Parathyroid Hormone mRNA Stability Research Articles

Kidney Failure Alters Parathyroid Pin1 Phosphorylation and Parathyroid Hormone mRNA-Binding Proteins, Leading to Secondary Hyperparathyroidism.

Secondary hyperparathyroidism (SHP) is a common complication of CKD that increases morbidity and mortality. In experimental SHP, increased parathyroid hormone (PTH) expression is due to enhanced PTH mRNA stability, mediated by changes in its interaction with stabilizing AUF1 and destabilizing KSRP. The isomerase Pin1 leads to KSRP dephosphorylation, but in SHP parathyroid Pin1 activity is decreased and hence phosphorylated KSRP fails to bind PTH mRNA, resulting in high PTH mRNA stability and levels. The up- and downstream mechanisms by which CKD stimulates the parathyroid glands remain elusive. Adenine-rich high-phosphate diets induced CKD in rats and mice. Parathyroid organ cultures and transfected cells were incubated with Pin1 inhibitors for their effect on PTH expression. Mass spectrometry was performed on both parathyroid and PTH mRNA pulled-down proteins. CKD led to changes in rat parathyroid proteome and phosphoproteome profiles, including KSRP phosphorylation at Pin1 target sites. Furthermore, both acute and chronic kidney failure led to parathyroid-specific Pin1 Ser16 and Ser71 phosphorylation, which disrupts Pin1 activity. Pharmacologic Pin1 inhibition, which mimics the decreased Pin1 activity in SHP, increased PTH expression ex vivo in parathyroid glands in culture and in transfected cells through the PTH mRNA-protein interaction element and KSRP phosphorylation. Kidney failure leads to loss of parathyroid Pin1 activity by inducing Pin1 phosphorylation. This predisposes parathyroids to increase PTH production through impaired PTH mRNA decay that is dependent on KSRP phosphorylation at Pin1-target motifs. Pin1 and KSRP phosphorylation and the Pin1-KSRP-PTH mRNA axis thus drive SHP.

Molecular Mechanisms of Parathyroid Disorders in Chronic Kidney Disease.

Secondary hyperparathyroidism (SHP) is a common complication of chronic kidney disease (CKD) that induces morbidity and mortality in patients. How CKD stimulates the parathyroid to increase parathyroid hormone (PTH) secretion, gene expression and cell proliferation remains an open question. In experimental SHP, the increased PTH gene expression is post-transcriptional and mediated by PTH mRNA–protein interactions that promote PTH mRNA stability. These interactions are orchestrated by the isomerase Pin1. Pin1 participates in conformational change-based regulation of target proteins, including mRNA-binding proteins. In SHP, Pin1 isomerase activity is decreased, and thus, the Pin1 target and PTH mRNA destabilizing protein KSRP fails to bind PTH mRNA, increasing PTH mRNA stability and levels. An additional level of post-transcriptional regulation is mediated by microRNA (miRNA). Mice with parathyroid-specific knockout of Dicer, which facilitates the final step in miRNA maturation, lack parathyroid miRNAs but have normal PTH and calcium levels. Surprisingly, these mice fail to increase serum PTH in response to hypocalcemia or uremia, indicating a role for miRNAs in parathyroid stimulation. SHP often leads to parathyroid hyperplasia. Reduced expressions of parathyroid regulating receptors, activation of transforming growth factor α-epidermal growth factor receptor, cyclooxygenase 2-prostaglandin E2 and mTOR signaling all contribute to the enhanced parathyroid cell proliferation. Inhibition of mTOR by rapamycin prevents and corrects the increased parathyroid cell proliferation of SHP. This review summarizes the current knowledge on the mechanisms that stimulate the parathyroid cell at multiple levels in SHP.

Post-transcriptional mechanisms regulating parathyroid hormone gene expression in secondary hyperparathyroidism.

Parathyroid hormone (PTH) regulates serum calcium levels and bone strength. Secondary hyperparathyroidism (SHP) is a common complication of chronic kidney disease (CKD) that correlates with morbidity and mortality. In experimental SHP, the increased PTH gene expression is due to increased PTH mRNA stability and is mediated by protein-PTH mRNA interactions. Adenosine-uridine-rich binding factor 1 (AUF1) stabilizes and K-homology splicing regulatory protein (KSRP) destabilizes PTH mRNA. The peptidyl-prolyl cis/trans isomerase Pin1 acts on target proteins, including mRNA-binding proteins. Pin1 leads to KSRP dephosphorylation, but in SHP, parathyroid Pin1 activity is decreased and phosphorylated KSRP fails to bind PTH mRNA, leading to increased PTH mRNA stability and levels. A further level of post-transcriptional regulation occurs through microRNA (miRNA). Dicer mediates the final step of miRNA maturation. Parathyroid-specific Dicer knockout mice that lack miRNAs in the parathyroid develop normally. Surprisingly, these mice fail to increase serum PTH in response to both hypocalcemia and CKD, indicating that parathyroid Dicer and miRNAs are essential for stimulation of the parathyroid. Human and rodent parathyroids share similar miRNA profiles that are altered in hyperparathyroidism. The evolutionary conservation of abundant miRNAs and their regulation in hyperparathyroidism indicate their significance in parathyroid physiology and pathophysiology. let-7 and miR-148 antagonism modifies PTH secretion invivo and invitro, suggesting roles for specific miRNAs in parathyroid function.This review summarizes the current knowledge on the post-transcriptional mechanisms of PTH gene expression in SHP and the central contribution of miRNAs to the high serum PTH levels of both primary hyperparathyroidism and SHP.

The peptidyl-prolyl isomerase Pin1 determines parathyroid hormone mRNA levels and stability in rat models of secondary hyperparathyroidism

Secondary hyperparathyroidism is a major complication of chronic kidney disease (CKD). In experimental models of secondary hyperparathyroidism induced by hypocalcemia or CKD, parathyroid hormone (PTH) mRNA levels increase due to increased PTH mRNA stability. K-homology splicing regulator protein (KSRP) decreases the stability of PTH mRNA upon binding a cis-acting element in the PTH mRNA 3' UTR region. As the peptidyl-prolyl isomerase (PPIase) Pin1 has recently been shown to regulate the turnover of multiple cytokine mRNAs, we investigated the role of Pin1 in regulating PTH mRNA stability in rat parathyroids and transfected cells. The data generated were consistent with Pin1 being a PTH mRNA destabilizing protein. Initial analysis indicated that Pin1 activity was decreased in parathyroid protein extracts from both hypocalcemic and CKD rats and that pharmacologic inhibition of Pin1 increased PTH mRNA levels posttranscriptionally in rat parathyroid and in transfected cells. Pin1 mediated its effects via interaction with KSRP, which led to KSRP dephosphorylation and activation. In the rat parathyroid, Pin1 inhibition decreased KSRP-PTH mRNA interactions, increasing PTH mRNA levels. Furthermore, Pin1-/- mice displayed increased serum PTH and PTH mRNA levels, suggesting that Pin1 determines basal PTH expression in vivo. These results demonstrate that Pin1 is a key mediator of PTH mRNA stability and indicate a role for Pin1 in the pathogenesis of secondary hyperparathyroidism in individuals with CKD.

Pin1 regulates parathyroid hormone mRNA stability

Secondary hyperparathyroidism often occurs in chronic kidney disease (CKD) and vitamin D deficiency, resulting in increased fractures and mortality. Understanding factors that stimulate parathyroid hormone (PTH) synthesis is important for devising methods to treat this condition. Previous work has demonstrated that murine Pth mRNA levels are regulated by proteins that bind AU-rich elements (AREs) within the 3' UTR region of Pth mRNA and influence Pth mRNA stability. In this issue of the JCI, Nechama et al. demonstrate that in murine secondary hyperparathyroidism associated with CKD or Ca deficiency, the activity of Pin1, a peptidyl-prolyl isomerase, is reduced (see the related article beginning on page 3102). Reduced Pin1 activity resulted in the phosphorylation and degradation of an ARE-binding protein, K-homology splicing regulator protein (KSRP), which normally enhances the degradation of Pth mRNA. The activity of other ARE-binding proteins, such as AU-rich binding factor 1 (AUF1), that increase Pth mRNA stability, was increased, thereby increasing PTH synthesis. This work suggests new ways by which to regulate PTH synthesis in secondary hyperparathyroidism.

The calcium-sensing receptor regulates parathyroid hormone gene expression in transfected HEK293 cells

BackgroundThe parathyroid calcium receptor determines parathyroid hormone secretion and the response of parathyroid hormone gene expression to serum Ca2+ in the parathyroid gland. Serum Ca2+ regulates parathyroid hormone gene expression in vivo post-transcriptionally affecting parathyroid hormone mRNA stability through the interaction of trans-acting proteins to a defined cis element in the parathyroid hormone mRNA 3'-untranslated region. These parathyroid hormone mRNA binding proteins include AUF1 which stabilizes and KSRP which destabilizes the parathyroid hormone mRNA. There is no parathyroid cell line; therefore, we developed a parathyroid engineered cell using expression vectors for the full-length human parathyroid hormone gene and the human calcium receptor.ResultsCo-transfection of the human calcium receptor and the human parathyroid hormone plasmid into HEK293 cells decreased parathyroid hormone mRNA levels and secreted parathyroid hormone compared with cells that do not express the calcium receptor. The decreased parathyroid hormone mRNA correlated with decreased parathyroid hormone mRNA stability in vitro, which was dependent upon the 3'-UTR cis element. Moreover, parathyroid hormone gene expression was regulated by Ca2+ and the calcimimetic R568, in cells co-transfected with the calcium receptor but not in cells without the calcium receptor. RNA immunoprecipitation analysis in calcium receptor-transfected cells showed increased KSRP-parathyroid hormone mRNA binding and decreased binding to AUF1. The calcium receptor led to post-translational modifications in AUF1 as occurs in the parathyroid in vivo after activation of the calcium receptor.ConclusionThe expression of the calcium receptor is sufficient to confer the regulation of parathyroid hormone gene expression to these heterologous cells. The calcium receptor decreases parathyroid hormone gene expression in these engineered cells through the parathyroid hormone mRNA 3'-UTR cis element and the balanced interactions of the trans-acting factors KSRP and AUF1 with parathyroid hormone mRNA, as in vivo in the parathyroid. This is the first demonstration that the calcium receptor can regulate parathyroid hormone gene expression in heterologous cells.

Regulation of PTH mRNA stability by the calcimimetic R568 and the phosphorus binder lanthanum carbonate in CKD

Secondary hyperparathyroidism is characterized by increased parathyroid hormone (PTH) mRNA stability that leads to increased PTH mRNA and serum PTH levels. PTH gene expression is reduced by the calcimimetic R568 and the oral phosphorus binder lanthanum carbonate (La). Changes in PTH mRNA stability are regulated by the binding of trans-acting stabilizing and destabilizing factors to a defined cis element in the PTH mRNA 3'-untranslated region (UTR). Adenosine-uridine (AU)-binding factor 1 (AUF1) is a PTH mRNA-stabilizing protein, and K-homology splicing regulatory protein (KSRP) is a destabilizing protein that targets mRNAs, including PTH mRNA, to degradation by the ribonuclease complex exosome. We now show that KSRP-PTH mRNA binding is decreased in parathyroids from rats with adenine-induced chronic kidney disease (CKD) where PTH mRNA is more stable. KSRP-PTH mRNA binding is increased by treatment with both R568 and La, correlating with decreased PTH gene expression. In vitro degradation assays using transcripts for PTH mRNA and rat parathyroid extracts reproduce the differences in mRNA stability in vivo. Accordingly, PTH mRNA is destabilized in vitro by parathyroid extracts from CKD rats treated with R568 or La compared with parathyroid extracts from untreated CKD rats. This destabilizing effect of R568 and La is dependent on KSRP and the PTH mRNA 3'-UTR. Therefore, the calcimimetic R568 and correction of serum phosphorus by La determine PTH mRNA stability through KSRP-mediated recruitment of a degradation complex to the PTH mRNA, thereby decreasing PTH expression.

The mRNA decay promoting factor K‐homology splicing regulator protein post‐transcriptionally determines parathyroid hormone mRNA levels

Serum calcium and phosphate concentrations and experimental chronic kidney failure control parathyroid hormone (PTH) gene expression post-transcriptionally through regulated binding of the trans-acting proteins AUF1 and upstream of N-ras (Unr) to an AU-rich element (ARE) in PTH mRNA 3'-untranslated region (3'UTR). We show that the mRNA decay promoting K-homology splicing regulator protein (KSRP) binds to PTH mRNA in intact parathyroid glands and in transfected cells. This binding is decreased in glands from calcium-depleted or experimental chronic kidney failure rats in which PTH mRNA is more stable compared to parathyroid glands from control and phosphorus-depleted rats in which PTH mRNA is less stable. PTH mRNA decay depends on the KSRP-recruited exosome in parathyroid extracts. In transfected cells, KSRP overexpression and knockdown experiments show that KSRP decreases PTH mRNA stability and steady-state levels through the PTH mRNA ARE. Overexpression of isoform p45 of the PTH mRNA stabilizing protein AUF1 blocks KSRP-PTH mRNA binding and partially prevents the KSRP mediated decrease in PTH mRNA levels. Therefore, calcium or phosphorus depletion, as well as chronic kidney failure, regulate the interaction of KSRP and AUF1 with PTH mRNA and its half-life. Our data indicate a novel role for KSRP in PTH gene expression.

Regulation of parathyroid hormone mRNA stability by calcium, phosphate and uremia

This review focuses on the regulation of parathyroid hormone gene expression by dietary-induced hypocalcemia, hypophosphatemia and uremia. Understanding the mechanism by which calcium and phosphate regulate parathyroid hormone gene expression is important for both normal physiology and in pathological states, especially chronic kidney disease. Calcium and phosphate regulate parathyroid hormone secretion, gene expression and, if prolonged, parathyroid cell proliferation. Chronic kidney disease is characterized by a high serum phosphate level that often leads to secondary hyperparathyroidism. In the rat, regulation of parathyroid hormone gene expression by calcium, phosphate and uremia is posttranscriptional, affecting mRNA stability. Differences in binding of protective trans-acting proteins to a conserved protein-binding cis-acting instability element in the parathyroid hormone mRNA 3'-untranslated region alter parathyroid hormone mRNA stability. Two trans-acting proteins - adenosine-uridine rich binding factor 1 and Up-stream of N-ras- stabilize parathyroid hormone mRNA in vivo and in vitro. Parathyroid hormone mRNA also interacts with mRNA decay-promoting proteins and ribonucleases that lead to parathyroid hormone mRNA degradation. Calcium, phosphate and uremia determine parathyroid hormone mRNA stability through the binding of the protective factors adenosine-uridine rich binding factor 1 and Up-stream of N-ras and the recruitment of a degradation complex that cleaves parathyroid hormone mRNA.

Regulation of PTH synthesis and secretion relevant to the management of secondary hyperparathyroidism in chronic kidney disease

Regulation of PTH synthesis and secretion relevant to the management of secondary hyperparathyroidism in chronic kidney disease. Small decreases in serum Ca(++) and more prolonged increases in serum phosphate (P) stimulate the parathyroid (PT) to secrete parathyroid hormone (PTH), while 1,25(OH)(2)-vitamin D(3) decreases PTH synthesis and secretion. A prolonged decrease in serum Ca(++) and 1,25(OH)(2)D(3), or increase in serum P, such as in patients with chronic renal failure, leads to the appropriate secondary increase in serum PTH. This secondary hyperparathyroidism involves increases in PTH gene expression, synthesis, and secretion, and, if chronic, to proliferation of the parathyroid cells. A low serum Ca(++) leads to an increase in PTH secretion, PTH mRNA stability, and parathyroid cell proliferation. Pi also regulates the parathyroid in a similar manner. The effect of Ca(++) on the parathyroid is mediated by a membrane Ca(2+) receptor (CaR). 1,25(OH)(2)D(3) decreases PTH gene transcription. Ca(2+) and P regulate the PTH gene post-transcriptionally by regulating the binding of parathyroid cytosolic proteins, trans factors, to a defined cis sequence in the PTH mRNA 3'-untranslated region (UTR), thereby determining the stability of the transcript. The parathyroid trans factors and cis elements have been defined.

Pathogenesis of parathyroid dysfunction in end-stage kidney disease

Small decreases in serum calcium (Ca(2+)) and more-prolonged increases in serum phosphate (Pi) stimulate the parathyroid (PT) to secrete parathyroid hormone (PTH). 1,25-Dihydroxyvitamin D(3) [1,25(OH)(2) D(3)] decreases PTH synthesis and secretion. The prolonged decrease in serum Ca(2+) and 1,25(OH)(2) D(3), or increase in serum Pi, observed in patients with chronic renal failure leads to a secondary increase in serum PTH. This secondary hyperparathyroidism involves increases in PTH gene expression, synthesis, and secretion and, if chronic, to proliferation of the PT cells. A low serum Ca(2+) leads to an increase in PTH secretion, PTH mRNA stability, and PT cell proliferation. Pi also regulates the PT in a similar manner. The effect of Ca(2+) on the PT is mediated by a membrane Ca(2+) receptor. 1,25(OH)(2) D(3) decreases PTH gene transcription. Ca(2+) and Pi regulate the PTH gene post transcriptionally by regulating the binding of PT cytosolic proteins, trans factors, to a defined cis sequence in the PTH mRNA 3'-untranslated region, thereby determining the stability of the transcript. The PT trans factors and cis elements have been defined.

Mechanisms of secondary hyperparathyroidism.

Small decreases in serum Ca(2+) and more prolonged increases in serum phosphate (P(i)) stimulate the parathyroid (PT) to secrete parathyroid hormone (PTH), and 1,25(OH)(2)D(3) decreases PTH synthesis and secretion. A prolonged decrease in serum Ca(2+) and 1,25(OH)(2)D(3), or increase in serum P(i), such as in patients with chronic renal failure, leads to the appropriate secondary increase in serum PTH. This secondary hyperparathyroidism involves increases in PTH gene expression, synthesis, and secretion, and if chronic, to proliferation of the PT cells. Low serum Ca(2+) leads to an increase in PTH secretion, PTH mRNA stability, and PT cell proliferation. P(i) also regulates the PT in a similar manner. The effect of Ca(2+) on the PT is mediated by a membrane Ca(2+) receptor. 1,25(OH)(2)D(3) decreases PTH gene transcription. Ca(2+) and P(i) regulate the PTH gene posttranscriptionally by regulating the binding of PT cytosolic proteins, trans factors, to a defined cis sequence in the PTH mRNA 3'-untranslated region, thereby determining the stability of the transcript. PT trans factors and cis elements have been defined.

Cis and trans acting factors in the regulation of parathyroid hormone (PTH) mRNA stability by calcium and phosphate

Calcium and phosphate regulate parathyroid hormone (PTH) mRNA stability through differences in binding of parathyroid proteins to an element in its 3′-untranslated region. One of the proteins is AUF1 (A+U-rich element binding factor 1). An in vitro degradation assay showed that transcripts for PTH and chimeric growth hormone (GH)–PTH 63 nt, but not for native GH, were stabilized by PT proteins from rats on low calcium diets and destabilized by proteins from rats on low phosphate diets, correlating with PTH mRNA levels in vivo. In transfection experiments the 63 nt binding element destabilized mRNAs of reporter genes and this was prevented by over-expression of AUF1. Our results identified a functional cis element in PTH mRNA. Differences in protein binding to this element determine PTH mRNA stability and its regulation by calcium and phosphate.

Pathogenesis of parathyroid dysfunction in end-stage renal disease.

The parathyroid functions to maintain normal calcium and phosphate homeostasis and is central to normal bone physiology. In end-stage renal disease (ESRD), there is a failure of these normal homeostatic mechanisms with the frequent development of secondary hyperparathyroidism, which contributes to the pathogenesis of renal bone disease. The phosphate retention of ESRD, together with the reduced serum calcium and 1,25-dihydroxycholecalciferol vitamin D(3) (1,25[OH](2)D(3)) concentrations are the known factors that determine the progression to secondary hyperparathyroidism. 1,25(OH)(2)D(3) markedly decreases parathyroid hormone (PTH) gene transcription, whereas the effects of calcium and phosphate are on PTH mRNA stability, PTH secretion, and parathyroid cell proliferation. The mechanisms of these effects are discussed in this review.

A Conserved cis-Acting Element in the Parathyroid Hormone 3′-Untranslated Region Is Sufficient for Regulation of RNA Stability by Calcium and Phosphate

Calcium and phosphate regulate parathyroid hormone (PTH) gene expression post-transcriptionally by changes in protein-PTH mRNA 3'-untranslated region (UTR) interactions, which determine PTH mRNA stability. We have identified the protein binding sequence in the PTH mRNA 3'-UTR and determined its functionality. The protein-binding element was identified by binding, competition, and antisense oligonucleotide interference. The sequence was preserved among species suggesting its importance. To study its functionality in the context of another RNA, a 63-base pair cDNA PTH sequence was fused to the growth hormone (GH) gene. There is no parathyroid (PT) cell line and therefore an in vitro degradation assay was used to determine the stability of transcripts for PTH, GH, and a chimeric GH-PTH 63 nucleotides with PT cytosolic proteins. The full-length PTH transcript was stabilized by PT proteins from rats fed a low calcium diet and destabilized by proteins from rats fed a low phosphate diet, correlating with PTH mRNA levels in vivo. These PT proteins did not affect the native GH transcript. However, the chimeric GH transcript was stabilized by low calcium PT proteins and destabilized by low phosphate PT proteins, similar to the PTH full-length transcript. Therefore, we have identified a PTH RNA-protein binding region and shown that it is sufficient to confer responsiveness to calcium and phosphate in a reporter gene. This defined element in the PTH mRNA 3'-UTR is necessary and sufficient for the regulation of PTH mRNA stability by calcium and phosphate.

Molecular mechanisms of secondary hyperparathyroidism.

Secondary hyperparathyroidism is a frequent complication of chronic renal failure (CRF) and a major factor in the pathogenesis of renal osteodystrophy. A high serum phosphate, decreased levels of serum 1,25(OH) 2 D 3 and the subsequently low serum calcium are the major metabolic abnormalities in CRF, which lead to the secondary hyperparathyroidism. At the level of parathyroid hormone (PTH) secretion there is insensi- tivity to the ambient serum calcium. PTH mRNA levels are increased by a post-transcriptional mechanism that involves the binding of PT cytosolic proteins to the PTH mRNA 3'-untranslated region (UTR). In a dietary model of secondary hyperparathyroidism due to hypocalcemia there is increased binding of parathyroid proteins to the 3'-UTR and decreased degradation as determined by an in vitro degradation assay. Changes in serum phosphate also dramatically regulate PTH mRNA stability. There is also regulation at the level of PT cell proliferation. PT cell proliferation is increased by experimental hypocal- cemia or hyperphosphatemia and decreased by hypo- phosphatemia and administered 1,25(OH) 2 D 3 . The un- derstanding of the molecular mechanisms involved in the genesis of secondary hyperparathyroidism will allow the design of new effective strategies in the management of this troubling condition.

Molecular mechanisms of secondary hyperparathyroidism.

Secondary hyperparathyroidism is a frequent complication of chronic renal failure (CRF) and a major factor in the pathogenesis of renal osteodystrophy. A high serum phosphate, decreased levels of serum 1,25(OH)2D3 and the subsequently low serum calcium are the major metabolic abnormalities in CRF, which lead to the secondary hyperparathyroidism. At the level of parathyroid hormone (PTH) secretion there is insensitivity to the ambient serum calcium. PTH mRNA levels are increased by a post-transcriptional mechanism that involves the binding of PT cytosolic proteins to the PTH mRNA 3'-untranslated region (UTR). In a dietary model of secondary hyperparathyroidism due to hypocalcemia there is increased binding of parathyroid proteins to the 3'-UTR and decreased degradation as determined by an in vitro degradation assay. Changes in serum phosphate also dramatically regulate PTH mRNA stability. There is also regulation at the level of PT cell proliferation. PT cell proliferation is increased by experimental hypocalcemia or hyperphosphatemia and decreased by hypophosphatemia and administered 1,25(OH)2D3. The understanding of the molecular mechanisms involved in the genesis of secondary hyperparathyroidism will allow the design of new effective strategies in the management of this troubling condition.

Identification of AUF1 as a Parathyroid Hormone mRNA 3′-Untranslated Region-binding Protein That Determines Parathyroid Hormone mRNA Stability

Parathyroid hormone (PTH) mRNA levels are post-transcriptionally increased by hypocalcemia and decreased by hypophosphatemia, and this is mediated by cytosolic proteins binding to the PTH mRNA 3'-untranslated region (UTR). The same proteins are also present in other tissues, such as brain, but only in the parathyroid is their binding regulated by calcium and phosphate. The function of the PTH mRNA 3'-UTR-binding proteins was studied using an in vitro degradation assay. Competition for the parathyroid-binding proteins by excess unlabeled 3'-UTR destabilized the full-length PTH transcript in this assay, indicating that these proteins protect the RNA from RNase activity. The PTH RNA 3'-UTR-binding proteins were purified by RNA affinity chromatography of rat brain S-100 extracts. The eluate from the column was enriched in PTH RNA 3'-UTR binding activity. Addition of eluate to the in vitro degradation assay with parathyroid protein extracts stabilized the PTH transcript. A major band from the eluate at 50 kDa was sequenced and was identical to AU-rich binding protein (AUF1). Recombinant AUF1 bound the full-length PTH mRNA and the 3'-UTR. Added recombinant AUF1 also stabilized the PTH transcript in the in vitro degradation assay. Our results show that AUF1 is a protein that binds to the PTH mRNA 3'-UTR and stabilizes the PTH transcript.

Mechanism of increased parathyroid hormone mRNA in experimental uremia: roles of protein RNA binding and RNA degradation.

Patients with chronic renal failure develop secondary hyperparathyroidism with increased synthesis and secretion of parathyroid hormone (PTH) resulting in severe skeletal complications. In rats with secondary hyperparathyroidism due to 5/6 nephrectomy, there are increased PTH mRNA levels, and this mechanism was studied. Parathyroid glands were microdissected from control and 5/6 nephrectomy rats and analyzed for PTH mRNA and control genes, and the nuclei were used for nuclear run-on experiments. The cytosolic proteins of the parathyroids were used to study PTH mRNA protein binding by ultraviolet cross-linking and the degradation of the PTH transcript in vitro. Nuclear run-ons showed that the increase in PTH mRNA levels was posttranscriptional. Protein binding to the PTH mRNA 3'-UTR determines PTH mRNA stability and levels. Parathyroid proteins from uremic rats bound PTH mRNA similar to control rats by ultraviolet cross-linking. To determine the effect of uremia on PTH mRNA stability, an in vitro RNA degradation assay was performed with parathyroid proteins from uremic rats. When parathyroid proteins from control rats were incubated with PTH mRNA, there was transcript degradation already at 30 min, reaching 50% at 60 min and 90% at 180 min. With uremic parathyroid proteins, the PTH mRNA was not degraded at all at 120 min and was moderately decreased at 180 min. This decrease in degradation by uremic parathyroid proteins suggests a decrease in parathyroid cytosolic endonuclease activity in uremia resulting in a more stable PTH transcript. The increased PTH mRNA levels would translate into increased PTH synthesis and serum PTH levels, which would lead to metabolic bone disease in many patients with chronic renal failure.