Microtubules mediate cellular 25-hydroxyvitamin D3 trafficking and the genomic response to 1,25-dihydroxyvitamin D3 in normal human monocytes.

The genomic actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) 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)2D3 has not been studied in viable cells. Our studies examined this interaction in normal human monocytes. Monocytes convert 25(OH)D3 to 1,25(OH)2D3 and to 24-hydroxylated metabolites more polar than 1,25(OH)2D3. Microtubule disruption totally abolished the ability of exogenous 1,25(OH)2D3 to suppress its own synthesis and to induce 24-hydroxylase mRNA and activity, without affecting either total 1,25(OH)2D3 uptake or maximal 1,25(OH)2D3-VDR binding. Thus, intact microtubules are essential for 1,25(OH)2D3-dependent modulation of gene transcription. Interestingly, microtubule disruption also decreased monocyte 1,25(OH)2D3 synthesis, not by decreasing the Vmax of monocyte mitochondrial 1α-hydroxylase but through an increase in the Km for 25(OH)D3. We examined 25(OH)D3 transport. Microtubule disruption did not affect total cellular 25(OH)D3 uptake but reduced its intracellular trafficking to the mitochondria. Thus, microtubules participate in intracellular 25(OH)D3 transport, and their integrity determines normal 1,25(OH)2D3 synthesis.

The most active metabolite of vitamin D, 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 )), 1 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) 2 D 3 -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) 2 D 3 -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) 2 D 3 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) 2 D 3 that are indistinguishable from those described in classical 1,25(OH) 2 D 3 target tissues (16), and the interactions of 1,25(OH) 2 D 3 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 3 (25(OH)D 3 ) to 1,25(OH) 2 D 3 (21). We have also shown that when peripheral blood monocytes were exposed to physiological concentrations of 1,25(OH) 2 D 3 , 1␣-hydroxylase activity is markedly suppressed. In addition, exogenous 1,25(OH) 2 D 3 promotes an induction of vitamin D catabolism by increasing 24-hydroxylase mRNA 2 and activity (22). Because both effects of the sterol require at least 2 h of exposure to 1,25(OH) 2 D 3 (22), it is likely that the inhibition of 1,25(OH) 2 D 3 production by 1,25(OH) 2 D 3 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) 2 D 3 . This report demonstrates for the first time that integrity of the microtubule network is critical for a normal genomic response to 1,25(OH) 2 D 3 and that an intracel-lular tubulin-25(OH)D 3 transport system mediates the delivery of 25(OH)D 3 to mitochondria, thus modulating the rate of 1,25(OH) 2 D 3 synthesis by monocytes. Culture of Human Monocytes-Peripheral blood was obtained from normal volunteers by venipuncture. Mononuclear leukocytes were isolated using a Ficoll-Hypaque gradient (Pharmacia Biotech Inc.). Cells were plated in six-well plates at a concentration of 7 ϫ 10 6 to 10 7 cells per well in 1 ml of RPMI 1640 containing 1% fatty acid-free albumin, 50,000 units/liter penicillin G sodium, 50,000 g/liter streptomycin sulfate, 10 mM HEPES, and 0.8 mM NaHCO 3 . After an incubation period of 18 h at 37°C, media and nonadherent cells were removed, and adherent cells were washed (once with 2 ml of phosphate-buffered saline (PBS) and twice with 1 ml of RPMI 1640 containing 0.1% fatty acid-free albumin). More than 95% of the adherent cells stained positively for macrophage-specific ␣-naphthyl acetate esterase activity (23). This adherent cell population was used in all studies.  20 l of media per well. Reactions were stopped after a 1-h incubation at 37°C by the addition of 1 ml of acetonitrile; 100 ng of radioinert 1,25(OH) 2 D 3 were added to the wells to monitor recoveries. Cells were scraped using a rubber policeman, and cells and media were collected. Wells were washed with 2 ml of acetonitrile:water (1:1), scraped, and pooled with the previously collected fraction. Samples were vortexed for 30 s and centrifuged at 2,500 ϫ g for 15 min. Vitamin D metabolites were obtained from the supernatants using C 18 cartridges (Fisher Scientific) and the extraction procedure developed by Reinhardt et al. (24). The vitamin D metabolite fraction eluting with acetonitrile was collected, dried under nitrogen, and purified by HPLC using a Zorbax Sil column (Phenomenex, Torrance, CA) with 3% isopropyl alcohol in methylene chloride as solvent. Average recoveries of radioinert 1,25(OH) 2 D 3 were higher than 75%. Immediately after the elution of the 1,25(OH) 2 D 3 peak, the column was stripped with 4 ml of methanol to quantitate the synthesis of metabolites more polar than 1,25(OH) 2 D 3 (polar metabolites). Tritium eluting in the 1,25(OH) 2 D 3 and more polar metabolite HPLC fractions was counted in a liquid scintillation counter (ICN Micromedic System Inc., Huntsville, AL). Radioactivity eluting with the methanol strip from wells incubated with media only was considered as nonspecific oxidation of 25(OH)D 3 and subtracted as background. Results were normalized per g of DNA measured using the ethidium bromide method (25). In each experiment, determinations were performed in triplicate. Exogenous 1,25(OH) 2 D 3 in Monocytes-Positive controls were adherent cells exposed for 4 h to 0 or 0.24 nM 1,25(OH) 2 D 3 . In the Pre group, adherent cells were exposed to colchicine (from 0 to 750 M as specified for each particular experiment) or vinblastine (50 M) for 30 min, washed and incubated in 1 ml of incubation media with 0 or 0.24 nM 1,25(OH) 2 D 3 for 4 h. In the Post group, adherent cells were first incubated with 0 or 0.24 nM 1,25(OH) 2 D 3 and then exposed to colchicine for 30 min.

The Effect of Microtubule Disrupting Agents on the Suppression of 1,25(OH) 2 D 3 Synthesis and the Stimulation of Vitamin D Catabolism by
The effect of the reversible microtubule disrupting agent, nocodazole, on the response to 1,25(OH) 2 D 3 was examined as follows. Adherent monocytes were treated with 0 or 10 M nocodazole for 30 min before a co-incubation with 0.24 nM 1,25(OH) 2 D 3 and 10 M nocodazole for 4 h. To test reversibility of the effect of nocodazole, adherent cells were exposed to 0 or 10 M nocodazole (dissolved in 1 l of dimethyl sulfoxide) for 4 1 ⁄2 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) 2 D 3 for 4 h.
In each independent experiment, the synthesis of 1,25(OH) 2 D 3 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.
Ribonuclease Protection Assay-RNA from monocytes was obtained using the commercially available rapid RNA isolation method, RNAzol (Tele Test Inc., Friendswood, TX).
Riboprobes for Human 24-Hydroxylase-A BamHI restriction fragment from the human 24-hydroxylase cDNA (generously provided by Dr. Hector DeLuca) corresponding to the bases 1657 to 1999 of the published sequence was subcloned in Bluescript KS (Stratagene). The antisense control template for human glyceraldehyde-3-phosphate dehydrogenase (pTRI-GAPDH) was purchased from Ambion (Austin, TX). DDeI digestion of the linearized pTRI-GAPDH resulted in a shortened template that allowed the synthesis of a 32 P-labeled transcript with a 150-nucleotide homology with human glyceraldehyde-3-phosphate dehydrogenase mRNA. Radiolabeled antisense RNAs were produced using T7 RNA polymerase and [␣ 32 P]CTP (Amersham). The size of the protected fragment for the human 24-hydroxylase was 342 base pairs.
Monocyte RNA samples were dissolved in 4 l of diethyl pyrocarbonate water and mixed with 26 l of hybridization buffer (80% formamide, 50 mM PIPES, pH 6.4, 400 mM NaCl, 1 mM EDTA) containing the 32 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 l of ribonuclease digestion mixture containing 2 g 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 g) and 20 l 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.
Total Cellular Uptake of 1,25(OH) 2 (26)  Immunofluorescence Staining for ␣-Tubulin-Peripheral blood mononuclear leukocytes were plated on coverslips in six-well plates following the protocols described for monocyte isolation. Adherent cells were exposed to 0, 10, 25, or 750 M colchicine for 30 min at 37°C. Cells were washed twice with PBS and incubated in fresh RPMI 1640 containing 1% fatty-acid-free albumin until fixation. For nocodazole treatment, monocytes were exposed to 0 or 10 M nocodazole for 30 min at 37°C and remained in contact with nocodazole until fixation. Cells were fixed in paraformaldehyde (4%) for 20 min at room temperature, washed three times (2% gelatin in phosphate-buffered saline), incubated for 20 min with blocking buffer (0.1 M NH 4 Cl, 0.2% gelatin, 0.05% Triton X-100), and stained for ␣-tubulin using a 1:1000 dilution of a monoclonal mouse anti-␣-tubulin (Sigma). After a 2 1 ⁄2-h incubation at room temperature, monocytes were washed and incubated for 1 h at room temperature in the dark with fluorescein isothiocyanate goatanti-mouse IgG (1:100) (Cappel). Antibodies were diluted with washing buffer containing 30% goat serum. After mounting with 50% glycerol in PBS, cells were examined for microtubular assembly using confocal microscopy. A minimum of 50 cells were scored for each experimental condition. Monocytes containing one or more filament-like structures were considered positive for polymerized tubulin and not completely disrupted.

D 3 or 25(OH)D 3 by Human Peripheral Monocytes-A modification of the procedure reported by Keenan and Holmes
Maximal Specific Binding of 1,25(OH) 2 D 3 to VDR-Adherent cells were incubated with or without colchicine (750 M for 30 min at 37°C). Cells were then washed and incubated with 0.5 nM [ 3 H]1,25(OH) 2 D 3 with (nonspecific binding) and without 125 nM radioinert 1,25(OH) 2 D 3 for 2 h. Cells were rinsed once with PBS containing 5 mg/ml fatty-acid-free albumin, then twice with PBS alone and placed on ice. After the addition of 2 ml of TEDK (10 mM Tris-HCl, pH 7.4, 1.5 mM EDTA, 400 mM KCl, and 5 mM dithiothreitol), the contents of each well were sonicated for 30 s. Samples were transferred to small tubes, and 500 l of charcoal was added. Tubes were kept on ice for 15 min and centrifuged at 2500 rpm for 15 min. Aliquots (0.5 ml) of the supernatant were mixed with 4.5 ml of scintillation fluid and counted for tritium. Results are expressed as femtomoles of 1,25(OH) 2 D 3 /g of DNA.
Subcellular 25(OH)D 3 Uptake-Subcellular uptake of 25(OH)D 3 was measured using a modification of the procedure described by Shany et al. (27). Adherent cells were incubated with or without colchicine (10 and 750 M). After a 30-min incubation at 37°C, cells were washed and incubated at 37°C for 1 h in 1 ml of RPMI 1640 containing 0.1% fatty-acid-free albumin, 0.1 Ci of [ 3 H]25(OH)D 3 , and 10 M ketoconazole, a cytochrome P-450 inhibitor, to block 25(OH)D 3 metabolism. Cells were rinsed once with phosphate-buffered saline and twice with RPMI 1640 containing 0.1% fatty acid-free albumin. Monocytes were disrupted by sonication in 1 ml of TMSS buffer, pH 7.4 (15 mM Tris acetate, 2 mM MgCl 2 , 2.5 mM succinate, and 250 mM sucrose) containing phenylmethylsulfonyl fluoride (10 M). Fifteen wells of untreated and colchicine-treated sonicated monocytes were pooled in 50-ml culture tubes, and final volumes were measured. 0.5 ml of these samples were counted for tritium, and total tritium in whole cell homogenates was calculated. For subcellular fractionation, homogenates were divided in 4 aliquots of equal volume. Aliquots were centrifuged at 700 ϫ g for 10 min at 4°C. The supernatants were centrifuged at 8500 ϫ g for 10 min at 4°C. Resultant supernatants were centrifuged at 10,000 ϫ g for 10 min at 4°C. Final supernatants were centrifuged at 100,000 ϫ g for 1 h at 4°C. The resultant pellets (700 ϫ g, containing mainly nuclei and cellular membranes; 8,500 ϫ g, mainly mitochondrial fraction; 10,000 ϫ g, residual mitochondrial fraction; 100,000 ϫ g, mainly microsomal fraction) were resuspended in 0.5 ml of ethanol, vortexed, and transferred to mini scintillation vials. Potential residual pellets were washed three times in 1 ml of hexane:isopropyl alcohol (95:5) and pooled with the ethanol fraction. Organic solvents were air-dried at room temperature, and the amount of tritium associated to each subcellular fraction was counted.
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.
K m and V max Determinations-Four different substrate concentrations (from 5 to 300 nM) of 25(OH)D 3 were assayed in triplicate to measure 1,25(OH) 2 D 3 synthesis. K m and V max were obtained from a linear regression analysis of the data using the double reciprocal plot of Lineweaver-Burk.
Subcellular Localization of Monocyte 1␣-Hydroxylase-Monocytes were incubated with or without 10 or 750 M colchicine for 30 min. Mitochondrial and microsomal fractions obtained as indicated above were incubated with 2 mM isocitrate and 1 mM NADPH, respectively, at 37°C, in an atmosphere containing 95% air, 5% CO 2 , for 1 h. The reaction was initiated by adding 5.0 nM [ 3 H]25(OH)D 3 dissolved in 200 l of the same buffer containing 5 mg/ml albumin. The final assay incubation volume was 1 ml. The reactions were stopped by the addition of 1 ml of acetonitrile. 100 ng of 1,25(OH) 2 D 3 was added to monitor recovery, and 1,25(OH) 2 D 3 production was measured as indicated above. Results are expressed as femtomoles of 1,25(OH) 2 D 3 /g of total cell DNA/h. Statistical Analysis-Statistical evaluation of the data was performed using Student's t test for unpaired observations.

Effect of Microtubule Disruption on the Response of Normal
Human Monocytes to 1,25(OH) 2 D 3 -We examined the effect of disruption of microtubular assembly on the ability of 1,25(OH) 2 D 3 to suppress its own production and to induce vitamin D catabolism. Fig. 1 shows that, in normal human monocytes, the ability of 1,25(OH) 2 D 3 to inhibit its own synthesis is impaired by colchicine in a dose-dependent manner. At a concentration of colchicine of 25 M, 1,25(OH) 2 D 3 was no longer able to suppress monocyte 1,25(OH) 2 D 3 production. The number of monocytes that stained positive for polymerized tubulin decreased from 91% in control preparations to 62, 22, and 9% after a 30-min exposure to concentrations of colchicine of 10, 25, and 750 M, respectively.
Because colchicine alone caused a dose-dependent reduction of 1,25(OH) 2 D 3 production by monocytes, we performed the experiments summarized in Fig. 2 to define the effects of microtubule disruption in the response of monocytes to 1,25(OH) 2 D 3 . The lowest (25 M) and highest (750 M) doses of colchicine, effective in blocking the ability of 1,25(OH) 2 D 3 to suppress its own production, were used to examine the effect of microtubule disruption on the ability of 1,25(OH) 2 D 3 to suppress its own synthesis (upper panel) and to induce vitamin D catabolism (generation of 24-hydroxylated metabolites more polar than 1,25(OH) 2 D 3 (polar metabolites); lower panel). In monocytes with intact microtubules (Controls), exposure to 0.24 nM 1,25(OH) 2 D 3 for 4 h reduced 1,25(OH) 2 D 3 synthesis to 55.2 Ϯ 1.3% and increased the generation of polar metabolites 7-fold above vehicle controls, respectively. However, when monocytes were treated with 25 or 750 M colchicine for 30 min before exposure to 0 (vehicle) or 0.24 nM 1,25(OH) 2 D 3 for 4 h (Fig. 2, Pre) exogenous 1,25(OH) 2 D 3 could no longer reduce 1,25(OH) 2 D 3 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) 2 D 3 synthesis.
To assess whether the lack of response to 1,25(OH) 2 D 3 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) 2 D 3 -VDR complex to the nucleus, monocytes were first incubated with 0 (vehicle) or 0.24 nM 1,25(OH) 2 D 3 for 4 h and then exposed to colchicine for 30 min. Measurements of synthesis of 1,25(OH) 2 D 3 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) 2 D 3 -VDR complex had interacted with the genome, and, despite the reduction in 1␣-hydroxylase activity in vehicle control monocytes, exogenous 1,25(OH) 2 D 3 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 Ϯ 3.1% reduction of 1,25(OH) 2  These results demonstrate that microtubular integrity is required for monocytes to respond to 1,25(OH) 2 D 3 . Further support for these findings came from measurements of steady state levels of 24-hydroxylase mRNA using a ribonuclease protection assay. Fig. 3 shows that intact monocytes responded to physiological concentrations of 1,25(OH) 2 D 3 with a marked induction of 24-hydroxylase mRNA. However, pretreatment of monocytes with colchicine impaired the ability of 1,25(OH) 2 D 3 to induce 24-hydroxylase gene transcription in a dose-dependent manner. A partial reduction of 24-hydroxylase mRNA levels was achieved with 10 M colchicine and, similar to the increase in the synthesis of polar metabolites, 25 M colchicine caused total inhibition of the 1,25(OH) 2 D 3 -mediated increase in 24-hydroxylase mRNA. When the 30-min treatment with colchicine (10, 25, and 750 M) followed the 4-h exposure to 0.24 nM 1,25(OH) 2 D 3 , the increase in 24-hydroxylase mRNA levels was identical with that observed in monocytes exposed to 1,25(OH) 2 D 3 alone. This suggests that a 30-min exposure to colchicine has no significant direct effect either in mRNA stability or in 1,25(OH) 2 D 3 -mediated transcription. To confirm that the effects of colchicine on the genomic action of 1,25(OH) 2 D 3 in monocytes were mediated by microtubule disruption only, we performed similar experiments with nocodazole, a reversible microtubule disrupting agent. Fig. 4 shows that the inability of 1,25(OH) 2 D 3 to induce 24-hydroxylase mRNA levels when microtubules are disrupted with 10 M 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. We next examined whether colchicine affected the formation of the 1,25(OH) 2 D 3 -VDR complex. In three independent experiments performed in triplicate, there was no significant difference in maximal specific binding of 1,25(OH) 2 D 3 to the VDR between untreated and colchicine-treated (750 M) monocytes (C: 0.21 Ϯ 0.02; T: 0.19 Ϯ 0.01 fmol/g of DNA, n ϭ 3).

Effect of Colchicine on 1,25(OH) 2 D 3 Uptake and Formation of the 1,25(OH) 2 D 3 -VDR Complex-To
Effect of Microtubule Disruption on Monocyte 1␣-Hydroxylase Activity-In Fig. 1, we showed that colchicine alone markedly reduced monocyte 1,25(OH) 2 D 3 synthesis, even at a 10 M concentration, a dose only partially effective in blocking the response to 1,25(OH) 2 D 3 . To examine whether this reduction on 1,25(OH) 2 D 3 synthesis by colchicine was the result of impaired protein synthesis, a known side effect of colchicine treatment (30) that could lead to a nonspecific reduction in the amount of 1␣-hydroxylase in monocytes, we measured the ef-  Table I shows that exposure to colchicine did not affect the V max of the enzyme but caused a 2-to 4-fold increase in the K m for 25(OH)D 3 . This indicated no alteration in the expression of monocyte 1␣-hydroxylase but a marked reduction in its apparent affinity for substrate, suggesting a role for microtubules in 25(OH)D 3 transport and/or its delivery to the enzyme. Fig. 5 shows that 10 M 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) 2 D 3 synthesis by human monocytes. techniques were employed, and the purity of the fractions was assessed using specific markers as shown in Table II. Table III shows that in monocytes with intact microtubules, most of the 25(OH)D 3 within the cell was associated with the mitochondrial fraction. Colchicine treatment reduced the uptake of 25(OH)D 3 by the mitochondrial fraction with a concomitant increase in the amount of 25(OH)D 3 in the cytosol and with no changes in the 25(OH)D 3 associated to microsomal or nuclear fractions.

Effect of Microtubule Disruption on the Uptake and Intracellular Transport of 25(OH)D 3 in Monocytes-
To test whether this reduction in the mitochondrial uptake of 25(OH)D 3 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 Ϯ 0.002; T: 0.020 Ϯ 0.002 mol/min/g of DNA, n ϭ 2) with colchicine treatment.

FIG. 4. Effect of nocodazole on 1,25(OH) 2 D 3 -mediated induction of 24-hydroxylase mRNA.
Monocytes were incubated in serumfree media with 0 or 10 M nocodazole for 30 min before a 4-h coincubation with nocodazole and 0 (Ϫ) or 0.24 nM 1,25(OH) 2 D 3 (ϩ) 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) 2 D 3 for 4 h (ϩ). 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 Ϯ S.E. from 4 independent experiments performed in duplicate.  5. Effect of nocodazole on monocyte 1␣-hydroxylase activity. Monocytes were incubated with 0 or 10 M 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) 2 D 3 production (reversed), after the 30-min exposure to 0 or 10 M nocodazole, nocodazole was removed and monocytes were allowed to recover microtubular assembly for 4 h before assaying 1␣hydroxylase activity.
Clearly, monocyte 1␣-hydroxylase, like the renal enzyme, is located exclusively in mitochondria.
To determine whether the reduction of 1,25(OH) 2 D 3 production by colchicine involved reduced 25(OH)D 3 transport to the mitochondria or directly impaired mitochondrial 25(OH)D 3 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 M colchicine. In both protocols, we found no effect of colchicine on mitochondrial 1,25(OH) 2 D 3 production ((a) control: 0.65 Ϯ 0.12 fmol/g of DNA/h; colchicine: 0.63 Ϯ 0.09, n ϭ 2; (b) control: 0.68 Ϯ 0.07; colchicine: 0.63 Ϯ 0.08 fmol of 1,25(OH) 2 D 3 /g of DNA/h). DISCUSSION 1,25(OH) 2 D 3 , 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) 2 D 3 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) 2 D 3 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) 2 D 3 . We found that disruption of microtubule integrity totally blocked the ability of exogenous 1,25(OH) 2 D 3 to suppress 1,25(OH) 2 D 3 production and to induce 24-hydroxylase mRNA and activity in normal human monocytes. The lack of response to exogenous 1,25(OH) 2 D 3 with microtubular disruption could not be attributed to a reduced uptake of 1,25(OH) 2 D 3 by monocytes or to a defective formation of the VDR-1,25(OH) 2 D 3 complex. Clearly, microtubular integrity is required after the formation of the 1,25(OH) 2 D 3 -VDR complex for monocytes to elicit a normal response to 1,25(OH) 2 D 3 . In addition, our studies could not demonstrate any direct action of microtubule disrupting agents on both genomic effects of 1,25(OH) 2 D 3 . The ability of exogenous 1,25(OH) 2 D 3 to suppress monocyte 1,25(OH) 2 D 3 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) 2 D 3 -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) 2 D 3 translocation in 1,25(OH) 2 D 3 action.
The present studies also demonstrated that disruption of microtubular integrity significantly decreased the ability of human monocytes to synthesize 1,25(OH) 2 D 3 . Since colchicine was reported to decrease protein synthesis (30), we examined whether this reduction in 1,25(OH) 2 D 3 generation was mediated by a decreased expression of monocyte 1␣-hydroxylase. Kinetic analysis demonstrated that disruption of microtubular assembly did not affect the V max of the enzyme but markedly reduced its apparent affinity for its substrate, 25(OH)D 3 . Further characterization of the mechanisms mediating this increase in the K m for 25(OH)D 3 showed that microtubule disruption did not affect whole cell uptake of 25(OH)D 3 but markedly decreased its intracellular delivery to the mitochondria, suggesting a role for microtubules in intracellular 25(OH)D 3 transport. We have also demonstrated that the reduction in mitochondrial 25(OH)D 3 uptake could not be attributed to a reduction in the number of mitochondria since succinate dehydrogenase activity was not decreased in colchicinetreated monocytes. The observation that 1␣-hydroxylase activity in isolated mitochondria was not affected by a dose of colchicine that effectively reduced 1,25(OH) 2 D 3 production by intact monocytes indicates no direct effect of colchicine on mitochondrial 25(OH)D 3 uptake or enzymatic activity. Thus, contrary to the well accepted theory of simple diffusion of 25(OH)D 3 due to its lipophilic nature, microtubules participate in the intracellular transport of 25(OH)D 3 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) 2 D 3 synthesis by monocytes with microtubular disruption, reduction in 1,25(OH) 2 D 3 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) 2 D 3 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) 2 D 3 generated regardless of its subcellular or extracellular location, and we found a marked decrease in 1,25(OH) 2 D 3 levels with microtubule disruption with no alteration in the V max 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 3 transport may partially explain the need for supraphysiological concentrations of 25(OH)D 3 to correct renal (38,39) and extrarenal (40, 41) 1,25(OH) 2 D 3 production in chronic renal failure. Our finding of a higher K m for 25(OH)D 3 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) 2 D 3 -VDR complex causing the abnormal response to 1,25(OH) 2 D 3 of chronic renal failure.
In summary, in normal monocytes, microtubules mediate intracellular transport of 25(OH)D 3 to the mitochondria and the translocation of the 1,25(OH) 2 D 3 -receptor complex to the nucleus. Disruption of microtubular integrity markedly impaired 1,25(OH) 2 D 3 synthesis by the mitochondrial 1␣-hydroxylase and totally blocked the ability of monocytes to respond to 1,25(OH) 2 D 3 which clearly indicate the physiological and/or pathophysiological relevance of the tubulin-transport system in humans.