Calcium and phosphate regulate PTH mRNA stability through differences in binding of parathyroid (PT) proteins to a minimal 63-nucleotide (nt) cis-acting instability element in its 3'-untranslated region. One of these proteins is adenosine-uridine-rich binding factor (AUF1), whose levels are not regulated in PT extracts from rats fed the different diets. However, two-dimensional gels showed posttranslational modification of AUF1 that included phosphorylation. There is no PT cell line, but in HEK 293 cells the 63-nt element is recognized as an instability element, and RNA interference for AUF1 decreased human PTH secretion in cotransfection experiments. Stably transfected cells with a chimeric GH gene containing the PTH 63-nt cis-acting element were used to study the signal transduction pathway that regulates AUF1 modification and chimeric gene mRNA stability. Cyclosporine A, the calcineurin inhibitor, regulated AUF1 posttranslationally, and this correlated with an increase in the stability of GH-PTH 63-nt mRNA but not of the control GH mRNA. Mice with genetic deletion of the calcineurin Abeta gene had markedly increased PTH mRNA levels that were still regulated by low calcium and phosphorus diets. Therefore, calcineurin regulates AUF1 posttranslationally in vitro and PTH gene expression in vivo but still allows its physiological regulation by calcium and phosphate.

Parathyroid hormone (PTH) gene expression is regulated post-transcriptionally by hypocalcemia and hypophosphatemia. This regulation is dependent upon binding of protective trans-acting factors to a specific element in the PTH mRNA 3-untranslated region (UTR). We have previously demonstrated that a 63-nucleotide (nt) AU-rich PTH mRNA element is sufficient to confer regulation of RNA stability by calcium and phosphate in an in vitro degradation assay (IVDA). The 63-nt element consists of a core 26-nt minimal binding sequence and flanking regions. We have now studied the functionality of this element in HEK293 cells using reporter genes and showed that it destabilizes mRNAs for green fluorescent protein (GFP) and growth hormone, similar to its effect in the IVDA. To understand how the cis-element functions as an instability element, we have analyzed its structure by RNase H, primer extension, and computer modeling. The results indicate 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 element demonstrated the importance of defined nucleotides for protein-RNA binding. In the GFP reporter system, the same mutations that prevented binding were also ineffective in destabilizing GFP mRNA in HEK293 cells. This is the first study of an AU-rich element that relates function to structure. The PTH mRNA 3-UTR cis-acting element is an open region that utilizes the distinct sequence pattern to determine mRNA stability by its interaction with trans-acting factors. PTH1 has a central role in maintaining normal calcium and phosphate homeostasis as well as bone strength. The parathyroids (PTs) are regulated by calcium, phosphate, and 1,25(OH) 2 vitamin D 3 (1). Hypocalcemia dramatically increases PTH mRNA levels, secretion, and, after prolonged stimulation, PT cell proliferation (2). The increase in PTH mRNA levels is post-transcriptional. Phosphate also regulates PTH secretion, gene expression, and PT cell proliferation (3, 4). Dietary induced phosphate depletion dramatically decreases PTH mRNA levels, and this is also post-transcriptional (3). We have shown that this post-transcriptional regulation is mediated by protein-RNA interactions involving protein binding to a specific element in the PTH mRNA 3Ј-UTR that determine PTH mRNA stability (2, 5). Protein binding to the PTH mRNA 3Ј-UTR was increased by hypocalcemia and decreased by hypophosphatemia. The regulation of binding by calcium and phosphate was observed only in the PT and not in other tissues of the same rats. We have identified AUF1 (heterogeneous nuclear ribonucleoprotein D) as a protein trans-acting factor that stabilizes the PTH mRNA (6). There is no PT 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 proces...

Calcium and phosphate regulate parathyroid hormone (PTH) mRNA stability through di¡erences in binding of parathyroid proteins to an element in its 3P P-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 overexpression of AUF1. Our results identi¢ed a functional cis element in PTH mRNA. Di¡erences in protein binding to this element determine PTH mRNA stability and its regulation by calcium and phosphate. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Background: Parathyroid hormone (PTH) gene expression is regulated post-transcriptionally through the binding of the trans-acting proteins AU rich binding factor 1 (AUF1), Upstream of Nras (Unr) and KH-type splicing regulatory protein (KSRP) to an AU rich element (ARE) in PTH mRNA 3'-UTR. AUF1 and Unr stabilize PTH mRNA while KSRP, recruiting the exoribonucleolytic complex exosome, promotes PTH mRNA decay.

No abstract