or 7, d, J
5.7 Hz), 5.61 (1H,
H7 or 6, dd, J
5.7 Hz, 2.5 Hz). UV: (100%
EtOH)
max 286 nm (
9, 400), 276 nm (
9, 300);
min 280 nm (
8, 800);
sh 296 nm (
5, 700), 266 nm (
7,000). HRMS: (CI, CH4) m/z
417.3366 (calcd. for C27H44O3 plus
H, 417.3370). Data for JP: 1H-NMR:
(CDCl3) d 0.65 (3H, C18-CH3, s),
0.92 (3H, C21-CH3, d, J
5.3 Hz), 1.21 (6H,
C26, 27-CH3, s), 1.30 (3H,
C19-CH3, s), 0.802.), 3.71 (1H,
H1, dd, J
2.8 Hz, 2.8 Hz), 3.94 (1H, H3,
dddd, J
10.9 Hz, 10.9 Hz, 5.5 Hz, 5.5 Hz), 5.34 (1H, H6 or
7, ddd, J
5.5 Hz, 2.7 Hz, 2.7 Hz), 5.95 (1H, H7 or
6, d, J
5.5 Hz). UV: (100% EtOH)
max 286 nm (
7, 800), 278 nm (
7, 900);
sh 296 nm (
5, 200), 270 nm (
6, 500).
HRMS: (CI, CH4) m/z 417.3351 (calcd. for
C27H44O3 plus H, 417.3370).
Animals and Cells
Riverside
White Leghorn cockerels (Hyline International, Lakeview, CA) were
obtained on the day of hatch and maintained on a vitamin D-supplemented
diet (1.0% calcium and 1.0% phosphorus; O. H. Kruse Grain and
Milling, Ontario, CA) for 56 weeks to prepare normal vitamin
D3-replete chicks for use in the transcaltachia studies.
All experiments employing animals were approved by the University of
California-Riverside Chancellors Committee on Animals in
Research.
Leuven
The human promyelocytic leukemia cell line (HL-60), the MCF-7 cell
line, the COS-7 cell line, and the MG-63 cells were obtained from the
American Type Culture Collection (Rockville, MD).
Houston
The ROS 17/2.8 cells (kindly provided originally by Dr. Gideon Rodan,
Merck, Sharp and Dohme, West Point, PA) were cultured in DMEM-Hams
F-12 1:1 medium containing 10% FCS (GIBCO BRL, Gaithersburg, MD). For
45Ca2+ uptake experiments, cells were seeded at
a density of 30,000 cells/ml into 3.5-cm dishes and grown to
approximately 50% confluency.
Calcium Uptake Assays
ROS 17/2.8 cells were assayed for Ca2+ uptake using
procedures described previously (16, 34).
Intestinal 45Ca2+
Transport (Transcaltachia)
Measurements of 45Ca2+ transport were
carried out in perfused chick duodena as previously described (8, 9, 34). In brief, normal vitamin D-replete chicks weighing approximately
500 g were anesthetized with Chloropent (Fort Dodge, IA; 0.3 ml
per 100 g), and the duodenal loop was surgically exposed. After
cannulation of the celiac artery and vein, the duodena was perfused
with modified Greys balanced salt solution (GBSS) + 0.9
mM Ca2+which was oxygenated with 95%
O2 and 5% CO2. A basal transport rate was
established by perfusion with control medium for 20 min after the
intestinal lumen was filled with 45Ca2+. The
tissue was then exposed to 1,25-(OH)2D3 or
analogs or reexposed to control medium for an additional 40 min. The
vascular perfusate was collected at 2-min intervals during the last 10
min of the basal and during the entire treatment period. Duplicate
100-µl aliquots were taken for determination of the
45Ca2+ levels by liquid scintillation
spectrometry. The results are expressed as the ratio of the
45Ca2+ appearing in the 40-min test period over
the average initial basal transport period.
Ligand-Binding Studies
The relative ability of each analog to compete with
[3H]1,25-(OH)2D3 for binding to
either the intestinal nuclear receptor for
1,25-(OH)2D3 from vitamin D-deficient chicks or
from a vitamin D-replete pig was carried out under in vitro
conditions according to our standard procedures (36, 57).
The data was plotted as
[competitor]/[[3H]1,25(OH)2D3]
vs.
1/[fraction bound]. The relative competitive index or
RCI was calculated as [slope of competitor]/[slope for
1,25(OH)2D3 ] x 100 as previously
described (36); such plots give linear curves characteristic for each
analog, the slopes of which are equal to the analogs competitive
index value (57). The competitive index value for each analog is then
normalized to a standard curve obtained with nonradioactive
1
,25(OH)2D3 as the competing steroid and
placed on a linear scale of relative competitive index (RCI), where the
RCI of 1
,25(OH)2D3 by definition is 100.
The relative ability of each analog to compete with
[3H]25(OH)D3 for binding to the human vitamin
D-binding protein was run using human DBP (Gc-Globulin, Sigma, St.
Louis, MO) as the binding protein according to our standard procedures
(36, 57). The data were plotted as
[competitor]/[[3H]25(OH)D3]
vs. 1/[fraction bound]. The relative competitive index or
RCI was calculated as [slope of competitor ]/[slope for
25(OH)D3] x 100. Note that although each analog was
assayed in competition with [3H]25(OH)D3, the
data are expressed as relative to the binding of
1,25(OH)2D3, with its RCI set to 100. Thus,
when the RCI of 1
,25(OH)2D3 = 100, the RCI
for 25(OH)D3 = 66,700.
Culture Conditions for HL-60, MCF-7, COS-7, and MG-63
Cells
HL-60 cells were seeded at 1.2 x 105 cells/ml,
and 1,25-(OH)2D3 or its analogs were added in
ethanol (final concentration <0.2%) in RPMI 1640 medium
supplemented with 10% heat-inactivated FCS (GIBCO), 100 U/ml
penicillin, and 100 U/ml of streptomycin (Boehringer). After 4 days of
culture in a humidified atmosphere of 5% CO2 in air at 37
C, the dishes were shaken to loosen any adherent cells, and all cells
were then assayed for differentiation by NBT reduction assay and for
proliferation by [3H]thymidine incorporation.
The COS-7 cells in Dulbeccos medium supplemented with 10% FCS were
seeded into six-well plates to reach 4060% confluence. After 24
h the medium was removed and refreshed with culture medium containing
2% dextran-coated charcoal-treated FCS. The cells were then
cotransfected with the pSG5hVDR expression plasmid (1.5 µg) and the
1,25(OH)2D3 responsive element (VDRE) linked
to the reporter plasmid (CT4)4TKGH (1.5 µg). Finally the
cells were exposed to different concentrations of
1
,25(OH)2D3 or analogs. The medium was
assayed for the expression of human GH using an in house RIA.
MCF-7 cells were cultured in Dulbeccos MEM nutrient mix F-12 (HAM)
medium supplemented with 10% heat-inactivated FCS, glutamine (2
mM), penicillin (100 U/ml) and streptomycin (0.1 mg/ml).
Cultures were maintained at 37 C in a humidified atmosphere of 5%
CO2 in air. MCF-7 cells were seeded at 5000 cells/well in
the above described medium in a 96-well microtiter plate in a final
volume of 0.2 ml per well. Triplicate cultures were performed. After
24 h, 1,25(OH)2D3 or analogs were added
in the appropriate concentrations for an incubation period of 72
h. Then 1 µCi of [3H]thymidine was added to each well,
and the cells were harvested after a 4-h incubation with a Packard
harvester and measured by the Packard Topcount System (Packard,
Meriden, CT).
The MG-63 cells were seeded at 5 x 103 cells/ml in 96-well flat bottomed culture plates (Falcon, Becton Dickinson, Franklin Lakes, NJ) in a volume of 200 µl of DMEM containing 2% of heat-inactivated charcoal-treated FCS and 1,25-(OH)2D3 or its analogs were added in ethanol (final concentration <0.2%). After 72 h of culture in a humidified atmosphere of 5% CO2 in air at 37 C the inhibition of proliferation was measured by [3H]thymidine incorporation and osteocalcin concentration in the medium was determined using a homologous human RIA (34).
NBT Reduction Assay
Superoxide production was assayed by NBT-reducing activity as
described previously (34).
Complimentary DNA Probes and Northern Blot Analyses
Total RNA was extracted from cell monolayers essentially as
previously described (39).
Plasmid Construction and Transfection of ROS 17/2.8 Cells
The rat OPN gene and 5'-flanking sequences have been previously
isolated and sequenced. Construction of the 1.7RI-Luc reporter gene
construct has been described previously (58). Sequence analysis,
transient transfection functional assays, and gel shifting assays have
confirmed that the 1.7RI fragment derived from the upstream region of
the rat OPN gene contains a proximal and a distal VDRE. Both VDREs have
been shown to bind the VDR-retinoid X receptor heterodimer, and their
composite response to 1,25(OH)2D3
stimulation is additive (A. L. Ridall et al.,
manuscript in preparation). Transient transfection in ROS 17/2.8 cells
was performed as described previously (16), with the exception that the
ß-galactosidase activity (monitored for transfection efficiency) was
assayed by using AMPGD or (3-(4-methoxy-spiro-[1,
2-dioxethane-3.2'-tricyclo-[3.3.1.13,7]decan]-4-yl)-phenyl-ß-D-galactopyranoside)
as a substrate, purchased as LumiGal (CLONTECH Laboratories, Inc., Palo
Alto, CA). Enzyme activity was monitored on a Turner Td-20 luminometer
(Turner Designs, Inc., Mt. View, CA).
Statistics
Statistical evaluation of the data was performed by Students
t test for unpaired observations.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported in part by UPHS Grant DK-0901230 (to A.W.N.) and DK-16595 (to W.H.O.), USPHS Grant DE-1031801 (to M.C.F.-C.), and the Belgian Fonds voor Geneeskundig Wetenschappelijk Onderzoek FGWO 3.0091.93 (to R.B.).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This paper is dedicated to the memory of Murray Carl Dormanen who
passed away in September of 1995.
2 Secosteroids are by definition compounds
in which one of the cyclopentanoperhydrophenanthrene rings of the
steroid ring structure is broken. In the case of vitamin D3
the 910 carbon-carbon bond of the B ring is broken generating a
seco-B steroid. See Fig. 1 for examples. The official IUPAC name for
vitamin D3 is
9,10-secocholesta-5,7,10(19 )-trien-3ß-ol.
3 The abbreviations used are
1,25-(OH)2D3, 1,25-dihydroxyvitamin
D3(analog C);
1,25-(OH)2-pre-D3,
1
,25-dihydroxy-previtamin-D3 (analog BC);
D3, vitamin D3; pre-D3,
pre-vitamin-D3;
1
,25(OH)2-d5-pre-D3
1
,25-dihydroxy-9,14,19,19,19-pentadeuterio-previtamin-D3
(analog HF);
1,25-(OH)2-d5-D3,
1
,25-dihydroxy-9,14,19,19,19-pentadeuterio-vitamin-D3,
(analog HG); 1,25-trans-T,
1
,25-dihydroxytachy-sterol3 (analog JB);
1,25-cis-isoT, 1
,25(OH)2-cis-isotachysterol (analog
JC); 1,25-trans-iso-T,
1
,25-dihydroxy-trans-isotachysterol3 (analog
JD); 1
,25(OH)2-7-DHC,
1
,25(OH)2-7-dehydrocholesterol (analog JM);
1
,25(OH)2-L,
1
,25(OH)2-lumisterol3 (analog
JN); 1
,25(OH)2-P
1
,25(OH)2-pyrocalciferol3 (analog
JO); 1
,25(OH)2-IP,
1
,25(OH)2-isopyrocalciferol3 (analog
JP); DBP, vitamin D-binding protein from human (h), rat (r),
or chick (c) serum; ICA, intestinal Ca2+ absorption; BCM,
bone Ca2+ mobilization; transcaltachia, the rapid hormonal
stimulation of intestinal Ca2+ absorption;
VDRnuc, nuclear receptor for
1
,25(OH)2D3; HRMS, high resolution mass
spectrometry; ROS 17/2.8 cells, an osteogenic sarcoma cell line from
rat; MCF-7 cell, a human breast adenocarcinoma cell line; COS-7 cells,
a SV40 transformed African green monkey kidney cell line; HL-60 cells,
a human promyelocytic cell line; MG-63 cells, a human osteosarcoma cell
line; GBSS, Greys balanced salt solution (see Materials and
Methods section for composition); NBT, 4-nitro blue tetrazolium;
OPN, osteopontin; rRNA, ribosomal RNA; VDRE, vitamin D response
element; PKC, protein kinase C.
Received for publication March 21, 1997. Revision received June 13, 1997. Accepted for publication June 17, 1997.
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REFERENCES |
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