(Received for publication, April 21, 1995)
From the
The genomic actions of 1,25-dihydroxyvitamin D (1,25(OH)
D
) are mediated by the
intracellular vitamin D receptor (VDR). Although immunocytochemistry
has shown that disruption of microtubular assembly prevents nuclear
access of the sterol-VDR complex, the role of microtubules in the
response to 1,25(OH)
D
has not been studied in
viable cells. Our studies examined this interaction in normal human
monocytes. Monocytes convert 25(OH)D
to
1,25(OH)
D
and to 24-hydroxylated metabolites
more polar than 1,25(OH)
D
. Microtubule
disruption totally abolished the ability of exogenous
1,25(OH)
D
to suppress its own synthesis and to
induce 24-hydroxylase mRNA and activity, without affecting either total
1,25(OH)
D
uptake or maximal
1,25(OH)
D
-VDR binding. Thus, intact
microtubules are essential for
1,25(OH)
D
-dependent modulation of gene
transcription. Interestingly, microtubule disruption also decreased
monocyte 1,25(OH)
D
synthesis, not by decreasing
the V
of monocyte mitochondrial
1
-hydroxylase but through an increase in the K
for 25(OH)D
. We examined 25(OH)D
transport. Microtubule disruption did not affect total cellular
25(OH)D
uptake but reduced its intracellular trafficking to
the mitochondria. Thus, microtubules participate in intracellular
25(OH)D
transport, and their integrity determines normal
1,25(OH)
D
synthesis.
The most active metabolite of vitamin D, 1,25-dihydroxyvitamin
D (1,25(OH)
D
)), (
)is a
potent steroid hormone. Similar to other steroids, its genomic actions
require binding to its intracellular receptor and interaction of the
1,25(OH)
D
-vitamin D receptor (VDR) complex with
specific vitamin D responsive regions in the
genome(1, 2) .
It has been suggested recently that hormone-free glucocorticoid receptors are located predominantly in the cytoplasm, and, after the addition of steroid, they are rapidly translocated to the nucleus (3, 4, 5, 6) . The transfer of glucocorticoid receptor into the nucleus involves translocation along microtubules as revealed by immunofluorescent studies (7) in a process that is driven by tubulin-associated dynein motors(8) .
In the case of vitamin D, there is some controversy as to whether
apoVDRs reside only in the nucleus like the thyroid hormone receptor (9) or whether they can undergo ligand-dependent translocation
like the glucocorticoid receptor(10, 11) . Using a
recently developed fluorescent ligand, Barsony et al.(12) were able to demonstrate the cytoplasmic localization of
the VDR in viable human skin fibroblasts, porcine kidney epithelial
cells, human breast cancer cells, and rat osteosarcoma cells,
supporting previous immunocytochemical findings in fixed human
fibroblasts (13) and osteoblasts(14) . Although
immunocytology has shown that cytoplasmic VDR co-localizes with tubulin
and that disruption of microtubular assembly blocks the translocation
of the 1,25(OH)D
-VDR complex into the nucleus (15) in microwave fixed fibroblasts, the role of microtubules
on VDR transport in viable cells has never been evaluated. We
hypothesized that if this intracellular transport system is of
physiological relevance, the genomic response to
1,25(OH)
D
should be impaired with alterations
in the structure or function of the microtubule network. We tested this
hypothesis in normal human monocytes.
Human monocytes express
receptors for 1,25(OH)D
that are
indistinguishable from those described in classical
1,25(OH)
D
target tissues(16) , and the
interactions of 1,25(OH)
D
with
monocytes-macrophages have critical implications for the regulation of
immune responses(17, 18, 19, 20) .
Our laboratory has demonstrated that peripheral blood monocytes from
normal individuals constitutively express 1
-hydroxylase, the
enzyme responsible for the conversion of 25-hydroxyvitamin D
(25(OH)D
) to
1,25(OH)
D
(21) . We have also shown that
when peripheral blood monocytes were exposed to physiological
concentrations of 1,25(OH)
D
, 1
-hydroxylase
activity is markedly suppressed. In addition, exogenous
1,25(OH)
D
promotes an induction of vitamin D
catabolism by increasing 24-hydroxylase mRNA (
)and
activity(22) . Because both effects of the sterol require at
least 2 h of exposure to
1,25(OH)
D
(22) , it is likely that the
inhibition of 1,25(OH)
D
production by
1,25(OH)
D
also involves a genomic mechanism. In
the present studies, we used this human monocyte model to assess the
physiological relevance of microtubule integrity in the response to
1,25(OH)
D
. This report demonstrates for the
first time that integrity of the microtubule network is critical for a
normal genomic response to 1,25(OH)
D
and that
an intracellular tubulin- 25(OH)D
transport system mediates
the delivery of 25(OH)D
to mitochondria, thus modulating
the rate of 1,25(OH)
D
synthesis by monocytes.
The effect of the reversible
microtubule disrupting agent, nocodazole, on the response to
1,25(OH)D
was examined as follows. Adherent
monocytes were treated with 0 or 10 µM nocodazole for 30
min before a co-incubation with 0.24 nM 1,25(OH)
D
and 10 µM nocodazole for 4 h. To test reversibility of the effect of
nocodazole, adherent cells were exposed to 0 or 10 µM nocodazole (dissolved in 1 µl of dimethyl sulfoxide) for 4 h;
cells were then washed, and the media were replaced by fresh
nocodazole-free incubation media. Monocytes were allowed to recover for
4 h. Control and nocodazole-treated monocytes were then exposed to 0.24
nM 1,25(OH)
D
for 4 h.
In each
independent experiment, the synthesis of 1,25(OH)D
and of polar metabolites by monocytes from the same individual
was measured in triplicate, and the steady state 24-hydroxylase mRNA
levels were quantified in duplicate using a ribonuclease protection
assay.
Monocyte RNA samples were dissolved in 4 µl of diethyl
pyrocarbonate water and mixed with 26 µl of hybridization buffer
(80% formamide, 50 mM PIPES, pH 6.4, 400 mM NaCl, 1
mM EDTA) containing the P-labeled riboprobes for
human 24-hydroxylase and glyceraldehyde-3-phosphate dehydrogenase. The
specific activity of the riboprobe for glyceraldehyde-3-phosphate
dehydrogenase was reduced 5-fold to obtain labeled, protected fragments
of similar radioactivity. After hybridization at 45 °C for 16 h,
the samples were mixed with 150 µl of ribonuclease digestion
mixture containing 2 µg of ribonuclease T1 in 10 mM Tris-HCl, pH 5.0, 300 mM NaCl, 5 mM EDTA, and
incubated at 37 °C for 15 min. Proteinase K (50 µg) and 20
µl of 5% SDS were added, and the samples were incubated for 15 min
at 37 °C. Following phenol:chloroform extraction and ethanol
precipitation, the samples were resolved on a 5% polyacrylamide gel.
The dried gel was exposed to x-ray film for 120 h, and the bands were
quantified by scanning densitometry.
To assess the purity of the subcellular fractions, we assayed succinic dehydrogenase (28) and NADPH-cytochrome c reductase (29) activities as mitochondrial and microsomal specific markers, respectively. Results were expressed as percent of total enzymatic activity in each subcellular fraction.
Figure 1:
Dose-dependent effect of colchicine
on the suppression of 1,25(OH)D
synthesis by
exogenous 1,25(OH)
D
. Adherent cells were
incubated in serum-free media with doses of colchicine from 0 to 750
µM for 30 min. Media were removed, and cells were washed
and incubated with 0 (vehicle) or 0.24 nM 1,25(OH)
D
for 4 h. Synthesis of
1,25(OH)
D
was measured as outlined under
``Experimental Procedures.'' Results are expressed as
femtomoles/µg of DNA/h. Values are mean ± S.E. of five
independent experiments performed in triplicate (*, differs from
control, p
0.05).
Because colchicine alone caused a dose-dependent
reduction of 1,25(OH)D
production by monocytes,
we performed the experiments summarized in Fig. 2to define the
effects of microtubule disruption in the response of monocytes to
1,25(OH)
D
. The lowest (25 µM) and
highest (750 µM) doses of colchicine, effective in
blocking the ability of 1,25(OH)
D
to suppress
its own production, were used to examine the effect of microtubule
disruption on the ability of 1,25(OH)
D
to
suppress its own synthesis (upper panel) and to induce vitamin
D catabolism (generation of 24-hydroxylated metabolites more polar than
1,25(OH)
D
(polar metabolites); lower
panel). In monocytes with intact microtubules (Controls), exposure
to 0.24 nM 1,25(OH)
D
for 4 h reduced
1,25(OH)
D
synthesis to 55.2 ± 1.3% and
increased the generation of polar metabolites 7-fold above vehicle
controls, respectively. However, when monocytes were treated with 25 or
750 µM colchicine for 30 min before exposure to 0
(vehicle) or 0.24 nM 1,25(OH)
D
for 4 h (Fig. 2, Pre) exogenous 1,25(OH)
D
could no longer reduce 1,25(OH)
D
synthesis nor enhance the production of polar metabolites. As
mentioned for the dose response to colchicine, in monocytes exposed to
colchicine alone (vehicle), there was a marked reduction of
1,25(OH)
D
synthesis.
Figure 2:
Effect of colchicine on the suppression of
1,25(OH)D
synthesis and induction of vitamin D
catabolism by exogenous 1,25(OH)
D
. Pre, adherent cells were incubated in serum-free media with 0 (Control), 25, or 750 µM colchicine for 30 min.
Media were removed, and cells were washed and incubated with 0
(vehicle) or 0.24 nM 1,25(OH)
D
for 4
h. Post, cells were exposed to 0 or 0.24 nM
1,25(OH)
D
for 4 h and then were incubated with
25 or 750 µM colchicine for 30 min. Synthesis of
1,25(OH)
D
(A) and polar metabolites (B) was measured as outlined under ``Experimental
Procedures.'' Results are expressed as femtomoles/µg of DNA/h.
Values are mean ± S.E. of three independent experiments
performed in triplicate.
To assess whether the
lack of response to 1,25(OH)D
with colchicine
treatment was the result of a direct effect of colchicine on monocyte
hydroxylases rather than the consequence of a defective access of the
1,25(OH)
D
-VDR complex to the nucleus, monocytes
were first incubated with 0 (vehicle) or 0.24 nM 1,25(OH)
D
for 4 h and then exposed to
colchicine for 30 min. Measurements of synthesis of
1,25(OH)
D
and polar metabolites (Fig. 2, Post) show, in vehicle controls, a similar decrease in
1
-hydroxylase as described in Pre experiments with no significant
changes in the apparent activity of the hydroxylases involved in
vitamin D catabolism. However, in these protocols, microtubule
disruption occurred after the 1,25(OH)
D
-VDR
complex had interacted with the genome, and, despite the reduction in
1
-hydroxylase activity in vehicle control monocytes, exogenous
1,25(OH)
D
could reduce its own synthesis and
increase vitamin D catabolism with a potency similar to that observed
in monocytes with intact microtubules. Specifically, a 54.2 ±
3.1% reduction of 1,25(OH)
D
synthesis and 743.2
± 24.1% induction of vitamin D catabolism by 0.24 nM 1,25(OH)
D
was observed in the presence of
25 µM colchicine, and 52.1 ± 3.1% and 701.2
± 24.2%, respectively, with 750 µM colchicine.
Similar blockage of the ability of exogenous
1,25(OH)D
to control
1,25(OH)
D
production and vitamin D catabolism
by monocytes was observed with another microtubule disrupting agent,
vinblastine, at 50 and 100 µM concentrations (data not
shown).
These results demonstrate that microtubular integrity is
required for monocytes to respond to 1,25(OH)D
.
Further support for these findings came from measurements of steady
state levels of 24-hydroxylase mRNA using a ribonuclease protection
assay. Fig. 3shows that intact monocytes responded to
physiological concentrations of 1,25(OH)
D
with
a marked induction of 24-hydroxylase mRNA. However, pretreatment of
monocytes with colchicine impaired the ability of
1,25(OH)
D
to induce 24-hydroxylase gene
transcription in a dose-dependent manner. A partial reduction of
24-hydroxylase mRNA levels was achieved with 10 µM colchicine and, similar to the increase in the synthesis of polar
metabolites, 25 µM colchicine caused total inhibition of
the 1,25(OH)
D
-mediated increase in
24-hydroxylase mRNA. When the 30-min treatment with colchicine (10, 25,
and 750 µM) followed the 4-h exposure to 0.24 nM 1,25(OH)
D
, the increase in 24-hydroxylase
mRNA levels was identical with that observed in monocytes exposed to
1,25(OH)
D
alone. This suggests that a 30-min
exposure to colchicine has no significant direct effect either in mRNA
stability or in 1,25(OH)
D
-mediated
transcription. To confirm that the effects of colchicine on the genomic
action of 1,25(OH)
D
in monocytes were mediated
by microtubule disruption only, we performed similar experiments with
nocodazole, a reversible microtubule disrupting agent. Fig. 4shows that the inability of 1,25(OH)
D
to induce 24-hydroxylase mRNA levels when microtubules are
disrupted with 10 µM nocodazole (74% of nocodazole-treated
monocytes stained negatively for polymerized tubulin) can be totally
reversed if monocytes are allowed to recover microtubular integrity
after removal of nocodazole from the incubation media.
Figure 3:
Effect of colchicine on
1,25(OH)D
-mediated induction of 24-hydroxylase
mRNA. Pre, monocytes were incubated in serum-free media with
0, 10, 25, or 750 µM colchicine for 30 min. Media were
removed, and cells were washed and incubated with 0(-) or 0.24
nM 1,25(OH)
D
(+) for 4 h. Total
RNA from monocytes was assayed for mRNA levels of 24-hydroxylase and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using
ribonuclease protection assay. A shows two representative
gels, in monocytes from the same individual. B depicts the
densitometric analysis of the 24-hydroxylase:glyceraldehyde-3-phosphate
dehydrogenase mRNA ratios. Values represent mean ± S.E. from 4
independent experiments performed in
duplicate.
Figure 4:
Effect of nocodazole on
1,25(OH)D
-mediated induction of 24-hydroxylase
mRNA. Monocytes were incubated in serum-free media with 0 or 10
µM nocodazole for 30 min before a 4-h co-incubation with
nocodazole and 0(-) or 0.24 nM 1,25(OH)
D
(+) for 4 h. The
reversibility of nocodazole effect (reversed) was examined by removing
nocodazole from the media and allowing monocytes to recover
microtubular assembly for 4 h before an exposure to 0.24 nM 1,25(OH)
D
for 4 h (+). Total RNA from
monocytes was assayed for mRNA levels of 24-hydroxylase and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using a
ribonuclease protection assay. A shows a representative gel in
monocytes from the same individual, and B depicts the
densitometric analysis of the 24-hydroxylase:glyceraldehyde-3-phosphate
dehydrogenase mRNA ratios. Values represent mean ± S.E. from 4
independent experiments performed in
duplicate.
We next examined whether colchicine affected the formation of the
1,25(OH)D
-VDR complex. In three independent
experiments performed in triplicate, there was no significant
difference in maximal specific binding of 1,25(OH)
D
to the VDR between untreated and colchicine-treated (750
µM) monocytes (C: 0.21 ± 0.02; T: 0.19 ±
0.01 fmol/µg of DNA, n = 3).
Fig. 5shows that 10 µM nocodazole causes a
similar reduction of monocyte 1-hydroxylase activity. If after
microtubule disruption, nocodazole was removed from the incubation
media and monocytes were allowed to recover microtubular assembly,
1
-hydroxylase activity returned to basal values, which supports
the fact that microtubular integrity is necessary for normal
1,25(OH)
D
synthesis by human monocytes.
Figure 5:
Effect of nocodazole on monocyte
1-hydroxylase activity. Monocytes were incubated with 0 or 10
µM nocodazole for 30 min, and 1
-hydroxylase activity
was measured as outlined under ``Experimental Procedures.''
To examine the reversibility of nocodazole effects on
1,25(OH)
D
production (reversed), after the
30-min exposure to 0 or 10 µM nocodazole, nocodazole was
removed and monocytes were allowed to recover microtubular assembly for
4 h before assaying 1
-hydroxylase
activity.
To test whether this reduction in
the mitochondrial uptake of 25(OH)D could be the result of
a decrease in the number of mitochondria with microtubule disruption,
we measured succinate dehydrogenase activity in the mitochondrial
fraction of untreated and colchicine-treated monocytes. We found no
changes in succinate dehydrogenase activity (C: 0.022 ± 0.002;
T: 0.020 ± 0.002 µmol/min/µg of DNA, n =
2) with colchicine treatment.
To determine whether the reduction of
1,25(OH)D
production by colchicine involved
reduced 25(OH)D
transport to the mitochondria or directly
impaired mitochondrial 25(OH)D
uptake, we measured
1
-hydroxylase activity in (a) mitochondrial fractions
isolated from intact and colchicine-treated monocytes and (b)
in isolated mitochondrial fractions from intact monocytes in the
presence of 0 or 10 µM colchicine. In both protocols, we
found no effect of colchicine on mitochondrial
1,25(OH)
D
production ((a) control:
0.65 ± 0.12 fmol/µg of DNA/h; colchicine: 0.63 ±
0.09, n = 2; (b) control: 0.68 ± 0.07;
colchicine: 0.63 ± 0.08 fmol of
1,25(OH)
D
/µg of DNA/h).
1,25(OH)D
, the most active form of
vitamin D, is a potent steroid hormone. Its actions extend beyond
calcium homeostasis to impact a variety of nonclassical targets
including the immune system. To elicit a biological response,
1,25(OH)
D
binds to its intracellular receptor
and translocates to the nucleus where it interacts with vitamin D
responsive elements in the genome(1, 2) . A
tubulin-mediated cytoplasm to nucleus transport system has been
demonstrated using immunocytochemistry in human fibroblasts and
osteoblast-like cells (13, 14, 15) ; however,
the physiological relevance of this transport system in the response to
1,25(OH)
D
has not been examined either in
normal or pathological states. In the present studies, we used our
human monocyte model to define the physiological role of microtubules
on the response to 1,25(OH)
D
. We found that
disruption of microtubule integrity totally blocked the ability of
exogenous 1,25(OH)
D
to suppress
1,25(OH)
D
production and to induce
24-hydroxylase mRNA and activity in normal human monocytes. The lack of
response to exogenous 1,25(OH)
D
with
microtubular disruption could not be attributed to a reduced uptake of
1,25(OH)
D
by monocytes or to a defective
formation of the VDR-1,25(OH)
D
complex.
Clearly, microtubular integrity is required after the formation of the
1,25(OH)
D
-VDR complex for monocytes to elicit a
normal response to 1,25(OH)
D
. In addition, our
studies could not demonstrate any direct action of microtubule
disrupting agents on both genomic effects of
1,25(OH)
D
. The ability of exogenous
1,25(OH)
D
to suppress monocyte
1,25(OH)
D
production and to induce
24-hydroxylase mRNA and activity was not affected when the microtubule
disrupting agent was added after exposure to the sterol for 4 h. These
results in viable human monocytes support previous reports in fixed
cells (13, 14) of cytoplasmic VDR localization. In
contrast to gene activation by the glucocorticoid
receptor(31) , microtubule integrity is mandatory for
1,25(OH)
D
-VDR-mediated modulation of the
transcription of vitamin D responsive genes. In addition to our
findings in vitro, the recent clinical demonstration of a
phenotype of vitamin D-resistant rickets type 1, caused by a defective
nuclear translocation of an otherwise normal VDR(32) ,
emphasizes the critical role of the tubulin transport system mediating
cytoplasmic to nuclear VDR-1,25(OH)
D
translocation in 1,25(OH)
D
action.
The
present studies also demonstrated that disruption of microtubular
integrity significantly decreased the ability of human monocytes to
synthesize 1,25(OH)D
. Since colchicine was
reported to decrease protein synthesis(30) , we examined
whether this reduction in 1,25(OH)
D
generation
was mediated by a decreased expression of monocyte 1
-hydroxylase.
Kinetic analysis demonstrated that disruption of microtubular assembly
did not affect the V
of the enzyme but markedly
reduced its apparent affinity for its substrate, 25(OH)D
.
Further characterization of the mechanisms mediating this increase in
the K
for 25(OH)D
showed that
microtubule disruption did not affect whole cell uptake of
25(OH)D
but markedly decreased its intracellular delivery
to the mitochondria, suggesting a role for microtubules in
intracellular 25(OH)D
transport. We have also demonstrated
that the reduction in mitochondrial 25(OH)D
uptake could
not be attributed to a reduction in the number of mitochondria since
succinate dehydrogenase activity was not decreased in
colchicine-treated monocytes. The observation that 1
-hydroxylase
activity in isolated mitochondria was not affected by a dose of
colchicine that effectively reduced 1,25(OH)
D
production by intact monocytes indicates no direct effect of
colchicine on mitochondrial 25(OH)D
uptake or enzymatic
activity. Thus, contrary to the well accepted theory of simple
diffusion of 25(OH)D
due to its lipophilic nature,
microtubules participate in the intracellular transport of
25(OH)D
to the mitochondrial 1
-hydroxylase of human
monocytes. Similarly, cytoskeletal components were shown to mediate the
transport of adrenal steroid precursors to the mitochondria, thus
limiting the rate of synthesis of adrenal steroids(33) . A
mitochondrial localization of monocyte 1
-hydroxylase was also
demonstrated in the chick myelomonocytic cell line HD11(27) ,
and the kinetics of the avian enzyme has marked similarities with the
1
-hydroxylase expressed in human pulmonary alveolar macrophages in
sarcoidosis(34, 35) .
Similar to our finding of
impaired 1,25(OH)D
synthesis by monocytes with
microtubular disruption, reduction in 1,25(OH)
D
production by vinblastine was demonstrated in renal tubules from
vitamin D-deficient chicks(36) . However, for the avian renal
enzyme, there was no actual reduction in total
1,25(OH)
D
synthesized but an impaired exit of
the sterol from the mitochondria and out of the renal epithelial cells.
On the contrary, in our human monocyte model, we measured the total
1,25(OH)
D
generated regardless of its
subcellular or extracellular location, and we found a marked decrease
in 1,25(OH)
D
levels with microtubule disruption
with no alteration in the V
of the enzyme.
Therefore, despite the similarities in subcellular localization and
regulation of the 1
-hydroxylase of human monocytes, it is clear
that the effects of microtubular disruption on the intracellular
transport of vitamin D metabolites vary with cell type, species, and
vitamin D status.
If the cytoskeletal abnormalities reported for
platelets in chronic uremia (37) are present in other cell
types such as renal epithelia or peripheral monocytes, it is likely
that a defective intracellular 25(OH)D transport may
partially explain the need for supraphysiological concentrations of
25(OH)D
to correct renal (38, 39) and
extrarenal(40, 41) 1,25(OH)
D
production in chronic renal failure. Our finding of a higher K
for 25(OH)D
in the
1
-hydroxylase of monocytes from hemodialysis patients (42) seems to support this hypothesis. These cytoskeletal
abnormalities may also affect the translocation of the
1,25(OH)
D
-VDR complex causing the abnormal
response to 1,25(OH)
D
of chronic renal failure.
In summary, in normal monocytes, microtubules mediate intracellular
transport of 25(OH)D to the mitochondria and the
translocation of the 1,25(OH)
D
-receptor complex
to the nucleus. Disruption of microtubular integrity markedly impaired
1,25(OH)
D
synthesis by the mitochondrial
1
-hydroxylase and totally blocked the ability of monocytes to
respond to 1,25(OH)
D
which clearly indicate the
physiological and/or pathophysiological relevance of the
tubulin-transport system in humans.