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Regulation of the parathyroid hormone gene by vitamin D, calcium and phosphate

1999; Elsevier BV; Volume: 56; Linguagem: Inglês

10.1046/j.1523-1755.1999.07310.x

1523-1755

Justin Silver, Cevdet Yalçındağ, Alin Sela-Brown, Rachel Kilav, Tally Naveh‐Many,

Alkaline Phosphatase Research Studies

Regulation of the parathyroid hormone gene by vitamin D, calcium, and phosphate. Secondary hyperparathyroidism is a frequent complication of chronic renal failure resulting in severe bone disease. Secondary hyperparathyroidism is composed of increased in parathyroid hormone (PTH) synthesis and secretion due to an increase in PTH gene expression and parathyroid cell proliferation. PTH gene expression is regulated by calcium, phosphate and 1,25-dihydroxy vitamin D (1,25(OH)2D). 1,25(OH)2D3 injected to rats leads to a dramatic decrease in PTH gene transcription without any increase in serum calcium. Hypocalcemia leads to a large increase in PTH mRNA levels which is post-transcriptional. Hypophosphatemia leads to a marked decrease in PTH gene expression that is also post-transcriptional. The mechanisms of the post-transcriptional effects of calcium and phosphate on the PTH gene have shown to be due to changes in protein–RNA interactions at the PTH mRNA 3′-UTR. Hypocalcemia leads to increased binding of parathyroid cytosolic proteins to the PTH mRNA 3′-UTR and hypophosphatemia to decreased binding of these proteins to the PTH mRNA 3′-UTR. The binding of the parathyroid proteins stabilizes the PTH RNA in an in vitro degradation assay. In rats with experimental uremia due to 5/6 nephrectomy, there is an increase in PTH mRNA levels due to a decrease in degradation of the PTH RNA as determined by this assay. The characterization of the parathyroid cytosolic proteins that interact with the PTH mRNA 3′-UTR may lead to a clearer understanding of how changes in serum calcium and phosphate result in secondary hyperparathyroidism. Regulation of the parathyroid hormone gene by vitamin D, calcium, and phosphate. Secondary hyperparathyroidism is a frequent complication of chronic renal failure resulting in severe bone disease. Secondary hyperparathyroidism is composed of increased in parathyroid hormone (PTH) synthesis and secretion due to an increase in PTH gene expression and parathyroid cell proliferation. PTH gene expression is regulated by calcium, phosphate and 1,25-dihydroxy vitamin D (1,25(OH)2D). 1,25(OH)2D3 injected to rats leads to a dramatic decrease in PTH gene transcription without any increase in serum calcium. Hypocalcemia leads to a large increase in PTH mRNA levels which is post-transcriptional. Hypophosphatemia leads to a marked decrease in PTH gene expression that is also post-transcriptional. The mechanisms of the post-transcriptional effects of calcium and phosphate on the PTH gene have shown to be due to changes in protein–RNA interactions at the PTH mRNA 3′-UTR. Hypocalcemia leads to increased binding of parathyroid cytosolic proteins to the PTH mRNA 3′-UTR and hypophosphatemia to decreased binding of these proteins to the PTH mRNA 3′-UTR. The binding of the parathyroid proteins stabilizes the PTH RNA in an in vitro degradation assay. In rats with experimental uremia due to 5/6 nephrectomy, there is an increase in PTH mRNA levels due to a decrease in degradation of the PTH RNA as determined by this assay. The characterization of the parathyroid cytosolic proteins that interact with the PTH mRNA 3′-UTR may lead to a clearer understanding of how changes in serum calcium and phosphate result in secondary hyperparathyroidism. The expression of the parathyroid hormone (PTH) gene is regulated by a number of factors, the most important in physiology being the serum calcium. However, the PTH gene is also powerfully regulated by other factors; those of most relevance to the nephrologist are vitamin D and serum phosphate. There has been recent progress in the understanding of how these factors regulate the PTH gene and these will be considered in the present review. The parathyroid cell is committed to the synthesis of PTH, implying that there are specific trans- and cis-factors acting on the PTH gene that are specific to the cell and gene. The gene itself is expressed once in the human genome and is a relatively simple gene. It has been cloned in a number of species and in all species it consists of three exons and two introns. These exons code for the 5′-untranslated region and the presequence of the gene, the pro-sequence and for the translated hormone and the 3′-untranslated region, respectively. The PTH gene is present in humans on the short arm of chromosome 11. The PTH promoter contains a canonical cAMP-responsive element 5′-TGACGTCA-3′ at position -81 with a single residue deviation1.Rupp E. Mayer H. Wingender E. The promoter of the human parathyroid hormone gene contains a functional cyclic AMP-response element.Nucleic Acid Res. 1990; 18: 5677-5683Crossref PubMed Scopus (21) Google Scholar. This element was fused to a reporter gene (CAT) and then transfected into different cell lines. Pharmacological agents that increase cyclic adenosine 3′,5′ monophosphate (cAMP) lead to an increased expression of the CAT gene, suggesting a functional role for the cAMP-responsive element (CRE). The role of this possible CRE in the context of the PTH gene in the parathyroid remains to be established. However, pharmacological agents that stimulate and inhibit PTH secretion and PTH mRNA levels in vitro in primary cultures of parathyroid cell certainly regulate these activities by their actions on the protein kinase A and C pathways. For example, dopamine, which stimulates cAMP synthesis, increases PTH secretion2.Brown E.M. Extracellular Ca2+ sensing, regulation of parathyroid cell function, and role of Ca2+ and other ions as extracellular (first) messengers.Physiol Rev. 1991; 71: 371-411Crossref PubMed Scopus (607) Google Scholar. Stimulation of the calcium-sensing receptor by hypercalcemia stimulates the protein kinase C pathway with an increase in inositol triphosphate and mobilization of intracellular calcium3.Brown E.M. Pollak M. Seidman C.E. Seidman J.G. Chou Y.W. Riccardi D. Hebert S.C. Calcium-ion-sensing cell-surface receptors.N Engl J Med. 1995; 333: 234-240Crossref PubMed Scopus (0) Google Scholar. There is a subsequent decrease in PTH secretion. Demay et al4.Demay M.B. Kiernan M.S. Deluca H.F. Kronenberg H.M. Sequences in the human parathyroid hormone gene that bind the 1,25-dihydroxyvitamin D3 receptor and mediate transcriptional repression in response to 1,25-dihydroxyvitamin D3.Proc Natl Acad Sci USA. 1992; 89: 8097-8101Crossref PubMed Scopus (347) Google Scholar identified DNA sequences in the human PTH gene that bind the 1,25(OH)2D3 receptor. Nuclear extracts containing the 1,25(OH)2D3 receptor were examined for binding to sequences in the 5′-flanking region of the human PTH gene. A 25-bp oligonucleotide containing the sequences from -125 to -101 from the start of exon 1 bound nuclear proteins, which were recognized by monoclonal antibodies against the 1,25(OH)2D3 receptor. The sequences in this region contained a single copy of a motif (AGGTTCA) that is homologous to the motifs repeated in the upregulatory 1,25(OH)2D3-response element of osteocalcin. When placed upstream to a heterologous viral promoter, the sequences contained in this 25-bp oligonucleotide mediated transcriptional repression in response to 1,25(OH)2-D3 in GH4C1 cells but not in ROS 17/2.8 cells. Therefore, this downregulatory element differs from upregulatory elements both in sequence composition and in the requirement for particular cellular factors other than the 1,25(OH)2D3 receptor (VDR) for repressing PTH transcription4.Demay M.B. Kiernan M.S. Deluca H.F. Kronenberg H.M. Sequences in the human parathyroid hormone gene that bind the 1,25-dihydroxyvitamin D3 receptor and mediate transcriptional repression in response to 1,25-dihydroxyvitamin D3.Proc Natl Acad Sci USA. 1992; 89: 8097-8101Crossref PubMed Scopus (347) Google Scholar. Farrow et al5.Farrow S.M. Hawa N.S. Karmali R. Hewison M. Walters J.C. O'riordan J.L. Binding of the receptor for 1,25-dihydroxyvitamin D3 to the 5′-flanking region of the bovine parathyroid hormone gene.J Endocrinol. 1990; 126: 355-359Crossref PubMed Scopus (15) Google Scholar,6.Hawa N.S. O'riordan J.L. Farrow S.M. Binding of 1,25-dihydroxyvitamin D3 receptors to the 5′-flanking region of the bovine parathyroid hormone gene.J Endocrinol. 1994; 142: 53-60Crossref PubMed Scopus (10) Google Scholar have identified DNA sequences upstream of the bovine PTH gene that bind the 1,25(OH)2D3 receptor. Liu et al7.Liu S.M. Koszewski N. Lupez M. Malluche H.H. Olivera A. Russell J. Characterization of a response element in the 5′-flanking region of the avian (chicken) parathyroid hormone gene that mediates negative regulation of gene transcription by 1,25-dihydroxyvitamin D3 and binds the vitamin D3 receptor.Mol Endocrinol. 1996; 10: 206-215Crossref PubMed Google Scholar have identified such sequences in the chicken PTH gene and demonstrated their functionality after transfection into the opossum kidney OK cell line. 1,25(OH)2D3 potently decreases the transcription of the PTH gene. This action was first demonstrated in vitro in bovine parathyroid cells in primary culture, where 1,25(OH)2D3 led to a marked decrease in PTH mRNA levels8.Silver J. Russell J. Sherwood L.M. Regulation by vitamin D metabolites of messenger ribonucleic acid for preproparathyroid hormone in isolated bovine parathyroid cells.Proc Natl Acad Sci USA. 1985; 82: 4270-4273Crossref PubMed Scopus (403) Google Scholar,9.Russell J. Silver J. Sherwood L.M. The effects of calcium and vitamin D metabolites on cytoplasmic mRNA coding for pre-proparathyroid hormone in isolated parathyroid cells.Trans Assoc Am Phys. 1984; 97: 296-303PubMed Google Scholar and a consequent decrease in PTH secretion10.Cantley L.K. Russell J. Lettieri D. Sherwood L.M. 1,25-Dihydroxyvitamin D3 suppresses parathyroid hormone secretion from bovine parathyroid cells in tissue culture.Endocrinology. 1985; 117: 2114-2119Crossref PubMed Scopus (180) Google Scholar. The physiological relevance of these findings was established by in vivo studies in rats11.Silver J. Naveh-Many T. Mayer H. Schmelzer H.J. Popovtzer M.M. Regulation by vitamin D metabolites of parathyroid hormone gene transcription in vivo in the rat.J Clin Invest. 1986; 78: 1296-1301Crossref PubMed Scopus (444) Google Scholar. Rats injected with amounts of 1,25(OH)2D3, which did not increase serum calcium had marked decreases in PTH mRNA levels, reaching 90% sequence identities with the type III Na+-Pi cotransporters, mouse and human PiT-1. Expression of rat PiT-1 in Xenopus oocytes revealed that it possessed Na+-dependent Pi cotransport activity. PiT-1 messenger RNA (mRNA) is widely distributed in rat tissues and was most abundant in brain, bone, and small intestine. The abundance of PiT-1 mRNA in the parathyroid was much greater in rats fed a low-Pi diet than in those fed a high-Pi diet. Therfore, the rat PiT-1 may contribute to the effects of Pi on parathyroid function. The contribution of hyperphosphatemia to the pathogenesis of the secondary hyperparathyroidism of chronic renal failure has been documented for many years27.Slatopolsky E. Bricker N.S. The role of phosphorus restriction in the prevention of secondary hyperparathyroidism in chronic renal disease.Kidney Int. 1973; 4: 141-145Abstract Full Text PDF PubMed Scopus (188) Google Scholar but it was never possible to separate the effect of hyperphosphatemia from the secondary decreases in serum calcium and 1,25(OH)2D328.Silver J. Kronenberg H.M. Parathyroid hormone – molecular biology and regulation.in: Bilezikian J.B. Raisz L.G. Rodan G.A. Principles of Bone Biology. Academic Press, San Diego1996Google Scholar. This was first established by the work of Kilav et al who succeeded in demonstrating that the effect of serum phosphate on PTH gene expression and serum PTH levels was independent of any changes in serum calcium or 1,25(OH)2D329.Kilav R. Silver J. Naveh-Many T. Parathyroid hormone gene expression in hypophosphatemic rats.J Clin Invest. 1995; 96: 327-333Crossref PubMed Google Scholar. Second generation vitamin D deficient weanling rats were fed a diet deficient in vitamin D and with a low calcium and a low phosphate content. After one night of this diet, the serum phosphate had markedly decreased with no changes in serum calcium or 1,25(OH)2D3. These rats with isolated hypophosphatemia had marked decreases in PTH mRNA levels and serum PTH. To demonstrate the effect of phosphate on the PT in vitro, it was imperative to maintain tissue architecture30.Almaden Y. Canalejo A. Hernandez A. Ballesteros E. Garcia-Navarro S. Torres A. Rodriguez M. Direct effect of phosphorus on parathyroid hormone secretion from whole rat parathyroid glands in vitro.J Bone Miner Res. 1996; 11: 970-976Crossref PubMed Scopus (282) Google Scholar, 31.Slatopolsky E. Finch J. Denda M. Ritter C. Zhong A. Dusso A. MacDonald P. Brown A.J. Phosphate restriction prevents parathyroid cell growth in uremic rats. High phosphate directly stimulates PTH secretion in vitro.J Clin Invest. 1996; 97: 2534-2540Crossref PubMed Scopus (439) Google Scholar, 32.Nielsen P.K. Feldt-Rasmusen U. Olgaard K. A direct effect of phosphate on PTH release from bovine parathyroid tissue slices but not from dispersed parathyroid cells.Nephrol Dial Transplant. 1996; 11: 1762-1768Crossref PubMed Scopus (134) Google Scholar. There was an effect in whole glands or tissue slices but not in isolated cells. We are studying the mechanisms whereby dietary phosphate and calcium regulate PTH gene expression. The clearest in vivo rat models for an effect of calcium and phosphate on PTH gene expression are hypocalcemia with a large increase in PTH mRNA levels and hypophosphatemia with a large decrease in PTH mRNA levels. In both instances the effect was post-transcriptional, as shown by nuclear transcript run on experiments. PT cytosolic proteins were found to bind in vitro transcribed PTH mRNA with three bands at about 50, 60 and 110 kDa24.Moallem E. Silver J. Kilav R. Naveh-Many T. RNA protein binding and post-transcriptional regulation of PTH gene expression by calcium and phosphate.J Biol Chem. 1998; 273: 5253-5259Crossref PubMed Scopus (249) Google Scholar. What was particularly interesting that this binding was increased with parathyroid proteins from hypocalcemic rats where PTH mRNA levels are increased, and decreased with PT proteins from hypophosphatemic rats, where PTH mRNA levels are decreased. There was protein binding to PTH mRNA with proteins from many tissues but only with PT proteins was this binding regulated by calcium and phosphate. Intriguingly, the binding was dependent upon the terminal 60 nt of the PTH transcript being present. Moallem and colleagues utilized an in vitro degradation assay to study the effects of hypocalcemic and hypophosphatemic parathyroid proteins of PTH mRNA stability24.Moallem E. Silver J. Kilav R. Naveh-Many T. RNA protein binding and post-transcriptional regulation of PTH gene expression by calcium and phosphate.J Biol Chem. 1998; 273: 5253-5259Crossref PubMed Scopus (249) Google Scholar. In this assay control rats' PT cytosolic proteins led to the degradation of a radiolabeled PTH transcript at about 40–60 min. Hypocalcemic PT proteins degraded the transcript only at 180 min, while hypophosphatemic PT proteins degraded the transcript already at 5 min Figure 1a. Moreover, the rapid degradation of PTH mRNA by hypophosphatemic proteins was totally dependent upon an intact 3′-untranslated region (UTR) and in particular the terminal 60 nt Figure 1b. Degradation with proteins from other tissues in these rats was not regulated by calcium or phosphate. Therefore, calcium and phosphate exert their effect on the parathyroid cell to change the properties of cytosolic proteins that bind specifically to the PTH mRNA 3′-UTR and determine its stability Figure 2.Figure 2Model of PTH mRNA including the 5′-UTR, the coding region and the 3′-UTR, and the parathyroid cytosolic proteins that interact with the 3′UTR. The parathyroid proteins contain both protective factors, measured by UV cross-linking, and degrading factors (endonucleases), measured by an in vitro degradation assay. In normal rats, the basal levels of PTH mRNA is determined by balance between the protective and degrading factors in the cytoplasm. In hypocalcemia, there is an increase in PTH mRNA associated with an increase in the binding of protective factors which leads to a more stable transcript. In hypophosphatemia, there is a decrease in protective factors, which leads to a less stable transcript and a decrease in PTH mRNA levels. In rats with chronic renal failure due to 5/6 nephrectomy, there is no change in the protective factors together with a decrease in endonuclease activity, resulting in increased PTH mRNA levels.(Reproduced with permission of the American Society of Nephrology [34]).View Large Image Figure ViewerDownload (PPT) Sela-Brown in our laboratory has now utilized affinity chromatography to isolate these RNA binding proteins. She recently presented preliminary information as to the identity of one of the PTH mRNA binding proteins (50 kDa protein on SDS Page gel) and demonstrated its functionality (abstract; Bone 23:S15519, 1998). The protein regulates PTH mRNA stability in degradation assays in vitro. This preliminary observation should now be supplemented by the identification of the other protein(s) (60 and 110 kDa) that form the protein complex on the PTH mRNA 3′-UTR33.Yalcindag C, Silver J, Naveh-Many T: Mechanism of increased PTH mRNA in experimental uremia: roles of protein RNA binding and RNA degradation. J Am Soc Nephrol (in press)Google Scholar. It might then be possible to understand how the different messages of changes in serum calcium and phosphate are transduced to the parathyroid cytosol. These proteins then bind to the PTH mRNA 3′-UTR and in particular the terminal 60 nt and determine its stability. A stable transcript, for instance after hypocalcemia, would then be translated into the hormone and available for rapid secretion. An unstable transcript, such as after hypophosphatemia, would be rapidly degraded and less PTH translated and secreted. The PTH gene is regulated by a number of factors. Calcitriol acts on the PTH gene to decrease its transcription; and this action is used in the management of patients with chronic renal failure. The main effect of calcium on PTH gene expression in vivo is for hypocalcemia to increase PTH mRNA levels and this is post-transcriptional. Phosphate also regulates PTH gene expression in vivo and this effect appears to be independent of phosphate's effects on serum calcium and 1,25(OH)2D3. The effect of phosphate is post-transcriptional. The effect of both calcium and phosphate on PTH gene expression is mediated by PT cytosolic proteins that bind to the PTH mRNA 3′-UTR and determine PTH mRNA stability.