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,25-Dihydroxyvitamin D31
Department of Biochemistry, Ohu University School of Dentistry, Koriyama 963, Japan
Address all correspondence and requests for reprints to: Noboru Horiuchi, Department of Biochemistry, Ohu University School of Dentistry, Koriyama 963, Japan.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-dihydroxyvitamin D3
[1,25-(OH)2D3] to 24,25-dihydroxyvitamin
D3 and 1,24,25-trihydroxyvitamin D3,
respectively. A full-length complementary DNA for mouse 24-OHase has
now been characterized. The complementary DNA consists of 3309 bp and
encodes a protein of 514 amino acids that shows 82% and 95% sequence
identity with the human and rat enzymes, respectively. Northern blot
analysis of tissues from mice injected with
1,25-(OH)2D3 (24 pmol/g) revealed that the
3.4-kb 24-OHase messenger RNA (mRNA) is most abundant in kidney and
intestine, with smaller amounts present in skin, thymus, and bone.
RT-PCR and Southern blot analysis detected 24-OHase mRNA in several
other tissues including lung, testis, spleen, pancreas, and heart.
Intraperitoneal injection of 1,25-(OH)2D3
induced dose- and time-dependent increases in both 24-OHase mRNA
abundance and enzyme activity in mouse kidney. Similarly,
1,25-(OH)2D3-induced increases in both 24-OHase
mRNA and activity were apparent in the duodenum. Although
1,25-(OH)2D3 increased the amount of 24-OHase
mRNA in skin, enzyme activity was not detected in this tissue.
Pretreatment of mice with cycloheximide (400 µg/g), an inhibitor of
protein synthesis, potentiated the increase in 24-OHase mRNA abundance,
but blocked the increase in 24-OHase activity, induced by
1,25-(OH)2D3 in kidney and duodenum, suggesting
that 24-OHase gene expression may be regulated not only by the vitamin
D receptor but also by a short-lived repressor protein. |
Introduction |
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,25-dihydroxyvitamin D3
\[1,25-(OH)2D3\], is an important regulator of
calcium metabolism and elicits most of its biological effects by
binding to a high-affinity receptor in target tissues, including
intestine, kidney, and bone (1, 2). In addition to its role in calcium
homeostasis, this steroid hormone is a potent inducer of
differentiation, proliferation, and biosynthetic activity in a variety
of malignant and nonmalignant cell types (2, 3). The concentration of
1,25-(OH)2D3 in plasma reflects the rate of
24,25-(OH)2D3 formation as well as the rates of
1,25-(OH)2D3 production and degradation.
Vitamin D 24-hydroxylase (24-OHase), a member of the cytochrome P-450
enzyme system (4), catalyzes the 24-hydroxylation of
1,25-(OH)2D3 and that of 25-hydroxyvitamin
D3 (25OHD3), which is the major circulating
form of vitamin D3 and the main precursor of vitamin
D3 metabolites. The hydroxylation reactions catalyzed by
1-hydroxylase and 24-OHase are sensitive to the vitamin D status of
the animals (1, 2, 4), and 1,25-(OH)2D3 (5, 6, 7)
as well as active analogs such as 22-oxacalcitriol (7) and EB1089 (8)
induce expression of the 24-OHase gene in a receptor-mediated manner.
Hydroxylation of 25OHD3 and
1,25-(OH)2D3 by 24-OHase is thought to be the
first step in the inactivation of vitamin D metabolites (9), given the
low biological activities of the respective products,
24,25-(OH)2D3 and
1,24,25-(OH)3D3 compared with those of
1,25-(OH)2D3. The intestines and kidneys appear
to be the major sites of 1,25-(OH)2D3
inactivation and 25OHD3 metabolism to produce polar
metabolites respectively, although most
1,25-(OH)2D3-responsive tissues show the
24-OHase gene expression. By contrast, a product of 24-OHase,
24,25-(OH)2D3, has been reported to elicit
several biological effects such as the role for normal hatchability of
chicken eggs (10) and increased bone formation in hypophosphatemic mice
(11). However, the biological importance of
24,25-(OH)2D3 remains controversial, because a
high affinity receptor for 24,25-(OH)2D3 has
not yet been established.
Full-length complementary DNAs (cDNAs) for 24-OHase have been cloned from humans (12) and rats (13). The promoter of the rat 24-OHase gene has been sequenced and contains a functional vitamin D-responsive elements (14, 15, 16). Characterization of the regulation of 24-OHase gene expression by other factors, such as PTH (6, 17) and phorbol ester (5), is required to increase our understanding of abnormalities in vitamin D metabolism associated with metabolic bone disorders (18). Recently, a portion of the cDNA corresponding to the open reading frame for 24-OHase was cloned from mice (19). Because detailed studies on the regulation of 24-OHase gene expression by 1,25-(OH)2D3 are usually performed in mice, we have now cloned and sequenced a full-length mouse 24-OHase cDNA. We have also determined the tissue distribution of 24-OHase messenger RNA (mRNA) and characterized the regulation of 24-OHase gene expression by 1,25-(OH)2D3 in mice.
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Materials and Methods |
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-32P]deoxycytidine triphosphate (dCTP) (110 TBq/mmol)
was obtained from ICN (Costa Mesa, CA), and
1,25-(OH)2\[26,27-3H\]D3 (6.0
TBq/mmol) was from Dupont NEN (Boston, MA). Crystalline
25OHD3 was from Philips Duphar (Amsterdam, Netherlands) and
crystalline 1,25-(OH)2D3 from Roussel UCLAF
(Romainville, France). Crystalline
24,25-(OH)2D3 was donated by H. Yamato (Kureha
Chemicals, Tokyo, Japan), and 1,24,25-(OH)3D3
was a gift from H. Ohkawa (Chugai Pharmaceutical, Gotemba, Japan). A
Fine-Pak SIL column was obtained from JASCO (Tokyo, Japan). Rodent chow
was obtained from Oriental Yeast (Tokyo, Japan). All other reagents and
chemicals were of analytic grade.
RNA isolation and cDNA library screening
Kidneys were removed from ddY mice 4 h after the injection
of 1,25-(OH)2D3 (24 pmol/g body mass, ip).
Total RNA was extracted from the kidneys with the use of guanidine
thiocyanate (20), and mRNA was isolated from the equivalent of 0.5
g of tissue with the Fasttrack mRNA isolation kit. The mRNA was
converted to single-strand cDNA with reverse transcriptase, and
double-stranded cDNA ligated with EcoRI adapters was
synthesized and purified by sucrose density gradient (5–20%)
centrifugation (21). Fractions containing cDNAs of 2–4 kb were
collected, and the purified cDNA molecules were cloned into 
gt10 to
create a library of 1 x 106 recombinants. The mouse
kidney cDNA library was screened with a 1.0-kb mouse 24-OHase cDNA
fragment generated by RT-PCR (7). The first round of screening yielded
three positive clones, and a clone with the largest insert was
subcloned into the pcDNA3 plasmid and sequenced by the dideoxy
chain-termination method (22).
5'-RACE
5'-RACE was performed with a kit of 5'-RACE system. Briefly,
first-strand cDNA was synthesized from mouse kidney total RNA with a
24-OHase-specific antisense primer [nucleotides (nt) 390–409] (Fig. 1
). The reaction mixture (24 µl) contained 1 µg total RNA, 100
nM primer, 20 mM Tris-HCl (pH 8.4), 50
mM KCl, 3 mM MgCl2, 10
mM dithiothreitol, 16 mM of each
deoxynucleoside triphosphate (dNTP), and 8 U SuperScript II reverse
transcriptase. After incubation for 30 min at 42 C, RNA was removed
with RNase H, and the cDNA was purified with a GlassMax DNA isolation
spin cartridge. An anchor sequence was added to the 3'-end of the cDNA
molecules with terminal deoxynucleotidyl transferase and dCTP, and the
products were subjected to PCR, under the same conditions as described
for RT-PCR analysis, with an anchor-specific primer (provided in kit)
and a 24-OHase-specific antisense primer (nt 348–367). The amplified
double-stranded cDNA products were cloned into pT7blueT-vector and
sequenced.
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Northern blot and RT-PCR analysis of 24-OHase mRNA
Total RNA was isolated from various tissues by extraction with
guanidine thiocyanate (20). To isolate total RNA from the calvaria,
tibia, femur, and skin, we first pulverized frozen tissue in a
stainless steel chamber chilled with dry ice. All tissues were
homogenized in a solution containing 4 M guanidine
thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5%
laurylsarcosine, and 0.1 M ß-mercaptoethanol. Total RNA
was extracted with phenol saturated with 0.1 M citrate
buffer (pH 4.3), layered onto CsCl (0.96 g/ml in 0.1 M
EDTA, pH 7.8) cushions, and centrifuged for 18 h at 400,000
x g and 20 C. The RNA pellets were resuspended in
DEPC-treated water and reprecipitated with 0.1 vol 3 M
sodium acetate (pH 5.2) and 2.5 vol ice-cold 99.5% (vol/vol) ethanol.
The RNA precipitates were washed with 75% (vol/vol) ethanol and
resuspended in DEPC-treated water.
For Northern blot analysis, total RNA was fractionated in a 1.2%
agarose gel containing formaldehyde and transferred to a
Hybond-N+ membrane. Blots were hybridized with a
full-length 24-OHase cDNA probe that had been labeled with
[-32P]dCTP with the use of the Megaprime DNA labeling
system. Hybridization was performed for 2 days at 42 C in 50%
formamide, 5x Denhardt’s solution, 0.5% SDS, and 5x SSPE (80
mM NaCl, 10 mM sodium phosphate, and 1
mM EDTA), after which the membranes were washed in 0.1x
SSPE at 20 C for 15 min and exposed to Hyperfilm at -80 C with
intensifying screens. Blots were subsequently hybridized with a probe
for cyclophilin mRNA, the gene for which is constitutively expressed as
a control for RNA loading. The amounts of 24-OHase and cyclophilin
mRNAs were quantified by densitometric scanning of the autoradiograms,
and the abundance of 24-OHase mRNA was normalized to that of
cyclophilin mRNA.
RT-PCR analysis of the tissue distribution of 24-OHase mRNA was performed with a GeneAmp RNA PCR kit. Briefly, 2 µg total RNA was incubated with 15 pmol 24-OHase antisense primer (nt 1524–1543) and 50 U Moloney murine leukemia virus reverse transcriptase for 30 min at 42 C in a total volume of 20 µl of RT buffer (50 mM KCl, 5 mM MgCl2 and 10 mM Tris-HCl, pH 8.3) containing 1 mM of each dNTP and 20 U RNase inhibitor (RNasin). The mixture was then heated at 99 C for 5 min and quickly chilled to 4 C. The resulting single-stranded cDNA (20 µl) was subjected to PCR amplification in a total volume of 100 µl containing PCR buffer (50 mM KCl, 2 mM MgCl2, and 10 mM Tris-HCl, pH 8.3), 200 µM of each dNTP, 15 pmol 24-OHase sense primer (nt 1049–1068), and 2.5 U Taq DNA polymerase. The amplification protocol comprised 20 cycles of denaturation for 1 min at 94 C, annealing for 1 min at 55 C, and extension for 2 min at 72 C. The PCR products were subjected to Southern blot analysis with a 32P-labeled full-length 24-OHase cDNA as probe. RT-PCR analysis of ß-actin mRNA was performed as a control to verify the amount and integrity of mRNA in the RNA preparations from the various tissues.
Measurement of 24-OHase activity
The kidney cortex was minced, and the duodenum was
scraped. They were then washed in ice-cold homogenization buffer (0.19
M sucrose, 25 mM sodium succinate, 2
mM MgCl2, 1 mM EDTA, 20
mM Tris-HEPES, pH 7.4) and homogenized in the same solution
(20 ml/g tissue). For measurement of renal 24-OHase activity,
25OH\[26,27-3H\]D3 (250 pmol, 80,000 cpm) or
1,25-(OH)2 \[26,27-3H\]D3 (250
pmol, 80,000 cpm), dissolved in 10 µl ethanol, was added to 1 ml
homogenate, and the mixture was incubated at 37 C for 10 min. For assay
of duodenal 24-OHase activity, the substrate was 1,25-(OH)2
\[26,27-3H\]D3 (250 pmol, 80,000 cpm), and the
incubation was performed at 37 C for 3 min. The reactions were stopped
by addition of 1 ml acetonitrile. Vitamin D metabolites were extracted
by C18 Sep-Pak as described previously (23). The respective
3H-labeled products, 24,25-(OH)2D3
and 1,24,25-(OH)3D3, were separated by
high-performance liquid chromatography on a Fine-Pak SIL column (250 by
3.9 mm internal diameter) with n-hexane-isopropanol-methanol \[90:5:5
(vol/vol) for kidney and 88:6:6 (vol/vol) for duodenum\] as solvent at
a flow rate of 1.5 ml/min. Both 24,25-(OH)2D3
and 1,24,25-(OH)3D3 were identified as
described previously (7). To monitor recovery, we added
3H-labeled 1,25-(OH)2D3 (3000 cpm)
or 25OHD3 (3000 cpm) to the kidney and duodenal reaction
mixtures, respectively, after termination of the reaction. The recovery
of each compound was 85–90% and was used to adjust the measured
amount of product. Enzyme activity is expressed in picomoles per
microgram of protein per min.
Statistical analysis
Data are presented as means ± SEM.
Differences between treated and untreated groups were assessed by
Student’s t test. Multiple comparisons were evaluated by
one-way ANOVA followed by Fisher’s protected least significant
difference. Statistical analysis was performed with a software package
(Statview 4.02; Abacus Concepts, Berkeley, CA). A P value of
<0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The sequence
surrounding the putative initiation codon conformed to the Kozak
consensus sequence (24). The predicted protein contains 514 amino acids
with a calculated molecular mass of 59,500 Da. The
NH2-terminal 35 amino acids of mouse 24-OHase show
characteristics of a mitochondrial signal sequence (25). The mature
enzyme was therefore assumed to comprise 479 amino acids, with a
calculated molecular mass of 55,450 Da. The mouse cDNA coding sequence
is 83% and 65% identical with those of the rat (13) and human (12)
24-OHase cDNAs, respectively. The deduced amino acid sequence of the
mouse enzyme shows identities of 95% and 82% with those of the rat
and human enzymes, respectively. The 21-amino acid heme-binding domain
in mouse 24-OHase is completely identical with that of the human
protein and differs by one amino acid from that of the rat enzyme. Five
AUUUA sequences are present in the 3'-noncoding region of the mouse
mRNA, and two AAUAAA sequences exist in the near 3'-terminal of the
mRNA.
Tissue distribution of mouse 24-OHase mRNA
Northern blot analysis showed that, among the tissues examined,
kidney and intestine, including the duodenum, jejunum, ileum, and
colon, of 1,25-(OH)2D3-treated mice contained
the largest amount of 24-OHase mRNA; skin contained a smaller, but
readily detectable, amount of the 3.4-kb mRNA (Fig. 2A).
Longer exposures of blots to x-ray film also revealed the presence of
24-OHase mRNA in thymus and bone. RT-PCR analysis confirmed the
Northern blot data and also detected small amounts of 24-OHase mRNA in
a variety of other tissues including lung, pancreas, testis, spleen,
and heart (Fig. 2B).
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). The amount of
24-OHase mRNA in kidney increased rapidly after injection of
1,25-(OH)2D3, reaching a maximum between 4–8
h, after which it decreased and then maintained a steady value between
18–72 h (Fig. 3A). Renal 24-OHase activity showed peaks at 8 and
30 h after 1,25-(OH)2D3 treatment and
remained increased at least until 72 h. The amount of 24-OHase
mRNA in duodenum also increased rapidly after
1,25-(OH)2D3 treatment, peaked at 4 h, and
thereafter rapidly decreased to basal values (Fig. 3B). In contrast,
duodenal 24-OHase activity showed peaks at 4 and 8 h after
1,25-(OH)2D3 injection, and thereafter
decreased, returning to the control level at 48 h. In skin,
24-OHase mRNA abundance was markedly increased between 4 and 6 h
after 1,25-(OH)2D3 administration, thereafter
decreased, and returned to basal values by 18 h (Fig. 3C).
However, 24-OHase activity was not detected in skin homogenates during
the time period examined. Administration of
1,25-(OH)2D3 to mice increased both 24-OHase
mRNA abundance and 24-OHase activity in kidney in a dose-dependent
manner 4 h after treatment (Fig. 4). The increase
in 24-OHase transcripts in response to
1,25-(OH)2D3 was significant at a dose of 1.2
pmol/g and was maximal at 3.6–24 pmol/g. The hormone,
1,25-(OH)2D3, also increased renal 24-OHase
activities in dose-dependent manners. The maximal effect of
1,25-(OH)2D3 on enzyme activity was apparent at
a dose of 24 pmol/g to 25OHD3 substrate and at doses
between 3.6–24 pmol/g to 1,25-(OH)2D3
substrate.
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, A and B). Indeed, in mice treated with both
cycloheximide and 1,25-(OH)2D3, the amount of
renal 24-OHase mRNA was significantly greater than that in mice treated
with 1,25-(OH)2D3 alone 2 h after hormone
administration. The 1,25-(OH)2D3-induced
increase in enzyme activity was prevented by treatment with
cycloheximide (Fig. 5C), indicating the effectiveness of the dose
administered in inhibiting protein synthesis. Cycloheximide
pretreatment also significantly enhanced the effect of
1,25-(OH)2D3 on 24-OHase mRNA accumulation in
mouse duodenum at both 2 and 4 h after hormone treatment (Fig. 6, A and B). In contrast, cycloheximide completely
inhibited the 1,25-(OH)2D3-induced increase in
enzyme activity (Fig. 6C). Finally, the amount of 24-OHase mRNA in the
skin of mice 4 h after administration of
1,25-(OH)2D3 did not differ between animals
pretreated with cycloheximide and those pretreated with vehicle (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As mentioned above, mouse 24-OHase transcripts were most abundant in kidney and intestine, both of which are important vitamin D target tissues. Furthermore, 24-OHase activity was detected only in homogenates of kidney and intestine. Given that both kidney and intestine are implicated in 25OHD3 and 1,25-(OH)2D3 catabolism, the increased abundance of the enzyme in these tissues likely plays an important role in the removal of vitamin D metabolites. Although bone is also a major target of 1,25-(OH)2D3, the relatively small amount of 24-OHase mRNA in this tissue suggests that it is not the predominant site of vitamin D catabolism in vivo. 1,25-(OH)2D3 is essential for the normal mineralization of new bone and is a potent inducer of bone resorption (1, 2), and it markedly increases the abundance of 24-OHase mRNA in rat osteoblasts (27). The 1,25-(OH)2D3-induced accumulation of 24-OHase mRNA in cells from two major target tissues of vitamin D such as rat osteoblasts (27) and human colon cancer cells (28) is apparent only in proliferating, not in differentiated, cells. Thus, our data may indicate that most osteoblasts in the bone of adult mice are differentiated and so do not respond to 1,25-(OH)2D3 with a large increase in 24-OHase mRNA.
Stimulating effect of 1,25-(OH)2D3 on 24-OHase gene activation is mediated by the vitamin D receptor (VDR), because the promoter of the gene has been sequenced and functional vitamin D-responsive elements have been mapped in the 5'-flanking region of the gene (4, 12, 13, 14). Thus, the abundance of 24-OHase mRNA in animals treated with 1,25-(OH)2D3 appears to be dependent on the concentrations of the VDR in the tissues. The thymus, in which the VDR is abundant (21), also contained small amount of 24-OHase mRNA.
After the intestine and kidney, the concentration of 24-OHase mRNA was highest in skin. Cultured human keratinocytes treated with 1,25-(OH)2D3 have been shown to contain 24-OHase mRNA (29); although transcripts of 3.4 and 1.0 kb were apparent in the human cells, we detected only the larger mRNA species in the skin of 1,25-(OH)2D3-treated mice. DNA synthesis in cultured human keratinocytes (30) and the cornified envelope formation of keratinocytes (31) are inhibited by 1,25-(OH)2D3. Both human keratinocytes and skin contain the VDR (32).
RT-PCR and Southern blot analysis also detected small amounts of 24-OHase mRNA in tissues, such as lung, pancreas, and testis, that are not considered classical targets of vitamin D. Alveolar macrophages from individuals with pulmonary sarcoidosis have been shown to produce 1,25-(OH)2D3 (33). Recent studies suggest that 1,25-(OH)2D3 may play a specific role in lung. The VDR is present in lung of fetal rats during the period of late gestation and detectable in neonates (34). Rat lung fibroblasts can produce 1,25-(OH)2D3, and the adjacent cells such as adult alveolar type II cells, which are responsible to 1,25-(OH)2D3, bear significant numbers of the VDR (35). Thus, induction of 24-OHase mRNA by 1,25-(OH)2D3 may be likely in lung tissue. Substantial numbers of VDRs are also present in the rat testis (36). In the pancreas, 1,25-(OH)2D3 clearly elicits biological effects including the stimulation of insulin secretion and restoration of glucose tolerance, which are mediated by the VDR (37), although the lungs and testis may not be target tissues of vitamin D. It is possible that, as in classical target tissues of vitamin D, 24-OHase mediates the catabolism of 1,25-(OH)2D3 in nonclassical target tissues as a means of terminating hormone action. Absence of activity in the skin as well as the bone and thymus may be due to the insensitive assay of 24-OHase activity using homogenates and tritiated substrate. If a highly sensitive method for 24-OHase such as RRA of 1,24,25-(OH)3D3 is used, the activity may be detected in these tissues. Alternatively, 1,25-(OH)2D3 inactivation by C-24 oxidation pathway may not be necessary in the skin.
We showed that 1,25-(OH)2D3 increased both 24-OHase mRNA abundance and enzyme activity in mice in a time- and dose-dependent manner. Most circulating 25OHD3 in plasma is absorbed into the kidneys and metabolized into polar compounds including 24,25-(OH)2D3. Because it is well established that 25OHD3 is a physiological substrate in the kidneys (1, 2), the metabolite was used as a substrate of 24-OHase in renal preparation. By contrast, 1,25-(OH)2D3 rather than 25OHD3 is considered to be a physiological substrate for 24-OHase in the duodenum (38). Furthermore, a similar dose-response curve of 1,25-(OH)2D3 on 24-OHase activity was obtained by different substrates such as [3H]25OHD3 and [3H]1,25-(OH)2D3 in kidney preparations. We therefore used [3H]1,25-(OH)2D3 for the assay of duodenal 24-OHase activity in time course experiments. The effect of 1,25-(OH)2D3 on 24-OHase gene expression was rapid in kidney, duodenum, and skin. The time course of the initial increase in enzyme activity was similar to that of 24-OHase mRNA accumulation in both kidney and duodenum, indicating that the mRNA is rapidly translated into protein. However, enzyme activity in kidney and duodenum showed a second peak, possibly attributable to induction of a different 24-OHase gene not detectable with our cDNA probe (39). The 1,25-(OH)2D3-induced increases in 24-OHase mRNA and activity of a pharmacological dose (24 pmol/g) persisted for a much longer period in the kidneys than duodenum or skin, suggesting the catabolism of large amounts of vitamin D3 metabolites in the kidneys before clearance from the body (1, 9). An alternative explanation is that cellular turnover may dictate decay of the 24-OHase message and activity following initial stimulation by 1,25-(OH)2D3, because the turnover in duodenum and skin is more rapid than that in the kidneys.
In the kidneys, proximal tubules, distal tubules, and collecting ducts contain VDR and its message, whereas 24-OHase mRNA and activity are only localized in proximal tubules of vitamin D-repleted animals (40, 41). Because we administered pharmacological doses of 1,25-(OH)2D3 into normal mice, 24-OHase localization along the nephron of the mice may differ from the observation of the previous reports. The 24-OHase activity is exclusively detected in the kidneys and intestine, indicating that the tissue distribution of the enzyme activity is strictly limited. It is likely that proximal tubules alone along the nephron would express the 24-OHase activity in mice administered a pharmacological dose of 1,25-(OH)2D3.
Rather than inhibiting the 1,25-(OH)2D3-induced increase in 24-OHase mRNA in kidney and duodenum, cycloheximide actually potentiated this effect of 1,25-(OH)2D3, indicating that de novo protein synthesis is not required for stimulation of 24-OHase gene transcription. Cycloheximide also markedly increases the abundance of mRNAs encoding stromelysin (42) and PTH-related peptide (43). Such effects of protein synthesis inhibitors on gene expression have been interpreted as indicating the existence of a short-lived repressor molecule, although administration of cycloheximide may reduce message expression and protein accumulation of other components of the enzyme complex such as ferredoxin and ferredoxin reductase. The 3'-untranslated region of mouse 24-OHase mRNA contains five AUUUA sequences. Such A + U-rich elements (AREs) are important determinants of mRNA turnover (44) and are targets of RNA-binding proteins such as AUF1 (45) that mediate rapid mRNA degradation. The half-life of an mRNA depends on the number of AREs, the binding affinity of AUF1 for the AREs, and the concentration of active AUF1 available for binding (46). The ß2-adrenergic receptor agonist isoproterenol increases the abundance of AUF1 mRNA and protein in DDT1-MF2 hamster smooth muscle cells, an effect that correlates with an increased rate of degradation of ß2-adrenergic receptor mRNA, which contains AREs in the 3'-noncoding region (47). Similar regulation of 24-OHase mRNA by a labile protein may explain the potentiating effect of cycloheximide on the 1,25-(OH)2D3-induced accumulation of 24-OHase mRNA in kidney and duodenum. The effect of cycloheximide on the accumulation of the 24-OHase mRNA differed in duodenum, kidney, and skin, suggesting that the difference may be due to altered concentrations of regulatory proteins for mRNA degradation among various tissues. Posttranscriptional regulation may be an important mechanism for determining the abundance of 24-OHase mRNA.
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Footnotes |
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Received December 3, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-dihydroxyvitamin D3 in rat kidney but
not in intestine. J Biol Chem 267:13757–13762,25-dihydroxyvitamin D3. Biochem
Pharmacol 48:2081–2090[CrossRef][Medline]
,25-dihydroxyvitamin D3-24-hydroxylase in
rat osteoblasts. Endocrinology 134:1794–1799-hydroxylase and
24-hydroxylase along the rat rephron. Proc Natl Acad Sci USA 78:1199–1203This article has been cited by other articles:
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M. Y. H. Zhang, X. Wang, J. T. Wang, N. A. Compagnone, S. H. Mellon, J. L. Olson, H. S. Tenenhouse, W. L. Miller, and A. A. Portale Dietary Phosphorus Transcriptionally Regulates 25-Hydroxyvitamin D-1{alpha}-Hydroxylase Gene Expression in the Proximal Renal Tubule Endocrinology, February 1, 2002; 143(2): 587 - 595. [Abstract] [Full Text] [PDF]
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D. Tiosano, Y. Weisman, and Z.'e. Hochberg The Role of the Vitamin D Receptor in Regulating Vitamin D Metabolism: A Study of Vitamin D-Dependent Rickets, Type II J. Clin. Endocrinol. Metab., May 1, 2001; 86(5): 1908 - 1912. [Abstract] [Full Text]
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H. S. Tenenhouse, J. Martel, C. Gauthier, M. Y. H. Zhang, and A. A. Portale Renal Expression of the Sodium/Phosphate Cotransporter Gene, Npt2, Is Not Required for Regulation of Renal 1{{alpha}}-Hydroxylase by Phosphate Endocrinology, March 1, 2001; 142(3): 1124 - 1129. [Abstract] [Full Text] [PDF]
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 X.-Y. Li, M. Boudjelal, J.-H. Xiao, Z.-H. Peng, A. Asuru, S. Kang, G. J. Fisher, and J. J. Voorhees 1,25-Dihydroxyvitamin D3 Increases Nuclear Vitamin D3 Receptors by Blocking Ubiquitin/Proteasome-Mediated Degradation in Human Skin Mol. Endocrinol., October 1, 1999; 13(10): 1686 - 1694. [Abstract] [Full Text]
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T. Kawane and N. Horiuchi Insulin-Like Growth Factor I Suppresses Parathyroid Hormone (PTH)/PTH-Related Protein Receptor Expression via a Mitogen-Activated Protein Kinase Pathway in UMR-106 Osteoblast-Like Cells Endocrinology, February 1, 1999; 140(2): 871 - 879. [Abstract] [Full Text]
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) or 3H-labeled 1,25-(OH)2D3
(
). Data are means ± SEM (n = 4). *,
P < 0.05; **, P < 0.01; ***,
P < 0.001 compared with control (vehicle-treated
mice).
