Human Calcium-sensing Receptor Gene

The calcium-sensing receptor (CASR), expressed in parathyroid chief cells, thyroid C-cells, and cells of the kidney tubule, is essential for maintenance of calcium homeostasis. Here we show parathyroid, thyroid, and kidney CASR mRNA levels increased 2-fold at 15 h after intraperitoneal injection of 1,25-dihydroxyvitamin D3(1,25(OH)2D3) in rats. Human thyroid C-cell (TT) and kidney proximal tubule cell (HKC) CASR gene transcription increased ∼2-fold at 8 and 12 h after 1,25(OH)2D3 treatment. The human CASR gene has two promoters yielding alternative transcripts containing either exon 1A or exon 1B 5′-untranslated region sequences that splice to exon 2 some 242 bp before the ATG translation start site. Transcriptional start sites were identified in parathyroid gland and TT cells; that for promoter P1 lies 27 bp downstream of a TATA box, whereas that for promoter P2, which lacks a TATA box, lies in a GC-rich region. In HKC cells, transcriptional activity of a P1 reporter gene construct was 11-fold and of P2 was 33-fold above basal levels. 10−8m 1,25(OH)2D3 stimulated P1 activity 2-fold and P2 activity 2.5-fold. Vitamin D response elements (VDREs), in which half-sites (6 bp) are separated by three nucleotides, were identified in both promoters and shown to confer 1,25(OH)2D3 responsiveness to a heterologous promoter. This responsiveness was lost when the VDREs were mutated. In electrophoretic mobility shift assays with either in vitrotranscribed/translated vitamin D receptor and retinoid X receptor-α, or HKC nuclear extract, specific protein-DNA complexes were formed in the presence of 1,25(OH)2D3 on oligonucleotides representing the P1 and P2 VDREs. In summary, functional VDREs have been identified in the CASR gene and provide the mechanism whereby 1,25(OH)2D up-regulates parathyroid, thyroid C-cell, and kidney CASR expression. The calcium-sensing receptor (CASR), expressed in parathyroid chief cells, thyroid C-cells, and cells of the kidney tubule, is essential for maintenance of calcium homeostasis. Here we show parathyroid, thyroid, and kidney CASR mRNA levels increased 2-fold at 15 h after intraperitoneal injection of 1,25-dihydroxyvitamin D3(1,25(OH)2D3) in rats. Human thyroid C-cell (TT) and kidney proximal tubule cell (HKC) CASR gene transcription increased ∼2-fold at 8 and 12 h after 1,25(OH)2D3 treatment. The human CASR gene has two promoters yielding alternative transcripts containing either exon 1A or exon 1B 5′-untranslated region sequences that splice to exon 2 some 242 bp before the ATG translation start site. Transcriptional start sites were identified in parathyroid gland and TT cells; that for promoter P1 lies 27 bp downstream of a TATA box, whereas that for promoter P2, which lacks a TATA box, lies in a GC-rich region. In HKC cells, transcriptional activity of a P1 reporter gene construct was 11-fold and of P2 was 33-fold above basal levels. 10−8m 1,25(OH)2D3 stimulated P1 activity 2-fold and P2 activity 2.5-fold. Vitamin D response elements (VDREs), in which half-sites (6 bp) are separated by three nucleotides, were identified in both promoters and shown to confer 1,25(OH)2D3 responsiveness to a heterologous promoter. This responsiveness was lost when the VDREs were mutated. In electrophoretic mobility shift assays with either in vitrotranscribed/translated vitamin D receptor and retinoid X receptor-α, or HKC nuclear extract, specific protein-DNA complexes were formed in the presence of 1,25(OH)2D3 on oligonucleotides representing the P1 and P2 VDREs. In summary, functional VDREs have been identified in the CASR gene and provide the mechanism whereby 1,25(OH)2D up-regulates parathyroid, thyroid C-cell, and kidney CASR expression. parathyroid hormone calcium-sensing receptor 25(OH)2D, 1,25-dihydroxyvitamin D 25(OH)2D3, 1,25-dihydroxyvitamin D3 vitamin D response element vitamin D receptor retinoid X receptor 5′-rapid amplification of cDNA ends primer extension electrophoretic mobility shift assay mouse osteopontin cortical thick ascending limb Dulbecco's modified Eagle's medium 1,4-piperazinediethanesulfonic acid reverse transcription human thyroid C-cell human kidney proximal tubule cell dithiothreitol fetal bovine serum untranslated region phenylmethylsulfonyl fluoride Maintenance of calcium homeostasis depends on a complex interplay between parathyroid hormone (PTH),1 the hormonally active metabolite of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D) and the extracellular calcium concentration itself (1Brown E.M. Becker K.L. Principles and Practice of Endocrinology and Metabolism. 3rd Ed. J. B. Lippincott Co., Philadelphia2001: 478-489Google Scholar, 2Bringhurst F.R. Demay M.B. Kronenberg H.M. Wilson J.D. Foster D.W. Kronenberg H.M. Larsen P.R. Williams Textbook of Endocrinology. 9th Ed. W. B. Saunders Co., Philadelphia1998: 1155-1209Google Scholar). PTH synthesis and secretion are negatively regulated by serum calcium and 1,25(OH)2D levels. In the kidney proximal tubule, the mitochondrial 25-hydroxyvitamin D-1α-hydroxylase, the key enzyme responsible for production of 1,25(OH)2D, is regulated by serum PTH, calcium and 1,25(OH)2D levels. Classic feedback loops operate such that PTH synthesis and secretion, and 1,25(OH)2D production, initially stimulated by reductions in circulating calcium and 1,25(OH)2D levels, are then shut off as the mineral ion and vitamin D metabolite concentrations normalize.The calcium-sensing receptor (CASR) that plays a critical role in this process is a glycoprotein with a predicted topology of a large extracellular domain, a seven-transmembrane domain, and an intracellular tail (3Brown E.M. Gamba G. Riccardi D. Lombardi M. Butter R. Kifor O. Sun A. Hediger M.A. Lytton J. Hebert S.C. Nature. 1993; 366: 575-580Crossref PubMed Scopus (2344) Google Scholar). This G protein-coupled receptor is expressed most abundantly in the parathyroid chief cells, along the length of the kidney tubule, and in thyroid C-cells. The CASR is activated by elevations in extracellular calcium concentration, leading to inhibition of PTH secretion and renal calcium reabsorption (4Brown E.M. Macleod R.J. Physiol. Rev. 2001; 81: 239-297Crossref PubMed Scopus (1216) Google Scholar).Potentially, two important regulators of CASR gene expression are extracellular calcium and 1,25(OH)2D. Two previous studies were unable to demonstrate an effect of extracellular calcium on parathyroid gland or whole kidney CASR mRNA in the rat in vivo (5Brown A.J. Zhang M. Finch J. Ritter C. McCracken R. Morrissey J. Slatopolsky E. Am. J. Physiol. 1996; 270: F454-F460PubMed Google Scholar, 6Rogers K.V. Dunn C.V. Conklin R.L. Hadfield S. Petty B.A. Brown E.M. Hebert S.C. Fox J. Endocrinology. 1995; 136: 499-504Crossref PubMed Google Scholar). This lack of modulation of CASR expression might be expected, given the constraints placed upon the CASR in tissues such as parathyroid gland or kidney, where it plays an essential role as a calciostat to sense very small changes in extracellular calcium concentration. Even modest alterations in the extracellular calcium set-point (this being defined as the extracellular calcium concentration for half-maximal stimulation of PTH secretion from the parathyroid gland or calcium reabsorption across the kidney tubule) brought about by changes in CASR synthesis could have major unwanted effects on overall calcium homeostasis.Previously, the effect of vitamin D status (depleted versusreplete) and/or treatment with 1,25(OH)2D3 on parathyroid and kidney CASR mRNA levels has been examined in rats. One study found that vitamin D-depleted rats had a 40% reduction in parathyroid CASR mRNA relative to replete animals and administration of 1,25(OH)2D3 to vitamin D-replete rats further enhanced parathyroid and kidney CASR mRNA levels (5Brown A.J. Zhang M. Finch J. Ritter C. McCracken R. Morrissey J. Slatopolsky E. Am. J. Physiol. 1996; 270: F454-F460PubMed Google Scholar). A second study found that administration of 1,25(OH)2D3 to rats up-regulated renal CASR mRNA levels in a dose- and time-dependent manner (7Yao J. Karnauskas A.J. Bushinsky D.A. Favus M.J. Bone. 1998; (abstr.): T265Google Scholar). One study failed to find evidence for vitamin D modulation of CASR expression (6Rogers K.V. Dunn C.V. Conklin R.L. Hadfield S. Petty B.A. Brown E.M. Hebert S.C. Fox J. Endocrinology. 1995; 136: 499-504Crossref PubMed Google Scholar), although for methodological reasons small differences in CASR mRNA levels might have been missed.The human CASR is encoded by six exons (exons 2–7) of the gene (8Pollak M.R. Brown E.M. Chou Y.-H.W. Hebert S.C. Marx S.J. Steinman B. Levi T. Seidman C.E. Seidman J.G. Cell. 1993; 75: 1297-1303Abstract Full Text PDF PubMed Scopus (897) Google Scholar, 9Pearce S.H.S. Trump D. Wooding C. Besser G.M. Chew S. Heath D. Hughes I. Thakker R.V. J. Clin. Invest. 1995; 96: 2683-2692Crossref PubMed Scopus (326) Google Scholar, 10Heath III, H. Odelberg S. Jackson C.E. Teh B.T. Hayward N. Larsson C. Buist N.R.M. Krapcho K.J. Hung B.C. Capuano I.V. Garrett J.E. Leppert M.F. J. Clin. Endocrinol. Metab. 1996; 81: 1312-1317Crossref PubMed Scopus (159) Google Scholar) located on chromosome 3q13.3–21 (11Janicic N. Soliman E. Pausova Z. Seldin M.F. Riviere M. Szpirer J. Szpirer C. Hendy G.N. Mamm. Genome. 1995; 6: 798-801Crossref PubMed Scopus (104) Google Scholar) with exon 2 encoding 242 nucleotides of the 5′-untranslated region (UTR), followed by the translation start site. Exons 1A and 1B encode alternative 5′-UTRs that splice to the common portion encoded by exon 2 (12Garrett J.E. Capuano I.V. Hammerland L.G. Hung B.C.P. Brown E.M. Hebert S.C. Nemeth E.F. Fuller F. J. Biol. Chem. 1995; 270: 12919-12925Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar, 13Chikatsu N. Fukumoto S. Takeuchi Y. Suzawa M. Obara T. Matsumoto T. Fujita T. J. Biol. Chem. 2000; 275: 7553-7557Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The gene sequence upstream of exon 1A has a TATA box, whereas the sequence upstream of exon 1B lacks a TATA box and is GC-rich. The precise transcriptional start sites of exons 1A and 1B have not been mapped, and functional cis-acting elements in the gene promoters have yet to be identified.In the present study, we have shown that 1,25(OH)2D3 up-regulates parathyroid, thyroid, and kidney CASR mRNA levels in vivo in the rat, and that 1,25(OH)2D3 up-regulates the endogenous CASR gene transcription in human thyroid and kidney cell lines. In addition, we have mapped the transcriptional start sites and identified functional vitamin D response elements (VDREs) in both promoters of the human CASR gene.DISCUSSIONWe have mapped the transcriptional start sites of promoters P1 and P2 of the human CASR gene. For P1 a TATA box is at nucleotide −26 and a CCAAT box is at −110 relative to the start site. For P2, the transcriptional start site lies between two Sp1 sites, but the mechanisms that control initiation site selection of such GC-rich promoters lacking a TATA box are not known. When transfected into COS-7 cells that do not express the CASR, both P1 and P2 demonstrated base-line transcriptional activity severalfold above that of the promoterless control. Whereas the activity of P1 in human proximal tubule cells (HKC) that do express the CASR was similar to that in COS-7 cells that do not express the CASR, that of P2 was markedly increased in the HKC cells, indicating that elements important for tissue-specific expression of the CASR gene are present in this promoter.Now that the CASR promoters have been defined, it is possible to focus on the regulation of the CASR at the transcriptional level. In the present study we have focused on the mechanism underlying the vitamin D stimulation of CASR expression (5Brown A.J. Zhang M. Finch J. Ritter C. McCracken R. Morrissey J. Slatopolsky E. Am. J. Physiol. 1996; 270: F454-F460PubMed Google Scholar, 7Yao J. Karnauskas A.J. Bushinsky D.A. Favus M.J. Bone. 1998; (abstr.): T265Google Scholar) and show it to be a transcriptional one. First, we have demonstrated that 1,25(OH)2D3 up-regulates parathyroid, thyroid, and kidney CASR mRNA levels in vivo. These observations confirm and extend previous findings (5Brown A.J. Zhang M. Finch J. Ritter C. McCracken R. Morrissey J. Slatopolsky E. Am. J. Physiol. 1996; 270: F454-F460PubMed Google Scholar, 7Yao J. Karnauskas A.J. Bushinsky D.A. Favus M.J. Bone. 1998; (abstr.): T265Google Scholar). Second, we showed that human thyroid C-cell and kidney proximal tubule cell CASR gene transcription is increased by 1,25(OH)2D3. Third, VDREs were identified in both promoters of the CASR gene; one is 380 bp upstream of the P1 transcriptional start site, and the other is 166 bp upstream of the P2 transcriptional start site. VDREs have been identified in several vitamin D-responsive genes and typically consist of two 6-bp half-sites separated by 3 bp (25Haussler M.R. Whitfield G.R. Haussler C.A. Hsieh J.-C. Thompson P.D. Selznick S.H. Dominguez C.E. Jurutka P.U. J. Bone Miner. Res. 1998; 13: 325-349Crossref PubMed Scopus (1212) Google Scholar, 26Toell A. Polly P. Carlberg C. Biochem. J. 2000; 352: 301-309Crossref PubMed Scopus (78) Google Scholar). The VDREs of the CASR conform to this arrangement; however, they are atypical in that the orientation of half-sites is inverted to that which is normally found. VDREs of this type are found in the 24-hydroxylase gene (27Ohyama Y. Ozono K. Uchida M. Shinki T. Kato S. Suda T. Yamamoto O. Noshiro M. Kato Y. J. Biol. Chem. 1994; 269: 10545-10550Abstract Full Text PDF PubMed Google Scholar, 28Hahn C.N. Kerry D.M. Omdahl J.L. May B.K. Nucleic Acids Res. 1994; 22: 2410-2416Crossref PubMed Scopus (64) Google Scholar, 29Zierold C. Darwish H.M. DeLuca H.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 900-902Crossref PubMed Scopus (145) Google Scholar).Up-regulation of the parathyroid and kidney CASR by 1,25(OH)2D would be physiologically relevant. In the parathyroid, up-regulation of the CASR by 1,25(OH)2D would make the gland more responsive to extracellular calcium and for any given calcium concentration PTH secretion would be reduced. This would reinforce the direct negative effect of 1,25(OH)2D on PTH gene transcription (30Silver J. Naveh-Many T. Mayer H. Schmelzer H.J. Popovtzer M.M. J. Clin. Invest. 1986; 78: 1296-1301Crossref PubMed Scopus (469) Google Scholar, 31Demay M.B. Kiernan M.S. DeLuca H.F. Kronenberg H.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8097-8101Crossref PubMed Scopus (364) Google Scholar). Several studies in which renal failure patients or aged populations were treated with 1,25(OH)2D3 have shown a decrease in the calcium suppression curve and significant decrease in the calcium set-point in some cases (32Delmez J.A. Tindira C. Grooms P. Dusso A. Windus D.W. Slatopolsky E. J. Clin. Invest. 1989; 83: 1349-1355Crossref PubMed Scopus (270) Google Scholar, 33Dunlay R. Rodriguez M. Felsenfeld A.J. Llach F. Kidney Int. 1989; 36: 1093-1098Abstract Full Text PDF PubMed Scopus (156) Google Scholar, 34Kwan J.T.C. Almond M.K. Beer J.C. Noonan K. Evans S.J.W. Cunningham J. Nephrol. Dial. Transplant. 1992; 7: 829-834Crossref PubMed Scopus (3) Google Scholar, 35Malberti F. Surian M. Cosci P. Nephrol. Dial. Transplant. 1992; 7: 822-828PubMed Google Scholar, 36Ledger G.A. Burritt M.F. Kao P.C. Fallon W.M. Riggs B.L. Khosla S. J. Clin. Endocrinol. Metab. 1994; 79: 211-216Crossref PubMed Scopus (69) Google Scholar). Parathyroid glands surgically removed from a patient with secondary hyperparathyroidism who had been treated with a 1,25(OH)2D3 analogue intravenously showed up-regulation of CASR expression relative to parathyroid glands removed from similar patients not so treated (37Shiraishi K. Tsuchida M. Wada T. Yoshihiro S. Takai K. Suga A. Kaneda Y. Naito K. Am. J. Nephrol. 2001; 21: 507-511Crossref PubMed Scopus (9) Google Scholar). Although decreases in maximum PTH secretion are likely the result of the direct negative effect of 1,25(OH)2D on PTH gene transcription, the improvement in parathyroid gland responsiveness to calcium could be the result in part of increased expression of the CASR.In the kidney, changes in serum calcium regulate production of 1,25(OH)2D by affecting the activity of the proximal tubule mitochondrial cytochrome P450 25-hydroxyvitamin D-1α-hydroxylase. In thyroparathyroidectomized rats in which PTH and phosphate were maintained at constant levels, an inverse correlation was seen between serum calcium and 1,25(OH)2D levels, suggesting that calcium regulates 1,25(OH)2D independently of PTH (38Trechsel V. Eisman J.A. Fischer J.A. Bonjour J.-P. Fleisch H. Am. J. Physiol. 1980; 239: E119-E124PubMed Google Scholar, 39Matsumoto T. Ikeda K. Morita K. Fukomoto S. Takahashi H. Ogata E. Am. J. Physiol. 1987; 253: E503-E507PubMed Google Scholar, 40Weisinger J.R. Favus M.J. Langman C.B. Bushinsky D.A. J. Bone Miner. Res. 1989; 4: 929-935Crossref PubMed Scopus (71) Google Scholar). Calcium directly regulates 1,25(OH)2D3production in the human proximal tubular (HKC) cell line (23Bland R. Walker E.A. Hughes S.V. Stewart P.M. Hewison M. Endocrinology. 1999; 140: 2027-2034Crossref PubMed Scopus (124) Google Scholar).The 25-hydroxyvitamin D-1α-hydroxylase enzyme is product-inhibited. Therefore, after production of 1,25(OH)2D, the enzyme will be inhibited by several mechanisms including the direct action of 1,25(OH)2D, the decreased level of serum PTH brought about by the action of 1,25(OH)2D on the PTH gene, and by the increased sensitivity to serum calcium brought about by increased proximal tubule expression of the CASR implied by the present study. In vitamin D deficiency, the reduced CASR expression would help to ensure a maximum efficiency of production of 1,25(OH)2D.In the distal nephron, the cortical thick ascending limb (CTAL) and distal convoluted tubule, the CASR plays a key role in regulating Ca2+ and Mg2+ reabsorption. In the CTAL, the paracellular transport of cations is driven by a lumen-positive voltage gradient set up by the activity of the apical Na+-K+-2Cl− cotransporter and K+ channel (see Ref. 41Brown E.M. Pollak M. Hebert S.C. Annu. Rev. Med. 1998; 49: 15-29Crossref PubMed Scopus (190) Google Scholar). A hormone such as PTH activates its receptor on the basolateral surface increasing intracellular cyclic AMP, which stimulates the Na+-K+-2Cl− cotransporter and cation reabsorption. Activation of the CASR on the basolateral surface inhibits adenylate cyclase, thereby inhibiting hormone-stimulated cation transport leading to increased divalent cation excretion. The CASR also participates in transcellular cation reabsorption in the distal convoluted tubule and increasing extracellular calcium or magnesium stimulates intracellular Ca2+ transients and inhibits adenylate cyclase activity inhibiting hormone (e.g.PTH)-stimulated cation uptake (42Bapty B.W. Dai L-J. Ritchie G. Jirik F. Canaff L. Hendy G.N. Quamme G.A. Kidney Int. 1998; 53: 583-592Abstract Full Text PDF PubMed Scopus (61) Google Scholar, 43Bapty B.W. Ritchie G. Canaff L. Hendy G.N. Quamme G.A. Am. J. Physiol. 1998; 275: F353-F360PubMed Google Scholar). Increased CASR expression in the CTAL and distal convoluted tubule in response to 1,25(OH)2D would stimulate calcium excretion.Indeed, the findings of the present study offer some insight into the special management problems of patients with autosomal dominant hypocalcemia caused by activating mutations in the CASR relative to other forms of hypoparathyroidism. Treatment with vitamin D metabolites fails to bring the serum calcium up toward the lower limit of normal, whereas calcium excretion is excessively stimulated potentially leading to nephrocalcinosis, nephrolithiasis, and renal damage (44Pearce S.H.S. Williamson C. Kifor O. Bai M. Coulthard M.G. Davies M. Lewis-Barned N. McCredie D. Powell H. Kendall-Taylor P. Brown E.M. Thakker R.V. N. Engl. J. Med. 1996; 335: 1115-1122Crossref PubMed Scopus (495) Google Scholar, 45Lienhardt A. Bai M. Lagarde J.-P. Rigaud M. Zhang Z. Jiang Y. Kottler M.-L. Brown E.M. Garabedian M. J. Clin. Endocrinol. Metab. 2001; 86: 5313-5323Crossref PubMed Scopus (111) Google Scholar). With the demonstration of VDREs in the CASR gene, the mechanism underlying the exuberant hypercalciuric response to vitamin D metabolites in autosomal dominant hypocalcemia patients now becomes clearer. The renal CASR is already too sensitive to divalent cations in these patients, and the situation is exacerbated when CASR expression is stimulated by 1α-hydroxylated vitamin D metabolite administration.Hypercalcemia blunts renal concentrating ability, in part through CASR-activated signaling that antagonizes arginine vasopressin actions. Vitamin D up-regulation of the renal CASR is likely to underlie the increased basal and vasopressin-elicited water and urea permeabilities in the inner medullary cortical ducts of rats made hypercalcemic with dihydrotachysterol that mimics 1,25(OH)2D action (46Sands J.M. Flores F.X. Kato A. Baum M.A. Brown E.M. Ward Hebert S.C. Am. J. Physiol. 1998; Google Scholar). in autosomal dominant hypocalcemia patients with activating CASR gene the mechanisms are to the vitamin leading to (44Pearce S.H.S. Williamson C. Kifor O. Bai M. Coulthard M.G. Davies M. Lewis-Barned N. McCredie D. Powell H. Kendall-Taylor P. Brown E.M. Thakker R.V. N. Engl. J. 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A. 2001; PubMed Scopus Google and the CASR gene, of the gene not to parathyroid A. J. Bone Miner. Res. 1996; Scholar, N. W. E. P. A. J. Clin. Endocrinol. Metab. 2000; PubMed Scopus Google Scholar), but parathyroid expression is reduced in both and secondary patients T. J. E. G. G. J. Clin. Endocrinol. Metab. 2000; PubMed Scopus Google Scholar, S. Clin. Endocrinol. 2000; 53: PubMed Scopus Google Scholar). the reduced CASR expression in be secondary to decreased expression. evidence for the of both the and CASR in parathyroid and/or from studies of and CASR gene with and/or secondary hyperparathyroidism T. A. P. E. S. J. G. H. Med. 1995; PubMed Scopus Google Scholar, T. Endocrinol. Metab. 2001; Full Text Full Text PDF PubMed Scopus (73) Google Scholar, M. T. T. S. M. A. K. Clin. Endocrinol. 2001; PubMed Scopus Google Scholar) and the parathyroid responsiveness to extracellular calcium in renal