Minerva Center for Calcium and Bone Metabolism, Nephrology Services, Hadassah University Hospital, Jerusalem, Israel 91120
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ABSTRACT |
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Hypophosphatemia leads to an increase
in Na+-Pi cotransporter (NaPi-2) mRNA levels.
This increase is posttranscriptional and correlates with a more stable
transcript mediated by the terminal 698 nt of the NaPi-2 mRNA. A 71-nt
binding element was identified with renal proteins from rats fed
control and low-Pi (Pi) diet. The binding of
Pi renal proteins to this transcript was increased compared with control proteins. The functionality of the cis
element was demonstrated by an in vitro degradation assay.
Pi renal proteins stabilized transcripts that included
the cis element compared with control renal extracts. The
full-length NaPi-2 transcript, but not control transcripts, was
stabilized by
Pi extracts. Insertion of the binding
element into green fluorescent protein (GFP) as a reporter gene
decreased chimeric GFP mRNA levels in transfection experiments.
Our results suggest that the protein-binding region of the NaPi-2 mRNA
functions as a cis-acting instability element. In
hypophosphatemia there is increased binding to the
cis-acting element and subsequent stabilization of NaPi-2 mRNA.
phosphate; messenger ribonucleic acid half-life; protein-ribonucleic acid interactions
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INTRODUCTION |
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PI
homeostasis is maintained by a well-defined membrane transport
system (31). In mammals, renal Pi reabsorption
is essential to Pi homeostasis. The renal tubule has an
intrinsic ability to adjust the reabsorption rate of Pi
according to need and availability of Pi (22).
The active reabsorption is mediated by the Na+-dependent
Pi transporters (NaPi). NaPi type IIa transporters (NaPi-2)
are expressed at the apical brush-border membrane (BBM) of the renal
proximal tubules and are responsible for the regulated reabsorption of
Pi in response to changes in dietary Pi
(16, 20). NaPi type IIb transporters are homologous to
NaPi type IIa but are found in a variety of other tissues such as
intestine, lung, prostate, and pancreas (4). Disruption of
the NaPi-2 gene in mice resulted in increased urinary Pi
excretion, an 85% loss in BBM Na+-Pi
cotransport, and significant hypophosphatemia (1). Mice homozygous for NaPi-2 gene deletion did not respond to Pi
depletion with an adaptive increase in Na+-Pi
cotransport or to parathyroid hormone (PTH) with a decrease in
transport. Therefore, NaPi-2 is the major regulator of renal Pi homeostasis. There is a rapid adaptive increase in
proximal tubule apical BBM Na+-Pi cotransport
activity and NaPi-2 protein abundance that is mediated by
microtubule-dependent translocation of presynthesized NaPi-2
cotransporter protein to the apical BBM (13).
Dexamethasone inhibits Na+-Pi cotransport, and
this is associated with a decrease in renal BBM NaPi-2 cotransporter
abundance and an increase in glucosylceramide content of the BBM
(15). Levi et al. (15) suggested that the increase in BBM glucosylceramide content plays a role in mediating the
effect of dexamethasone on Na+-Pi cotransport
activity. PTH and a high-Pi diet lead to a decrease in
renal proximal tubular Na+-Pi cotransport,
which correlates with a decrease in the number of NaPi-2 transporters
in the BBM because of their routing to the lysosome and subsequent
degradation (6, 7, 23, 24). Several interacting proteins
have been identified that may contribute either to its apical
distribution or its subapical/lysosomal traffic (5, 22).
However, chronic Pi depletion not only increases the
transfer of preformed NaPi-2 to the apical BBM but also increases the
level of the NaPi-2 mRNA and protein level (9, 30). The adaptive response to Pi depletion also occurs in vitro in
opossum kidney cells, where it has been shown to be a
posttranscriptional effect (2, 17, 21). In vivo in rats we
previously showed (9) by nuclear transcript run-on
experiments that the effect of chronic Pi depletion is
mainly posttranscriptional, although others also found a
transcriptional effect (8). We studied the mechanisms
involved in the posttranscriptional effect and found that this was
dependent on protein-RNA interactions (19). Cytosolic
renal proteins showed increased binding to the NaPi-2 mRNA
5'-untranslated region (UTR), and this was associated with increased
translation of NaPi-2 in vitro (19). Renal proteins from
rats fed a low-Pi diet (Pi) stabilized the
NaPi-2 transcript in vitro, and this was dependent on the presence of
the terminal 698 nt at the 3' end of the mRNA. In the present studies
we have defined the region in the NaPi-2 mRNA 698 nt that mediates the binding of
Pi proteins. The functionality of the NaPi-2
mRNA protein-binding sequence was demonstrated in an in vitro
degradation assay with renal proteins from control and low-phosphate
rats. In addition, the protein binding region was inserted into a green fluorescent protein (GFP) reporter gene that was transfected into human
embryonic kidney (HEK)293 cells to study its effect on GFP expression.
The sequence was shown to be an instability region in both systems.
Therefore, the increase in binding of
Pi renal proteins
to this region stabilizes the NaPi-2 mRNA.
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EXPERIMENTAL PROCEDURES |
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Experimental animals. Weanling male Sabra rats were fed a normal-phosphate (0.3%), normal-calcium (0.6%) or a low-phosphate (0.02%), normal-calcium (0.6%) diet (Teklad) for 3 wk. This low-phosphate diet resulted in a serum phosphate of 4.1 ± 0.5 mg/dl (control = 9.6 ± 0.9 mg/dl) and a serum calcium of 12.3 ± 0.7 mg/dl (control = 10.6 ± 0.5 mg/dl). After 3 wk, the kidneys were removed under pentobarbital sodium anesthesia and blood samples were taken for measurements of serum calcium and phosphate in a Roche autoanalyzer. Tissues for protein extracts were used immediately as described below.
Plasmids for RNA transcription.
The full-length 2,464-nt NaPi-2 transcript (GenBank accession no.
L13257) was prepared with T7 RNA polymerase from linearized plasmid
construct containing the cDNA in pSPORT (a gift from J. Biber) as
previously described (19). The 698 bp of the
NaPi-2 cDNA, spanning the region of 1746-2464 of the NaPi-2 cDNA,
were subcloned into Bluescript II KS (Stratagene, La Jolla, CA) as previously described (19). RNAs for the 698, 461, 362, 315, 231, and 144 nt were transcribed from this construct linearized with NotI, MscI, StyI,
SphI, AvaII, and BbvII, respectively,
with T3 RNA polymerase. The 450 transcript was prepared by
restriction of the plasmid containing the 698-bp cDNA with
BbvII and SphI that removed 172 bp and religation
of the plasmid. The 95-nt transcript was prepared by subcloning a
corresponding PCR product prepared with the forward oligonucleotide
5'AGTCTTCCTGGAGGAGCTT3' and a reverse oligo 5'TCTGGACCTGCAGCCTAGA3'.
The PCR fragment was ligated into pGEM-T Easy vector (Promega, Madison,
WI) and linearized with NcoI for the 95-nt transcript and
with BcnI for the 71-nt transcript. RNA was transcribed with
SP6 RNA Pol. The transcript for the transferrin receptor (Tfr)
contained 250 nt of the Tfr 3'-UTR that included three iron responsive
elements (IREs).
In vitro RNA synthesis. Radiolabeled RNA probes for RNA electrophoretic mobility shift assay (REMSA), and in vitro degradation assays were prepared from linearized templates with the appropriate RNA polymerase in a transcription reaction containing 1 µg DNA, 0.5 mM ATP, CTP, and GTP, 8 µM UTP, 2 µM UTP, 500 U/ml RNase inhibitor (Promega) and [32P]UTP (800 Ci/mmol, 20 mCi/ml). Samples were incubated at 37°C for 1 h, purified on Sephadex G-50 columns, and aliquots were taken for scintillation counting. The specific activity of the RNA probe was 0.5-1.0 × 106 cpm/ng. For competition experiments RNA was transcribed similarly in the presence of the 4 nt at 1 mM. Unlabeled RNAs were quantified by spectrophotometry at 260 nm/280 nm and visualization on agarose gels.
Protein purification for REMSA and in vitro degradation assays.
Kidneys were removed from the rats and immediately washed in cold PBS.
For binding assays, kidney protein extracts were prepared as described
previously (19) by suspending the tissue in buffer D containing 20 mM HEPES, 20% glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM PMSF, and 0.5 mM DTT and homogenizing with a Polytron. Total
protein was extracted by repeated freezing and thawing of the samples
and centrifugation for 15 min at 12,000 g. Protein extracts
were immediately frozen at 80°C in aliquots.
REMSA. Labeled RNA transcripts (10,000 cpm) spanning different regions of the NaPi RNA were incubated with renal protein extracts, in a final volume of 20 µl containing 4 µg tRNA, 10 mM HEPES, 3 mM MgCl2, 40 mM KCl, 5% glycerol, and 1 mM DTT (binding buffer) for 10 min at 4°C. Addition of heparin (5 mg/ml) did not affect binding and the size of the complex. For competition experiments unlabeled RNA was added as indicated. The specificity of binding was further demonstrated by the addition of unlabeled nonrelated transcripts that did not affect binding. The samples were run for 3 h at 4°C on a native 4% polyacrylamide gel (polyacrylamide-bisacrylamide, 70:1) in a cold room. RNA-protein binding was visualized by autoradiography of the dried gels.
In vitro cell free degradation assay. In vitro degradation was performed essentially as described previously (18, 19). Radiolabeled RNA transcripts (0.3 × 106 cpm) were incubated with 20-60 µg of cytoplasmic extract and 80 U/ml RNasin to prevent nonspecific RNA degradation, in a total volume of 40 µl at room temperature. At each time point 6 µl was transferred to a tube containing 300 µl of TRI reagent (Molecular Research Center, Cincinnati, OH), and 10 µg of tRNA and RNA was extracted. Samples were run on formaldehyde-agarose gels, transferred to Hybond membranes (Amersham, Little Chalfont, UK), and autoradiographed. The remaining undegraded transcripts at the different time points were quantified by densitometry.
Construction of chimeric GFP constructs containing cDNAs of
various regions of NaPi-2 mRNA for transfection experiments.
The 461-, 362-, 315-, 144-, 95-, and 71-bp cDNAs corresponding to
different regions of the NaPi-2 mRNA terminal 698 nt (Fig. 1) were excised by restriction enzymes
and inserted into the multiple cloning site of pEGFP-C1 (Clontech, Palo
Alto, CA), at the 3' end of GFP cDNA.
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Transient transfection experiments.
The plasmids (1 µg DNA/well/24-well plate) were transiently
transfected into HEK293 cells by calcium phosphate precipitation. Twenty-four hours after transfection total RNA was extracted by TRI
reagent and analyzed for GFP mRNA levels by Northern blot. GFP protein
was measured by immunofluorescence microscopy. Expression of
cotransfected cytomegalovirus (CMV)--galactosidase plasmid demonstrated transfection efficiency.
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RESULTS |
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Characterization of binding of renal cytosolic proteins to defined
region in NaPi-2 mRNA.
To study protein-RNA interaction we performed REMSA with renal proteins
from control rats or rats fed a low-Pi diet for 3 wk and transcripts
representing different regions of the NaPi-2 mRNA. A transcript that
excluded the terminal 698 nt of the NaPi-2 mRNA did not bind
Pi renal proteins by REMSA (not shown). A transcript of
698 nt that contained the terminal coding region and the 3'-UTR (Fig.
1B) formed a shifted complex with renal cytosolic proteins from
Pi rats (Fig. 1A). To define the
Pi protein-binding region in the 698-nt NaPi-2
transcript, we transcribed RNAs representing smaller regions in the
mRNA and analyzed them for binding with the
Pi renal
proteins. Shortening of the transcript by excluding successively larger
regions of the 3'-UTR to transcripts of 461, 362, 315 (Fig. 1,
A and B), and 231 nt (Figs. 1B and
2) did not affect binding. All these
transcripts bound proteins, resulting in a shift of the free probe that
was dose dependent. The larger shifted complexes when more protein is
added may represent the binding of additional subunits of the protein
RNA complex. Further shortening of the transcript to 144 nt resulted in
no binding (Fig. 1, A and B). This suggests that
the binding region is between 144 and 231 nt (Fig. 1B).
Therefore, we transcribed RNA that deleted the 164 nt between the 151- and 315-nt transcripts (Fig. 1B,
461 nt). This
461-nt
transcript did not bind renal proteins (Fig. 1, A and
B). These results indicate that the binding region is between 151 and 231 nt in the 698 nt represented in Fig. 1B.
Transcripts of 95 (not shown) and 71 nt in this region (Fig.
1A) were sufficient for protein binding (Fig. 1,
A and B), defining the minimal protein binding
region that corresponds to 1889-1960 of the NaPi-2 cDNA at the
junction of the coding region and the 3'-UTR. Renal proteins from
control rats showed significantly reduced binding to the 71 (not
shown)-, 95-, and 231-nt transcripts (Fig. 1C). The
decreased binding may have been in part due to some degradation of the
transcript by the renal proteins from control rats. This is evident by
the partially degraded excess free probe that remains after binding with control renal proteins but not with
Pi proteins.
With
Pi proteins the increased binding utilizes all the
probe (Fig. 1C).
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Renal cytosolic proteins from Pi
stabilize NaPi-2 transcript in an in vitro degradation assay at the
protein binding region.
We previously showed (19) that the increase in renal
NaPi-2 mRNA levels in hypophosphatemic rats can be reproduced by an in
vitro degradation assay. In this assay labeled transcripts for the
NaPi-2 mRNA are incubated with cytosolic renal proteins from control or
Pi rats. The amounts of transcripts remaining with time
are determined, and this has been shown to represent the degradatory
processes that occur in vivo. We showed that the full-length 2,464-nt
NaPi-2 transcript and the terminal 698 nt were stabilized by
Pi proteins, correlating with mRNA levels in vivo.
However, a transcript of the 5' 1,043 nt that does not include the
698-nt binding region was not stabilized in this assay (19). We have now performed the in vitro degradation assay
with the shorter transcripts that we have now shown are relevant for binding. We analyzed the transcripts of 461, 231, and 95 nt (Fig. 1B) that showed increased binding to the
Pi
renal proteins compared with control proteins. These three transcripts
were stabilized in five different in vitro degradation assay
experiments (Fig. 3A). The
half-time (t1/2) for the degradation of the
95-nt transcript was ~2 h with renal proteins from control rats and
~5 h with proteins from
Pi rats. The
t1/2 for both the 231- and the 461-nt
transcripts was <0.5 h with control proteins and >2 h with
Pi proteins. Despite the differences in degradation time
between the shorter transcript of 95 nt (2 h) and the longer
transcripts (0.5 h) in this assay, all of the transcripts were
stabilized by the
Pi proteins compared with proteins from
control rats. In contrast, a nonrelevant transcript for Tfr showed the
same degradation rate with both control and
Pi renal
proteins (Fig. 3B) similar to the 5' 1,043-nt NaPi-2 transcript (19). These results indicate that the protein
binding region of the NaPi-2 mRNA is also the target region that
determines NaPi-2 mRNA stability in response to a
Pi
diet. We also studied renal proteins from rats fed a low-calcium diet
for 10 days, in which serum phosphate was increased, serum calcium
decreased, and PTH levels markedly increased with no change in NaPi-2
mRNA levels (9). The in vitro degradation time course of
the 461-nt NaPi-2 transcript with proteins from control and low-calcium
rat kidneys was the same (Fig. 3D). This result demonstrates
that the in vitro studies accurately reflect the in vivo changes in NaPi-2 mRNA and underlines the stabilizing effect of
Pi
renal proteins in this assay.
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Protein-binding region decreases levels of reporter gene mRNA and
protein in transfected cells.
The correlation between binding and stabilization of the NaPi-2
transcript by Pi renal proteins suggests that the protein binding protects the NaPi-2 transcript in hypophosphatemia, resulting in increased NaPi-2 mRNA and protein levels in vivo. To study the
instability effect of the protein-binding region, we inserted different
cDNAs of the NaPi-2 transcript at the 3' end of the GFP cDNA in an
expression vector driven by a CMV promoter. The wild-type and chimeric
constructs were transiently transfected into HEK293 cells. These cells
have the same protein-binding pattern to the NaPi-2 transcript as does
protein from rat kidney tissue (not shown). At 24 h GFP mRNA
levels were analyzed by Northern blots and GFP protein levels by
immunofluorescence. Chimeric transcripts containing 461, 362, and 95 nt
decreased GFP mRNA and protein levels (Fig.
4), with the effects of 461 > 95 > 362 nt. These transcripts all contained the NaPi-2 mRNA
protein-binding region (Fig. 1). Figure 4B shows a
representative gel for GFP mRNA levels performed in triplicate for each
chimeric construct. Quantification of four separate experiments showed
that the GFP mRNA levels were decreased with the NaPi-2 461-, 95-, and
362-nt inserts by 70-90%. Insertion of the 144 nt that did not
bind the
Pi renal proteins did not affect GFP mRNA.
Correction after cotransfection with a
-gal expression plasmid
confirmed that the results were not due to differences in transfection
efficiency (not shown). The chimeric GFP cDNAs all used the same CMV
promoter, suggesting that the differences in expression are not a
transcriptional effect. Furthermore, when actinomycin D was added to
cells transfected with the GFP plasmids containing such insertions,
there was no difference in GFP mRNA decay (not shown). Therefore, the
effect of the protein-binding element of the NaPi-2 mRNA to decrease GFP levels is posttranscriptional at the level of mRNA stability. The
amount of GFP protein in the transfected cells correlated with GFP mRNA
levels (Fig. 4C). However, the amount of GFP protein expression as shown by immunofluorescence provides a qualitative rather
than a quantitative representation of the amount of GFP protein. The
measurement of GFP mRNA in the Northern blot is the direct indication
of the effect of the NaPi-2 inserts on transcript levels and stability.
The transfection experiments together with the in vitro degradation and
binding experiments suggest that the junctional region between the 3'
end of the coding region and the 5' end of the 3'-UTR is important for
binding and regulation of NaPi-2 RNA stability.
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DISCUSSION |
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Renal phosphate homeostasis involves the coordinate regulation of
Pi reabsorption by both intrinsic renal sensing as well as
the response to hormonal signals such as PTH (14). The
regulated Pi reabsorption is through the NaPi-2
cotransporter whose gene is Npt2. The regulation of the NaPi-2-mediated
Na+Pi cotransport by dietary Pi
occurs primarily at the posttranscriptional level (21,
26), despite the identification of several cis-acting elements in the promoter region (8, 27, 28). We previously showed (19) that Pi cytoplasmic renal
proteins stabilized the NaPi-2 transcript in an in vitro degradation
assay, which was dependent on an intact NaPi-2 3'-end. We also showed
that there was increased translation of NaPi-2 in vitro in reticulocyte
lysate assays and in vivo in pulse chase experiments with
[35S]methionine-injected rats. This increased translation
correlated with increased binding of
Pi renal proteins to
the 5'-UTR by UV cross-linking gels. Therefore, the
Pi
diet regulates NaPi-2 gene expression by affecting protein-RNA
interactions. In addition, Levi et al. (14) showed that at
short time intervals (2 h) when rats were transferred from a high to a
Pi diet there was an increase in NaPi-2 protein with no
change in mRNA levels, indicating an effect at the level of translation
or protein stability. In the chronic
Pi model there is an
increase in mRNA levels that our results suggest is posttranscriptional
(19). We have now defined the region in the NaPi-2 mRNA at
the junction of the coding region and the 3'-UTR that binds
Pi renal proteins by REMSA. UV cross-linking gels do not
detect the binding to this region (19). REMSA is a more
sensitive assay for RNA-protein binding and more closely represents the
interactions in vivo because it utilizes nondenaturing conditions. The
binding of renal proteins to the defined NaPi-2 region was increased by
Pi renal proteins. A transcript for this region was
stabilized by
Pi renal proteins in an in vitro
degradation assay, similar to the full-length NaPi-2 transcript. The
stabilizing effect of
Pi renal proteins correlates with
NaPi-2 mRNA levels in vivo. Therefore, this defined region in the
NaPi-2 transcript is a cis-acting instability element. Under
normal conditions there is limited binding of cytosolic proteins to
this cis-acting element, and this determines the
steady-state levels of NaPi-2 mRNA and NaPi-2 protein. In
hypophosphatemia there is increased binding to this element, which
protects the RNA from degradation resulting in an increase in NaPi-2
mRNA levels.
To demonstrate the destabilizing properties of the defined cis-acting element, we inserted the cDNA coding for the element into the 3'-end of a GFP reporter gene and studied its effect on GFP mRNA and protein levels by transient transfections in HEK293 cells. These cells have the same protein binding pattern to the NaPi-2 transcript as does protein from rat kidney tissue (not shown), indicating that these RNA binding proteins are preserved. Therefore, the HEK293 cells were used to study the correlations between binding and degradation. The expression of the chimeric GFP-NaPi-2 cis-element was reduced compared with the wild-type GFP, confirming that the cis-acting element is a destabilizing element.
The NaPi-2 cis-acting element is not homologous to any other
reported cis-acting element. Defined elements in many mRNAs
have been characterized that function in RNA stability, translation, and localization (3). The paradigm for such elements is
the IRE, which is present in a number of mRNAs that code for proteins that are regulated by iron (12, 29). PTH gene expression
is also regulated at the posttranscriptional level by Pi,
but in the opposite direction from NaPi-2 (10). In the
parathyroid low Pi results in posttranscriptional decrease
in PTH mRNA levels, which is dependent on a 26-nt cis-acting
element in the PTH mRNA 3'-UTR (11). A Pi
diet leads to less binding of parathyroid cytosolic proteins to this
element and a more rapid degradation of the PTH mRNA. A low-calcium
diet leads to increased binding of parathyroid cytosolic proteins to
this element and a more stable PTH transcript. One of the cytosolic
proteins that bind the defined cis element in the
parathyroid is AU-rich binding protein (AUF1), which has been shown to
stabilize the PTH transcript in the in vitro degradation assay
(25). The cytosolic proteins that bind to the NaPi-2
transcript are as yet unknown. The definition and functional
characterization of the NaPi-2 cis-acting element may help
in the isolation of renal cytosolic proteins that respond to
Pi diet and bind to and stabilize the NaPi-2 mRNA.
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ACKNOWLEDGEMENTS |
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This work was supported in part by grants from the Israel Academy of Sciences, the US-Israel Binational Foundation (BSF), the Hadassah Research Fund for Women's Health (to T. Naveh-Many), and the Minerva Foundation.
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FOOTNOTES |
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Address for reprint requests and other correspondence: T. Naveh-Many, Minerva Center for Calcium and Bone Metabolism, Nephrology Services, Hadassah Hebrew Univ. Hospital, PO Box 12000, Jerusalem, Israel 91120 (E-mail: tally{at}huji.ac.il).
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.
First published December 10, 2002;10.1152/ajprenal.00332.2002
Received 16 September 2002; accepted in final form 3 December 2002.
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