(Received for publication, July 11, 1995; and in revised form, August 4, 1995)
From the
We demonstrate here that RNA levels of 25-hydroxyvitamin
D-24-hydroxylase (24-(OH)ase), a key catabolic enzyme for
1,25-dihydroxyvitamin D
, are increased by a highly
selective retinoid X receptor (RXR) ligand, LG100268, in mice within
hours. Correspondingly, upon LG100268 treatment, kidney 24-(OH)ase
enzymatic activity increases 5-10-fold. The endogenous retinoid
hormones, all-trans-retinoic acid and 9-cis-retinoic
acid, and the synthetic retinoic acid receptor-selective compound,
TTNPB, also stimulate 24-(OH)ase. Additionally, we show that LG100268
stimulates transcription of a luciferase reporter plasmid driven by
24-(OH)ase promoter sequences in the presence of RXR in CV-1 cell
cotransactivation assays. This first demonstration of a gene that is
regulated in the intact animal through an RXR-mediated pathway confirms
earlier hypotheses that RXR is a bona fide hormone receptor.
Regulation of a key gene in the vitamin D signaling pathway by a
retinoid transducer may provide a molecular basis for some of the
documented biological effects of vitamin A on bone and vitamin D
metabolism.
Retinoic acid receptors (RARs) ()have been shown to
act as hormone receptors by virtue of the fact that they bind
all-trans-retinoic acid (tRA) with high affinity and activate
target genes in the presence of tRA in cell-based cotransactivation
assays and in vivo (reviewed in (1) ). Retinoid X
receptors (RXRs) are also thought to act as hormone receptors (reviewed
in (1) ) since they bind 9-cis-retinoic acid (9cRA)
with high affinity (2, 3) and have been shown to
stimulate transcription from distinct elements known as RXR-responsive
elements in promoter sequences of the apolipoprotein A1 (apoA1) and
cellular retinol-binding protein II (CRBPII) genes in cell-based
cotransactivation assays in response to 9cRA(2, 3) ,
tRA(4, 5) , or RXR-selective
ligands(6, 7) . However, these genes have not been
shown to be regulated by retinoids in vivo. While another gene
product (growth hormone) has been shown to increase in cultured cells
treated with RXR- and RAR-selective ligands(8) , there are
currently no known biological target genes of RXR in the intact animal.
RXRs are thought to form homodimers upon interaction with ligand (1) to activate putative RXR-selective genes, such as CRBPII
and apoA1, through RXR-responsive elements in the promoters of those
genes(2, 3, 4, 5, 6, 7) .
RXR is also known to participate in heterodimers with a number of other
intracellular receptors, including the vitamin D receptor (VDR), RAR,
or thyroid hormone receptor to activate a variety of target genes,
which are stimulated by 1,25-dihydroxyvitamin D (1,25-(OH)
D
), tRA, or thyroid hormone,
respectively(1) . RXR is thought to act as a silent partner in
these interactions, although the effects of liganding of RXR under
these circumstances have not been thoroughly explored. Additionally,
recent studies describe instances of stimulation of gene transcription
by hormone-occupied RXR interacting with a supposedly unoccupied orphan
receptor(9, 10) .
To study the consequences of
liganding of RXR in vivo, we began by testing the effects that
an RXR-selective compound might have on the activity of a vitamin
D-inducible gene. 25-Hydroxyvitamin D-24-hydroxylase
(24-(OH)ase) is one such gene product that is induced by
1,25-(OH)
D
(11) , acting through VDR-RXR
heterodimers that bind to a vitamin D response element within the
promoter of the gene(12, 13) . The effects of in
vivo administration of various retinoids on the levels of the
24-(OH)ase gene product were examined. We found that RXR-selective
ligands are able to stimulate 24-(OH)ase RNA levels and enzymatic
activity within hours in normal or vitamin D-deficient mice. These
results describe a novel RXR-target gene, which is also the first
demonstration of such a gene in the intact animal.
Figure 2:
Effects of 1,25-(OH)D
and retinoid treatment of vitamin D-deficient mice on kidney
24-(OH)ase RNA levels. Panel a, 20 µg of total kidney RNA
from female Balb/c mice fed a normal diet treated with sesame oil
vehicle (lanes 1-3) or from mice fed a diet that renders
them 1,25-(OH)
D
-deficient, treated with vehicle (D-def veh, lanes 4-6), with 0.25 µg/kg
1,25-(OH)
D
(lanes 7-9), 0.25
µg/kg 1,25-(OH)
D
plus 30 mg/kg LG100268 (lanes 10-12), or with 30 mg/kg LG100268 (lanes
13-16) for 8 days with once daily doses. Panel b,
20 µg of total kidney RNA from mice fed a normal diet treated with
vehicle (lanes 1-3) or mice fed a vitamin D-deficient
diet treated with either vehicle (lanes 4-6), 0.25
µg/kg 1,25-(OH)
D
(lanes
7-9), 30 mg/kg LG100268 (lanes 13-15), or
with 0.25 µg/kg 1,25-(OH)
D
in combination
with 30 mg/kg LG100268 (lanes 10-12) for 32 h with 5
doses. Blots were hybridized with r24-(OH)ase and hGAPDH probes.
Average-fold induction was determined by comparison of quantitated
bands from drug-treated vitamin D-deficient groups with those of the
vehicle-treated vitamin D-deficient groups.
Figure 3:
Effects of retinoid treatment of normal
fed mice on 24-(OH)ase, RAR, apoA1, and CRBPII expression. Mice
(three to four per dosing group) were treated with two doses 4 h apart
by oral gavage in sesame oil; sacrifice occurred
3 h after the
last dose. Twenty µg of kidney (panela), 30
µg of kidney (panel b), 20 µg of liver (panelc, lanes 1-16), or 20 µg of intestine (panelc, lanes 17-24) total RNA was
subjected to Northern analyses. Mice were treated with sesame oil
vehicle (a and b, lanes 1-4, lanes
17-19; c, lanes 1-4, 17, 21),
LG100268 (a and b, lanes 5-8; lanes 20-22; c, lanes 5-8, 18,
22), 9cRA (a and b, lanes 9-12; lanes 23-25; c, lanes 9-12, 19,
23), tRA (a and b, lanes 13-16; c, lanes 13-16, 20, 24), or TTNPB (a, lanes 26-28). Blots were hybridized with the indicated
probes. Loading was equivalent in all lanes in panelc (GAPDH hybridization not
shown).
Mice were treated with the RXR-selective compound, LG100268 (Fig. 1), a documented highly specific, high affinity binder and
activator of RXRs(14) , and kidney 24-(OH)ase RNA levels were
monitored by Northern analysis ( Fig. 2and 3). Vitamin
D-deficient mice were treated with 1,25-(OH)D
or vehicle with and without LG100268. After an 8-day regimen with
once daily doses, 1,25-(OH)
D
-treated mice
exhibited increased levels of kidney 24-(OH)ase RNA (
6-fold), as
expected (11) (Fig. 2a, lanes
7-9) versus vehicle-treated mice (Fig. 2a, lanes 4-6). LG100268 in
combination treatment with 1,25-(OH)
D
resulted
in an almost complete inhibition (Fig. 2a, lanes
10-12) of the increase in RNA levels that was observed with
1,25-(OH)
D
alone (Fig. 2a, lanes 7-9). Surprisingly, LG100268 treatment alone
resulted in a
2-fold increase in 24-(OH)ase RNA levels (Fig. 2a, lanes 13-16) in comparison
with the vitamin D-deficient mice that received vehicle (Fig. 2a, lanes 4-6). Interestingly, the
effect of LG100268 to repress 1,25-(OH)
D
induction of 24-(OH)ase RNA observed after 8 days of treatment
was not observed after dosing periodically for 32 h (Fig. 2b, lanes 10-12) in comparison
with 1,25-(OH)
D
-treated mice (Fig. 2b, lanes 7-9). However, the
induction of 24-(OH)ase RNA by LG100268 alone was observed at 32 h (Fig. 2b, lanes 13-15), and the -fold
induction was greater (
3.3-fold) than the 2-fold induction seen at
8 days (Fig. 2a, lanes 13-16). Since the
repression effect of LG100268 on 1,25-(OH)
D
induction of 24-(OH)ase occurred only after longer time periods,
a direct negative effect of the RXR ligand on the ability of the
VDR-RXR heterodimer to stimulate 24-(OH)ase seemed unlikely. An
alternate possibility might be that liganded RXR does not
heterodimerize with VDR; complete depletion of the pool of unliganded
RXR available to VDR may take longer than 32 h. However, since LG100268
induces 24-(OH)ase RNA in the absence of 1,25-(OH)
D
at both short and long time periods, it may also act via a
VDR-independent mechanism to stimulate 24-(OH)ase. Since
24-hydroxylation is the initial enzymatic step in the catabolism of
1,25-(OH)
D
, increased 24-(OH)ase activity over
time might be expected to result in lower cellular concentrations of
1,25-(OH)
D
and, therefore, less VDR activity on
its target genes, including 24-(OH)ase.
Figure 1: Structure of LG100268. The chemical name is 6-[1(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopropyl]nicotinic acid.
To test this hypothesis we set out to determine whether the induction of 24-(OH)ase by LG100268 also occurred in the non-vitamin D-deficient state. Mice were fed a normal diet and treated with LG100268 and various other retinoids. TTNPB, a known RAR-selective ligand of high potency(23, 24) , was tested to determine if the retinoid effect on 24-(OH)ase was limited to RXR-selective ligands. Mice were also treated with 9cRA and tRA to determine if the increased levels of 24-(OH)ase RNA observed upon treatment with the synthetic RXR-selective compound could also be elicited by the naturally occurring endogenous retinoid hormones. Three- to five-fold increases in kidney 24-(OH)ase RNA levels were detected from mice treated for a period of 8 h with two doses (spaced 4 h apart) of LG100268, 9cRA, or tRA in comparison with vehicle-treated mice (Fig. 3a, lanes 1-25). While no increase was detected with the potent RAR-selective ligand TTNPB at 1 µg/kg (data not shown), TTNPB at 10 µg/kg did elicit a 3-fold increase in 24-(OH)ase RNA levels (Fig. 3a, lanes 26-28). Potency comparisons between the various retinoids based upon their elicited efficacies (-fold inductions of RNA levels) are not possible without taking into consideration the complicated differences in bioavailability, metabolism, and excretion of the compounds in vivo.
While LG100268 is immaculately specific for RXR as
assessed by both ligand binding assays (Kversus [
H]9cRA for RXRs = 3
nM; for RARs >10 µM) and in cell-based
cotransactivation assays ( (14) and see Fig. 4, lanes 1-4) to ensure that LG100268 was acting as an
RXR-selective ligand in vivo in our system, Northern analysis
was performed on the RAR
gene product. RAR
transcripts have
been shown to be up-regulated by ligands that bind to RAR, but not by
RXR-selective compounds, in F9 cells(8) . Fig. 3b shows that while 9cRA (lanes 9-12) and tRA (lanes 13-16) effect 1.5-2-fold increases in the
levels of kidney RAR
RNA, LG100268 does not (lanes
5-8). Therefore, LG100268 exhibits the properties of an
RXR-selective compound in this system. Since RXR-selective (LG100268)
and RAR-selective (TTNPB) compounds, as well as the endogenous
retinoids 9cRA and tRA, increase 24-(OH)ase RNA levels, both RXR and
RAR are able to participate in the induction of 24-(OH)ase.
Figure 4:
Retinoids stimulate transcription from
human 24-(OH)ase promoter sequences in the presence of retinoid
receptors. RXR (lanes 1, 2, 5, 6, 10, and 12),
RAR
(lanes 3, 4, and 7), VDR (lanes 9 and 11), or RAR
and RXR
(lane 8)
expression plasmids (13, 22) were cotransfected along
with CRBPII (2, 22) (lanes 1 and 2),
TRE-pal (22) (lanes 3 and 4), 24-(OH)-6000 (13) (lanes 5-8), 24-(OH)-400 (13) (lanes 9 and 10), or 24-(OH)-400)-1 (13) (lanes 11 and 12) containing reporter
plasmids. -Fold induction was determined by dividing the normalized
response (2, 22) at saturating concentrations of
ligand by the normalized response with 10
M ligand or with no added ligand.
Certain
genes including apoA1 and CRBPII have been postulated to be activated
through RXR-driven pathways by virtue of the fact that reporter
plasmids driven by sequences from within their promoters have been
shown to be stimulated by 9cRA or RXR-selective compounds in the
presence of cotransfected RXR through a defined element in cell-based
cotransactivation
assays(2, 3, 4, 5, 6, 7) .
However, since this initial work 4 years ago, there have not been any
published reports showing that these genes are regulated in
vivo. To test whether these genes are regulated in vivo in our system, Northern analyses were performed using RNA from
liver (apoA1) and intestine (CRBPII) of the mice treated with two or
five doses of the various retinoids for 8 or 32 h, respectively (Fig. 3c). ApoA1 (Fig. 3c, lanes1-16) and CRBPII (Fig. 3c, lanes 17-24) RNA levels did not change with retinoid
treatment on the 8- or 32-h schedule in comparison with untreated
animals. Therefore, in BALB/c mice under the conditions of these
experiments no significant up-regulation of apoA1 and CRBPII was
observed with retinoid treatment. Additionally, apoA1 RNA levels from
rat liver also were not observed to change after dosing of rats with
three different concentrations of RXR-selective compound for durations
ranging from 4 h to 5 days. ()Therefore, to date, 24-(OH)ase
is the only gene that has been shown to be regulated by RXR-selective
ligands in the intact animal.
Circulating levels of LG100268 and
9cRA were determined by performing pharmacokinetic studies on mice
treated with LG100268 or 9cRA. Two doses of 30 mg/kg LG100268, 3 mg/kg
LG100268, or 10 mg/kg 9cRA were given 4 h apart, mice were sacrificed,
and blood was obtained at various time points. Table 1shows that
doses of 30 mg/kg LG100268 resulted in plasma concentrations of
1-13 µM LG100268 throughout the duration of the
study. Ten-fold lower doses of LG100268 (3 mg/kg) effected the same
-fold increase in 24-(OH)ase RNA levels as did higher LG100268 doses of
12 mg/kg (Fig. 3a) and 30 mg/kg (data not shown) and
resulted in circulating LG100268 levels below the limits of
quantitation of the assay (<250 nM). Doses of 10 mg/kg of
9cRA resulted in plasma concentrations of
100-600 nM 9cRA at the indicated time points. Ten-fold lower doses of 9cRA (1
mg/kg) also effected induction of 24-(OH)ase RNA (Fig. 3a); circulating levels were not measured at that
dose. Therefore, the determined circulating levels of LG100268 and 9cRA
after administration of effective doses for exertion of a biological
effect (i.e. increase of 24-(OH)ase RNA) are in the low
nanomolar range.
To ascertain if the observed increases in
24-(OH)ase RNA corresponded with an increase in kidney 24-hydroxylase
enzyme activity, metabolic conversion assays were performed. Table 2shows that enzymatic conversion of 25-(OH)D to
24,25-(OH)
D
in kidney extracts from mice
treated with LG100268 (3 mg/kg twice daily for 8 h or 3.5 days) was
5-10-fold greater than the conversion observed in kidney extracts
from normal untreated mice. The retinoids utilized in this study,
including LG100268, are the only agents other than
1,25-(OH)
D
that have been shown to stimulate
24-(OH)ase activity. 1,25-(OH)
D
has been
reported to increase 24-(OH)ase activity by 4-20-fold, depending
on the species examined(25) . Therefore, the increase in kidney
24-(OH)ase RNA levels exerted by ligands that bind to RXR and RAR is
reflected in a corresponding bioeffect, i.e. the kidney
24-(OH)ase enzymatic activity increases. Ultimately, elevated levels of
24-(OH)ase over time might be expected to decrease cellular
concentrations of 1,25-(OH)
D
, leading to
reduced activity of VDR on its target genes, including 24-(OH)ase. This
hypothesis is borne out by the inhibition by LG100268 of
1,25-(OH)
D
-induced 24-(OH)ase RNA levels after
8 days of treatment (Fig. 2a) and may provide a
molecular basis for some of the accumulated observations over the past
50 years describing vitamin A effects on bone that mimic vitamin D
deficiency conditions (26, 27, 28, 29, 30) and on
other evidence showing a decrease in serum 1,25-(OH)
D
levels with high concentrations of vitamin
A(30, 31, 32) .
To test whether the
retinoids are able to stimulate transcription from 24-(OH)ase promoter
sequences and to confirm that RXR is required for that activation,
cotransfection experiments were performed in CV-1 cells with a
promoterless luciferase vector containing human 24-(OH)ase promoter
sequences(13) . Saturating concentrations of 9cRA or LG100268
elicited 7-10-fold inductions of luciferase activity in CV-1
cells that had been cotransfected with an expression vector producing
RXR and a reporter plasmid containing 6 kb of 24-(OH)ase promoter
sequence (13) (Fig. 4, lanes 5 and 6) while 1.5-2-fold induction was observed without
transfected receptor, representative of endogenous RXR present in CV-1
cells (data not shown). 9cRA was also able to stimulate promoter
activity in the presence of RAR
(
9-fold (Fig. 4, lane 7)) or RXR
and RAR
(
14-fold (Fig. 4, lane 8)). 1,25-(OH)
D
induced transcription (
10-fold) from a 400-bp 24-(OH)ase
promoter sequence in the presence of VDR, as previously demonstrated (lane 9)(13) . This reporter was also activated by
9cRA in the presence of RXR (lane 10). A reporter vector with
the 400-bp 24-(OH)ase promoter sequence containing three mutations in
the upstream half-site of the vitamin D responsive element no longer
binds to VDR, and the 1,25-(OH)
D
-induced
transactivation through the promoter sequence is abolished (13) (Fig. 4, lane 11). 9cRA was able to
stimulate luciferase activity in the presence of RXR via the mutant
promoter (Fig. 4, lane 12) as well as it did through
the wild type promoter sequence (Fig. 4, lane 10).
Therefore, retinoid ligands acting through their respective receptors
are able to stimulate transcription from the 24-(OH)ase promoter in a
heterologous system utilizing cis element(s) that appear to be
distinct from or overlapping with that utilized by VDR. Hence,
effectors of vitamin A signal transduction, including the RXR-selective
ligand, LG100268, acting through the nuclear retinoid receptors, induce
a key gene involved in vitamin D signaling, thereby adding a new level
of complexity to the cross-talk between the two systems.