1 Departments of Pediatrics and Physiology and 2 Department of Orthopedic Surgery, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, Tucson, Arizona 85724
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ABSTRACT |
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The
phosphate-regulating gene with homologies to endopeptidases on the X
chromosome (PHEX) is a member of the neutral endopeptidase family,
which is expressed predominantly on the plasma membranes of mature
osteoblasts and osteocytes. Although it is known that the loss of PHEX
function results in X-linked hypophosphatemic rickets, characterized by
abnormal bone matrix mineralization and renal phosphate wasting, little
is known about how PHEX is regulated. We therefore sought to determine
whether the murine PHEX gene is regulated by glucocorticoids (GCs),
which are known to influence phosphate homeostasis and bone metabolism.
Northern blot analysis revealed increased PHEX mRNA expression in
GC-treated suckling mice (1.5-fold) and in rat osteogenic sarcoma
(UMR-106) cells (2.5-fold). An increase was also seen in PHEX promoter
activity in transiently transfected UMR-106 cells with GC treatment.
Analysis of nested promoter deletions revealed that an atypical GC
response element was located between 337 and
315 bp. Mutational
analysis and electrophoretic mobility shift assays further identified
326 to
321 bp as a site involved in GC regulation. Supershift
analyses and electrophoretic mobility shift assay competition studies
indicated that the core binding factor
1-subunit transcription
factor is able to bind to this region and may therefore play a role in
the GC response of the murine PHEX gene.
dexamethasone; core binding factor 1-subunit; Hyp
mouse; transcriptional regulation
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INTRODUCTION |
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THE PHOSPHATE-REGULATING gene with homologies to endopeptidases on the X chromosome (PHEX) is a neutral endopeptidase found predominately on the plasma membrane of osteoblasts and osteocytes (1, 8, 16, 20). Loss of function mutations in the PHEX gene result in X-linked hypophosphatemic rickets (XLH) in humans and, in the animal model of XLH, the Hyp mouse (28). Manifestations of XLH can vary widely and include vitamin D-resistant rickets, which results in growth retardation, lower body skeletal abnormalities, bone and joint pain, and decreased range of motion (28). Biochemical characteristics include high serum alkaline phosphatase levels (28) and hypophosphatemia resulting from decreased phosphate reabsorption in the renal proximal tubules (5, 22, 23, 34). Although the precise physiological function of PHEX is unclear, studies on the Hyp mouse have implicated PHEX in phosphate homeostasis and bone mineralization.
The role of PHEX in phosphate homeostasis has been characterized by means of the Hyp mouse model. The renal phosphate leak seen in the Hyp mouse and in XLH is a result of decreased expression of the sodium-dependent phosphate cotransporter (NaPi-IIa) in the proximal tubules (5, 21, 34). Additional experiments with the murine model of XLH revealed that the phosphate wasting is the result of a humoral factor, rather than a primary renal defect (15, 24), and that this circulating factor must be modified by PHEX.
Conversely, the role of PHEX in bone mineralization is not as clear. Clinically, skeletal features of XLH in humans and in Hyp mice include shortened stature, osteomalacia in trabecular and cortical bone, recurrent dental abscesses, and late dentition (28). The osteomalacia seen is a result of slowed bone remodeling, with a significant delay in osteoid mineralization and reduced resorption (28). This defective mineralization seen in XLH has also been seen in vitro in cultured, immortalized osteoblasts (38) and in primary cultures of bone marrow cells (20). Recent studies by Miao et al. (20) on Hyp and normal mice demonstrate that the observed osteomalacia is associated with altered expression of several bone matrix proteins. This altered expression occurred both transcriptionally, with Hyp mice having decreased levels of bone sialoprotein and vitronectin mRNA, and posttranscriptionally, with biglycan and fibrillin immunoreactive protein levels being elevated in Hyp mice while mRNA levels remained unchanged (20).
Although the physiological results of inactivating mutations in PHEX are known and it is clear that PHEX is involved in regulating bone mineralization and phosphate homeostasis, little is known about how the PHEX gene and protein are regulated. Ecarot et al. (8) have shown that vitamin D3 is capable of downregulating both PHEX mRNA and protein in cultured primary osteoblasts and in the osteoblast-like cell line MC3T3-E1. However, the effect of vitamin D3 on PHEX expression in vivo has yet to be determined. More recently, PHEX mRNA was shown to be significantly increased in bones of hypophysectomized rats treated with insulin-like growth factor I (40). Furthermore, the endopeptidase activity of purified PHEX protein has been shown to be inhibited by inorganic phosphate, inorganic pyrophosphate, and osteocalcin, with the effects of the latter being reversible by Ca2+ (3).
We recently cloned and characterized the murine PHEX gene promoter and now have the ability to use this as a tool to investigate transcriptional regulation of the PHEX gene (12). Computer analysis of the promoter region predicted several putative glucocorticoid (GC) response elements (12). GCs are known to regulate renal sodium-dependent phosphate reabsorption and to exert both positive and negative effects on bone growth and metabolism. In the present investigation, we sought to determine the following: 1) whether PHEX is regulated by GCs in vivo and in an in vitro cell culture model (UMR-106 cells), 2) whether this regulation is at the level of gene transcription, and 3) whether the cloned murine promoter contains cis-acting elements that mediate this response to GC.
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MATERIALS AND METHODS |
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Experimental animals.
Male C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were used in
groups of five. Twelve-day-old suckling mice were subcutaneously injected with methylprednisolone (MP; Solu-Medrol, Upjohn, Kalamazoo, MI) at a dose of 30 µg/g body wt or equal volumes of saline once every 12 h for a total of four injections. Animals were supplied with food and water ad libitum. The animals were killed 3 h after the last injection (at 14 days of age) by CO2 narcosis
followed by cervical dislocation. Calvaria were removed, flash frozen
in liquid nitrogen, and stored at 70°C. All animal procedures were approved in advance by the University of Arizona Institutional Animal
Care and Use Committee.
Chemicals and reagents. Lipofectamine, 10× Tris-boric acid-EDTA (TBE), 20× SSC, 100 mM sodium pyruvate, and T4 DNA ligase were purchased from GIBCO BRL (Bethesda, MD). High-glucose DMEM and fetal bovine serum were from Irvine Scientific (Santa Ana, CA). Taq polymerase, restriction enzymes, and dual luciferase assay kit were from Promega (Madison, WI). DNA oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA). All other reagents, unless otherwise indicated, were purchased from Fisher Scientific (Pittsburgh, PA) or Sigma (St. Louis, MO).
Northern blot analyses.
Total RNA was prepared from mouse calvaria or UMR-106 cells with TRIzol
Reagent (GIBCO) according to the manufacturer's protocol. Ten
micrograms of total RNA were fractionated on a 1% formaldehyde gel and
downward transferred to a Zeta-Probe GT nylon membrane (Bio-Rad,
Hercules, CA). An antisense PHEX DNA probe was generated by using the
PCR EZ strip kit (Ambion, Austin, TX). The probe was synthesized by
using PHEX reverse primer 5'-TGTCATGTTCAGCTCGAGAG-3', with a PHEX cDNA
template from +924 to +1,541 bp [numbered with respect to the
identified transcriptional start site (12)] and [-32P]dATP (NEN, Boston, MA). Membranes were
hybridized with radiolabeled probe in Ultrahyb buffer (Ambion), washed
according to the manufacturer's protocol, and then exposed to X-ray
film (Pierce, Rockford, IL) at
70°C. Resulting films were analyzed
with a Bio-Rad GS-700 Imaging Densitometer with Quantity One software.
PHEX hybridization band intensities were normalized for 18 s on
the ethidium bromide-stained membrane.
Reporter gene constructs.
Nested promoter deletions, with the same 3' ends, were generated
between 542 and
133 bp of the PHEX promoter (12) by
using the Exo-Size Deletion Kit (New England Biolabs, Beverly, MA). All
deletion constructs were confirmed by sequencing. Site-directed scanning mutations were introduced five bases at a time between
336
and
317 bp by two-step PCR, as previously described
(13). The resulting mutant constructs were cloned into
pGL3-basic (Promega), as previously described (12). The
primers used to construct the mutant promoter constructs were forward
542 primer, 5'-ACTACTGGTACCATTAAGCTCACAC-3'; reverse +104 primer,
5'-AGCCAATCTAGATGTCTGAACTGTC-3'; mutant 1 forward and
reverse, 5'-ATCCTCAGGACTAGTCTGAAATAAC-3' and 5'-GTTATTTCAGACTAGTCCTGAGGAT-3', respectively; mutant
2 forward and reverse, 5'-CAGGAAGCTGAGTCCATAACCACTT-3'
and 5'-AAGTGGTTATGGACTCAGCTTCTTG-3', respectively;
mutant 3 forward and reverse,
5'-AGCTGCTGAACGCCACACTTTAGGG-3' and 5'-CCCTAAAGTGTGGCGTTCAGCAGCT-3', respectively; and
mutant 4 forward and reverse,
5'-CTGAAATAACACAGGTAGGGAAACA-3' and
5'-TGTTTCCCTACCTGTGTTATTTCAG-3', respectively. Bold type indicates mutated bases, which were mutated A
C and G
T.
Cell culture and transient transfection.
Rat osteogenic sarcoma cells (UMR-106) were obtained from the American
Type Culture Collection (CRL-1661) and were cultured according to its
guidelines. The cells (passages 7-13) were seeded on
24-well plates and cotransfected, by means of liposome-mediated transfection, at 70-80% confluency with 0.5 µg of reporter
vector DNA and 0.015 µg of pRL-TK vector (encoding Renilla
luciferase, used as an internal standard; Promega) in serum-free media.
Twelve hours after transfection, cells were allowed to recover for
24 h in medium with 10% fetal calf serum followed by 12 h in
serum-free medium. Cells were then treated with 107 M
dexamethasone (DEX; Sigma), 10 µM mifepristone (RU-486, an antagonist of the GC receptor; Sigma) (36), DEX+RU-486, or
equal amounts of vehicle (ethanol) in medium with charcoal-stripped serum. Twenty-four hours after treatment, cells were harvested, and
dual luciferase assays were performed with equal amounts of cellular
lysate. To assess the effect of DEX on endogenous PHEX expression in
UMR-106 cells, untransfected cells were cultured in 100-mm plates and
treated as described above. These experiments were repeated at least
three times with separate cell populations on different days. In each
experiment, two (for 100-mm plates) or three dishes (for 24-well
plates) were considered as n = 1, and the results from
these dishes were averaged. Additionally, firefly luciferase activity
was normalized for Renilla luciferase activity in each cell extract.
Electrophoretic mobility shift assays.
Nuclear protein for electrophoretic mobility shift assays (EMSAs) was
prepared from UMR-106 cells as previously described (33).
Double-stranded synthetic olignucleotides were end labeled with
[-32P]dATP (NEN). For each probe used for EMSAs,
20,000 counts/min of probe was incubated with 4 µg of nuclear
protein, 4 µl of 5× binding buffer [(in mM) 100 HEPES, pH 7.5, 5 EDTA, 50 (NH4)2SO4, 5 dithiothreitol, and 150 KCl, as well as 1% Tween 20 (wt/vol); Roche
Molecular Biochemicals, Indianapolis, IN], 1 µg poly [d(I-C), Roche
Molecular Biochemicals], and H2O to 20 µl. For
competition studies, 100× of cold probe was added to the reaction.
Reactions were incubated at room temperature for 15-20 min. Five
microliters of DNA loading buffer [60% 0.25× TBE, 40% glycerol,
0.2% bromphenol blue (wt/vol)] were added, and the reaction was
loaded on a 6% DNA retardation gel (Invitrogen, Carlsbad, CA) and
electrophoresed at 250 V in 0.5× TBE. Gels were dried and then exposed
to X-ray film (Pierce) at
70°C. For supershift experiments, 2 µg
of anti-core binding factor (Cbf)
1 antibody (Santa Cruz
Biotechnology, Santa Cruz, CA) was added to the reaction and incubated
at room temperature for 45 min before electrophoresis. Oligos used for
EMSAs were PHEX
332 to
306 bp, 5'-AGCTGCTGAAATAACCACTTTAGGGAA-3';
mutant 3,
5'-AGCTGCTGAACGCCACACTTTAGGGASA-3'; mutant
4, 5'-AGCTGCTGAAATAACACAGGTAGGGAA-3'; and Cbf
1,
5'-CCCGTATTAACCACAATAAAACTCG-3' (Geneka Biotechnology, Montreal, Canada). Bold type indicates mutated bases in mutant 3 and mutant 4 oligos and the core binding sequence in
the Cbfal oligo.
Statistical analysis. Statistical significance was determined by Student's t-test or ANOVA followed by Fisher's paired least significant difference, by using the Statview software package version 4.53 (SAS Institute, Cary, NC), and expressed as means ± SE.
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RESULTS |
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GC regulation of PHEX in mice.
To determine the ability of exogenous GCs to regulate PHEX in vivo,
12-day-old mice were treated with pharmacological doses of MP or an
equal volume of saline (vehicle). Total RNA was extracted from calvaria
and subjected to Northern blot analysis with a PHEX cDNA-specific
probe. Results revealed a 1.5-fold increase in PHEX mRNA
expression in MP-treated mice compared with controls (Fig. 1A).
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GC regulation of PHEX in UMR-106 cells.
Northern blot analysis of rat osteogenic sarcoma UMR-106 cells treated
with 107 M DEX or vehicle revealed a 2.51-fold increase
in PHEX mRNA expression in DEX-treated cells compared with ethanol
(vehicle)-treated cells (P < 0.005, n = 3; Figs. 1, B and C).
Responsiveness of the murine PHEX promoter to GCs.
We have previously cloned and shown functionality of the murine PHEX
promoter in UMR-106 cells (12). Preliminary experiments designed to test for GC responsiveness of the previously characterized PHEX promoter fragments (12) showed that the 542/+104
construct was responsive to DEX whereas the
133/+104 construct was
not. To better define the region of the promoter responsible for the observed DEX response, a series of nested deletions were constructed between
542/+104 and
133/+104. DEX treatment of transiently transfected UMR-106 cells showed no increase in luciferase activity with the
133/+104 and
315/+104 constructs; however, the remaining three constructs,
338/+104,
354/+104, and
542/+104 showed
a 60-85% increase compared with pGL3-basic (P < 0.05, n = 7-10; Fig.
2). Data are expressed as "fold"
increase due to variation in basal promoter activity levels within
individual constructs among experiments. However, the fold increases in
promoter activity with DEX treatment remained consistent among
experiments.
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Mutational analysis of the PHEX promoter region
338 to
315 bp.
To determine which base pairs between
338 and
315 bp were
responsible for the observed DEX response, A
C and G
T mutations were introduced five bases at a time between
336 and
316 bp by PCR.
All the mutations were introduced in the
542/+104 construct for ease
of construction. The four mutant promoter constructs, mutant
1, mutant 2, mutant 3, and mutant
4, (Fig. 4 and Table 1) along with the
542/+104 construct
and pGL3-basic were transfected into UMR-106 cells, which were then
treated with DEX or vehicle. Analysis of luciferase activity revealed
that the DEX response of the mutant 3 construct was
significantly reduced (P < 0.02, n = 4) from the twofold increase in the
542/+104 construct to levels not
significantly different from pGL3-basic (Fig. 4). The remaining
constructs, mutant 1, mutant 2, and mutant
4, showed trends toward reduction in DEX-stimulated activity,
compared with the wild-type
542/+104 construct, that were not
statistically different from the
542/+104 construct but were
different from pGL3-basic (P < 0.05, n = 4-5; Fig. 4).
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EMSA analysis of the PHEX promoter region from
332 to
306 bp.
To assess the ability of transcription factors to bind to the region
between
326 and
321 bp (encompassing mutant 3), which when mutated showed a loss of DEX response, EMSAs were conducted. Nuclear extracts obtained from UMR-106 cells were incubated with an
oligonucleotide probe spanning
332 to
306 bp, which resulted in two
shifted bands (Fig. 5). Both of these
bands were competed with a 100-fold excess of cold probe (Fig. 5,
lane 3). However, these bands were not competed with an
excess of cold mutant 3 probe (Fig. 5, lane 4).
Comparison of
325 to
315 bp (the region encompassing mutant
3) of the PHEX promoter with known osteoblast-specific transcription factor binding sites showed that mutations 3 and 4 contain a putative osteoblast-specific, cis-acting element
(OSE2) (6) to which the transcription factor Cbf
1 has
been shown to bind (7). Therefore, to determine whether
any of the observed shifted bands corresponded to Cbf
1, we competed
the PHEX wild-type probe with an excess of cold probe specific for
Cbf
1. The Cbf
1 probe was able to compete out both bands seen with
the PHEX wild-type probe (Fig. 5, lane 6). To
further confirm the binding of Cbf
1 to the PHEX probe, an
anti-Cbf
1 antibody was used. The addition of the anti-Cbf
1
antibody completely removed the highest, shifted band (Fig. 5,
lane 7), indicating that Cbf
1 was binding to the probe.
Additionally, incubation of the Cbf
1-specific oligonucleotide probe
with nuclear extract shifted bands of identical molecular weight to
those shifted by the PHEX-specific probe, with the upper band being
supershifted by the anti-Cbf
1 antibody (Fig. 5, lane 12).
Additionally, because it appeared that Cbf
1 was capable of binding
to the OSE2 site spanning mutations 3 and 4,
competition with 100× cold mutant 4 probe, which contains
the three 3' bases of the OSE2 site, was conducted. Results of this
competition revealed that mutations made between
321 and
316 bp
(mutant 4) could only partially compete the PHEX wild-type
probe (Fig. 5, lane 5). Furthermore, the affinity of
the nuclear protein for the PHEX probe was seen to be lower than that
for the Cbf
1 probe (Fig. 5) when equal amounts of protein were
incubated with equal probe activities.
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DISCUSSION |
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We undertook the present study to determine whether GCs, which are known to have important implications in phosphate homeostasis bone and metabolism, are capable of regulating the PHEX gene. We found that PHEX mRNA isolated from calvaria of suckling mice treated with methlyprednisolone was elevated 1.5-fold compared with that of vehicle-treated mice (Fig. 1). Additionally, PHEX mRNA expression in the rat osteogenic sarcoma cell line UMR-106 treated with DEX was seen by Northern blot analysis to be elevated ~2.5-fold (Fig. 1). The physiological relevance of this observed upregulation of PHEX by GCs is unclear. Studies on the Hyp mouse showed that PHEX is involved in phosphate homeostasis through transcriptional regulation of the renal NaPi-IIa gene. Furthermore, NaPi-IIa has been shown to be downregulated by GCs at a posttranscriptional level in an age-dependent fashion (9). Because of these differences in the way that gene expression is effected (up- vs. downregulation) and at what level GC regulation occurs (transcriptional vs. posttranscriptional), it seems unlikely that GC-mediated effects on PHEX expression play a role in NaPi-IIa gene expression. However, GC studies in Hyp and normal littermates will ultimately determine whether GC regulation of the PHEX gene, in turn, alters renal phosphate reabsorption by means of changes in NaPi-IIa expression.
GCs have been reported to exert both positive and negative effects on osteoblasts, the predominant site of PHEX expression. It is well characterized that long-term clinical use of GCs results in serious skeletal side effects, most notably osteoporosis. Weinstein et al. (37) and Silvestrini et al. (32) have both indicated a role for osteoblast apoptosis in GC-induced osteoporosis. However, no correlation has yet been made between PHEX expression and osteoporosis. Additionally GCs were administered over a much longer time period in the studies showing GC-induced osteoblast apoptosis than we used in the present study, making precise comparisons difficult.
GCs have also been shown to exert positive effects on cells of the osteoblast lineage. The addition of GCs to cultured osteoblasts has been shown to promote differentiation (25, 31), along with increasing the size and amount of bone nodule formation (2, 11, 19, 31). This increase in nodule formation has further been shown to be greater in less mature than in more mature osteoblastic cells, with the former requiring GCs to differentiate (35). Therefore, it is likely that the increase in endogenous PHEX mRNA expression seen in UMR-106 cells and the activity of the longer promoter constructs is the result of GC-induced maturation of the relatively immature UMR-106 cells. This is consistent with the observations that PHEX expression increases as osteoblasts differentiate (8) and that DEX increases nodule formation in UMR-106 cells (11). The observation that there are two populations of osteoblastic cells that respond differently to GCs would also explain the difference in GC response seen between the in vitro and in vivo studies. UMR-106 cells, which are a homogenous population of relatively immature osteoblasts, show a 2.5-fold increase in PHEX expression with GC treatment, whereas a more modest 1.5-fold increase in expression is seen in mice calvaria, which contain a more heterogeneous population of osteoblasts at different stages of maturation.
Analysis of PHEX promoter constructs transfected into UMR-106 cells
showed that treatment with 107 M DEX significantly
increased (P
0.006) promoter-driven luciferase activity from the
542/+104 construct compared with the promoterless luciferase vector pGL3-basic. This twofold increase in promoter activity is consistent with the observed DEX increase in endogenous PHEX expression in UMR-106 cells. To define the region of the promoter
responsible for upregulation by GCs, a series of nested deletions of
the
542/+104 promoter construct were produced and assayed for GC
responsiveness. Results revealed that the
337/+104 construct
responded to DEX, whereas the
315/+104 construct, which is only 23 bases shorter, was unresponsive. This indicated that a
cis-element responsible for GC upregulation was likely
between
337 and
315 bp of the PHEX gene promoter. Analysis of the
DNA sequence from
337 to
315 bp revealed the lack of a classical GC
response element(s); however, the ability of RU-486 to abolish the GC
response indicated an effect mediated by the GC receptor.
To further define the bases responsible for GC regulation,
site-directed mutations were introduced into the 542/+104 promoter construct between
336 and
316 bp (Table 1). Analysis of these promoter mutants revealed that only mutations introduced in
mutant 3, between
326 and
321 bp, resulted in a
significant (P < 0.05, n = 4)
reduction in DEX-induced luciferase activity compared with the
wild-type
542/+104 construct. When the bases mutated in the mutant 3 construct along with three bases flanking each side
of the mutation were scanned with Matinspector (Genomatrix, Munich, Germany; http://genomatix.gsf.de/) for putative transcription factor
binding sites, no potential cis-acting elements were
identified. As PHEX is predominantly expressed in mature osteoblasts
and osteocytes (8, 10, 20, 29), these bases were further
examined manually for potential osteoblast-specific transcription
factor binding sites. A single OSE2 site (6), to which the
transcription factor Cbf
1 has been shown to bind (7),
was found to span one-half of mutant 3 and one-half of
mutant 4. Cbf
1 is a member of the runt gene family with
homology to the DNA-binding domain of the Drosophila runt
gene (26) and has been shown to be necessary for
osteoblast differentiation (7, 14, 27). Furthermore, Cbf
1 is capable of regulating the osteoblast genes osteocalcin, osteopontin, and
1(I) collagen (7, 39) and is itself
regulated by 1,25(OH)2 vitamin D3
(7), GCs (4), and parathyroid hormone (30). Interestingly, these three hormones are also
important in bone metabolism and phosphate homeostasis.
To determine whether this putative Cbf1-binding site was capable of
binding Cbf
1, an EMSA was conducted utilizing a probe from
332 to
306 bp, encompassing the bases mutated in the mutant 3 construct. The EMSA revealed that the PHEX
332 to
306 bp probe shifted two bands when incubated with UMR-106 nuclear protein. Both of
these bands were competed with 100× cold PHEX and Cbf
1-specific probes but neither were competed with the PHEX mutant 3 probe. Furthermore, the Cbf
1-specific probe shifted bands identical to those of the PHEX
332 to
306 bp probe. Additionally, the affinity of the nuclear proteins for the Cbf
1-specific probe was
much greater than for the PHEX probe. To determine whether one or more
of the shifted bands contained the Cbf
1 protein, supershift analyses
were conducted with an anti-Cbf
1 antibody. With addition of
anti-Cbf
1 antibody, the upper shifted band seen with the PHEX probe
was abolished, whereas the same band with the Cbf
1-specific probe
was shifted to a higher molecular weight. The inability of the
anti-Cbf
1 antibody to supershift the upper PHEX probe band is likely
the result of disruption of the much weaker interaction of Cbf
1 with
the PHEX probe compared with the Cbf
1-specific probe.
Taken together, the EMSA data and the results with promoter
mutant 3 suggest that Cbf1 may play a role in the GC
response of the PHEX gene. This would appear to be in contrast with the results of Liu S et al. (17), who reported that
overexpression of Cbf
1 had no effect on luciferase activity driven
by the murine PHEX promoter. However, these authors only evaluated the
ability of Cbf
1 to stimulate promoter expression and did not appear
to consider the possibility of repression of PHEX expression by
Cbf
1. The ability of Cbf
1 to repress PHEX expression would be
consistent with the recent observations by Liu et al.
(18), which showed that late-stage osteoblast maturation
was blocked in transgenic mice overexpressing Cbf
1, along with those
of Ecarot et al. (8) and Miao et al. (20),
who showed PHEX expression is highest in fully differentiated
osteoblasts and osteocytes. In light of these findings, it is possible
that Cbf
1 may serve to repress PHEX expression in nonmature
osteoblasts. Therefore, attempts to alter PHEX promoter expression, in
an osteoblastic cell line that endogenously expresses Cbf
1, by
overexpressing Cbf
1 would likely result in no change in promoter
activity. Additionally, studies by Chang et al. (4) showed
that GCs decreased immunoreactive Cbf
1 protein in primary rat
osteoblasts. Therefore, our findings along with those of Liu et al.
(18) and Chang et al. (4) suggest that the
GC-induced increase in PHEX expression in suckling mice and UMR-106
cells, along with increased PHEX promoter activity in GC-treated
UMR-106 cells, may result from removal of Cbf
1 from the promoter,
which may act to repress PHEX expression.
In conclusion, we have demonstrated that the rodent PHEX gene is
regulated by GCs through the GC receptor and that this regulation is,
at least in part, mediated at the transcriptional level. Furthermore, we have identified position 326 to
321 bp in the murine PHEX promoter as a site involved in this regulation. Additionally EMSA analysis of the GC responsive region indicates that the osteoblast transcription factor Cbf
1 is likely involved. Future studies will
define the precise role Cbf
1 plays in the expression of the PHEX
gene and its regulation by GCs.
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ACKNOWLEDGEMENTS |
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This investigation was funded by National Institute of Diabetes and Digestive and Kidney Diseases Grant 2RO1-R37-DK-33209-17 and the W. M. Keck Foundation.
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FOOTNOTES |
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* E. R. Hines and J. F. Collins contributed equally to this work.
Address for reprint requests and other correspondence: F. K. Ghishan, Dept. of Pediatrics, Steele Memorial Children's Research Ctr., Univ. of Arizona Health Sciences Ctr., 1501 N. Campbell Ave., Tucson, AZ 85724 (E-mail: fghishan{at}peds.arizona.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
March 5, 2002;10.1152/ajprenal.00357.2001
Received 7 December 2001; accepted in final form 28 January 2002.
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