Lucie Canaff

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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Invest. 1998; PubMed Scopus Google Scholar). the findings of the present study, it would be predicted that levels of CASR secondary to enhanced vitamin D would be found in the hypercalciuric rat the increased calcium excretion. in renal CASR mRNA levels were found in the hypercalciuric rats relative to rats (7Yao J. Karnauskas A.J. Bushinsky D.A. Favus M.J. Bone. 1998; (abstr.): T265Google of CASR as in the hyperparathyroidism of of the CASR gene M.R. Chou W. Marx S.J. Steinman B. Hendy G.N. Brown E.M. Seidman C.E. Seidman J.G. J. Clin. Invest. 1994; PubMed Scopus Google Scholar) or in the cases in which of the CASR gene with atypical hyperparathyroidism M. B. Hendy G.N. J. Bone Miner. Res. 1999; Scholar, T. E. Bai M. P. G. P. S. P. Brown E.M. B. J. J. Clin. Endocrinol. Metab. 2000; PubMed Scopus Google Scholar) or hyperparathyroidism B. Hendy G.N. Marx S.J. 81: PubMed Scopus Google Scholar), have the between parathyroid calcium and Although of the CASR gene is not a significant in parathyroid Y. Pollak M.R. Brown E.M. A. J. Clin. Endocrinol. Metab. 1995; PubMed Google Scholar, Odelberg S. J. Heath III, H. J. Clin. Endocrinol. Metab. 1995; PubMed Google Scholar, S. A. W. C. Kidney Int. 1998; 53: Full Text PDF PubMed Scopus Google Scholar, F. A. E. L. E. E. P. P. C. J. Bone Miner. Res. 1999; PubMed Scopus Google Scholar), more of the parathyroid glands of patients with and secondary hyperparathyroidism show reduced CASR expression 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, O. P. M. P. Kifor I. Hebert S. Brown E.M. J. Clin. Endocrinol. Metab. 1996; 81: PubMed Google Scholar, F. U. L. M. M. Larsson C. J. Endocrinol. Metab. PubMed Scopus Google Scholar, F. A. K. Larsson C. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, J. P. B. M. Y. E. T. Kidney Int. Full Text PDF PubMed Scopus Google Scholar). mutations in genes the calcium set-point by expression of the CASR F. A. P. E. S. E. P. P. Kifor O. Brown E.M. A. C. J. Clin. Endocrinol. Metab. 2000; PubMed Scopus Google Scholar, G.N. A. L.G. G.A. Principles of Bone Ed. Google Scholar). for this from a mouse in which a is the control of the PTH gene region Y. Y. K. E. S. A. Kifor O. T. M. F. G. C. Brown E.M. R. A. J. Clin. Invest. 2001; PubMed Scopus Google Scholar). Parathyroid gland CASR expression is the calcium set-point is to the parathyroid and serum calcium and PTH levels are the specific genes and precise mechanisms in of parathyroid CASR expression are not effects of vitamin D metabolites on the parathyroid gland are with in which is of the or its the mouse of these parathyroid (see Ref. D. J. R. Hendy G.N. D. Proc. Natl. Acad. Sci. U. S. 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

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by endocrine tumors of parathyroids, pancreatic islets, and anterior pituitary. The MEN1 gene encodes a nuclear protein called menin. In MEN1 carriers inactivating mutations give rise to a truncated product consistent with menin acting as a tumor suppressor gene. However, the role of menin in tumorigenesis and its physiological functions are not known. Here, we show that menin inactivation by antisense RNA antagonizes transforming growth factor type beta-mediated cell growth inhibition. Menin interacts with Smad3, and antisense menin suppresses transforming growth factor type beta-induced and Smad3-induced transcriptional activity by inhibiting Smad3/4-DNA binding at specific transcriptional regulatory sites. These results implicate a mechanism of tumorigenesis by menin inactivation.

The calcium-sensing receptor (CASR) is a plasma membrane G protein coupled receptor that is expressed in the parathyroid hormone (PTH) producing chief cells of the parathyroid gland and the cells lining the kidney tubule. By virtue of its ability to sense small changes in circulating calcium concentration ([Ca(2+)](o)) and to couple this information to intracellular signaling pathways that modify PTH secretion or renal cation handling, the CASR plays an essential role in maintaining mineral ion homeostasis. Inherited abnormalities of the CASR gene located on chromosome 3p13.3-21 can cause either hypercalcemia or hypocalcemia depending upon whether they are inactivating or activating, respectively. Heterozygous loss-of-function mutations give rise to familial (benign) hypocalciuric hypercalcemia (FHH) in which the lifelong hypercalcemia is asymptomatic. The homozygous condition manifests itself as neonatal severe hyperparathyroidism (NSHPT), a rare disorder characterized by extreme hypercalcemia and the bony changes of hyperparathyroidism which occur in infancy. The disorder autosomal dominant hypocalcemia (ADH) is due to gain-of-function mutations in the CASR gene. ADH may be asymptomatic or present with neonatal or childhood seizures. A common polymorphism in the intracellular tail of the CASR, Ala to Ser at position 986, has a modest effect on the serum calcium concentration in healthy individuals.

Increased expression of the calcium-sensing receptor (CASR), which controls blood calcium homeostasis, leads to a decrease in the extracellular calcium set-point, thereby reducing parathyroid hormone secretion and renal calcium reabsorption and increasing calcitonin secretion resulting in reduced circulating calcium levels. Critically ill patients with elevated proinflammatory cytokine levels commonly have hypocalcemia, although the mechanism is not known. After intraperitoneal injection of interleukin (IL)-6 in the rat, circulating levels of parathyroid hormone, 1,25-dihydroxyvitamin D, and calcium fell within hours and remained low at 24 h. Expression of CASR (mRNA and protein) increased within hours in parathyroid, thyroid, and kidney and remained elevated at 24 h. The CASR gene has two promoters (P1 and P2) yielding transcripts having alternative 5′-untranslated regions but encoding the same receptor protein. Activities of P1 and P2 promoter/luciferase reporter constructs were stimulated ∼2–3-fold by IL-6 in proximal tubule HKC cells and TT thyroid C-cells. Studies with P1 deleted and mutated promoter-reporter and Stat1 and/or Stat3 dominant-negative constructs showed that a Stat1/3 element downstream of the P1 start site accounted for the IL-6 induction. There are no Stat elements in the P2 promoter, but Sp1/3 elements are clustered at the transcription start site. A series of transfection P2 promoter-reporter analyses showed that Sp1 together with Stat1/3 was critical for IL-6 responsiveness of P2. By oligonucleotide precipitation assay, IL-6 rapidly promoted a complex containing both Sp1/3 and Stat1/3 on the Sp1/3 elements. In conclusion, Stat1/3 directly controls promoter P1, and the Stats indirectly regulate promoter P2 via Sp1/3 in response to IL-6. By this mechanism, the cytokine likely contributes to altered extracellular calcium homeostasis. Increased expression of the calcium-sensing receptor (CASR), which controls blood calcium homeostasis, leads to a decrease in the extracellular calcium set-point, thereby reducing parathyroid hormone secretion and renal calcium reabsorption and increasing calcitonin secretion resulting in reduced circulating calcium levels. Critically ill patients with elevated proinflammatory cytokine levels commonly have hypocalcemia, although the mechanism is not known. After intraperitoneal injection of interleukin (IL)-6 in the rat, circulating levels of parathyroid hormone, 1,25-dihydroxyvitamin D, and calcium fell within hours and remained low at 24 h. Expression of CASR (mRNA and protein) increased within hours in parathyroid, thyroid, and kidney and remained elevated at 24 h. The CASR gene has two promoters (P1 and P2) yielding transcripts having alternative 5′-untranslated regions but encoding the same receptor protein. Activities of P1 and P2 promoter/luciferase reporter constructs were stimulated ∼2–3-fold by IL-6 in proximal tubule HKC cells and TT thyroid C-cells. Studies with P1 deleted and mutated promoter-reporter and Stat1 and/or Stat3 dominant-negative constructs showed that a Stat1/3 element downstream of the P1 start site accounted for the IL-6 induction. There are no Stat elements in the P2 promoter, but Sp1/3 elements are clustered at the transcription start site. A series of transfection P2 promoter-reporter analyses showed that Sp1 together with Stat1/3 was critical for IL-6 responsiveness of P2. By oligonucleotide precipitation assay, IL-6 rapidly promoted a complex containing both Sp1/3 and Stat1/3 on the Sp1/3 elements. In conclusion, Stat1/3 directly controls promoter P1, and the Stats indirectly regulate promoter P2 via Sp1/3 in response to IL-6. By this mechanism, the cytokine likely contributes to altered extracellular calcium homeostasis. The calcium-sensing receptor (CASR) 3The abbreviations used are: CASR, calcium-sensing receptor; PTH, parathyroid hormone; 1,25(OH)2D, 1,25-dihydroxyvitamin D; IL, interleukin; TT cells, human thyroid C-cells; HKC, human proximal tubule cells; IL-6R, interleukin-6 receptor; JAK, Janus kinase; STAT, signal transducers and activators of transcription; Sp1/3, specificity protein 1/3; MAPK, mitogen-activated protein kinase; SRF, serum-response factor; EMSA, electrophoretic mobility shift assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DMEM, Dulbecco's modified Earle's medium; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; UTR, untranslated region; oligo, oligonucleotide. is expressed in the parathyroid hormone (PTH) producing chief cells of the parathyroid gland, the calcitonin-producing C-cells of the thyroid, and the cells lining the kidney tubule. The CASR, a plasma membrane G protein-coupled receptor, senses small changes in circulating calcium concentration and modulates intracellular pathways that alter PTH and calcitonin secretion or renal cation handling, thereby playing an essential role in blood mineral ion homeostasis. The relationship between extracellular ionized calcium and PTH concentrations is represented by an inverse sigmoidal curve. The activity and/or expression level of the CASR dictates the extracellular calcium set point (defined as the extracellular calcium concentration at which PTH secretion from the parathyroid gland or calcium reabsorption across the kidney tubule is half-maximal). Increases in extracellular calcium directly stimulate calcitonin secretion. The importance of the CASR in orchestrating the endocrine control of blood calcium concentrations has been underscored by the identification of naturally occurring mutations in the CASR gene that cause human disease. Inactivating mutations result in hypercalcemia, and activating mutations result in hypocalcemia (1Pollak M.R. Brown E.M. Chou Y.-W.H. Hebert S.C. Marx S.J. Steinmann B. Levi T. Seidman C.E. Seidman J.G. Cell. 1993; 75: 1297-1303Abstract Full Text PDF PubMed Scopus (916) Google Scholar, 2Pollak M.R. Brown E.M. Estep H.L. McLaine P.N. Kifor O. Park J. Hebert S.C. Seidman C.E. Seidman J.G. Nat. Genet. 1994; 8: 303-307Crossref PubMed Scopus (539) Google Scholar, 3Hendy G.N. D'Souza-Li L. Yang B. Canaff L. Cole D.E.C. Hum. Mutat. 2000; 16: 281-296Crossref PubMed Scopus (238) Google Scholar). Hypocalcemia is common in critically ill patients, especially those with sepsis and major burn injury (4Zivin J.R. Gooley T. Zager R.A. Ryan M.J. Am. J. Kidney Dis. 2001; 37: 689-698Abstract Full Text PDF PubMed Scopus (192) Google Scholar), and in nonacutely ill patients undergoing surgery (5Lepage R. Legare G. Racicot C. Brossard J.H. Lapointe R. Dagenais M. D'Amour P. J. Clin. Endocrinol. Metab. 1999; 84: 2654-2658Crossref PubMed Google Scholar). The mechanisms underlying the hypocalcemia are not known. Several factors may be involved, including decreased secretion of PTH and resistance to the action of PTH in kidney and bone. The metabolism and function of vitamin D are impaired. Calcitonin precursors are increased in the circulation of critically ill patients with sepsis and could contribute to the hypocalcemia (6Lind L. Carlstedt F. Rastad J. Stiernstrom H. Stridsberg M. Ljungren O. Wide L. Larsson A. Hellman P. Ljunghall S. Crit. Care Med. 2000; 28: 93-99Crossref PubMed Scopus (138) Google Scholar, 7Muller B. Becker K.L. Kranzlin M. Schachinger H. Huber P.R. Nylen E.S. Snider R.H. White J.C. Schmidt-Gayk H. Zimmerli W. Ritz R. Eur. J. Clin. Investig. 2000; 30: 823-831Crossref PubMed Scopus (93) Google Scholar). 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Care Med. 2000; 28: 93-99Crossref PubMed Scopus (138) Google Scholar) and correlate with a poor prognosis (11Remick D.G. Bolgos G.R. Siddiqui J. Nemzek J.A. Shock. 2002; 17: 463-467Crossref PubMed Scopus (373) Google Scholar, 12Ohzato H. Monden M. Yoshizaki K. Ogata A. Nishimoto N. Gotoh M. T. T. 1993; PubMed Scopus Google Scholar, C.E. M. Google Scholar, S. K. PubMed Scopus Google Scholar, B. B. W. J. PubMed Scopus Google Scholar, C. R.A. Clin. PubMed Scopus Google Scholar). Carlstedt P. L. Rastad J. 1999; PubMed Scopus Google Scholar) have shown that in parathyroid cells with IL-6 PTH secretion at N. M. Kifor O. D. Brown E.M. G.L. Crit. Care Med. 2000; 28: PubMed Scopus Google Scholar) that in parathyroid CASR levels were in a of burn injury in which circulating cytokine levels be to be analyses are to more the mechanisms the IL-6 levels to altered calcium the that as increase CASR expression in those for the control of calcium thereby to hypocalcemia and showed in in the that parathyroid, thyroid, and kidney CASR and protein increased intraperitoneal injection of L. G.N. J. Full Text Full Text PDF PubMed Scopus Google Scholar). was with decreased circulating PTH, 1,25-dihydroxyvitamin D and The CASR gene has two promoters (P1 and P2) yielding alternative transcripts containing or 5′-untranslated that to containing the start Brown E.M. Hebert S.C. F. J. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, N. S. M. T. T. T. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, L. G.N. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). that both the CASR gene promoters have elements that by L. G.N. J. Full Text Full Text PDF PubMed Scopus Google Scholar). In this that by CASR expression in parathyroid, thyroid and kidney reducing PTH secretion and renal calcium and increasing calcitonin contributes to altered calcium homeostasis. have that IL-6 CASR expression in and for the the mechanisms the cytokine CASR gene promoter The resulting in CASR expression could be to the hypocalcemia of critically ill IL-6 was from The human thyroid TT was from the and the human proximal kidney tubule cells were a of Dulbecco's modified Earle's serum and were from and were from and were from and were from and and Sp1 response elements were from constructs were as human Sp1 in human H. S. J. 1999; PubMed Scopus Google Scholar), human in and human Stat3 in Stats and were the constructs as with the The were and and are in and were a containing and vitamin were in and were the Care and were at or with or was by and the serum was and at The were with the were and the parathyroid and thyroid were and were for calcium and PTH PTH and and was from cells or to the of CASR the CASR a of a CASR L. M. M. G.N. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) was and and the glyceraldehyde-3-phosphate that a the was After of the the were in with a and the were used with a was with of by with a L. Canaff L. N. Cole D.E.C. G.N. Hum. Mutat. 2001; 18: PubMed Scopus Google Scholar). were on and to transcription were a G.N. PubMed Scopus (25) Google Scholar). were from to HKC or TT cells with IL-6 or serum in were PBS, at and with After on were at were with of DTT, in and at were at in DTT, PMSF, of or of and for was with to the of containing gene or no were and The were as human CASR a in human CASR in human CASR a in human a from HKC and human GAPDH, a from HKC and in The were with of in and for a of h. In of were used for were to or the signal was with the and of the of transcription was by of the or cells were in PMSF, for at The were at for at and the were at were and were in with in for and with CASR an L. Yang B. Canaff L. M. M. Brown E.M. Cole D.E.C. G.N. J. Clin. Endocrinol. Metab. 2002; PubMed Scopus Google Scholar), a CASR to or the the same a was as with the for with the which was were by the were with a as CASR of the reporter as containing the P1 promoter, and the of to is the A of the of the reporter gene in has been The and were for A in which a Stat element in is mutated was the as with the as S. M. Canaff L. O. G.N. Hum. Genet. PubMed Scopus Google Scholar). The used were as and are in used for CASR gene promoter P1 P2 P1 and P2 in a The of the reporter as containing the P2 promoter, and the of to of the gene in has been The was by for in which Sp1 and two the transcription are mutated was by the 1999; Scopus Google Scholar) as The used were as with a site in with mutated in to and with mutated in and with site in to The were and with and to that was with and the to cells were in with TT cells were in with and and B. mitogen-activated protein cells were in to and with or P1 or P2 promoter constructs and the to cells were for stimulated or not with and of the or were to the to After were for and cells were in in DMEM, cells and The cells were with of with of CASR promoter and of The cells were in and with or cytokine for h. The cells were in and in of on The were for and by activity was in a of and activity was to of HKC were stimulated with IL-6 for of PBS, and at for at were in a in of DTT, PMSF, and were by for at in PMSF, DTT, and After a at were for PMSF, was and were at of were for on with of in were or and were for at of were and for a used for are in were at in which were and used for and/or promoters P1 a was at the a was at the P2 a was at the P2 a was at the element of the human A gene is a was at the element of the human A gene is in a cells were with IL-6 for to and by in DTT, with and was by were with of to the or mutated CASR P1 Stat1/3 or CASR P2 Sp1 elements for for h. of and of protein) were to and with and of with in a The were at for and were and with and were to with Stat1 or Stat3 or Sp1 or are expressed as The from the in IL-6 response studies were to of The of from was the and transfection were by A of was to a PTH, 1,25(OH)2D, and in the of IL-6 on extracellular calcium homeostasis, the cytokine was to and circulating PTH, 1,25(OH)2D, and calcium levels were a After a intraperitoneal injection of IL-6 in serum PTH, 1,25(OH)2D, and calcium levels were decreased at and 24 and Kidney CASR in in circulating PTH, and calcium levels by IL-6 could be to altered CASR expression in for of extracellular calcium homeostasis, CASR levels by the After the injection of IL-6 in parathyroid, thyroid, and kidney CASR levels level to at and the levels were elevated at 24 The to levels were and and of no on CASR levels not and Kidney CASR in the changes in CASR levels were in changes in CASR protein was on of thyroid and the the CASR in both and The is and is D and The mobility are likely to be S. M. Canaff L. O. G.N. Hum. Genet. PubMed Scopus Google Scholar). After injection of IL-6 in thyroid and kidney CASR protein levels and related to levels by 24 and of no on CASR protein levels not CASR protein levels were not of the of Increases CASR the changes in CASR levels changes at the transcription were on of human TT cells and HKC cells with and IL-6 for and h. CASR gene and transcripts were stimulated and gene transcription in both not gene transcription was by IL-6. IL-6 CASR gene Activities of CASR P1 and P2 by the of IL-6 on the activity of CASR gene constructs were used in which human CASR P1 or P2 promoters transcription of a reporter gene HKC cells, the of P1 and P2 were and that of the and In the TT thyroid the of P1 and P2 were and that of the The of IL-6 stimulated reporter activity of P1 and P2 constructs in a in HKC and and TT cells not with an increase at the proinflammatory cytokine the activity of both CASR gene IL-6 the Activities of and CASR P1 and P2 regions of the CASR promoters that responsiveness to a series of constructs of the P1 promoter with and or the P2 promoter with and a reporter gene were The or deleted constructs were HKC TT that were stimulated or not with IL-6. the P1 activity increased with constructs to to the the of a between and IL-6 stimulated the activity of constructs the P2 constructs containing of the P2 transcription start IL-6 promoter activity and IL-6 receptor of an IL-6 and a signal which is the for the cytokine of IL-6 to receptor and that and to the and to elements thereby activating gene transcription 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, Jr., Nat. 2002; PubMed Scopus Google Scholar). Stat1 and Stat3 IL-6 The can to the the activity of a of transcription factors as the but other that response elements. transcription factors like serum-response and Sp1/3 can to factors by IL-6 and regulate gene expression S. G. F. J. PubMed Scopus Google Scholar). Several to IL-6 in the CASR of the CASR gene with K. K. K. B. M. A. M. M. T. PubMed Scopus Google Scholar) STAT, SRF, and Sp1 elements in P1 and/or P2 promoters and/or the 5′-untranslated regions in CASR promoter P1 is a Stat1 element in and in CASR promoter P2 are no Stat elements of are Sp1 elements that at the transcription start site. The the IL-6 of the CASR P1 and P2 activity was for activity and/or activity of the CASR the P1 and P2 and deleted constructs were HKC cells TT that were stimulated or not with IL-6 in the or of the the CASR P1 constructs in the of the promoter activity was that in the of the for the CASR P2 constructs in the of the promoter activity was that in the of the to IL-6 although the activity level for P1 and was in the than in the of the the was no A and the to activity of the CASR not by IL-6. Stat1 and Stat3 CASR P1 via the Stat in the role of in the IL-6 of the CASR P1 promoter, or mutated at the Stat1 element in were or with a or a HKC cells and the cells were stimulated or not with IL-6. the a increase in activity was stimulated by and this increase was by of or The of the by IL-6 was reduced to that of the and with or the the Stat1 element in is very for the IL-6 of promoter P1, and Stat1 or Stat3 can to the IL-6 Sp1 for and CASR P2 the role of Sp1 in the activity and IL-6 of the CASR P2 promoter, the or constructs the in which the Sp1 clustered at the transcription site were were or with an Sp1 expression or with an expression HKC cells that were stimulated or not with IL-6. of the Sp1 with or to in activity of of with or to in activity of The of and were stimulated by IL-6 or with of The of these promoter constructs by IL-6 was by with The was and of Sp1 or no and the was to IL-6 not Sp1 is essential for activity and is critical for the IL-6 of the CASR P2 Stats for the IL-6 of the CASR P2 P2 promoter has no Stats could be IL-6 by with other transcription factors that to response elements in the the role of Stats in the IL-6 of the CASR P2 promoter, the or constructs were or with or expression HKC cells that were stimulated or not with IL-6. of of the dominant-negative constructs with to a in the with IL-6 The was activity Stat1 and Stat3 are critical for the IL-6 of the CASR P2 on a Stat in of the CASR were with Stat1 elements in CASR P1 and HKC with the CASR gene element and the in which were by Stat1 and/or Stat3 were with and Stat1/3 with The of the reduced the of the of an oligonucleotide was not on the Stat1 element in CASR gene P1 not on Sp1 at the of CASR P2 were with a Sp1 element or a of Sp1 elements a of and downstream of the transcription site of the CASR gene P2 promoter and HKC The that with CASR gene Sp1 elements and Sp1 element electrophoretic and were in a by the of Sp1 or The of the CASR P2 reduced the of the and CASR gene Sp1 in a of an oligonucleotide not or an oligonucleotide a protein no not on a Sp1 element of the transcription site of the CASR gene P2 promoter not of a Stat on the CASR P1 Stat1/3 Stat1 or Stat3 to the P1 Stat1/3 element oligonucleotide precipitation were of HKC cells with IL-6 for increasing of a complex the CASR P1 Stat1/3 element and Stat1 or Stat3 of the was and at with no increase at not Sp1 was in the complex no on mutated CASR gene of an on the CASR P2 Sp1 Stat1 or Stat3 Sp1 or is to the P2 Sp1/3 element oligonucleotide precipitation were of HKC cells with IL-6 for increasing of a complex the CASR P1 Sp1 elements and Sp1 and and Stat1 and Stat3 of the was and at with no increase at not no on mutated CASR gene that the with P1 and P2 and mutated were the same HKC for of by the In this have the mechanisms underlying the of CASR expression by the proinflammatory IL-6. In of IL-6 to to in serum PTH, 1,25(OH)2D, and calcium that were a that IL-6 parathyroid, thyroid, and kidney CASR levels and for the thyroid and kidney which of could be that the CASR protein levels were thyroid and kidney proximal tubule CASR gene transcription was increased by IL-6 in of both CASR gene P1 and P2 transcripts were In renal proximal tubule HKC and thyroid TT cells, IL-6 stimulated activity of P1 and P2 reporter gene constructs and IL-6 by the IL-6 receptor that of an and a signal which is the for the cytokine of IL-6 to receptor and that and to the and to elements thereby activating gene transcription 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, Jr., Nat. 2002; PubMed Scopus Google Scholar). There are of the with Stat1 and Stat3 the that IL-6 The can to the the activity of a of transcription factors as the but other that response elements S. G. F. J. PubMed Scopus Google Scholar). transcription factors like SRF, and Sp1/3 G. 1999; PubMed Scopus Google Scholar) to factors by IL-6 and regulate gene the is for the of IL-6 by activating Stats and/or other the CASR gene promoters with that response element to and elements in to Stat elements. The could the of the that to these elements. in this of the that although of the to activity of both P1 and P2 was not in a major in the IL-6 of of the of the Stat1 and Stat3 are the that IL-6 The studies with and mutated constructs of both CASR P1 and P2 promoters on a Stat1/3 element in and Sp1/3 elements at the transcription start site of promoter P2 as critical for IL-6 of the CASR The of IL-6 with of Stat1 and/or Stat3 dominant-negative constructs and the P1 promoter the of these Stats for the The of IL-6 with of an Sp1 dominant-negative and the P2 promoter constructs the of Sp1 for the downstream even Stat elements are not in the P2 promoter, of Stat1 or Stat3 dominant-negative constructs the IL-6 of the P2 In with HKC and Stat1/3 on the Stat1 element in on Stat1 element within promoter The that Stats on this element the from the promoter-reporter that the IL-6 of the P1 promoter the Stat Stats were as and this cytokine is a Stat1 and of is on Stat elements in to as in the S. C. Cell. 17: PubMed Scopus Google Scholar). of the by IL-6 of Stat1 and Stat3 with the CASR P1 promoter Stat element was by an oligonucleotide precipitation In with HKC Sp1/3 and on the of Sp1 elements the transcription start site of promoter P2. In with the promoter-reporter in which of an Sp1 dominant-negative the IL-6 of promoter this the importance of the Sp1 in the by the In the Sp1 elements are for the activity of the that both Stat and Sp1 elements in that the of the is to between the Stat and Sp1 at response elements. this with Stat1 and Sp1 for of the gene M.R. M.J. J. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar) and with Stat3 and Sp1 for the protein gene Cell. 18: PubMed Google Scholar). this is not the with the CASR P2 promoter as has no Stat elements. the of the IL-6 of P2 promoter reporter constructs by Stat1 or Stat3 dominant-negative constructs a role for the Stats in the of this promoter by IL-6. of the was in the of not Sp1 and but Stat1 and Stat3 in a complex at the Sp1 element in the CASR P2 A mechanism has been for cytokine of other gene promoters that Stat elements but have Sp1 elements. IL-6 of the gene via between Stat3 and Sp1 S. B. J. J. J. PubMed Scopus Google Scholar), and Stat3 with Sp1 the by of the of gene S. Endocrinol. PubMed Scopus Google Scholar). In studies the altered CASR expression that the endocrine control of blood calcium may be in occurring in critically ill patients in other circulating proinflammatory cytokine levels are In these blood that although the of increased The mechanisms that are may a critical that the of calcium and in and the that the in serum calcium may be as in critically ill patients a of hypocalcemia is with a The the to the of small that and the CASR, be in critically P. J. and for critical of the and for for the cells, and and of for the

Familial hypocalciuric hypercalcemia (FHH) is caused by heterozygous loss-of-function mutations in the calcium-sensing receptor (CASR), in which the lifelong hypercalcemia is generally asymptomatic. Homozygous loss-of-function CASR mutations manifest as neonatal severe hyperparathyroidism (NSHPT), a rare disorder characterized by extreme hypercalcemia and the bony changes of hyperparathyroidism, which occur in infancy. Activating mutations in the CASR gene have been identified in several families with autosomal dominant hypocalcemia (ADH), autosomal dominant hypoparathyroidism, or hypocalcemic hypercalciuria. Individuals with ADH may have mild hypocalcemia and relatively few symptoms. However, in some cases seizures can occur, especially in younger patients, and these often happen during febrile episodes due to intercurrent infection. Thus far, 112 naturally-occurring mutations in the human CASR gene have been reported, of which 80 are unique and 32 are recurrent. To better understand the mutations causing defects in the CASR gene and to define specific regions relevant for ligand-receptor interaction and other receptor functions, the data on mutations were collected and the information was centralized in the CASRdb (www.casrdb.mcgill.ca), which is easily and quickly accessible by search engines for retrieval of specific information. The information can be searched by mutation, genotype-phenotype, clinical data, in vitro analyses, and authors of publications describing the mutations. CASRdb is regularly updated for new mutations and it also provides a mutation submission form to ensure up-to-date information. The home page of this database provides links to different web pages that are relevant to the CASR, as well as disease clinical pages, sequence of the CASR gene exons, and position of mutations in the CASR. The CASRdb will help researchers to better understand and analyze the mutations, and aid in structure-function analyses.