RNA-Protein Binding and Post-transcriptional Regulation of Parathyroid Hormone Gene Expression by Calcium and Phosphate*

Eli Moallem, Rachel Kilav, Justin SilverDagger , and Tally Naveh-Many

From the Minerva Center for Calcium and Bone Metabolism, Nephrology Services, Hadassah University Hospital, Jerusalem il-91120, Israel

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Parathyroid hormone (PTH) regulates serum calcium and phosphate levels, which, in turn, regulate PTH secretion and mRNA levels. PTH mRNA levels are markedly increased in rats fed low calcium diets and decreased after low phosphate diets, and this effect is post-transcriptional. Protein-PTH mRNA binding studies, with parathyroid cytosolic proteins, showed three protein-RNA bands. This binding was to the 3'-untranslated region (UTR) of the PTH mRNA and was dependent upon the terminal 60 nucleotides. Parathyroid proteins from hypocalcemic rats showed increased binding, and proteins from hypophosphatemic rats decreased binding, correlating with PTH mRNA levels. There is no parathyroid cell line; however, a functional role was provided by an in vitro degradation assay. Parathyroid proteins from control rats incubated with a PTH mRNA probe led to an intact transcript for 40 min; the transcript was intact with hypocalcemic proteins for 180 min and with hypophosphatemic proteins only for 5 min. A PTH mRNA probe without the 3'-UTR, or just the terminal 60 nucleotides, incubated with hypophosphatemic proteins, showed no degradation at all, indicating that the sequences in the 3'-UTR determine PTH mRNA degradation. Hypocalcemia and hypophosphatemia regulate PTH gene expression post-transcriptionally. This correlates with binding of proteins to the PTH mRNA 3'-UTR, which determines its stability.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

PTH1 is the major hormone that regulates calcium homeostasis and also has an important role in regulating bone strength. In turn, the synthesis and secretion of PTH is finely regulated by the serum calcium concentration, with hypocalcemia resulting in a marked increase in serum PTH, PTH mRNA levels, and parathyroid cell number (1, 2). Changes in extracellular calcium are sensed by the PT cell by a cell membrane sensor, which then leads to a change in intracellular calcium and inositol triphosphate concentrations (3). How these factors then determine the levels of PTH secretion, gene expression, and PT cell proliferation is not clear.

A negative calcium regulatory element in the atrial natriuretic peptide gene, with a homologous sequence in the PTH gene (4) has been shown to bind a protein (ref1), which was known to activate several transcription factors (5). Because no parathyroid cell line is available, these studies were performed in nonparathyroid cell lines, so their relevance to physiologic PTH gene regulation remains to be established. A post-transcriptional effect of calcium on PTH gene expression in primary cultures of bovine parathyroid cells has been demonstrated (6), which correlated with binding of parathyroid proteins to the 5'- and 3'-untranslated regions (UTRs) of bovine PTH mRNA probes (7).

PTH leads to a decrease in serum phosphate by increasing renal phosphate excretion, and in turn, serum phosphate has a direct effect to increase PTH secretion and PTH mRNA levels (8-11). Dietary induced hypophosphatemia leads to a dramatic decrease in PTH gene expression, and this effect is posttranscriptional (8).

We have now studied the mechanism whereby hypocalcemia and hypophosphatemia in vivo regulate PTH mRNA levels. We show that the effect of hypocalcemia to increase PTH mRNA levels in vivo is post-transcriptional, similar to the effect of hypophosphatemia to decrease PTH mRNA levels, which is also post-transcriptional (8). PT cellular proteins bind to PTH mRNA and specifically to the 3'-UTR. PT proteins from hypocalcemic rats led to increased protein-RNA binding and hypophosphatemic PT proteins decreased this binding. In vitro mRNA degradation studies show that the PTH mRNA 3'-UTR mediates the rapid degradation of the transcript by parathyroid proteins from hypophosphatemic rats and stabilization of the transcript by parathyroid proteins from hypocalcemic rats.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Animals-- Weanling male Sabra rats were fed a normal calcium (0.6%), normal phosphate (0.3%) diet, a low calcium (0.02%) normal phosphate (0.3%) diet, or a low phosphate (0.02%) normal calcium (0.6%) diet (Teklad, IL) for different time intervals. This low phosphate diet at 2 weeks results in a serum phosphate of 4.0 ± 0.4 mg/dl (control = 9.8 ± 1.2 mg/dl) and serum calcium of 12.6 ± 0.6 mg/dl (control = 10.6 ± 0.4 mg/dl) (8).

At timed intervals the thyroparathyroid tissue, microdissected parathyroid and thyroid, and other tissues were removed under pentobarbital anesthesia, and blood samples were taken for serum calcium and PTH measurements. The thyroparathyroid tissue for RNA extraction was frozen in liquid nitrogen and stored at -70 °C until analysis. To confirm the accuracy of the microdissection, Northern blots were performed and showed that the microdissected parathyroids had PTH mRNA, and the isolated thyroid had no PTH mRNA even after a prolonged exposure. Tissues for nuclear run-on assays and protein extracts were used immediately as described below.

Measurement of Cellular mRNA Levels-- RNA was extracted from rat parathyroid tissue, and the levels of PTH mRNA were measured by Northern blots after extraction with TRI reagent (Molecular Research Center, Inc., Cincinnati, OH) as described previously (12). The integrity of the RNA and the uniformity of RNA transfer to the membrane were determined by UV visualization of the ribosomal RNA bands of the gels and the filters. Hybridization was to a random primed rat PTH cDNA probe and 18 S rRNA as a control. The autoradiograms were scanned by a densitometer and quantitated as optical density units.

Serum Measurements-- Serum Ca2+ and phosphate were measured in a Roche autoanalyzer. Serum PTH levels were measured with a rat Allegro immunoradiometric assay (Nichols, San Clemente, CA).

Nuclear Run-on Transcription Assay-- Nuclei were prepared from pooled thyroparathyroid tissue of 10 rats, and nuclear run-on transcription assays were performed as described previously (8). RNA was extracted and resuspended in 300 µl of hybridization buffer (7% SDS, 10% polyethylene glycol (8,000), 1.5% SSPE). Aliquots of RNA from treated and untreated samples were counted in a scintillation counter, and an equal number of counts from each condition (1-2 × l06 cpm) were hybridized to linearized cDNAs (5 µg) for PTH, calcitonin, actin, rat glyceraldehyde-3-phosphate dehydrogenase, and pBR322 DNA (8), which were immobilized to Hybond filters using a slot blot apparatus. Hybridization was performed at 65 °C for 72 h. The filters were washed three times at room temperature in 2 × SSC, 0.2% SDS for 5 min, then washed once at 55 °C for 15 min. The blots were exposed to Agfa CURIX-RP2 film at -70 °C with intensifying screens for 14 days. In addition, the filters were exposed for 24 h to a bio-imaging plate and quantified by a bio-imaging analyzer BAS2000 (Fuji Photo Film, Japan).

Plasmid Constructs and Labeling of RNA-- Rat PTH cDNA was recloned by inserting the PstI/PstI rat PTH cDNA fragment from PT43 plasmid into Bluescript II KS+ plasmid, which contains T3/T7 promoters (Stratagene, La Jolla, CA). This fragment spans the region of the PTH cDNA from 32 to 817 base pairs and includes most of the 5'-UTR and all the 3'-UTR, including the long poly(A) tail (13). A clone in which the 5' of the PTH cDNA insert was adjacent to the T3 promoter was used for sense RNA synthesis (see Fig. 4). RNA was transcribed from plasmids that had been linearized using different restriction enzymes followed by phenol extraction and ethanol precipitation. The template for probe A was prepared by digesting the plasmid with SmaI, probe B by digestion with DraI, probe C by digestion with XbaI, probe D by digestion with BstXI, and probe E by digestion with NcoI. The 3'-UTR template for probe F was prepared by a polymerase chain reaction as described previously (12) of the PTH cDNA fragment using two oligonucleotides complimentary to the 5' and 3' ends of the 3'-UTR. The 5' primer that included the T3 polymerase primer sequence (underlined) was 5'- ATTAACCCTCACTAAAGGGATGCTGACGTATTC-3'. The 3' primer was 5'-GATCATTAAACTTTA-3'. The polymerase chain reaction product was run on a 2% agarose gel, and the single product of the expected size of 247 base pairs was purified using a DNA isolation kit (Biological Industries, Kibbutz Beit Haemek, Israel) and used for RNA transcription.

Radiolabeled RNA probes for UV cross-linking were prepared from linearized templates using T3 RNA polymerase in a transcription reaction containing 1 µg of DNA, 0.5 mM each ATP, CTP, GTP, 8 µM UTP, 2 µM bromo-UTP, 500 units/ml RNase inhibitor (Boehringer Mannheim, Germany), and [32P]UTP (800 Ci/mmol, 20 mCi/ml). Samples were incubated at 37 °C for 1 h and 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 RNA degradation assays the RNA was labeled without bromo-UTP. For competition experiments and for RNA translation, unlabeled PTH mRNA was transcribed similarly in the presence of 1 mM each of the four nucleotides. The RNA was visualized on a 2% agarose gel, and the transcription level was estimated.

Cytoplasmic Protein Purification-- Cytoplasmic proteins (S100) were extracted by the method of Dignam et al. (14). Different tissues were removed from the rats and immediately washed in cold phosphate-buffered saline. Parathyroid proteins were prepared from microdissected parathyroids or from thyroparathyroid tissue. The tissue was cut with a scalpel, suspended in 5 volumes of buffer A containing 10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride, and incubated on ice for 10 min. After centrifugation at 600 × g for 10 min (4 °C) the pellet was resuspended in 2 volumes of buffer A and homogenized by a Polytron. The homogenate was centrifuged at 600 × g for 10 min, and the supernatant was carefully decanted, mixed with 0.1 volume of buffer B containing 0.3 M HEPES, 1.4 M KCl, and 0.03 M MgCl2, and centrifuged (4 °C) at 100,000 × g for 1 h (Beckman type TL-100). The high speed supernatant (S100) was dialyzed for 20 h at 4 °C against 50 volumes of buffer D containing 20 mM HEPES, 20% glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM DTT. For RNA degradation assays and in vitro translation the S100 fraction was prepared by homogenizing the tissue with a Polytron in 2 volumes of 10 mM Tris/HCl, pH 7.4, 0.5 mM DTT, 10 mM KCl, and 1.5 mM MgCl2. 0.1 volume of the extraction buffer (1.5 mM KCl, 15 mM MgCl2, 100 mM Tris/HCl, pH 7.4, 5 mM DTT) was added, and the homogenate was centrifuged at 14,000 × g for 2 min to pellet the nuclei. The supernatant was centrifuged at 100,000 × g for 1 h at 4 °C. Cytoplasmic extracts were immediately frozen at -80 °C in aliquots. Protein concentration was determined by optical density densitometry (595-µm wavelength) using a Bradford reagent (Bio-Rad).

UV Cross-linking Assay-- 1 ng of RNA probe (0.5-1.0 × 106 cpm) was incubated with different amounts (10-30 µg) of cytoplasmic protein extracts (S100) in a final volume of 20 µl containing 10 mM HEPES, 3 mM MgCl2, 40 mM KCl, 5% glycerol, and 1 mM DTT (binding buffer). After 30 min at room temperature, heparin was added to a final concentration of 5 mg/ml, to eliminate nonspecific binding, and the samples were irradiated at 2.5 J/cm2 with a UV light source of 312 nm. RNase A-XII (Sigma) was then added for 15 min at 37 °C to a final concentration of 1 mg/ml to digest unprotected RNA. The samples were heated for 5 min at 65 °C after addition of 5 µl of Leamlli sample buffer containing 50% glycerol, 10% SDS, 0.4 M Tris, pH 6.8, 0.5 M DTT, and a small amount of bromphenol blue. The samples were then loaded on a SDS 10% polyacrylamide electrophoresis gel. RNA-protein binding was visualized by autoradiography. A molecular weight marker (Bio-Rad) was also run on the gel for size estimation of the protein-RNA bands. In some experiments proteinase K (200 µg/ml) was added. For competition experiments unlabeled RNA was added.

In Vitro Cell-free Degradation Assay-- In vitro cell free degradation was performed essentially as described previously (15-17). Radiolabeled RNA transcripts (0.3 × 106 cpm) were incubated with 40 µg of cytoplasmic extract and 80 units/ml RNasin to prevent nonspecific RNA degradation in a total volume of 40 µl at room temperature. At each time point 6 µl were transferred to a tube containing 300 µl of TRI reagent (Molecular Research Center), and RNA was extracted. Samples were run on formaldehyde-agarose gels, transferred to Hybond membranes (Amersham, UK), and autoradiographed. The remaining undegraded transcripts at the different time points were quantified by densitometry.

In Vitro Translation Assay-- A RNA transcript (1 µg) for the full-length PTH mRNA (probe A) was translated using a rabbit reticulocyte lysate system (Promega, Madison, WI) according to the manufacturer's instructions using [35S]methionine (10 mCi/ml, Amersham). Translation was conducted in the presence or absence of cytosolic parathyroid proteins (30 µg). At timed intervals aliquots of the reaction were removed, analyzed on SDS-polyacrylamide gel electrophoresis, and autoradiographed.

Statistical Analysis-- Results were analyzed by one-way analysis of variance with the post hoc Bonferroni multiple comparisons test to determine the significance of differences between means. Probability values under 5% were determined as significant. The results are presented as the mean ± S.E.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Hypocalcemia Increases PTH mRNA Levels Post-transcriptionally-- To study the mechanism of the effect of hypocalcemia on PTH gene expression, weanling rats were fed a low calcium diet, and the levels of serum calcium, PTH, and PTH mRNA were determined at different times. Serum calcium had decreased from 8.9 to 7.5 mg/dl after 3 days on the low calcium diet with an increase in serum PTH. PTH mRNA levels, as measured by Northern blots (2), were increased from day 7, and at day 21 both PTH and PTH mRNA levels had increased ~10-fold (Fig. 1). There was no change in a control gene 18 S RNA (not shown). The effect of hypocalcemia to increase PTH mRNA was also demonstrated by in situ hybridization, where it was evident that there was an in increase in PTH mRNA uniformly throughout the PT gland, involving all the PT cells (not shown). To differentiate whether the increase in PTH mRNA levels was transcriptional or post-transcriptional, nuclear transcript run-on experiments were performed on thyroparathyroid nuclei from rats fed either a control diet or a low calcium diet for 3, 7, and 21 days after weaning. The nuclear transcription rates for the PTH gene, as well as a number of other genes expressed in this tissue, showed that there was no difference between the nuclei from control and hypocalcemic rats at day 21 (Fig. 2), as well as at days 3 and 7 (not shown). At day 21, the ratio of transcription rates in hypocalcemic rats:control rats for PTH was 1.5, for calcitonin 3.0, for actin 1.5, for glyceraldehyde-3-phosphate dehydrogenase 2.5, which show that PTH transcription was the same or less than that of control genes studied. The same results were shown in two repeat experiments. The results of the nuclear run-on experiments therefore indicate that the major effect of hypocalcemia to increase PTH gene expression is post-transcriptional. In addition, we have previously shown that PTH mRNA levels are markedly decreased in hypophosphatemic rats and that this effect is also post-transcriptional (8).


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Fig. 1.   Effect of a low calcium diet on serum PTH, serum calcium, and PTH mRNA after 1, 3, 7, and 21 days. Weanling rats were fed a low calcium diet (0.02%) until study. PTH mRNA levels (optical density (OD) units) were determined from Northern blots of total thyroparathyroid RNA. Each point represents the mean ± S.E. of four rats, and when the S.E. was smaller than the symbol it was not shown. All points are significantly different from day 1 at p < 0.01 by analysis of variance with the Bonferroni post hoc test apart from PTH mRNA at day 3, which was not different from day 1.


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Fig. 2.   Nuclear transcript run-ons for PTH, calcitonin (CT), actin, glyceraldehyde-3-phosphate dehydrogenase (GPDN), and pBR322 of rats fed a normal (ND NCa) or low calcium (-D -Ca) diet for 21 days after weaning. The thyroparathyroid tissue from 10 rats was pooled in each group.

Parathyroid Cytoplasmic Proteins Bind to the PTH mRNA with Increased Binding from Hypocalcemic Rats and Decreased Binding from Hypophosphatemic Rats-- Post-transcriptional regulation is often determined by proteins binding to mRNAs and altering mRNA stability. To study the mechanism of the post-transcriptional regulation of PTH mRNA by dietary calcium and phosphate, we performed protein-RNA binding experiments. In vitro synthesized radiolabeled PTH mRNA riboprobes spanning the full-length mRNA were incubated with cytoplasmic proteins isolated from rat parathyroids as well as from other tissues, cross-linked by UV light, digested with RNase A, to digest the RNA that was not protected by protein, and run on a SDS-polyacrylamide gel. When no extract was added, all the RNA was digested and no bands were detected (not shown). Incubation of parathyroid protein extracts with PTH mRNA probe resulted in three specific protected bands (Fig. 3). The sizes of the proteins, which were estimated by running molecular mass markers, were approximately 110, 60, and 50 kDa (see Fig. 3). When the reaction mix was treated with proteinase K, there were no bands detected (not shown), indicating that protein binding had protected the radiolabeled PTH mRNA sequences. Excess unlabeled PTH mRNA competed for binding of the radiolabeled PTH mRNA (see Fig. 5, last three lanes). These results indicate that there is specific binding of protein(s) to the PTH mRNA.


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Fig. 3.   UV cross-linking of S100 parathyroid, brain, and liver protein extracts to full-length PTH mRNA. The proteins were UV cross-linked to 32P-labeled full-length PTH mRNA, run on a SDS-polyacrylamide gel, and visualized by autoradiography. A, parathyroid cytoplasmic proteins (10 µg) from rats fed a normal diet, a low calcium diet, or a low phosphate diet for 14 days were used. The size of molecular mass markers is shown. B, UV cross-linking of cytoplasmic proteins from parathyroid and brain from rats fed a normal or low phosphate diet, and liver from rats fed a normal or a low calcium diet.

To determine if this binding has a role in the regulation of PTH mRNA levels by calcium and phosphate, RNA protein binding was analyzed with proteins from hypocalcemic and hypophosphatemic rats. Parathyroid proteins from rats fed a normal diet, a low calcium diet, or a low phosphate diet for 2 weeks were incubated with PTH mRNA probe and analyzed by UV cross-linking. RNA protein binding was increased by proteins from the hypocalcemic rats and decreased by proteins from the hypophosphatemic rats (Fig. 3A). Proteins isolated from rat brain and liver (Fig. 3B), thyroid, muscle, spleen, and heart (not shown) also showed the same binding pattern as the parathyroid. Proteins from brain of hypophosphatemic rats showed the same levels of binding as did control rats (Fig. 3B). The intensity of binding to proteins from brain was consistently higher than other tissues. Proteins from liver of hypocalcemic rats did not lead to increased RNA binding (Fig. 3B). There was also no difference in binding of proteins from thyroid of hypophosphatemic and hypocalcemic rats (not shown). These results show that the increased binding to PTH mRNA in hypocalcemia and the decreased binding in hypophosphatemia are specific to parathyroid proteins and do not occur in other tissues studied.

The Region of the PTH mRNA That Binds Cytosolic Proteins Is the 3'-Untranslated Region, and the Binding Is Dependent on Its Terminal 60 Nucletotides-- To determine which region of the PTH mRNA bound the cytoplasmic proteins, shorter PTH mRNA constructs were prepared. The PTH cDNA consists of three exons (13, 18-20). Exon I codes for the 5'-UTR, exon II for most of the prepro sequence of prepro-PTH mRNA, and exon III for the PTH structural gene and the 3'-UTR. A restriction map of the rat PTH cDNA is shown in Fig. 4, as well as the sites of restriction enzyme cleavage used to prepare the different RNA probes: probe A, the full-length PTH mRNA; probe B, consisting of exon I, II, and part of exon III designed to exclude 60 nucleotides of the 3'-UTR; probe C, consisting of exon I, II, and part of exon III designed to exclude the complete 3'-UTR; probe D, consisting of exons I and II; probe E, consisting of exon I. Probe F for the 3'-UTR RNA was transcribed from a polymerase chain reaction product (Fig. 4). A radiolabeled riboprobe of part of the PTH mRNA spanning the 5'-UTR, the coding region of the gene, and most of the 3'-UTR excluding about 60 nucleotides (probe B) did not bind any proteins (Figs. 4 and 6). Probe C, representing exons I, II, and the translated region of exon III; probe D, representing exons I and II; and probe E, representing the 5'-UTR of exon I, also showed no binding (not shown). However, a probe representing only the 3'-UTR (probe F) bound parathyroid cytoplasmic proteins with the same pattern as the full-length mRNA, indicating that this was the region to which the proteins bound. The sizes of the proteins were the same size as when the full-length probe was studied (Figs. 3, 5, and 6). The specificity of the binding to the 3'-UTR was confirmed by competition experiments (Fig. 5). The addition of increasing amounts of unlabeled RNA which excluded the terminal 60 nucleotides of the 3'-UTR did not compete with protein binding of the full-length PTH mRNA at 100 × concentration, and showed a moderate competition at higher concentrations (Fig. 5, lanes 2-4). However, the full-length unlabeled RNA competed for binding even at low concentrations (10 ×) (Fig. 5, last three lanes). This confirms the specificity of the binding to the 3'-UTR and shows that the terminal 60 nucleotides are necessary for binding.


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Fig. 4.   Diagram of the PTH cDNA and the PTH mRNA probes used for RNA-protein binding assays. RNA probes (A-E) and the restriction enzyme cleavage sites used for their generation by T3 RNA polymerase are indicated. Probe A represents the full-length PTH mRNA and was prepared by linearization with SmaI; probe B represents the full-length PTH mRNA without the terminal 60 nucleotides by linearization with DraI; probe C represents exons I, II, and the translated region of exon III prepared by linearization with XbaI; probe D represents exon I and most of exon II by linearization with BstXI; probe E represents exon I and a small portion of exon II by linearization with NcoI. Probe F represents the 3'-UTR and was transcribed from a polymerase chain reaction product containing the T3 polymerase primer sequence. The number of nucleotides of each PTH mRNA probe is given. The intensity of binding of cytoplasmic parathyroid proteins to the RNA probes, as detected by UV cross-linking analysis, is indicated at the right as no detectable binding (-) and positive binding (+).


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Fig. 5.   Competition experiment for parathyroid protein binding to the full-length PTH mRNA probe with unlabeled PTH RNA. Parathyroid proteins were incubated with the full-length PTH mRNA probe and increasing amounts of unlabeled PTH mRNA without the terminal 60 nucleotides of the 3'-UTR, or with the unlabeled full-length RNA and analyzed by UV cross-linking.

Fig. 6 demonstrates that a probe excluding the terminal 60 nucleotides of the 3'-UTR (probe B) did not bind any parathyroid proteins. In addition, a 3'-UTR probe (probe F) showed the same binding pattern size as the full-length probe, with three RNA-protein bands of the same size (Fig. 6). Moreover, protein-RNA binding to the 3'-UTR with parathyroid proteins from hypocalcemic rats was markedly increased (Fig. 6). Proteins from rats fed a low phosphate diet for 2 weeks showed decreased binding (Fig. 6). Proteins from brain, liver, and thyroid of hypocalcemic and hypophosphatemic rats did not lead to changes in binding to the 3'-UTR (not shown). These results are the same as for the full-length probe (Fig. 3, A and B). Identical results were found in three repeat experiments using protein pools from different rats. It should be noted that the intensity of the three bands differed among experiments; however, the specificity of the binding was constant and was always regulated by dietary calcium and phosphate with proteins from the parathyroid.


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Fig. 6.   UV cross-linking of parathyroid protein extracts to different regions of the PTH mRNA. 20 µg of thyroparathyroid protein extracts were UV cross-linked to 32P-labeled PTH mRNA probe B, which represents the PTH mRNA without the 60 nucleotides at the 3' end (lanes 1 and 2), and probe F, which is the 3'-UTR (lanes 3-5). The protein extracts were from rats fed a normal diet for 14 days (lanes 1 and 3), rats fed a low calcium diet for 14 days (lanes 2 and 4), and rats fed a low phosphate diet for 14 days (lane 5). The arrows indicate the specific bands protected by the protein. The positions of molecular mass markers (kDa) are indicated on the right.

Quantification of the PTH mRNA-Protein Binding-- To characterize the differences in binding of parathyroid proteins from rats on the different diets to PTH mRNA, the binding was performed with increasing concentrations of the full-length RNA probe. Binding was analyzed by UV cross-linking experiments and the autoradiograms quantified by densitometry of the three bands together (Fig. 7). The proteins from hypocalcemic rats showed greater binding than controls at all RNA probe concentrations. The binding of proteins from hypocalcemic rats reached a plateau at 1.0 ng of RNA. The proteins from rats fed a low phosphate diet showed decreased binding at all probe concentrations studied (Fig. 7). A plateau of RNA-protein binding was not reached with the control and low phosphate proteins, because increases in the amount of probe above 2 ng resulted in high levels of background, which prevented the accurate measurement of binding. Attempts to decrease the specific activity of the probe did not improve the resolution. For this reason, it was not possible to determine the maximum binding of the control and low phosphate protein preparations.


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Fig. 7.   RNA concentration binding curve. UV cross-linking of thyroparathyroid proteins from control, hypocalcemic (low calcium), and hypophosphatemic (low Pi) rats to increasing concentrations of full-length PTH mRNA. Protein RNA binding (optical density (OD) units) was measured at different concentrations of RNA probe (ng). The points represent the mean ± S.E. of three experiments, but the S.E. are smaller than the symbols. The binding at 0.5 ng is the mean of two experiments.

The PTH Transcript Half-life in an in Vitro RNA Degradation Assay Is Increased by Parathyroid Proteins from Hypocalcemic Rats and Decreased by Parathyroid Proteins from Hypophosphatemic Rats-- Degradation assays were performed by incubating PTH RNA transcripts with cytoplasmic extracts at room temperature and measuring the amount of undegraded transcript remaining at different time intervals. After addition of the proteins from rats on a control diet to the full-length PTH mRNA, the transcript was still intact at 40 min (Fig. 8, A and B). Hypocalcemic parathyroid proteins led to an intact transcript for a much longer period (180 min). When proteins from hypophosphatemic rats were added, the decrease was dramatic and already evident at 5 min (Fig. 8, A and B). Cytosolic proteins from the livers of these hypocalcemic and hypophosphatemic rats did not show any differences in PTH transcript degradation (not shown). The mRNA transcripts without the 3'-UTR (Fig. 4, probe C) and without the terminal 60 nucleotides of the 3'-UTR (Fig. 4, probe B), were not degraded at all until 180 min, with proteins from rats fed normal, low calcium (not shown) as well as a low phosphate diet (Fig. 8C). This is in contrast to the rapid degradation of the full-length transcript (probe A) by the low phosphate parathyroid proteins (Fig. 8C). These results suggest that there are instability regions in the PTH mRNA 3'-UTR which are degraded by parathyroid cytosolic proteins, and appreciably more with proteins from hypophosphatemic rats than hypocalcemic and control rats. The instability region is the same sequence of RNA, which is necessary for protein binding, suggesting that the binding protects the RNA from degradation.


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Fig. 8.   In vitro degradation of PTH mRNA by parathyroid cytosolic proteins. A, gel electrophoresis of full-length PTH mRNA riboprobe incubated with cytosolic proteins from rats fed control, hypocalcemic (-Ca) and hypophosphatemic (-P) diets for different time periods. B, time response curves of intact full-length PTH mRNA after incubation with parathyroid cytosolic proteins as in A. Each point represents the mean ± S.E. of three to four different experiments, apart from -Ca at 240 and 300 min, which is the mean of two experiments. At some points the S.E. is less than the size of the graphic symbols. The PTH transcript is degraded very rapidly by proteins from -P rats, and remains intact for a longer time period with proteins from -Ca rats. C, mapping a region in the PTH 3'-UTR that mediates degradation by proteins from -P rats. PTH mRNA probes used are defined in the methods and Fig. 3. They are intact (probe A), without the 3'-UTR (probe C), and without the 3'-terminal 60 nucleotides of the 3'-UTR (probe B).

In Vitro Translation of the PTH mRNA Is Not Regulated by Hypocalcemic and Hypophosphatemic Parathyroid Proteins-- Translation of the PTH mRNA in vitro in a rabbit reticulocyte lysate assay resulted in a single product of molecular mass 17 kDa (not shown). There was no difference in the amount of PTH protein synthesized in the presence of parathyroid cytosol proteins from normal, hypocalcemic, and hypophosphatemic rats, indicating that in this assay there is no translational regulation by dietary calcium and phosphate. The translation was performed for 0.5 and 2.0 h to ensure that a difference at a short time interval would be detected.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The present studies have defined the time sequence with which weanling rats develop hypocalcemia and increase their serum PTH and PTH mRNA levels. To determine whether the increased PTH gene expression was transcriptional or post-transcriptional, nuclear transcript run-ons were performed. Transcription rates were the same in nuclei from rats fed a normal or low calcium diet, in contrast to the 10-fold increase in PTH mRNA levels. Therefore, the effect of hypocalcemia to increase PTH mRNA levels in vivo is predominantly post-transcriptional. The marked decrease in PTH mRNA levels in hypophosphatemic rats is also post-transcriptional (8). Further studies were performed to determine the role of protein-RNA interactions in these post-transcriptional effects.

A full-length radiolabeled PTH cDNA riboprobe incubated with rat parathyroid cytoplasmic proteins as well as other tissues resulted in three specific protein-RNA bands after UV cross-linking of the RNA-protein complexes. UV cross-linking enables the sizes of the proteins which bind the RNA to be estimated. In this method RNase treatment leaves only a small portion of the RNA, which is protected by the bound protein. This enables a reasonably accurate estimate of the size of the bound proteins, which were about 50, 60, and 110 kDa. This raises the possibility that the larger 110-kDa protein represents a heterodimer or a homodimer of the smaller proteins.

Parathyroid proteins from hypocalcemic rats had an increase in RNA binding, and proteins from hypophosphatemic rats decreased binding. The changes in binding may represent both changes in the affinity and the amount of the RNA binding proteins, but to differentiate between these two possibilities requires a more purified protein preparation. It is clear from these studies that proteins from hypocalcemic rats bind PTH mRNA much more than proteins from hypophosphatemic rats. Proteins from brain, liver, muscle, spleen, and thyroid showed the same binding pattern as did parathyroid proteins, but in contrast the binding was not affected by the different diets.

The region of the PTH mRNA that bound cytoplasmic proteins was determined by using different PTH cDNA constructs, and it was found to be specific to the 3'-UTR of the RNA. Furthermore, a probe that lacked 60 nucleotides of the 3' end of the mRNA did not bind proteins. This suggests that this region is important for protein binding. Further studies are needed to determine if this is the binding site. The 3'-UTR itself was sufficient for protein binding and showed the same binding pattern as the full-length mRNA probe. Proteins from hypocalcemic rats increased the binding to the 3'-UTR, and proteins from hypophosphatemic rats decreased the binding to the 3'-UTR, as for the full-length probe. These results confirm the importance of this region for protein binding.

There is no parathyroid cell line to measure PTH mRNA half-life in vitro; however, the effect of parathyroid cytosolic proteins to degrade PTH mRNA can be studied by an in vitro degradation assay. This assay has been used to define the post-transcriptional regulation of other mRNAs, such as vascular endothelial growth factor by hypoxia (17, 21). After addition of the proteins from rats on a control diet to the full-length PTH mRNA, the transcript was still intact at 40 min. After proteins from hypophosphatemic rats were added, the decrease was dramatic and evident as early as 5 min, reaching 20% of the initial transcript. Hypocalcemic parathyroid proteins led to an intact transcript up to 180 min. The mRNA transcript without the 3'-UTR was not degraded at all, with proteins from rats fed normal as well as a low phosphate diet. These results suggest that there are instability regions in the PTH mRNA 3'-UTR, which are degraded by parathyroid cytosolic proteins. In addition to proteins that degrade RNA, the cytosolic protein preparation includes the proteins that bind to the PTH mRNA to stabilize it. The mRNA half-life in vivo and in vitro is determined by both these degrading and stabilizing proteins. Parathyroid proteins from hypocalcemic rats protect the PTH mRNA from degradation, possibly by the increased binding to the 3'-UTR. Hypophosphatemic parathyroid proteins led to a rapid degradation of the PTH mRNA, which may be related to the decreased binding to the 3'-UTR. Deletion of the 3'-UTR stabilizes the RNA even in the presence of hypophosphatemic proteins. There was no difference in the degradation of PTH mRNA with cytosolic proteins from the livers of these hypocalcemic and hypophosphatemic rats, indicating the specificity of the parathyroid's response to hypophosphatemia and hypocalcemia.

There are well characterized paradigms of protein-RNA interactions determining mRNA stability (22). An example is the effect of iron on the transferrin receptor mRNA stability and ferritin translation (23-25). There are also several genes which have hormone-induced changes in mRNA stability such as thyrotropin beta -subunit (26), growth hormone (27), vitellogenin (28), and vasopressin (29). To this group should now be added the regulation of PTH mRNA by proteins from hypocalcemic and hypophosphatemic rats, where the RNA-binding trans-acting factors remain to be identified.

Serum calcium and phosphate determine PTH mRNA levels by regulating the binding of proteins to the 3'-UTR of the PTH mRNA. Identification of the proteins that determine PTH mRNA stability will help us to understand how calcium and phosphate regulate the PTH gene.

    ACKNOWLEDGEMENTS

We thank Miriam Offner for excellent technical assistance, and Dr. Yoel Israeli and colleagues for helpful discussions.

    FOOTNOTES

* This work was supported in part by a grant from the Israel Academy of Sciences (to T. N.-M.).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.

Dagger To whom correspondence should be addressed: Nephrology Services, Hadassah University Hospital, P. O. Box 12000, Jerusalem, Israel 91120. Tel.: 972 2 6436778; Fax: 972 2 6421234; E-mail: silver{at}cc.huji.ac.il.

1 The abbreviations used are: PTH, parathyroid hormone; PT, parathyroid; UTR, untranslated region; DTT, dithiothreitol.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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