Regulation of Parathyroid Hormone mRNA Stability by Calcium and Phosphate
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Abstract
Calcium and phosphate regulate parathyroid hormone (PTH) secretion, gene expression and if prolonged also parathyroid proliferation.
The regulation of PTH gene expression by Ca2+ and Pi is post-transcriptional, affecting mRNA stability.
The regulation of PTH mRNA stability is mediated by the binding of protective trans acting factors to a cis acting instability element in the 3’UTR. In hypocalcemia there is increased binding that protects the PTH mRNA from degradation by cytosolic ribonucleases resulting in higher levels of PTH mRNA.
In hypophosphatemia there is decreased binding, with a subsequent increased degradation of the PTH mRNA resulting in low levels of PTH mRNA.
We have identified an AU rich protein binding sequence in the PTH mRNA 3'-UTR and determined its functionality.
A 63 nt PTH mRNA 3' UTR element was inserted into a growth hormone (GH) reporter transcript and led to destabilization of the GH transcript.
Moreover, this element was sufficient to reproduce the regulation of the reporter transcript’s stability by calcium and phosphate in an in vitro degradation assay (IVDA).
The PTH mRNA 63 nt element destabilized mRNA for the reporter genes GFP and growth hormone, in transient transfection experiments in HEK293 cells, similar to its effect in the IVDA.
Therefore, the PTH RNA-protein binding region is a destabilizing element and is sufficient to confer responsiveness to calcium and phosphate of PTH mRNA. We have identified one of the PTH mRNA 3'-UTR trans acting proteins as AU rich binding factor 1 (AUF1).
Recombinant AUF1 stabilized the PTH mRNA transcript in the IVDA with parathyroid proteins.
In parathyroid extracts from rats fed the different diets there was no difference in AUF1 protein levels by Western blots despite differences in RNA – protein binding. However, 2D gels showed post-translational modification of AUF1 in these extracts, suggesting that calcium and phosphate alter the AUF1 protein and thus its ability to bind and stabilize the PTH mRNA.
Regulation of the Parathyroid Gland by Calcium and Phosphate
PTH has a central role in maintaining normal calcium (Ca2+) and phosphate (Pi) homeostasis as well as bone strength.
Dietary induced hypocalcemia markedly increases PTH secretion, mRNA levels and after prolonged stimulation, parathyroid cell proliferation.
PTH then acts to correct serum calcium by mobilizing calcium from bone and increasing renal reabsorption of calcium. Dietary induced hypocalcemia leads to a 10X increase in PTH mRNA levels.
We have shown that the increase in PTH mRNA levels is post-transcriptional affecting mRNA stability.
The serum inorganic phosphate is also regulated within a narrow range. 90% of the inorganic phosphate in the serum is ultrafiltrable by the kidneys where there is a sensitive regulation of renal reabsorption by both intrinsic and hormonal (PTH) mechanisms.
PTH decreases serum phosphate by increasing renal phosphate excretion.
Serum phosphate in turn, has a direct effect to increase PTH secretion, PTH mRNA levels and parathyroid cell proliferation.
In careful in vivo studies we were able to show that the effect of phosphate on the parathyroid was independent of any changes in serum calcium and 1,25(OH)2D3.
This was confirmed by in vitro studies that showed that phosphate had a direct effect on the parathyroid.
For the in vitro effect of increasing phosphate concentrations on PTH secretion it was imperative to maintain tissue architecture.
There was an effect in whole glands or tissue slices but not in isolated cells.
This effect was confirmed by Slatopolsky and his colleagues and Olgaard et al, in parathyroid tissue of different sources, showing that phosphate regulates the parathyroid directly.
In vivo studies showed that dietary induced hypophosphatemia leads to a dramatic decrease in PTH gene expression and this effect is post-transcriptional as is the effect of hypocalcemia to increase PTH mRNA levels.
There is a ~60-fold difference in PTH mRNA levels between hypocalcemic and hypophosphatemic rats, and we used these dietary models as tools to define the mechanism of the post-transcriptional regulation of PTH gene expression.
We have shown that the post-transcriptional regulation by dietary induced hypocalcemia and hypophosphatemia is mediated by protein-RNA interactions involving protein binding to a specific element in the PTH mRNA 3’-UTR that determine PTH mRNA stability. After a low calcium diet there is increased binding to the PTH mRNA 3’-UTR that protects the RNA from degradation resulting in increased PTH mRNA levels.
After a low phosphate diet there is less binding that allows more degradation and leads to the decrease in PTH mRNA levels.
Protein Binding and PTH mRNA Stability
To define the mechanism of the post-transcriptional regulation of PTH gene expression, weanling rats were fed a control diet (0.6% calcium, 0.3% phosphate) or diets deficient in calcium (0.02% calcium, 0.3% phosphate)(low calcium) or phosphate (0.6% calcium, 0.02% phosphate) (low phosphate) for 2 weeks.
A low calcium diet resulted in a decrease in serum calcium levels, an increase in serum PTH levels and a 10-fold increase in PTH mRNA levels.
A low phosphate diet led to a decrease in serum phosphate levels and PTH levels and a 6-fold decrease in PTH mRNA levels compared to the rats fed a control diet.
Nuclear run on experiments showed that these effects were post-transcriptional.
Post-transcriptional regulation of gene expression is often associated with protein-RNA interactions that regulate mRNA stability.
There was specific binding of parathyroid cytosolic protein extracts to the PTH mRNA transcript using UV cross-linking and RNA mobility shift assays (REMSA).
The binding was to the full-length PTH mRNA transcript or to a transcript for the 3' terminal region of the PTH mRNA 3'-UTR but not to transcripts that did not include this region.
Protein binding to the PTH mRNA 3’-UTR was increased by hypocalcemia and decreased by hypophosphatemia by REMSA and UV cross-linking gels, in correlation with PTH mRNA levels.
The PTH mRNA binding proteins were also present in other tissues but the binding was regulated by Ca2+ and Pi only in the parathyroid and not in other tissues of the same rats.
UV cross-linking of parathyroid proteins to transcripts for the full-length and the 3’-UTR showed 3 protein-RNA bands of ~110, 70 and 50 kDa, and the regulation of binding was evident in all 3 bands.
There is no parathyroid cell line.
Therefore we have utilized a cell-free mRNA in vitro degradation assay (IVDA) to demonstrate the functionality of the parathyroid cytosolic proteins in determining the stability of the PTH transcript.
This assay has been shown to authentically reproduce cellular decay processes.
Parathyroid proteins were incubated with a 32P labeled full-length PTH mRNA transcript and at timed intervals samples were removed and run on agarose gels to measure the amount of labeled intact PTH transcript remaining with time. Incubation with parathyroid proteins led to gradual degradation of the PTH transcript, which was a result of degrading and protective factors present in the cytosolic extract.
The transcript was intact until 40 min. with parathyroid proteins from control rats, however, with hypocalcemic parathyroid proteins the transcript was not degraded until 180 min. and with hypophosphatemic proteins the transcript was degraded already at 5 min, correlating with mRNA levels in vivo.
Therefore, the IVDA reproduces the in vivo stabilizing effect of low Ca2+ and destabilizing effects of low Pi on PTH mRNA levels.
A transcript that did not include the 3’-UTR or just the 60 terminal nt protein-binding region of the PTH mRNA 3’-UTR was more stable than the full-length PTH transcript and was intact for more than 180 min even with the low Pi parathyroid proteins, suggesting that the binding region is an instability element.
Moreover, Ca2+ (not shown) and Pi did not regulate the stability of the truncated transcript that did not include the protein-binding region. Therefore, the regulation by parathyroid proteins from low Ca2+ and Pi rats in the IVDA is dependent upon the protein-binding sequence in the PTH mRNA 3’-UTR.
Identification of the PTH mRNA 3'-UTR Binding Proteins and Their Function
The PTH mRNA binding proteins were purified by PTH RNA 3’-UTR affinity chromatography.
One of the purified proteins was sequenced and was identical to AU-rich binding factor (AUF1) (hnRNP D) which is central to the stability of other mRNAs.
Recombinant AUF1 bound the PTH mRNA 3’-UTR with a single band at 50 kDa, which corresponds to 1 of 3 protein-RNA bands found with cytosolic parathyroid proteins by UV cross-linking gels.
Binding of recombinant AUF1 to the PTH mRNA transcript was also demonstrated by RNA electrophoretic mobility shift assays.
Antibodies to AUF1 resulted in super shift of the PTH RNA-parathyroid extract complex, demonstrating that AUF1 is part of the protein-RNA complex.
Brewer et al have cloned this RNA-binding protein which binds with high affinity to a variety of A+U-rich elements (AREs) in the 3’-UTRs of a number of mRNAs.
These include mRNAs for cytokines, oncoproteins, and G protein coupled receptors, where AUF1 is associated with instability of these mRNAs.
Three classes of AREs have been characterized, two of which contain several scattered or overlapping copies of the pentanucleotide AUUUA.
The class III AREs lack the AUUUA motif but require a U-rich sequence and possibly other unknown determinants.
It has been shown that the AUUUA motif is not required for AUF1 binding.
The PTH mRNA 3’-UTRs are rich in A and U ranging from 68-74% of the nucleotides. The PTH mRNA 3’-UTR binding element is a type III ARE that does not contain any AUUUA sequences.
There are four isoforms for AUF1 that are generated by alternative splicing of the AUF1 transcript. The activity of AUF1 may be determined by the presence of other proteins in the binding complex, such as αCP1 and 2 in stabilizing the α-globin mRNA and in the stabilization of other mRNAs. To study the effect of AUF1 on PTH RNA stability, recombinant AUF1 was added to the IVDA.
Addition of recombinant p40AUF1 and p37AUF1 isoforms of AUF1 stabilized the PTH transcript dose-dependently even by hypophosphatemic proteins, that without AUF1 led to rapid degradation of the full-length PTH transcript.
Addition of eluate from the PTH 3' UTR affinity column together with p40AUF1 stabilized the PTH transcript at doses that alone had no effect.
Other control proteins, bovine serum albumin (BSA) and dynein light chain (Mr 8000) (LC8), that also binds to the PTH mRNA 3’-UTR had no effect.
This result supports the regulatory role of AUF1 in PTH mRNA stability. The mechanism by which AUF1 stabilizes PTH mRNA is not clear.
Low calcium and Pi did not affect the level of AUF1 in the parathyroids by western blots (unpublished results). Preliminary results show that calcium and phosphate lead to differences in post-translational modifications, possibly phosphorylation, rather than in the amount of AUF1 protein (not shown).
Two additional PTH mRNA binding proteins were identified by RNA affinity chromatography (not shown), hnRNP K. and Up stream of nras (UNR).
These proteins bind PTH transcripts by binding assays using recombinant proteins.
Specific antibodies to each of the identified proteins led to a super shift of the PTH mRNA 3’-UTR binding complex (not shown), demonstrating that they are part of the protein RNA complex.
An additional protein, dynein light chain (Mr 8000) or LC8 was identified by Northwestern expression cloning and was shown to mediate the binding of the PTH mRNA to microtubules.
LC8 may have a role in the intracellular localization of PTH mRNA in the parathyroid cell rather than in the stability of PTH mRNA.
Identification of the Minimal cis Acting Protein Binding Element in the PTH mRNA 3’-UTR
The PTH cDNA consists of three exons coding for the 5'-UTR (exon I), the prepro region of PTH (exon II), and the structural hormone together with the 3'-UTR (exon III).
The rat 3’-UTR is 239 nt long out of the 712 nt of the full-length PTH RNA.
The 3’-UTR is 42% conserved between human and rat, while the coding region is 78% conserved.
Binding and competition experiments by REMSA and UV cross-linking gels identified a 26 nt element as the minimal sequence sufficient for parathyroid protein binding.
Sequence analysis of the 26 nt element in the PTH mRNA of different species revealed high conservation of the rat element in the PTH mRNA 3’-UTRs of the murine (23 of 26 nt), human (19 of 26 nt) and canine (19 of 26 nt) species, with human and canine being identical.
The human and rat are 73% identical in the 26 nt element, and only 42% identical in their 3’-UTR.
The canine and rat are 73% identical in their 26 nt element and 50% identical in their 3’-UTR. The human and canine are 100% identical in the 26 nt of the element and only 70% identical in their 3’-UTR.
Comparison of the 26 nt sequence in rat and mouse showed 89% identity, however, their 3’-UTRs show a comparable degree of identity. All in all, this analysis suggests that the binding element may represent a functional unit that has been evolutionarily conserved, but sequencing of the 3’-UTR in many other species is needed to establish this conclusion.
To study the functionality of the protein-binding element in the context of another RNA, a 63 bp fragment coding for the 26 nt of the PTH mRNA 3’- UTR and flanking nt, was fused to growth hormone (GH) reporter gene. RNAs were transcribed in vitro and transcripts subjected to IVDA with parathyroid proteins.
The chimeric GH-PTH 63 nt transcript, as the full-length PTH transcript, was stabilized by parathyroid proteins from rats fed a low calcium diet and destabilized by parathyroid proteins of a low P diet.
The native GH transcript was more stable than PTH and the chimeric RNAs and was not affected by parathyroid proteins from the different diet.
Therefore, the PTH RNA protein-binding region destabilized the GH transcript in the presence of parathyroid proteins.
Furthermore, this element conferred responsiveness of GH to changes in parathyroid proteins by calcium and phosphate.
The results demonstrate that the protein binding region of the PTH mRNA 3’-UTR is both necessary and sufficient for determining RNA stability and for the response to Ca2+ and Pi. We also studied the function of the PTH element in cells, using the heterologous cell line HEK293.
cDNAs coding for the protein binding region of the PTH mRNA 3’-UTR were inserted at the 3’ end of two reporter cDNAs.
63 bp of the PTH 3’UTR, coding for the cis element were inserted into a GH expression construct driven by a S16 ribosomal protein gene promoter and a larger fragment of 100 bp into GFP expression construct driven by a CMV promoter. The plasmids were transiently transfected into HEK293 cells.
At 24 h mRNA levels were measured by Northern blot, and protein levels of GFP by immunofluorescence, and secreted GH by radioimmunoassay.
There was a marked reduction in the expression of the chimeric genes containing the PTH elements compared to the wt genes.
A truncated PTH 40 nt element had no effect on GH reporter gene expression.
The 100 and 63 nt transcripts, like the full-length PTH RNA, bound parathyroid proteins by UV and REMSA, but the truncated element did not bind parathyroid proteins.
The different constructs for each reporter gene all used the same promoter; therefore these results suggest that insertion of the PTH 3’-UTR element decreased the stability of the reporter transcripts and not their transcription levels.
Measurement of mRNA decay after inhibition of transcription by the addition of DRB to the transfected cells, confirmed that the decrease in mRNA levels was post-transcriptional.
These results are in agreement with the decreased stability of the GH chimeric transcript in the IVDA with parathyroid proteins.
The Structure of the PTH cis Acting Element
The IVDA and the transfection experiments demonstrate the functional importance of the RNA protein binding region in the PTH 3’-UTR. RNA utilizes sequence and structure for its regulatory functions. To understand how the cis element functions as an instability element we have analyzed its structure by RNase H, primer extension, mutation analysis and computer modeling.
The results indicates that the PTH mRNA 3’-UTR and in particular the region of the cis element are dominated by significant open regions with little folded base pairing.
Mutation analysis of the 26 nt core binding element demonstrated the importance of defined nts for protein-RNA binding. The same mutations that prevented binding were also ineffective in destabilizing reporter GFP mRNA in HEK293 cells.
The PTH mRNA 3’-UTR cis acting element is therefore an open region that utilizes the distinct sequence pattern to determine mRNA stability by its interaction with trans acting RNA binding factors.
Conclusions
Dietary-induced hypocalcemia and hypophosphatemia regulate PTH gene expression post-transcriptionally and this is dependent upon the binding of parathyroid cytosolic proteins to instability regions in the PTH mRNA 3’-UTR.
The binding of these cytosolic proteins to the PTH mRNA is increased in hypocalcemia and decreased in hypophosphatemia correlating with PTH mRNA levels in vivo.
There is no parathyroid cell line and the stability of PTH transcripts was studied by an IVDA.
Parathyroid proteins from hypocalcemic rats lead to an increase in PTH RNA stability in the IVDA and hypophosphatemic proteins to a marked decrease in stability.
One of the PTH mRNA binding proteins is AUF1 that stabilizes the PTH mRNA. We have identified a conserved 26 nt element in the PTH mRNA 3’-UTR as the minimal protein binding sequence.
The functionality of a 63 nt element that included the 26 nt was studied in reporter RNAs.
The stability of the chimeric RNAs was studied in the IVDA with parathyroid proteins of low Ca and P rats and in transfection experiments in HEK 293 cells.
This element destabilized the reporter genes and was sufficient to confer responsiveness to calcium and phosphate in the IVDA with parathyroid proteins. The results demonstrate a functional cis element in the PTH mRNA 3’-UTR that upon binding to trans acting proteins determines PTH mRNA stability. Our model suggests that differences in binding of the trans acting factors to this element determine PTH mRNA stability and its regulation by Ca2+ and Pi.
The increased protein binding to the PTH mRNA 3' UTR cis element after a low calcium diet results in protection of the PTH mRNA and an increase in PTH mRNA levels.
After a low phosphate diet there is less binding, more degradation and a decrease in PTH mRNA levels.
