Human Calcium-sensing Receptor GeneVITAMIN D RESPONSE ELEMENTS IN PROMOTERS P1 AND P2 CONFER TRANSCRIPTIONALRESPONSIVENESS TO 1,25-DIHYDROXYVITAMIN D*

Received for publication, February 22, 2002, and in revised form, May 13, 2002Published, JBC Papers in Press, May 29, 2002, DOI 10.1074/jbc.M201804200

Lucie Canaff‡ and Geoffrey N. Hendy§

From the Departments of Medicine, Physiology, and Human Genetics, McGill University and Royal Victoria Hospital,Montreal, Quebec H3A 1A1, Canada

The calcium-sensing receptor (CASR), expressed inparathyroid chief cells, thyroid C-cells, and cells of thekidney tubule, is essential for maintenance of calciumhomeostasis. Here we show parathyroid, thyroid, andkidney CASR mRNA levels increased 2-fold at 15 h afterintraperitoneal injection of 1,25-dihydroxyvitamin D3(1,25(OH)2D3) in rats. Human thyroid C-cell (TT) andkidney proximal tubule cell (HKC) CASR gene transcrip-tion increased �2-fold at 8 and 12 h after 1,25(OH)2D3treatment. The human CASR gene has two promotersyielding alternative transcripts containing either exon1A or exon 1B 5�-untranslated region sequences thatsplice to exon 2 some 242 bp before the ATG translationstart site. Transcriptional start sites were identified inparathyroid gland and TT cells; that for promoter P1 lies27 bp downstream of a TATA box, whereas that for pro-moter P2, which lacks a TATA box, lies in a GC-richregion. In HKC cells, transcriptional activity of a P1reporter gene construct was 11-fold and of P2 was 33-fold above basal levels. 10�8 M 1,25(OH)2D3 stimulated P1activity 2-fold and P2 activity 2.5-fold. Vitamin D re-sponse elements (VDREs), in which half-sites (6 bp) areseparated by three nucleotides, were identified in bothpromoters and shown to confer 1,25(OH)2D3 responsive-ness to a heterologous promoter. This responsivenesswas lost when the VDREs were mutated. In electro-phoretic mobility shift assays with either in vitro tran-scribed/translated vitamin D receptor and retinoid Xreceptor-�, or HKC nuclear extract, specific protein-DNA complexes were formed in the presence of1,25(OH)2D3 on oligonucleotides representing the P1and P2 VDREs. In summary, functional VDREs havebeen identified in the CASR gene and provide the mech-anism whereby 1,25(OH)2D up-regulates parathyroid,thyroid C-cell, and kidney CASR expression.

Maintenance of calcium homeostasis depends on a complex

interplay between parathyroid hormone (PTH),1 the hormon-ally active metabolite of vitamin D, 1,25-dihydroxyvitamin D(1,25(OH)2D) and the extracellular calcium concentration itself(1, 2). PTH synthesis and secretion are negatively regulated byserum calcium and 1,25(OH)2D levels. In the kidney proximaltubule, the mitochondrial 25-hydroxyvitamin D-1�-hydroxy-lase, 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 andsecretion, and 1,25(OH)2D production, initially stimulated byreductions in circulating calcium and 1,25(OH)2D levels, arethen shut off as the mineral ion and vitamin D metaboliteconcentrations normalize.

The calcium-sensing receptor (CASR) that plays a criticalrole in this process is a glycoprotein with a predicted topologyof a large extracellular domain, a seven-transmembrane do-main, and an intracellular tail (3). This G protein-coupledreceptor is expressed most abundantly in the parathyroid chiefcells, along the length of the kidney tubule, and in thyroidC-cells. The CASR is activated by elevations in extracellularcalcium concentration, leading to inhibition of PTH secretionand renal calcium reabsorption (4).

Potentially, two important regulators of CASR gene expres-sion are extracellular calcium and 1,25(OH)2D. Two previousstudies were unable to demonstrate an effect of extracellularcalcium on parathyroid gland or whole kidney CASR mRNA inthe rat in vivo (5, 6). This lack of modulation of CASR expres-sion might be expected, given the constraints placed upon theCASR in tissues such as parathyroid gland or kidney, where itplays an essential role as a calciostat to sense very smallchanges in extracellular calcium concentration. Even modestalterations in the extracellular calcium set-point (this beingdefined as the extracellular calcium concentration for half-maximal stimulation of PTH secretion from the parathyroidgland or calcium reabsorption across the kidney tubule)brought about by changes in CASR synthesis could have majorunwanted effects on overall calcium homeostasis.

Previously, the effect of vitamin D status (depleted versusreplete) and/or treatment with 1,25(OH)2D3 on parathyroidand kidney CASR mRNA levels has been examined in rats. One* This work was supported in part by Canadian Institutes of Health

Research (CIHR) Grant MT-9315 and by a Kidney Foundation of Can-ada grant (to G. N. H.) The costs of publication of this article weredefrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submittedto the GenBankTM/EBI Data Bank with accession number(s) AY116081and AY116082.

‡ Recipient of a doctoral fellowship from the CIHR and a NationalCancer Institute of Canada research studentship.

§ To whom correspondence should be addressed: Calcium ResearchLaboratory, Rm. H4.67, Royal Victoria Hospital, 687 Pine Ave. W.,Montreal, Quebec H3A 1A1, Canada. Tel.: 514-843-1632; Fax: 514-843-1712; E-mail: geoffrey.hendy@mcgill.ca.

1 The abbreviations used are: PTH, parathyroid hormone; CASR,calcium-sensing receptor; 1,25(OH)2D, 1,25-dihydroxyvitamin D;1,25(OH)2D3, 1,25-dihydroxyvitamin D3; VDRE, vitamin D responseelement; VDR, vitamin D receptor; RXR, retinoid X receptor; 5�-RACE,5�-rapid amplification of cDNA ends; PE, primer extension; EMSA,electrophoretic mobility shift assay; mOP, mouse osteopontin; CTAL,cortical thick ascending limb; DMEM, Dulbecco’s modified Eagle’s me-dium; Pipes, 1,4-piperazinediethanesulfonic acid; RT, reverse tran-scription; TT, human thyroid C-cell; HKC, human kidney proximaltubule cell; DTT, dithiothreitol; FBS, fetal bovine serum; UTR, untrans-lated region; PMSF, phenylmethylsulfonyl fluoride.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 33, Issue of August 16, pp. 30337–30350, 2002© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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study found that vitamin D-depleted rats had a 40% reductionin parathyroid CASR mRNA relative to replete animals andadministration of 1,25(OH)2D3 to vitamin D-replete rats fur-ther enhanced parathyroid and kidney CASR mRNA levels (5).A second study found that administration of 1,25(OH)2D3 torats up-regulated renal CASR mRNA levels in a dose- andtime-dependent manner (7). One study failed to find evidencefor vitamin D modulation of CASR expression (6), although formethodological reasons small differences in CASR mRNA lev-els might have been missed.

The human CASR is encoded by six exons (exons 2–7) of thegene (8–10) located on chromosome 3q13.3–21 (11) with exon 2encoding 242 nucleotides of the 5�-untranslated region (UTR),followed by the translation start site. Exons 1A and 1B encodealternative 5�-UTRs that splice to the common portion encodedby exon 2 (12, 13). The gene sequence upstream of exon 1A hasa TATA box, whereas the sequence upstream of exon 1B lacksa TATA box and is GC-rich. The precise transcriptional startsites of exons 1A and 1B have not been mapped, and functionalcis-acting elements in the gene promoters have yet to beidentified.

In the present study, we have shown that 1,25(OH)2D3 up-regulates parathyroid, thyroid, and kidney CASR mRNA levelsin vivo in the rat, and that 1,25(OH)2D3 up-regulates the en-dogenous CASR gene transcription in human thyroid and kid-ney cell lines. In addition, we have mapped the transcriptionalstart sites and identified functional vitamin D response ele-ments (VDREs) in both promoters of the human CASR gene.

EXPERIMENTAL PROCEDURES

Materials—Protocols for obtaining human parathyroid tissue wereapproved by the local ethics committee. The human genomic library(945203, in �DASH II) was from Stratagene (La Jolla, CA). The COS-7and human medullary thyroid carcinoma TT cell lines were from theAmerican Type Culture Collection (Rockville, MD), and the humanproximal kidney tubule cells (HKC-8) were a gift of Dr. Martin Hewison(University of Birmingham, Birmingham, UK). Dulbecco’s modifiedEagle’s medium (DMEM), Ham’s F-12 medium, fetal bovine serum(FBS), and antibiotics were from Invitrogen Canada (Burlington, On-

tario, Canada). [�-32P]Adenosine 5�-triphosphate and [�-32P]dUTP werefrom ICN Biomedicals (Baie d’Urfe, Quebec, Canada). Restriction en-zymes, polynucleotide kinase, and Moloney murine leukemia virus re-verse transcriptase were from MBI Fermentas (Burlington, Ontario,Canada). Hybond membranes and Ready-to-Go beads were from Amer-sham Biosciences (Baie d’Urfe, Quebec, Canada).

Animals and Experimental Procedures—Normal male Sprague-Daw-ley rats (Charles River Laboratories, Inc., St. Constant, Quebec, Can-ada) weighing 180–200 g when received, were fed a standard rodentchow (Ralston Purina Co., LaSalle, Quebec, Canada) containing 1.01%calcium, 0.74% phosphorus, and 3.3 IU/g vitamin D3. All animal exper-iments were carried out in compliance with, and were approved by, theinstitutional Animal Care and Use Committee. Rats were injected in-traperitoneally, at 24, 15, 12, 8, and 4 h before death, with either vehicle(propylene glycol, 0.2 ml/100 g body weight) or 1,25(OH)2D3 (250 pmol/100 g body weight) (14, 15). The rats were anesthetized with pentobar-bital, the kidneys were taken, and the parathyroid and thyroid glandswere microdissected separately and quick-frozen.

Ribonuclease Protection Assay of Rat CASR mRNA—For the CASRriboprobe, a 232-bp fragment of a rat CASR cDNA (16) was PCRamplified (forward primer, 5�-ACCTTGAGTTTTGTTGCCCA-3� (inexon 3); reverse primer, 5�-GGAATGGTGCGGAGGAAGGATT-3� (inexon 4)) and cloned into the PCR2.1 vector. For the actin riboprobe thatprotects a 126-base transcript, the pTRI-�-actin-125 rat vector (AmbionInc., Austin, TX) was used. After linearization of the vectors, the anti-sense probes were in vitro transcribed with T7 polymerase incorporat-ing [�-32P]UTP using a MAXIscript kit and the gel-purified riboprobeswere used with an RPA III kit (Ambion Inc.). Each probe (2.5 � 105

cpm) was hybridized overnight with 2–25 �g of total RNA followed bydigestion with a ribonuclease A:T1 mix (17). Protected fragments wereresolved on 6% acrylamide denaturing gels and exposed to x-ray film.

Nuclear Run-on Transcription Assays—Relative transcription rateswere measured using a nuclear run-on assay (18). Nuclei were preparedfrom 10–20 � 106 HKC or TT cells incubated with either 10�8 M

1,25(OH)2D3 or ethanol carrier alone. Cells were scraped into ice-coldPBS, pH 7.4, pelleted at 4 °C, and lysed with Nonidet P-40 lysis buffer(0.3 M sucrose, 60 mM KCI, 15 mM NaC1, 15 mM HEPES, pH 7.5, 2 mM

EDTA, 0.5 mM EGTA, 0.15 mM spermine, 0.5 mM spermidine, 14 mM

�-mercaptoethanol, 0.2% Nonidet P-40). After 8 min on ice, nuclei werepelleted at 800 � g. They were rinsed once with 1 ml of nuclei storagebuffer (50% glycerol, 20 mM Tris, pH 7.9, 75 mM NaC1, 0.5 mM EDTA,0.85 mM DTT, 0.125 mM phenylmethylsulfonyl fluoride (PMSF)), snap-frozen in liquid nitrogen, and stored at �80 °C until assay. Run-on

FIG. 1. Induction of parathyroid, thyroid, and kidney CASR mRNA by 1,25(OH)2D3. Rats were injected intraperitoneally with 100 ng of1,25(OH)2D3, sacrificed at the times shown, and CASR and actin mRNA levels of panel A, parathyroid gland; panel B, thyroid gland; and panel C,kidney measured by ribonuclease protection assay as described under “Experimental Procedures.” Autoradiographs of representative ribonucleaseprotection analysis signals are shown for each time point. CASR and actin mRNA levels were assessed by densitometry and each value is themean � S.E. (n � 3). Asterisks indicate a significant difference (p � 0.05) from the time 0 value.

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reactions were carried out at 30 °C in 300 mM NH4(SO4)2, 100 mM

Tris-HC1, pH 7.9, 4 mM MgCl2, 4 mM MnCl2, 50 mM NaCl, 0.4 mM

EDTA, 1.2 �M DTT, 0.1 mM PMSF, 10 mM creatine phosphate, 29%glycerol, 150 �Ci of [32P]UTP, 3000 Ci/mmol (ICN, Mississauga, On-tario, Canada), 1.5 mM each of CTP, ATP, and GTP (MBI Fermentas) for45 min. RNA was extracted with TRIzol (Invitrogen Canada) accordingto manufacturer’s instructions. Five �g of plasmid DNA containingspecific gene inserts or no insert were NaOH-denatured and slot-blotted(HybriSlot, Invitrogen Canada) onto Nytran membranes. The gene-specific plasmids were: 1) human CASR exon 1A, a 280-bp AseI-StuIfragment cloned in pBluescript II KS; 2) human CASR exon 1B, a230-bp NotI-StuI fragment cloned in pBluescript II KS; 3) human CASRexon 2, a 227-bp StuI-NcoI fragment cloned in pBluescript II KS; 4)human 24-hydroxylase, a 304-bp fragment RT-PCR-amplified fromHKC RNA (forward primer, 5�-CTGATGACCGACGGTGAGACTC-3�;reverse primer 5�-AGCCCGTAGGCTTCGTTGCGATG-3�) and TA-cloned in pCR2.1; 5) rat cyclophilin, a 800-bp BamHI restriction frag-ment cloned in plasmid pCD15.8.1 (19) (kindly provided by Dr. GregorSutcliffe, The Scripps Research Institute, La Jolla, CA). The mem-branes were hybridized with 2 � 107 cpm 32P-labeled transcripts in 50%formamide, 50 mM Hepes, pH 7.3, 0.75 M NaC1, 2 mM EDTA, 0.5% SDS,10� Denhardt’s, and 20 �g/ml salmon sperm DNA for a minimum of40 h. In any single experiment, equal numbers of counts were used forall conditions. Membranes were exposed to autoradiographic film, andquantitation of the relative rates of transcription was achieved bydensitometry.

Screening of the Human Genomic DNA Library—Human CASR geneprobes were generated by PCR amplifying normal human leukocyteDNA with primer Exon 1AF (5�-AGGCACCTGGCTGCAGCCAGGAAG-3�) and Exon 1BR (5�-GGTCTCCACGAGGATGAGCTCTGG-3�) togenerate probe 1 of 784 bp and with primers Exon 2F (5�-GTGGCTT-CCAAAGACTCAAGG-3�) and Exon 2R (5� AGACAGCTAGGAGTTTG-GAGG-3�) to generate probe 2 of 184 bp. The products were cloned intoTA cloning vectors, maxipreps made, inserts excised, and randomprimer-labeled with [�-32P]dCTP. Each probe was used to screen onemillion clones of a human genomic library. Prehybridization andhybridization were performed in 40% formamide, 5� SSC, 25 mM sod-ium phosphate, pH 7.4, 1% SDS, 2 mM EDTA, 1� Denhardt’s, and 200�g/ml salmon sperm DNA at 42 °C. Filters were washed twice with 2�SSC, 0.1% SDS at 50 °C. Positive plaques were purified by secondaryand tertiary screening, and several positive plaques were obtained thatrepresented two independent clones designated �ghCASR1 and �ghC-ASR2 (for probe 1) and two independent clones designated �ghCASR3and �ghCASR4 (for probe 2).

Restriction Enzyme Mapping and DNA Sequence Analysis—Recom-binant clones were digested with HindIII, and selected restriction frag-ments were subcloned into pBluescript II KS for DNA sequencing.

RNA Extraction—Total RNA was prepared from cells or tissues usingTRIzol (Invitrogen Canada) according to the manufacturer’sinstructions.

5�-RACE Amplification—This was performed with a 5�-RACE system(Invitrogen Canada) according to the manufacturer’s instructions. Fivemicrograms of human TT cell total RNA were reversed transcribed withSuperscript II using a CASR exon 2-specific reverse primer (5�-ACAT-CATGCAGAGGCCTGGTGTGATGC-3�). The first strand cDNA waspurified with a GlassMAX DNA isolation spin cartridge and homopoly-mer tailed with dCTP and terminal deoxynucleotidyltransferase. Thetailed cDNA was amplified with Taq polymerase and a 5�-RACE anchorprimer specific for the homopolymer tail and either an exon 1A (5�-TGCCGCAAGACCTCGGTGCTGGCA-3�) or exon 1B (5�-CTATGC-CAAGGTCACGGTCTTGGA-3�) reverse primer. PCR conditions wereinitial denaturation (94 °C, 40 s) then 58 °C, 45 s, and 72 °C, 1 min for30 cycles. PCR products were Topo-TA cloned into PCR2.1 andsequenced.

Primer Extension Analysis of CASR mRNA—One pmol of primer thathad been labeled with [�-32P]ATP using T4 polynucleotide kinase wasincubated with 10 �g of either human TT cell or human parathyroidadenoma RNA or yeast tRNA (negative control) overnight at 60 °C in 10mM Pipes, pH 6.4, 0.4 M NaCl, 1 mM EDTA. The RNA templates werethen reversed transcribed using 200 units of Moloney murine leukemiavirus-reverse transcriptase. The extension products were phenol/chlo-roform-extracted, ethanol-precipitated, and analyzed on a 6% denatur-ing polyacrylamide gel in parallel with an unrelated sequencing reac-tion as a marker.

Ribonuclease Protection Analysis of Human CASR mRNA—For theriboprobes, portions of human genomic DNA were PCR amplified andTA-cloned into the PCR2.1 vector. The linearized vectors were in vitrotranscribed with T7 polymerase incorporating [�-32P]UTP using a

FIG. 2. Induction of CASR gene transcription by 1,25(OH)2D3.Nuclear run-on assays were performed as described under “Experimen-tal Procedures” on nuclear extracts of TT cells, panel A, and HKC cells,panel B. Autoradiographs of representative experiments repeated threetimes are shown. Densitometry was performed and relative transcrip-tion rates calculated taking CASR exon 1A as 100%.

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FIG. 3. Physical map of human DNA encoding the 5� part of the CASR gene. Panel A, four independent bacteriophage recombinant clones,�ghCASR1–4, were isolated after screening a human genomic DNA as described under “Experimental Procedures.” A map of the 5� portion of theCASR gene is shown with the positions of library screening probes marked. Panel B, promoter P1, exon 1A, promoter P2, exon 1B, and exon 2showing the alternative splicing of exon 1A and 1B to exon 2. The positions of primers used for the 5�-RACE (open arrow) and primer extension(closed arrow) analyses are shown. Key restriction enzyme sites are shown (HindIII; Bs, BssHII; Es, EspI).

FIG. 4. Mapping the CASR P1 pro-moter transcription start site. PanelA, 5�-RACE; 5 �g human TT cell totalRNA were reversed transcribed using anexon 2 specific primer as described under“Experimental Procedures.” After PCRamplification with an exon 1A-specificprimer a 548-bp product was obtained(lane 2). DNA size markers (lane 1) and aPCR blank (lane 3) are shown. Panel B,primer extension; ten micrograms humanTT cell total RNA or yeast tRNA (control)were annealed with an excess of polynucle-otide kinase-labeled oligonucleotide andthe extension reaction performed as de-scribed under “Experimental Procedures.”The products were analyzed on a denatur-ing gel. A DNA sequence ladder served assize markers (lanes 1–4) and the arrowindicates the extension product of 271 nu-cleotides obtained with the human thyroidTT cell mRNA (lane 5). The yeast tRNAcontrol was run in lane 6. Panel C, RNaseprotection analysis. Upper panel, sche-matic representation of probes used. Lowerpanel, products were analyzed on a dena-turing gel. Lanes 1 and 2, undigested P1Aand P1B probes; lanes 3–5, RNase I-digestedP1A probe; lanes 6–8, RNase I-digested P1Bprobe; lanes 3 and 6, PT, parathyroid RNA;lanes 4 and 7, TT cell RNA; lanes 5 and 8,yeast tRNA.

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MAXIscript kit (Ambion). The gel-purified antisense probes (2 � 105

cpm) were hybridized overnight with 20 �g of TT cell DNase I-treatedRNA followed by digestion with a ribonuclease A:T1 mix. Protectedfragments were resolved on 6% polyacrylamide urea gels, which weredried and exposed to x-ray film overnight.

Human CASR Gene Promoter Constructs—To construct the P1-lucif-erase reporter plasmid, a 2.2-kb HindIII fragment containing exon 1Aand 5�-flanking sequence was cloned into pBluescript KS such that thepolylinker KpnI and SmaI sites flanked the 5� and 3� ends of the insert,respectively. By RT-PCR, a 468-bp fragment extending from a BssHIIsite in exon 1A to nucleotide �1 (nucleotide �1 is the A of the ATGinitiation codon) in exon 2 was amplified from TT cell RNA. The forwardprimer was 5�-CGGGCCTCCAAGCAGCGCGCTGTGGA-3� (the natu-rally occurring BssHII site is in boldface type), and the reverse primerwas 5�-ACGATCCCGGGGGTTCTGCCGTCTCTCCAGGGCA-3� (theadded SmaI site is in boldface type). The PCR product was digestedwith BssHII and SmaI and cloned into the BssHII/SmaI-digested pBlu-escript KS plasmid described above. This construct was digested withKpnI/SmaI and the CASR P1 insert cloned into pGL3 basic to generateconstruct PI-VDRE WT.

To construct the P2-luciferase reporter plasmid, a 2.0-kb HindIIIfragment containing exon 1B was cloned into pBluescript KS such thatthe polylinker KpnI and SmaI sites flanked the 5� and 3� ends of theinsert, respectively. By RT-PCR, a 350-bp fragment extending from aBssHII site in exon 1B to nucleotide �1 (see above) in exon 2 wasamplified from TT cell RNA. The forward primer was 5�-GAGCGGGCT-GCGCGCAGTCCTGAG-3� (the naturally occurring BssHII site is inboldface type), and the reverse primer was 5�-ACGATCCCGGGGGT-TCTGCCGTCTCTCCAGGGCA-3� (the added SmaI site is in boldfacetype). The PCR product was digested with BssHII and SmaI and clonedinto the BssHII/SmaI-digested pBluescript KS plasmid describedabove. To complete the 5� portion of the P2 promoter, a 331-bp fragmentwas amplified using �ghCASR2 DNA. The forward primer was 5�-ACTGAGGTACCGTAAGAGTTTGGGCACGCGAT-3� (the added KpnIsite is in boldface type), and the reverse primer was 5�-GACCCTGAA-GAGTCAGCTAAGCCTCTCTG-3� (the naturally occurring EspI site isin boldface type). The PCR product was digested with KpnI and SmaI

and cloned into the KpnI/EspI-digested pBluescript KS plasmid de-scribed above. The entire P2-containing insert was excised with KpnIand SmaI and cloned into pGL3 basic to generate construct P2-VDREWT.

The P1-VDRE MUT and P2-VDRE MUT constructs were generatedusing the QuikChange XL site-directed mutagenesis kit (Stratagene,San Diego, CA). For each mutagenesis, a pair of complementary prim-ers were designed with the mutant base pair in the middle. The primersused were: P1 forward (5�-TGCTTTAGCATTTGCTCATTTCCTTCTT-TTACCCTGTATTTGAGGGA-3�), P1 reverse (5�-TCCCTCAAATACAG-GGTAAAAGAAGGAAATGAGCAAATGCTAAAGCA-3�), P2 forward(5�-CTCGGGGAACCGAAGACGCGCTTTCAGCGATTCTGAAGAGT-CAGCTAAGC-3�), P2 reverse (5�-GCTTAGCTGACTCTTCAGAATCG-CTGAAAGCGCGTCTTCGGTTCCCCGAG-3�). The VDR/RXR half-sites are in boldface type, and the mutated nucleotides are underlined.The primers were annealed to the appropriate template, 12 rounds ofextension were performed with Pfu Turbo DNA polymerase, and thetemplate was digested with DpnI. The reactions were used to transformXL10-Gold Ultracompetent cells, which incorporate nicked DNA andrepair it. The correctness of all constructs was confirmed by sequenceanalysis.

To construct the P1-heterologous promoter luciferase reporter plas-mids, 104-bp products containing the VDRE were amplified using P1-VDRE WT and P1-VDRE MUT as templates with forward primer P1F(5�-atgctGCTAGCACCAGATTTTGCCCCTTCACTG-3�) and reverseprimer P1R (5�-tctgaCTCGAGATGGCCAAGTTCTGCCCATTTG-3�),where the nucleotides in boldface type are an NheI or XhoI site and thelowercase nucleotides are those added to ensure complete restrictionenzyme cleavage. The PCR products were digested with NheI and XhoIand cloned into the pGL3-Promoter vector (Promega) upstream of theSV40 promoter to generate constructs SV-40 P1-VDRE WT and SV-40PI-VDRE MUT.

To construct the P2-heterologous promoter luciferase reporter plas-mids, 104-bp products containing the VDRE were amplified using P2-VDRE WT and P2-VDRE MUT as templates with forward primer P2F(5�-atgctGCTAGCTGGGGACCCGAGCCCGCCTGTG-3�) and reverseprimer P2R (5�-tctgaCTCGAGTCACCGTCTCCTTAGCCCGCAG-3�),

FIG. 5. Sequence of the human CASR gene P1 promoter and exon 1A. The nucleotide sequence was determined as described under“Experimental Procedures” from subcloned HindIII genomic fragments shown in Fig. 3. The positions of primer P1-RACE used for 5�-RACE andprimer P1-PE used for primer extension analyses are indicated. The transcription initiation site mapped by primer extension and RNase is markedas �1. TATA, CAAT and VDRE homologies are in bold and boxed. HindIII sites and the complete sequence of exon 1A are in bold. The beginningof partial exon 1A sequence obtained from a previously reported human CASR cDNA (12) is indicated by the open arrowhead. The closed arrowheadindicates the longest cDNA obtained by 5�-RACE in the present study. The asterisk (*) marks a putative transcription start site obtained by5�-RACE in a previous study (13).

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FIG. 6. Mapping the human CASR P2 promoter transcription start site. Panel A, 5�-RACE; 5 �g human TT cell total RNA were reversedtranscribed using an exon 2 specific primer as described under “Experimental Procedures.” After PCR amplification with an exon 1B-specificprimer a 158-bp product was obtained (lane 2). DNA size markers (lane 1) and a PCR blank (lane 3) are shown. Panel B, primer extension; 10 �ghuman TT cell or parathyroid total RNA or yeast tRNA (control) were annealed with an excess of labeled oligonucleotide P2-PE and the extensionreaction performed as described under “Experimental Procedures”. The products were analyzed on a denaturing gel. A DNA sequence ladder servedas size markers (lanes 1–4) for the extension product of 153 nucleotides (arrow) obtained with the human thyroid TT cell (lane 5) and parathyroid(lane 7) RNA. The yeast tRNA control is in lane 6. Panel C, RNase protection analysis. Upper panel, schematic representation of probes used. Lowerpanel, products were analyzed on denaturing gel. Lanes 1 and 2, undigested P2A and P2B probes; lanes 3–5, RNase I-digested P2A probe; lanes6–8, RNase I-digested P2B probe; lanes 3 and 9, PT, parathyroid RNA; lanes 4 and 8, TT cell RNA; lanes 5 and 6, yeast tRNA; lanes 10–13, DNAsequence ladder.

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where the nucleotides in boldface type are an NheI or XhoI site and thelowercase nucleotides are those added to ensure complete restrictionenzyme cleavage. The PCR products were digested with NheI and XhoIand cloned into the pGL3-Promoter vector upstream of the SV40 pro-moter to generate constructs SV-40 P2-VDRE WT and SV-40 P2-VDREMUT.

Cell Culture—COS-7 and HKC cells were grown in DMEM supple-mented with 10% FBS. TT cells were cultured in RPMI 1640 mediumwith 10% FBS and 5% horse serum. All maintenance medium contained100 units/ml penicillin, 100 �g/ml streptomycin, and 250 ng/ml ampho-tericin B.

Transfection and Reporter Assay—For transient transfection, cellswere trypsinized, plated in six-well dishes in DMEM plus 10% FBS(1–4 � 105 cells/well) and incubated overnight. The next day, cells weretransfected with 30 �g/well Superfect reagent with 1 �g of CASRpromoter construct and 0.5 �g/well pCH110. The following day, cellswere serum-starved in DMEM overnight and cultured with or without10�8 M 1,25(OH)2D3 for 10h. The cells were washed in PBS and lysed in250 �l of lysis buffer (1% Triton X-100, 15 mM MgSO4, 4 mM EGTA, 1mM DTT, 25 mM glycyl glycine) on ice. Luciferase activity was measuredin an EG&G Berthold luminometer using 45 �l of cell lysate andD-luciferin. Luciferase activity was normalized to �-galactosidaseactivity.

Statistics—Data are expressed as mean � S.E. The results from thein vivo 1,25(OH)2D3 response studies were initially subjected to analy-sis of variance. The significance of differences from base line was thendetermined using Dunnett’s multiple comparison test. For the nuclearrun-on assays and luciferase transient transfection assays, compari-sons were performed by Student’s t test. A p value of �0.05 was takento indicate a statistically significant difference.

In Vitro Transcription and Translation—Plasmids VDR/pSG5 andRXR�/pSG5 (20) were transcribed with T7 RNA polymerase and trans-lated in vitro with the TNT reticulocyte lysate system according to themanufacturer’s instructions.

Nuclear Extracts of HKC Cells—Cells were washed, scraped into 1 mlof PBS, and centrifuged at 1500 � g for 10 min at 4 °C. Cell pellets wereprocessed in a loose Dounce tissue homogenizer in two volumes of buffer

(25 mM Tris, pH 7.9, 0.3 mM DTT, 420 mM NaCl, 10 mM EDTA, 0.5 mM

PMSF, and protease inhibitors). Nuclear pellets were obtained by cen-trifugation (25,000 � g for 20 min at 4 °C), resuspended in 20 mM

HEPES, pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM

EDTA, 0.5 mM PMSF, 0.5 mM DTT, and Dounce homogenized. After a20-min centrifugation at 25,000 � g, nuclear extracts (supernatants)were dialyzed for 5 h against 20 nM HEPES, pH 7.9, 20% glycerol, 0.1M KCl, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT. Protein content wasdetermined and aliquots stored at �80 °C.

Oligonucleotides and Antibodies—Oligonucleotides used were as fol-lows: CASRP1-VDRE-WT (5�-AGGGTAGGAGAAGGAGCTGAGCAA-T-3�), CASRPI-VDRE-MUT (5�-AGGGTAAAAGAAGGAAATGAGCA-AT-3�), CASRP2-VDRE-WT (5�-CTTCAGGGTCGCTGAGGGCGCGT-CT-3�), CASRP2-VDRE-MUT (5�-CTTCAGAATCGCTGAAAGCGCG-TCT-3�), and MOP-WT (5�-GTACAAGGTTCACGAGGTTCACGTC-3�).Only the sense strand is shown; half-sites are in boldface type, andmutant nucleotides are underlined. Antibodies against the VDR (VDRN-20, VDR C-20) and the RXR� (RXR�N197, RXR�D-20) were fromSanta Cruz Biotechnology.

Electrophoretic Mobility Shift Assay—Two micrograms of nuclearextract or VDR/RXR in vitro translated products were incubated for 20min on ice in the absence or presence of 10�8 M 1,25(OH)2D3 and 1 �gof poly(dI�dC) in binding buffer (25 mM Tris-HCl, pH 8.0, 50 mM KCl,15% glycerol, 0.5 mM DTT). Antibodies were then added or not, andsamples were incubated for 20 min at room temperature. Five fmol of32P-end-labeled double-stranded oligonucleotides were added and incu-bated for an additional 20 min. Samples were electrophoresed at 8 V/cmon 6% nondenaturing polyacrylamide gels equilibrated in 0.25 M Tris,pH 8.3, 1.9 M glycine, 10 mM EDTA, dried, and autoradiographed.

RESULTS

1,25(OH)2D3 Up-regulates Parathyroid, Thyroid, and KidneyCASR mRNA Levels in Vivo—As previous studies aimed atassessing whether 1,25(OH)2D3 regulates CASR mRNA levelsin vivo have produced inconsistent results, we conducted ourown analysis. After a single intraperitoneal injection of

FIG. 7. Sequence of the human CASR gene P2 promoter, exon 1B, and part of intron 1. The nucleotide sequence was determined asdescribed under “Experimental Procedures” from a subcloned HindIII genomic fragment indicated in Fig. 3. The positions of primer P2-RACE usedfor 5�-RACE and primer P2-PE used for primer extension analyses are indicated. The transcription initiation site mapped by 5�-RACE, primerextension and RNase protection is marked as �1. VDRE and Sp1 homologies are in bold and boxed. Hind III sites and the complete sequence ofexon 1B are in bold. The beginning of partial exon 1B sequence obtained from a previously reported human CASR cDNA (12) is indicated by theopen arrowhead. The asterisk (*) marks a putative transcription start site obtained by 5�-RACE in a previous study (13).

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1,25(OH)2D3 in rats, parathyroid, thyroid, and kidney CASRmRNA levels rose to 2–2.5-fold over basal level at 15 h and hadreturned to base line at 24 h (Fig. 1). Injection of vehicle had noeffect on CASR mRNA levels (data not shown).

1,25(OH)2D3 Increases CASR Gene Transcription—The re-sults of nuclear run-on assays performed on extracts of humanTT and HKC cells cultured with and without 1,25(OH)2D3 for 8and 12 h, respectively, are shown in Fig. 2. CASR gene exon 1A,exon 1B, and exon 2 transcripts were all stimulated 2-fold, aswas 24-hydroxylase gene transcription in both cell types. Cy-clophilin gene transcription was unaffected by 1,25(OH)2D3.

Cloning of CASR Genomic Clones—Several positive phageplaques were identified after the initial screening of the humangenomic library. Two independent overlapping clones isolatedwith probe 1 (exons 1A and 1B) were designated �ghCASR1

and �ghCASR2, and two overlapping clones isolated with probe2 (exon 2) were designated �ghCASR3 and �ghCASR4 (Fig. 3,A and B). Insert sizes were 18–20 kb. The 2.2- and 1.9-kbHindIII fragments encoding exons 1A and 1B, respectively,were subcloned and completely sequenced. These were used togenerate CASR promoter/luciferase reporter gene constructs(see below).

Characterization of Transcription Initiation Sites—A previ-ous study (13) using only 5�-RACE found a putative start sitefor exon 1A some 105 bp downstream of a putative TATA box.This suggested that the actual start site might lie upstream ofthat previously identified. We used 5�-RACE, primer extension,and RNase protection analyses of human parathyroid and TTcell RNA to map the sites of transcription initiation at the5�-ends of exons 1A and 1B. 5�-RACE of TT cell RNA and an

FIG. 8. Human CASR gene promoter constructs. Panel A, promoter P1, exon 1A, promoter P2, exon 1B, and exon 2 showing the alternativesplicing of exons 1A and 1B to exon 2. The positions of primers used to amplify CASR cDNA or genomic sequences (see text) for the CASR genepromoter luciferase reporter gene constructs are marked and key restriction enzyme sites are shown (H, HindIII; B, BssHII; Es, EspI). Panel B,portions of human CASR gene promoters P1 or P2 (without and with mutated VDREs) with appropriate 5�-UTR sequences were cloned upstreamof the luciferase reporter gene in pGL3 basic as described under “Experimental Procedures” to create P1-VDRE WT through P2-VDRE MUT.Portions of promoters P1 and P2 with the VDREs (wild-type or mutated) were cloned upstream of the SV40 promoter and the luciferase reportergene in the pGL3-Promoter vector to create SV40 P1-VDRE WT through SV40 P2-VDRE MUT.

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exon 1A primer yielded products of greater than 500 bp (Fig.4A). After subcloning and sequencing several subclones, thelongest was found to be 548 bp. Although this extended thesequence of exon 1A some 19 bp upstream of that previouslydetermined (13), it was still 86 bp from the TATA box.

Primer extension analysis with TT cell RNA yielded a singlemajor extension product of 271 bases (Fig. 4B). This places thetranscriptional start site at the A nucleotide 27 bp downstreamof the TATA box within a TTATTCT sequence (Fig. 5), whichmatches the consensus for this type of start site (21). RNaseprotection analysis of human parathyroid and TT cell RNAwith two different riboprobes generated protected products of292 and 170 bases from the longer and shorter riboprobes,respectively (Fig. 4C). In both cases, the result was fully con-sistent with that obtained with the primer extension analysis.

5�-RACE of TT cell RNA and an exon 1B primer yieldedproducts of �150 bp (Fig. 6A). After subcloning and sequencingseveral subclones, the longest was found to be 158 bp. Thisextended the sequence of exon 1B some 9 bp upstream of thatdetermined previously (13). Primer extension analysis with TTcell and parathyroid RNA yielded in each case a single majorextension product of 153 bases (Fig. 6B). This coincided pre-cisely with the assignment from the 5�-RACE. RNase protec-tion analysis of human parathyroid and TT cell RNA with twodifferent riboprobes generated protected products of 138 and107 bases from the longer and shorter riboprobes, respectively(Fig. 6C). These results were fully consistent with those ob-tained with the other techniques. Thus, for exon 1B all threemethods used identified the same transcription start site. TheP2 promoter lacks a TATA box and is GC-rich, with one Sp1site located 11 bp upstream and a second Sp1 site located 3 bpdownstream of the transcription start site (Fig. 7).

Human CASR P1 and P2 Promoters Are Active in HumanProximal Tubule Kidney Cells—To analyze their transcrip-tional activities, constructs were prepared in which P1 and P2promoters drive transcription of the luciferase gene. We havenoted previously that addition of 5�-UTR exonic sequence mayhelp to promote optimal transcriptional activity in such con-structs (22). Therefore, in the present constructs, exon 1A orexon 1B was included after promoter P1 or P2 sequences,respectively, and exon 2 sequence to just before the ATG was

included in both (Fig. 8).The HKC-8 human proximal tubule cell line that expresses

the CASR and VDR (23) was used for the transient transfectionstudies. When transfected into COS-7 (African green monkeykidney) cells that do not express the CASR, both P1 and P2constructs demonstrated a transcriptional activity 7–8-foldthat of the pGL3 control (Fig. 9). When transfected into HKCcells, the activities of P1 and P2 were 11- and 33-fold that ofcontrol, respectively (Fig. 9).

Transcriptional Activities of P1 and P2 Are Up-regulated by1,25(OH)2D3—Addition of 10�8 M 1,25(OH)2D3 during the tran-sient transfection experiments stimulated transcriptional ac-tivity of P1 2-fold and of P2 2.5-fold (Fig. 10A). Custom pro-gramming of Mat Inspector version 2.2 (Genomatix Software)(24) with consensus VDR/RXR half-sites (see Table II in Ref.25) revealed potential VDREs in both P1 and P2 promoters.Promoter-luciferase reporter constructs were prepared inwhich the VDREs were mutated (see Fig. 8). When these con-structs were transfected into HKC cells, the 1,25(OH)2D3 -stim-ulated component of the transcriptional activity was lost (Fig.10A).

PI and P2 VDREs Confer 1,25(OH)2D3 Responsiveness to aHeterologous Promoter—Portions of the P1 and P2 promoterscontaining their respective VDREs (either wild type or mu-tated) were cloned upstream of the SV40 promoter driving theluciferase reporter gene in pGL3-Promoter vector (see Fig. 8).Wild-type P1 and P2 VDRE sequences conferred 1,25(OH)2D3

responsiveness on the heterologous promoter when transientlytransfected into HKC cells (Fig. 10B). The fold stimulation withthe vitamin D metabolite corresponds exactly to that obtainedwith the P1 and P2 VDREs in the context of their naturalpromoters (Fig. 10, compare A and B). Constructs with mutatedVDREs had lost the ability to confer 1,25(OH)2D3 responsive-ness to the heterologous promoter (Fig. 10B).

Similar Protein-DNA Complexes Form on the P1, P2, andMouse Osteopontin VDREs with VDR and RXR—EMSAs wereconducted with oligonucleotides representing the well charac-terized mOP VDRE, the P1 VDRE, or the P2 VDRE and VDRand RXR� prepared by in vitro transcription/translation (Fig.11). The protein-DNA complexes formed with the P1, P2, andmOP VDREs had similar electrophoretic mobilities, their for-mation was stimulated by ligand, and they were shifted in asimilar fashion by addition of antibodies against either theVDR or RXR� (Fig. 11). The VDR antibody N-20 reduced theintensity of the complex, whereas the VDR antibody C-20 su-pershifted it. The RXR� antibody 197 reduced the intensity ofthe complex, whereas the RXR� antibody D-20 supershifted it(Fig. 11). Addition of unlabelled oligonucleotides reduced theintensity of the labeled mOP, P1, and P2 VDRE-protein com-plexes in a similar manner (data not shown). When the EMSAswere conducted with labeled mutated mOP, P1, and P2 VDREs,the normal DNA-protein complexes failed to form (data notshown).

When EMSAs were conducted with HKC nuclear extract,similar ligand-induced protein-DNA complexes were formedwith the mOP and P2 VDRE oligonucleotides, which wereshifted by addition of VDR or RXR� antibodies (Fig. 12). Sim-ilar findings were obtained with the P1 VDRE oligonucleotideand the HKC nuclear extract (data not shown).

DISCUSSION

We have mapped the transcriptional start sites of promotersP1 and P2 of the human CASR gene. For P1 a TATA box is atnucleotide �26 and a CCAAT box is at �110 relative to thestart site. For P2, the transcriptional start site lies between twoSp1 sites, but the mechanisms that control initiation site se-lection of such GC-rich promoters lacking a TATA box are not

FIG. 9. Relative transcriptional activity of human CASR P1and P2 promoters in CASR-expressing (HKC) and non-express-ing (COS-7) cells. Cells were transfected with either pGL3, P1(P1-VDRE WT), or P2 (P2-VDRE WT) and luciferase activity measured asdescribed under “Experimental Procedures.”

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known. When transfected into COS-7 cells that do not expressthe CASR, both P1 and P2 demonstrated base-line transcrip-tional activity severalfold above that of the promoterless con-trol. Whereas the activity of P1 in human proximal tubule cells(HKC) that do express the CASR was similar to that in COS-7cells that do not express the CASR, that of P2 was markedlyincreased in the HKC cells, indicating that elements importantfor tissue-specific expression of the CASR gene are present inthis promoter.

Now that the CASR promoters have been defined, it is pos-sible to focus on the regulation of the CASR at the transcrip-tional level. In the present study we have focused on the mech-anism underlying the vitamin D stimulation of CASRexpression (5, 7) and show it to be a transcriptional one. First,we have demonstrated that 1,25(OH)2D3 up-regulates parathy-roid, thyroid, and kidney CASR mRNA levels in vivo. Theseobservations confirm and extend previous findings (5, 7). Sec-ond, we showed that human thyroid C-cell and kidney proximaltubule cell CASR gene transcription is increased by1,25(OH)2D3. Third, VDREs were identified in both promotersof the CASR gene; one is 380 bp upstream of the P1 transcrip-tional start site, and the other is 166 bp upstream of the P2

transcriptional start site. VDREs have been identified in sev-eral vitamin D-responsive genes and typically consist of two6-bp half-sites separated by 3 bp (25, 26). The VDREs of theCASR conform to this arrangement; however, they are atypicalin that the orientation of half-sites is inverted to that which isnormally found. VDREs of this type are found in the 24-hydrox-ylase gene (27–29).

Up-regulation of the parathyroid and kidney CASR by1,25(OH)2D would be physiologically relevant. In the parathy-roid, up-regulation of the CASR by 1,25(OH)2D would make thegland more responsive to extracellular calcium and for anygiven calcium concentration PTH secretion would be reduced.This would reinforce the direct negative effect of 1,25(OH)2D onPTH gene transcription (30, 31). Several studies in which renalfailure patients or aged populations were treated with1,25(OH)2D3 have shown a decrease in the calcium suppressioncurve and significant decrease in the calcium set-point in somecases (32–36). Parathyroid glands surgically removed from apatient with secondary hyperparathyroidism who had beentreated with a 1,25(OH)2D3 analogue intravenously showedup-regulation of CASR expression relative to parathyroidglands removed from similar patients not so treated (37). Al-

FIG. 10. Human CASR P1 and P2 promoters both have VDREs. HKC cells were transfected with the indicated constructs (see Fig. 8) in theabsence (�) or presence (�) of 10�8 M 1,25(OH)2D3. A, a comparison of the ability of P1 and P2 wild type and mutant VDREs to confertranscriptional responsiveness to 1,25(OH)2D3. B, a comparison of the ability of P1 and P2 wild type and mutant VDREs to confer transcriptionalresponsiveness to 1,25(OH)2D3 to a heterologous (SV40) promoter.

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though decreases in maximum PTH secretion are likely theresult of the direct negative effect of 1,25(OH)2D on PTH genetranscription, the improvement in parathyroid gland respon-siveness to calcium could be the result in part of increasedexpression of the CASR.

In the kidney, changes in serum calcium regulate productionof 1,25(OH)2D by affecting the activity of the proximal tubulemitochondrial cytochrome P450 25-hydroxyvitamin D-1�-hy-droxylase. In thyroparathyroidectomized rats in which PTHand phosphate were maintained at constant levels, an inversecorrelation was seen between serum calcium and 1,25(OH)2Dlevels, suggesting that calcium regulates 1,25(OH)2D inde-pendently of PTH (38–40). Calcium directly regulates1,25(OH)2D3 production in the human proximal tubular (HKC)cell line (23).

The 25-hydroxyvitamin D-1�-hydroxylase enzyme is prod-uct-inhibited. Therefore, after production of 1,25(OH)2D, theenzyme will be inhibited by several mechanisms including thedirect action of 1,25(OH)2D, the decreased level of serum PTHbrought about by the action of 1,25(OH)2D on the PTH gene,and by the increased sensitivity to serum calcium broughtabout by increased proximal tubule expression of the CASRimplied by the present study. In vitamin D deficiency, the

reduced CASR expression would help to ensure a maximumefficiency 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 rolein regulating Ca2� and Mg2� reabsorption. In the CTAL, theparacellular transport of cations is driven by a lumen-positivevoltage gradient set up by the activity of the apical Na�-K�-2Cl� cotransporter and K� channel (see Ref. 41). A hormonesuch as PTH activates its receptor on the basolateral surfaceincreasing intracellular cyclic AMP, which stimulates the Na�-K�-2Cl� cotransporter and cation reabsorption. Activation ofthe CASR on the basolateral surface inhibits adenylate cyclase,thereby inhibiting hormone-stimulated cation transport lead-ing to increased divalent cation excretion. The CASR also par-ticipates in transcellular cation reabsorption in the distal con-voluted tubule and increasing extracellular calcium ormagnesium stimulates intracellular Ca2� transients and in-hibits adenylate cyclase activity inhibiting hormone (e.g. PTH)-stimulated cation uptake (42, 43). Increased CASR expressionin the CTAL and distal convoluted tubule in response to1,25(OH)2D would stimulate calcium excretion.

Indeed, the findings of the present study offer some insightinto the special management problems of patients with auto-

FIG. 11. Comparison of protein-DNA complexes formed in gel retardation assays with oligonucleotides representing mOP, theCASR P2 and P1 VDREs, and in vitro transcribed/translated VDR and RXR�. Electrophoretic mobility shift assays were conducted asdescribed under “Experimental Procedures” and antibodies against VDR or RXR� were added as shown. The VDR/RXR� containing complex isindicated by an arrow (3).

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somal dominant hypocalcemia caused by activating mutationsin the CASR relative to other forms of hypoparathyroidism.Treatment with vitamin D metabolites fails to bring the serumcalcium up toward the lower limit of normal, whereas calciumexcretion is excessively stimulated potentially leading tonephrocalcinosis, nephrolithiasis, and renal damage (44, 45).With the demonstration of VDREs in the CASR gene, themechanism underlying the exuberant hypercalciuric responseto vitamin D metabolites in autosomal dominant hypocalcemiapatients now becomes clearer. The renal CASR is already toosensitive to divalent cations in these patients, and the situationis exacerbated when CASR expression is stimulated by 1�-hydroxylated vitamin D metabolite administration.

Hypercalcemia blunts renal concentrating ability, in partthrough CASR-activated signaling that antagonizes argininevasopressin actions. Vitamin D up-regulation of the renalCASR is likely to underlie the increased basal and vasopressin-elicited water and urea permeabilities in the inner medullarycortical ducts of rats made hypercalcemic with dihydrotachys-terol that mimics 1,25(OH)2D action (46). However, in autoso-mal dominant hypocalcemia patients with activating CASRgene mutations, the normal counter-regulatory mechanismsare clearly often insufficient to protect against the vitaminD-stimulated hypercalciuria leading to nephrocalcinosis (44,45).

Altered regulation of CASR expression by vitamin D may becritical in genetic hypercalciuria contributing to stone forma-tion. In kindreds predisposed to idiopathic hypercalciuria andcalcium nephrolithiasis, linkage of the trait to chromosome12q12–14 markers near the VDR locus was found (47). Thesame investigators found that markers flanking and within the

CASR locus on chromosome 3q13.3–21 were not linked to idi-opathic hypercalciuria (48). Linkage studies in a genetic hyper-calciuric stone-forming rat model, which demonstrates many ofthe features of human hypercalciuric nephrolithiasis, sug-gested a quantitative trait locus on chromosome 7q in a partthat encodes the VDR (49). The hypercalciuric rat demon-strates increased sensitivity of the VDR to 1,25(OH)2D3, lead-ing to a defect in renal calcium absorption (50). From thefindings of the present study, it would be predicted that ele-vated levels of CASR expression, secondary to enhanced vita-min D action, would be found in the hypercalciuric rat model,causing the increased urinary calcium excretion. Indeed,greater increases in 1,25(OH)2D3-stimulated renal CASRmRNA levels were found in the hypercalciuric rats relative tonormal rats (7).

Loss of CASR function, as occurs in the inherited disorderneonatal severe hyperparathyroidism because of homozygousinactivation of the CASR gene (51) or in the cases in whichheterozygous inactivation of the CASR gene causes familialhypocalciuric hypercalcemia with atypical hyperparathyroid-ism (52, 53) or familial isolated hyperparathyroidism (54), haveestablished the link between impaired parathyroid calciumsensing and dysregulated proliferation. Although somatic mu-tation of the CASR gene is not a significant factor in parathy-roid tumorigenesis (55–58), more than half of the parathyroidglands of patients with primary and severe uremic secondaryhyperparathyroidism show reduced CASR expression (13, 59–62). Thus, mutations in growth-regulating genes may second-arily alter the calcium set-point by decreasing expression of theCASR (63, 64). Evidence for this comes from a mouse model inwhich a cyclin D1 transgene is under the control of the PTH

FIG. 12. Comparison of protein-DNA complexes formed in gel retar-dation assays with oligonucleotidesrepresenting mOP and the CASR P2VDREs and HKC nuclear extract.Electrophoretic mobility shift assays wereconducted as described under “Experi-mental Procedures” and antibodiesagainst VDR or RXR� were added as indi-cated. The VDR/RXR� containing-com-plexes formed on the mOP and P2 oligo-nucleotides is indicated by an arrow (3).

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gene regulatory region (65). Parathyroid gland CASR expres-sion is reduced, the calcium set-point is shifted to the right,parathyroid enlargement occurs and serum calcium and PTHlevels are increased. However, the specific genes and precisemechanisms involved in down-regulation of parathyroid CASRexpression are not known.

The antiproliferative effects of vitamin D metabolites on theparathyroid gland are well documented. Patients with inher-ited disorders in which there is homozygous inactivation of theVDR or its ligand (and the corresponding mouse models ofthese disorders) manifest marked parathyroid hyperplasia (seeRef. 66 and references therein). However, like the CASR gene,somatic mutation of the VDR gene does not contribute to par-athyroid tumorigenesis (67, 68), but parathyroid VDR expres-sion is reduced in both primary and secondary hyperparathy-roid patients (69, 70). Thus the reduced CASR expression may,in part, be secondary to decreased VDR expression. Additionalevidence for the involvement of both the VDR and CASR incontrolling parathyroid function and/or growth comes fromstudies showing association of VDR and CASR gene polymor-phisms with primary and/or secondary uremic hyperparathy-roidism (71–73) and the parathyroid responsiveness to extra-cellular calcium in end stage renal disease (74, 75).

In summary, we have identified the transcriptional startsites of promoters P1 and P2 of the human CASR gene anddemonstrated functional VDREs in both promoters. Thus,1,25(OH)2D acting via the VDR is important for the tonicexpression of parathyroid and kidney CASR and helps to main-tain the normal functions of these tissues. Alterations in thevitamin D regulation of CASR expression are likely to contrib-ute to the development of primary and secondary hyperpara-thyroidism. Our studies provide the basis for further workinvestigating the link between vitamin D action and alteredCASR expression in the development of hyperparathyroid andhypercalciuric states.

Acknowledgments—We thank Drs. Bernard Turcotte and John H.White for insights into aspects of these studies and critical review of themanuscript. We thank Dr. Lise Binderup (Leo Pharmaceutical Prod-ucts, Ballerup, Denmark) and Dr. Milan Uskokovic (Hoffman LaRocheInc., Nutley, NJ) for providing 1,25(OH)2D3, Dr. Martin Hewison (Uni-versity of Birmingham, Birmingham, UK) for the HKC cells, Dr. GregorSutcliffe (The Scripps Research Institute, La Jolla, CA) for the cyclo-philin plasmid, and Martine Girard for technical assistance with theanimal studies.

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Lucie Canaff and Geoffrey N. HendyTO 1,25-DIHYDROXYVITAMIN D

PROMOTERS P1 AND P2 CONFER TRANSCRIPTIONAL RESPONSIVENESS Human Calcium-sensing Receptor Gene: VITAMIN D RESPONSE ELEMENTS IN

doi: 10.1074/jbc.M201804200 originally published online May 29, 20022002, 277:30337-30350.J. Biol. Chem. 

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