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Human Calcium-sensing Receptor Gene

VITAMIN D RESPONSE ELEMENTS IN PROMOTERS P1 AND P2 CONFER TRANSCRIPTIONAL RESPONSIVENESS TO 1,25-DIHYDROXYVITAMIN D*
  • Lucie Canaff
    Footnotes
    Affiliations
    From the Departments of Medicine, Physiology, and Human Genetics, McGill University and Royal Victoria Hospital, Montreal, Quebec H3A 1A1, Canada
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  • Geoffrey N. Hendy
    Correspondence
    To whom correspondence should be addressed: Calcium Research Laboratory, Rm. H4.67, Royal Victoria Hospital, 687 Pine Ave. W., Montreal, Quebec H3A 1A1, Canada. Tel.: 514-843-1632; Fax: 514-843-1712;
    Affiliations
    From the Departments of Medicine, Physiology, and Human Genetics, McGill University and Royal Victoria Hospital, Montreal, Quebec H3A 1A1, Canada
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  • Author Footnotes
    * This work was supported in part by Canadian Institutes of Health Research (CIHR) Grant MT-9315 and by a Kidney Foundation of Canada grant (to G. N. H.)The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EBI Data Bank with accession number(s) AY116081 and AY116082.
    ‡ Recipient of a doctoral fellowship from the CIHR and a National Cancer Institute of Canada research studentship.
Open AccessPublished:May 29, 2002DOI:https://doi.org/10.1074/jbc.M201804200
      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.
      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 response element
      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 medium
      Pipes
      1,4-piperazinediethanesulfonic acid
      RT
      reverse transcription
      TT
      human thyroid C-cell
      HKC
      human kidney proximal tubule cell
      DTT
      dithiothreitol
      FBS
      fetal bovine serum
      UTR
      untranslated region
      PMSF
      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 (
      • Brown E.M.
      ,
      • Bringhurst F.R.
      • Demay M.B.
      • Kronenberg H.M.
      ). 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 (
      • Brown E.M.
      • Gamba G.
      • Riccardi D.
      • Lombardi M.
      • Butter R.
      • Kifor O.
      • Sun A.
      • Hediger M.A.
      • Lytton J.
      • Hebert S.C.
      ). 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 (
      • Brown E.M.
      • Macleod R.J.
      ).
      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 (
      • Brown A.J.
      • Zhang M.
      • Finch J.
      • Ritter C.
      • McCracken R.
      • Morrissey J.
      • Slatopolsky E.
      ,
      • Rogers K.V.
      • Dunn C.V.
      • Conklin R.L.
      • Hadfield S.
      • Petty B.A.
      • Brown E.M.
      • Hebert S.C.
      • Fox J.
      ). 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 (
      • Brown A.J.
      • Zhang M.
      • Finch J.
      • Ritter C.
      • McCracken R.
      • Morrissey J.
      • Slatopolsky E.
      ). 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 (
      • Yao J.
      • Karnauskas A.J.
      • Bushinsky D.A.
      • Favus M.J.
      ). One study failed to find evidence for vitamin D modulation of CASR expression (
      • Rogers K.V.
      • Dunn C.V.
      • Conklin R.L.
      • Hadfield S.
      • Petty B.A.
      • Brown E.M.
      • Hebert S.C.
      • Fox J.
      ), 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 (
      • Pollak 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.
      ,
      • Pearce S.H.S.
      • Trump D.
      • Wooding C.
      • Besser G.M.
      • Chew S.
      • Heath D.
      • Hughes I.
      • Thakker R.V.
      ,
      • Heath 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.
      ) located on chromosome 3q13.3–21 (
      • Janicic N.
      • Soliman E.
      • Pausova Z.
      • Seldin M.F.
      • Riviere M.
      • Szpirer J.
      • Szpirer C.
      • Hendy G.N.
      ) 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 (
      • Garrett J.E.
      • Capuano I.V.
      • Hammerland L.G.
      • Hung B.C.P.
      • Brown E.M.
      • Hebert S.C.
      • Nemeth E.F.
      • Fuller F.
      ,
      • Chikatsu N.
      • Fukumoto S.
      • Takeuchi Y.
      • Suzawa M.
      • Obara T.
      • Matsumoto T.
      • Fujita T.
      ). 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.

      EXPERIMENTAL PROCEDURES

      Materials

      Protocols for obtaining human parathyroid tissue were approved by the local ethics committee. The human genomic library (945203, in λDASH II) was from Stratagene (La Jolla, CA). The COS-7 and human medullary thyroid carcinoma TT cell lines were from the American Type Culture Collection (Rockville, MD), and the human proximal kidney tubule cells (HKC-8) were a gift of Dr. Martin Hewison (University of Birmingham, Birmingham, UK). Dulbecco's modified Eagle's medium (DMEM), Ham's F-12 medium, fetal bovine serum (FBS), and antibiotics were from Invitrogen Canada (Burlington, Ontario, Canada). [γ-32P]Adenosine 5′-triphosphate and [α-32P]dUTP were from ICN Biomedicals (Baie d'Urfé, Quebec, Canada). Restriction enzymes, polynucleotide kinase, and Moloney murine leukemia virus reverse transcriptase were from MBI Fermentas (Burlington, Ontario, Canada). Hybond membranes and Ready-to-Go beads were from Amersham Biosciences (Baie d'Urfé, Quebec, Canada).

      Animals and Experimental Procedures

      Normal male Sprague-Dawley rats (Charles River Laboratories, Inc., St. Constant, Quebec, Canada) weighing 180–200 g when received, were fed a standard rodent chow (Ralston Purina Co., LaSalle, Quebec, Canada) containing 1.01% calcium, 0.74% phosphorus, and 3.3 IU/g vitamin D3. All animal experiments were carried out in compliance with, and were approved by, the institutional Animal Care and Use Committee. Rats were injected intraperitoneally, 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) (
      • Soliman E.
      • Canaff L.
      • Fox J.
      • Hendy G.N.
      ,
      • Canaff L.
      • Bennett H.P.J.
      • Hou Y.
      • Seidah N.G.
      • Hendy G.N.
      ). The rats were anesthetized with pentobarbital, the kidneys were taken, and the parathyroid and thyroid glands were microdissected separately and quick-frozen.

      Ribonuclease Protection Assay of Rat CASR mRNA

      For the CASR riboprobe, a 232-bp fragment of a rat CASR cDNA (
      • Canaff L.
      • Petit J.-L.
      • Kisiel M.
      • Watson P.H.
      • Gascon-Barré M.
      • Hendy G.N.
      ) was PCR amplified (forward primer, 5′-ACCTTGAGTTTTGTTGCCCA-3′ (in exon 3); reverse primer, 5′-GGAATGGTGCGGAGGAAGGATT-3′ (in exon 4)) and cloned into the PCR2.1 vector. For the actin riboprobe that protects a 126-base transcript, the pTRI-β-actin-125 rat vector (Ambion Inc., Austin, TX) was used. After linearization of the vectors, the antisense probes were in vitro transcribed with T7 polymerase incorporating [α-32P]UTP using a MAXIscript kit and the gel-purified riboprobes were used with an RPA III kit (Ambion Inc.). Each probe (2.5 × 105 cpm) was hybridized overnight with 2–25 μg of total RNA followed by digestion with a ribonuclease A:T1 mix (
      • D'Souza-Li L.
      • Canaff L.
      • Janicic N.
      • Cole D.E.C.
      • Hendy G.N.
      ). Protected fragments were resolved on 6% acrylamide denaturing gels and exposed to x-ray film.

      Nuclear Run-on Transcription Assays

      Relative transcription rates were measured using a nuclear run-on assay (
      • Mouland A.J.
      • Hendy G.N.
      ). Nuclei were prepared from 10–20 × 106 HKC or TT cells incubated with either 10−8m1,25(OH)2D3 or ethanol carrier alone. Cells were scraped into ice-cold PBS, 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 β-mercaptoethanol, 0.2% Nonidet P-40). After 8 min on ice, nuclei were pelleted at 800 × g. They were rinsed once with 1 ml of nuclei storage buffer (50% glycerol, 20 mm Tris, pH 7.9, 75 mm NaC1, 0.5 mmEDTA, 0.85 mm DTT, 0.125 mmphenylmethylsulfonyl fluoride (PMSF)), snap-frozen in liquid nitrogen, and stored at −80 °C until assay. Run-on reactions were carried out at 30 °C in 300 mmNH4(SO4)2, 100 mmTris-HC1, pH 7.9, 4 mm MgCl2, 4 mmMnCl2, 50 mm NaCl, 0.4 mm EDTA, 1.2 μm DTT, 0.1 mm PMSF, 10 mmcreatine phosphate, 29% glycerol, 150 μCi of [32P]UTP, 3000 Ci/mmol (ICN, Mississauga, Ontario, Canada), 1.5 mmeach of CTP, ATP, and GTP (MBI Fermentas) for 45 min. RNA was extracted with TRIzol (Invitrogen Canada) according to manufacturer's instructions. Five μg of plasmid DNA containing specific 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-bpAseI-StuI fragment cloned in pBluescript II KS; 2) human CASR exon 1B, a 230-bp NotI-StuI fragment cloned in pBluescript II KS; 3) human CASR exon 2, a 227-bpStuI-NcoI fragment cloned in pBluescript II KS; 4) human 24-hydroxylase, a 304-bp fragment RT-PCR-amplified from HKC 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 fragment cloned in plasmid pCD15.8.1 (
      • Danielson P.E.
      • Forss-Petter S.
      • Brow M.A.
      • Calaretta L.
      • Douglass J.
      • Milner R.J.
      • Sutcliffe J.G.
      ) (kindly provided by Dr. Gregor Sutcliffe, The Scripps Research Institute, La Jolla, CA). The membranes were hybridized with 2 × 107 cpm 32P-labeled transcripts in 50% formamide, 50 mm Hepes, pH 7.3, 0.75m NaC1, 2 mm EDTA, 0.5% SDS, 10× Denhardt's, and 20 μg/ml salmon sperm DNA for a minimum of 40 h. In any single experiment, equal numbers of counts were used for all conditions. Membranes were exposed to autoradiographic film, and quantitation of the relative rates of transcription was achieved by densitometry.

      Screening of the Human Genomic DNA Library

      Human CASR gene probes were generated by PCR amplifying normal human leukocyte DNA with primer Exon 1AF (5′-AGGCACCTGGCTGCAGCCAGGAAG-3′) and Exon 1BR (5′-GGTCTCCACGAGGATGAGCTCTGG-3′) to generate probe 1 of 784 bp and with primers Exon 2F (5′-GTGGCTTCCAAAGACTCAAGG-3′) and Exon 2R (5′ AGACAGCTAGGAGTTTGGAGG-3′) to generate probe 2 of 184 bp. The products were cloned into TA cloning vectors, maxipreps made, inserts excised, and random primer-labeled with [α-32P]dCTP. Each probe was used to screen one million clones of a human genomic library. Prehybridization and hybridization were performed in 40% formamide, 5× SSC, 25 mm sodium phosphate, pH 7.4, 1% SDS, 2 mm EDTA, 1× Denhardt's, and 200 μg/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 secondary and tertiary screening, and several positive plaques were obtained that represented two independent clones designated λghCASR1 and λghCASR2 (for probe 1) and two independent clones designated λghCASR3 and λghCASR4 (for probe 2).

      Restriction Enzyme Mapping and DNA Sequence Analysis

      Recombinant clones were digested withHindIII, and selected restriction fragments were subcloned into pBluescript II KS for DNA sequencing.

      RNA Extraction

      Total RNA was prepared from cells or tissues using TRIzol (Invitrogen Canada) according to the manufacturer's instructions.

      5′-RACE Amplification

      This was performed with a 5′-RACE system (Invitrogen Canada) according to the manufacturer's instructions. Five micrograms of human TT cell total RNA were reversed transcribed with Superscript II using a CASR exon 2-specific reverse primer (5′-ACATCATGCAGAGGCCTGGTGTGATGC-3′). The first strand cDNA was purified with a GlassMAX DNA isolation spin cartridge and homopolymer tailed with dCTP and terminal deoxynucleotidyltransferase. The tailed cDNA was amplified with Taq polymerase and a 5′-RACE anchor primer specific for the homopolymer tail and either an exon 1A (5′-TGCCGCAAGACCTCGGTGCTGGCA-3′) or exon 1B (5′-CTATGCCAAGGTCACGGTCTTGGA-3′) reverse primer. PCR conditions were initial denaturation (94 °C, 40 s) then 58 °C, 45 s, and 72 °C, 1 min for 30 cycles. PCR products were Topo-TA cloned into PCR2.1 and sequenced.

      Primer Extension Analysis of CASR mRNA

      One pmol of primer that had been labeled with [γ-32P]ATP using T4 polynucleotide kinase was incubated with 10 μg of either human TT cell or human parathyroid adenoma RNA or yeast tRNA (negative control) overnight at 60 °C in 10 mm Pipes, pH 6.4, 0.4m NaCl, 1 mm EDTA. The RNA templates were then reversed transcribed using 200 units of Moloney murine leukemia virus-reverse transcriptase. The extension products were phenol/chloroform-extracted, ethanol-precipitated, and analyzed on a 6% denaturing polyacrylamide gel in parallel with an unrelated sequencing reaction as a marker.

      Ribonuclease Protection Analysis of Human CASR mRNA

      For the riboprobes, portions of human genomic DNA were PCR amplified and TA-cloned into the PCR2.1 vector. The linearized vectors were in vitro transcribed with T7 polymerase incorporating [α-32P]UTP using a MAXIscript kit (Ambion). The gel-purified antisense probes (2 × 105 cpm) were hybridized overnight with 20 μg of TT cell DNase I-treated RNA followed by digestion with a ribonuclease A:T1 mix. Protected fragments were resolved on 6% polyacrylamide urea gels, which were dried and exposed to x-ray film overnight.

      Human CASR Gene Promoter Constructs

      To construct the P1-luciferase reporter plasmid, a 2.2-kb HindIII fragment containing exon 1A and 5′-flanking sequence was cloned into pBluescript KS such that the polylinker KpnI and SmaI sites flanked the 5′ and 3′ ends of the insert, respectively. By RT-PCR, a 468-bp fragment extending from a BssHII site in exon 1A to nucleotide −1 (nucleotide +1 is the A of the ATG initiation codon) in exon 2 was amplified from TT cell RNA. The forward primer was 5′-CGGGCCTCCAAGCAGCGCGCTGTGGA-3′ (the naturally occurring BssHII site is in boldface type), and the reverse primer was 5′-ACGATCCCGGGGGTTCTGCCGTCTCTCCAGGGCA-3′ (the added SmaI site is in boldface type). The PCR product was digested with BssHII and SmaI and cloned into theBssHII/SmaI-digested pBluescript KS plasmid described above. This construct was digested withKpnI/SmaI and the CASR P1 insert cloned into pGL3 basic to generate construct PI-VDRE WT.
      To construct the P2-luciferase reporter plasmid, a 2.0-kbHindIII fragment containing exon 1B was cloned into pBluescript KS such that the polylinker KpnI and SmaI sites flanked the 5′ and 3′ ends of the insert, respectively. By RT-PCR, a 350-bp fragment extending from aBssHII site in exon 1B to nucleotide −1 (see above) in exon 2 was amplified from TT cell RNA. The forward primer was 5′-GAGCGGGCTGCGCGCAGTCCTGAG-3′ (the naturally occurringBssHII site is in boldface type), and the reverse primer was 5′-ACGATCCCGGGGGTTCTGCCGTCTCTCCAGGGCA-3′ (the addedSmaI site is in boldface type). The PCR product was digested with BssHII and SmaI and cloned into theBssHII/SmaI-digested pBluescript KS plasmid described above. To complete the 5′ portion of the P2 promoter, a 331-bp fragment was amplified using λghCASR2 DNA. The forward primer was 5′-ACTGAGGTACCGTAAGAGTTTGGGCACGCGAT-3′ (the addedKpnI site is in boldface type), and the reverse primer was 5′-GACCCTGAAGAGTCAGCTAAGCCTCTCTG-3′ (the naturally occurringEspI site is in boldface type). The PCR product was digested with KpnI and SmaI and cloned into theKpnI/EspI-digested pBluescript KS plasmid described above. The entire P2-containing insert was excised withKpnI and SmaI and cloned into pGL3 basic to generate construct P2-VDRE WT.
      The P1-VDRE MUT and P2-VDRE MUT constructs were generated using the QuikChange XL site-directed mutagenesis kit (Stratagene, San Diego, CA). For each mutagenesis, a pair of complementary primers were designed with the mutant base pair in the middle. The primers used were: P1 forward (5′-TGCTTTAGCATTTGCTCATTTCCTTCTTTTACCCTGTATTTGAGGGA-3′), P1 reverse (5′-TCCCTCAAATACAGGGTAAAAGAAGGAAATGAGCAAATGCTAAAGCA-3′), P2 forward (5′-CTCGGGGAACCGAAGACGCGCTTTCAGCGATTCTGAAGAGTCAGCTAAGC-3′), P2 reverse (5′-GCTTAGCTGACTCTTCAGAATCGCTGAAAGCGCGTCTTCGGTTCCCCGAG-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 of extension were performed withPfu Turbo DNA polymerase, and the template was digested withDpnI. The reactions were used to transform XL10-Gold Ultracompetent cells, which incorporate nicked DNA and repair it. The correctness of all constructs was confirmed by sequence analysis.
      To construct the P1-heterologous promoter luciferase reporter plasmids, 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 reverse primer P1R (5′-tctgaCTCGAGATGGCCAAGTTCTGCCCATTTG-3′), where the nucleotides in boldface type are an NheI or XhoI site and the lowercase nucleotides are those added to ensure complete restriction enzyme cleavage. The PCR products were digested withNheI and XhoI and cloned into the pGL3-Promoter vector (Promega) upstream of the SV40 promoter to generate constructs SV-40 P1-VDRE WT and SV-40 PI-VDRE MUT.
      To construct the P2-heterologous promoter luciferase reporter plasmids, 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 reverse primer P2R (5′-tctgaCTCGAGTCACCGTCTCCTTAGCCCGCAG-3′), where the nucleotides in boldface type are an NheI or XhoI site and the lowercase nucleotides are those added to ensure complete restriction enzyme cleavage. The PCR products were digested withNheI and XhoI and cloned into the pGL3-Promoter vector upstream of the SV40 promoter to generate constructs SV-40 P2-VDRE WT and SV-40 P2-VDRE MUT.

      Cell Culture

      COS-7 and HKC cells were grown in DMEM supplemented with 10% FBS. TT cells were cultured in RPMI 1640 medium with 10% FBS and 5% horse serum. All maintenance medium contained 100 units/ml penicillin, 100 μg/ml streptomycin, and 250 ng/ml amphotericin B.

      Transfection and Reporter Assay

      For transient transfection, cells were trypsinized, plated in six-well dishes in DMEM plus 10% FBS (1–4 × 105 cells/well) and incubated overnight. The next day, cells were transfected with 30 μg/well Superfect reagent with 1 μg of CASR promoter construct and 0.5 μg/well pCH110. The following day, cells were serum-starved in DMEM overnight and cultured with or without 10−8m1,25(OH)2D3 for 10h. The cells were washed in PBS and lysed in 250 μl of lysis buffer (1% Triton X-100, 15 mm MgSO4, 4 mm EGTA, 1 mm DTT, 25 mm glycyl glycine) on ice. Luciferase activity was measured in an EG&G Berthold luminometer using 45 μl of cell lysate and d-luciferin. Luciferase activity was normalized to β-galactosidase activity.

      Statistics

      Data are expressed as mean ± S.E. The results from the in vivo 1,25(OH)2D3response studies were initially subjected to analysis of variance. The significance of differences from base line was then determined using Dunnett's multiple comparison test. For the nuclear run-on assays and luciferase transient transfection assays, comparisons were performed by Student's t test. A p value of <0.05 was taken to indicate a statistically significant difference.

      In Vitro Transcription and Translation

      Plasmids VDR/pSG5 and RXRα/pSG5 (
      • Ferrara J.
      • McCuaig K.
      • Hendy G.N.
      • Uskokovic M.
      • White J.H.
      ) were transcribed with T7 RNA polymerase and translated in vitro with the TnT reticulocyte lysate system according to the manufacturer's instructions.

      Nuclear Extracts of HKC Cells

      Cells were washed, scraped into 1 ml of PBS, and centrifuged at 1500 × g for 10 min at 4 °C. Cell pellets were processed 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 mmEDTA, 0.5 mm PMSF, and protease inhibitors). Nuclear pellets were obtained by centrifugation (25,000 × gfor 20 min at 4 °C), resuspended in 20 mm HEPES, pH 7.9, 25% glycerol, 0.42 m NaCl, 1.5 mmMgCl2, 0.2 mm EDTA, 0.5 mm PMSF, 0.5 mm DTT, and Dounce homogenized. After a 20-min centrifugation at 25,000 × g, nuclear extracts (supernatants) were dialyzed for 5 h against 20 nmHEPES, pH 7.9, 20% glycerol, 0.1 m KCl, 0.2 mmEDTA, 0.5 mm PMSF, 0.5 mm DTT. Protein content was determined and aliquots stored at −80 °C.

      Oligonucleotides and Antibodies

      Oligonucleotides used were as follows: CASRP1-VDRE-WT (5′-AGGGTAGGAGAAGGAGCTGAGCAAT-3′), CASRPI-VDRE-MUT (5′-AGGGTAAAAGAAGGAAATGAGCAAT-3′), CASRP2-VDRE-WT (5′-CTTCAGGGTCGCTGAGGGCGCGTCT-3′), CASRP2-VDRE-MUT (5′-CTTCAGAATCGCTGAAAGCGCGTCT-3′), and MOP-WT (5′-GTACAAGGTTCACGAGGTTCACGTC-3′). Only the sense strand is shown; half-sites are in boldface type, and mutant nucleotides are underlined. Antibodies against the VDR (VDR N-20, VDR C-20) and the RXRα (RXRαΔN197, RXRαD-20) were from Santa Cruz Biotechnology.

      Electrophoretic Mobility Shift Assay

      Two micrograms of nuclear extract or VDR/RXR in vitro translated products were incubated for 20 min on ice in the absence or presence of 10−8m 1,25(OH)2D3 and 1 μg of 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, and samples were incubated for 20 min at room temperature. Five fmol of 32P-end-labeled double-stranded oligonucleotides were added and incubated for an additional 20 min. Samples were electrophoresed at 8 V/cm on 6% nondenaturing polyacrylamide gels equilibrated in 0.25 mTris, pH 8.3, 1.9 m glycine, 10 mm EDTA, dried, and autoradiographed.

      RESULTS

      1,25(OH)2D3 Up-regulates Parathyroid, Thyroid, and Kidney CASR mRNA Levels in Vivo

      As previous studies aimed at assessing whether 1,25(OH)2D3regulates CASR mRNA levels in vivo have produced inconsistent results, we conducted our own analysis. After a single intraperitoneal injection of 1,25(OH)2D3 in rats, parathyroid, thyroid, and kidney CASR mRNA levels rose to 2–2.5-fold over basal level at 15 h and had returned to base line at 24 h (Fig. 1). Injection of vehicle had no effect on CASR mRNA levels (data not shown).
      Figure thumbnail gr1
      FIG. 1Induction of parathyroid, thyroid, and kidney CASR mRNA by 1,25(OH)2D3. Rats were injected intraperitoneally with 100 ng of 1,25(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 ribonuclease protection analysis signals are shown for each time point. CASR and actin mRNA levels were assessed by densitometry and each value is the mean ± S.E. (n = 3).Asterisks indicate a significant difference (p < 0.05) from the time 0 value.

      1,25(OH)2D3 Increases CASR Gene Transcription

      The results of nuclear run-on assays performed on extracts of human TT and HKC cells cultured with and without 1,25(OH)2D3 for 8 and 12 h, respectively, are shown in Fig. 2. CASR gene exon 1A, exon 1B, and exon 2 transcripts were all stimulated >2-fold, as was 24-hydroxylase gene transcription in both cell types. Cyclophilin gene transcription was unaffected by 1,25(OH)2D3.
      Figure thumbnail gr2
      FIG. 2Induction of CASR gene transcription by 1,25(OH)2D3. Nuclear run-on assays were performed as described under “Experimental Procedures” on nuclear extracts of TT cells, panel A, and HKC cells, panel B. Autoradiographs of representative experiments repeated three times are shown. Densitometry was performed and relative transcription rates calculated taking CASR exon 1A as 100%.

      Cloning of CASR Genomic Clones

      Several positive phage plaques were identified after the initial screening of the human genomic library. Two independent overlapping clones isolated with probe 1 (exons 1A and 1B) were designated λghCASR1 and λghCASR2, and two overlapping clones isolated with probe 2 (exon 2) were designated λghCASR3 and λghCASR4 (Fig. 3, A and B). Insert sizes were 18–20 kb. The 2.2- and 1.9-kb HindIII fragments encoding exons 1A and 1B, respectively, were subcloned and completely sequenced. These were used to generate CASR promoter/luciferase reporter gene constructs (see below).
      Figure thumbnail gr3
      FIG. 3Physical map of human DNA encoding the 5′ part of the CASR gene. Panel A, four independent bacteriophage recombinant clones, λghCASR1–4, were isolated after screening a human genomic DNA as described under “Experimental Procedures.” A map of the 5′ portion of the CASR gene is shown with the positions of library screening probes marked. Panel B, promoter P1, exon 1A, promoter P2, exon 1B, and exon 2 showing 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).

      Characterization of Transcription Initiation Sites

      A previous study (
      • Chikatsu N.
      • Fukumoto S.
      • Takeuchi Y.
      • Suzawa M.
      • Obara T.
      • Matsumoto T.
      • Fujita T.
      ) using only 5′-RACE found a putative start site for exon 1A some 105 bp downstream of a putative TATA box. This suggested that the actual start site might lie upstream of that previously identified. We used 5′-RACE, primer extension, and RNase protection analyses of human parathyroid and TT cell RNA to map the sites of transcription initiation at the 5′-ends of exons 1A and 1B. 5′-RACE of TT cell RNA and an exon 1A primer yielded products of greater than 500 bp (Fig.4A). After subcloning and sequencing several subclones, the longest was found to be 548 bp. Although this extended the sequence of exon 1A some 19 bp upstream of that previously determined (
      • Chikatsu N.
      • Fukumoto S.
      • Takeuchi Y.
      • Suzawa M.
      • Obara T.
      • Matsumoto T.
      • Fujita T.
      ), it was still 86 bp from the TATA box.
      Figure thumbnail gr4
      FIG. 4Mapping the CASR P1 promoter transcription start site. Panel A, 5′-RACE; 5 μg human TT cell total RNA were reversed transcribed using an exon 2 specific primer as described under “Experimental Procedures.” After PCR amplification with an exon 1A-specific primer a 548-bp product was obtained (lane 2). DNA size markers (lane 1) and a PCR blank (lane 3) are shown. Panel B, primer extension; ten micrograms human TT cell total RNA or yeast tRNA (control) were annealed with an excess of polynucleotide kinase-labeled oligonucleotide and the extension reaction performed as described under “Experimental Procedures.” The products were analyzed on a denaturing gel. A DNA sequence ladder served as size markers (lanes 1–4) and the arrow indicates the extension product of 271 nucleotides obtained with the human thyroid TT cell mRNA (lane 5). The yeast tRNA control was run inlane 6. Panel C, RNase protection analysis.Upper panel, schematic representation of probes used.Lower panel, products were analyzed on a denaturing gel.Lanes 1 and 2, undigested P1A and P1B probes; lanes 3–5, RNase I-digested P1A probe;lanes 6–8, RNase I-digested P1B probe; lanes 3and 6, PT, parathyroid RNA; lanes 4 and 7, TT cell RNA; lanes 5 and 8, yeast tRNA.
      Primer extension analysis with TT cell RNA yielded a single major extension product of 271 bases (Fig. 4B). This places the transcriptional start site at the A nucleotide 27 bp downstream of the TATA box within a TTATTCT sequence (Fig.5), which matches the consensus for this type of start site (
      • Carey M.
      ). RNase protection analysis of human parathyroid and TT cell RNA with two different riboprobes generated protected products of 292 and 170 bases from the longer and shorter riboprobes, respectively (Fig. 4C). In both cases, the result was fully consistent with that obtained with the primer extension analysis.
      Figure thumbnail gr5
      FIG. 5Sequence 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 . The positions of primer P1-RACE used for 5′-RACE and primer P1-PE used for primer extension analyses are indicated. The transcription initiation site mapped by primer extension and RNase is marked as +1. TATA, CAAT and VDRE homologies are inbold and boxed. HindIII sites and the complete sequence of exon 1A are in bold. The beginning of partial exon 1A sequence obtained from a previously reported human CASR cDNA (
      • Garrett J.E.
      • Capuano I.V.
      • Hammerland L.G.
      • Hung B.C.P.
      • Brown E.M.
      • Hebert S.C.
      • Nemeth E.F.
      • Fuller F.
      ) is indicated by the open arrowhead. Theclosed arrowhead indicates the longest cDNA obtained by 5′-RACE in the present study. The asterisk (*) marks a putative transcription start site obtained by 5′-RACE in a previous study (
      • Chikatsu N.
      • Fukumoto S.
      • Takeuchi Y.
      • Suzawa M.
      • Obara T.
      • Matsumoto T.
      • Fujita T.
      ).
      5′-RACE of TT cell RNA and an exon 1B primer yielded products of ∼150 bp (Fig. 6A). After subcloning and sequencing several subclones, the longest was found to be 158 bp. This extended the sequence of exon 1B some 9 bp upstream of that determined previously (
      • Chikatsu N.
      • Fukumoto S.
      • Takeuchi Y.
      • Suzawa M.
      • Obara T.
      • Matsumoto T.
      • Fujita T.
      ). Primer extension analysis with TT cell and parathyroid RNA yielded in each case a single major extension product of 153 bases (Fig. 6B). This coincided precisely with the assignment from the 5′-RACE. RNase protection analysis of human parathyroid and TT cell RNA with two different riboprobes generated protected products of 138 and 107 bases from the longer and shorter riboprobes, respectively (Fig. 6C). These results were fully consistent with those obtained with the other techniques. Thus, for exon 1B all three methods used identified the same transcription start site. The P2 promoter lacks a TATA box and is GC-rich, with one Sp1 site located 11 bp upstream and a second Sp1 site located 3 bp downstream of the transcription start site (Fig.7).
      Figure thumbnail gr6
      FIG. 6Mapping the human CASR P2 promoter transcription start site. Panel A, 5′-RACE; 5 μg human TT cell total RNA were reversed transcribed using an exon 2 specific primer as described under “Experimental Procedures.” After PCR amplification with an exon 1B-specific primer 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 μg human TT cell or parathyroid total RNA or yeast tRNA (control) were annealed with an excess of labeled oligonucleotide P2-PE and the extension reaction performed as described under “Experimental Procedures”. The products were analyzed on a denaturing gel. A DNA sequence ladder served as 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. Lower panel, products were analyzed on denaturing gel. Lanes 1 and 2, undigested P2A and P2B probes; lanes 3–5, RNase I-digested P2A probe; lanes 6–8, 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, DNA sequence ladder.
      Figure thumbnail gr7
      FIG. 7Sequence of the human CASR gene P2 promoter, exon 1B, and part of intron 1. The nucleotide sequence was determined as described under “Experimental Procedures” from a subcloned HindIII genomic fragment indicated in . The positions of primer P2-RACE used for 5′-RACE and primer P2-PE used for primer extension analyses are indicated. The transcription initiation site mapped by 5′-RACE, primer extension and RNase protection is marked as +1. VDRE and Sp1 homologies are in bold and boxed. Hind III sites and the complete sequence of exon 1B are in bold. The beginning of partial exon 1B sequence obtained from a previously reported human CASR cDNA (
      • Garrett J.E.
      • Capuano I.V.
      • Hammerland L.G.
      • Hung B.C.P.
      • Brown E.M.
      • Hebert S.C.
      • Nemeth E.F.
      • Fuller F.
      ) is indicated by the open arrowhead. The asterisk (*) marks a putative transcription start site obtained by 5′-RACE in a previous study (
      • Chikatsu N.
      • Fukumoto S.
      • Takeuchi Y.
      • Suzawa M.
      • Obara T.
      • Matsumoto T.
      • Fujita T.
      ).

      Human CASR P1 and P2 Promoters Are Active in Human Proximal Tubule Kidney Cells

      To analyze their transcriptional activities, constructs were prepared in which P1 and P2 promoters drive transcription of the luciferase gene. We have noted previously that addition of 5′-UTR exonic sequence may help to promote optimal transcriptional activity in such constructs (
      • Canaff L.
      • Bevan S.
      • Wheeler D.G.
      • Mouland A.J.
      • Rehfuss R.P.
      • White J.H.
      • Hendy G.N.
      ). Therefore, in the present constructs, exon 1A or exon 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).
      Figure thumbnail gr8
      FIG. 8Human CASR gene promoter constructs. Panel A, promoter P1, exon 1A, promoter P2, exon 1B, and exon 2 showing the alternative splicing 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 gene promoter 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 upstream of 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 reporter gene in the pGL3-Promoter vector to create SV40 P1-VDRE WT through SV40 P2-VDRE MUT.
      The HKC-8 human proximal tubule cell line that expresses the CASR and VDR (
      • Bland R.
      • Walker E.A.
      • Hughes S.V.
      • Stewart P.M.
      • Hewison M.
      ) was used for the transient transfection studies. When transfected into COS-7 (African green monkey kidney) cells that do not express the CASR, both P1 and P2 constructs demonstrated a transcriptional activity 7–8-fold that of the pGL3 control (Fig.9). When transfected into HKC cells, the activities of P1 and P2 were 11- and 33-fold that of control, respectively (Fig. 9).
      Figure thumbnail gr9
      FIG. 9Relative transcriptional activity of human CASR P1 and P2 promoters in CASR-expressing ( HKC ) and non-expressing (COS-7) cells. Cells were transfected with either pGL3, P1(P1-VDRE WT), or P2 (P2-VDRE WT) and luciferase activity measured as described under “Experimental Procedures.”

      Transcriptional Activities of P1 and P2 Are Up-regulated by 1,25(OH)2D3

      Addition of 10−8m 1,25(OH)2D3 during the transient transfection experiments stimulated transcriptional activity of P1 2-fold and of P2 2.5-fold (Fig.10A). Custom programming of Mat Inspector version 2.2 (Genomatix Software) (
      • Quandt K.
      • Frech K.
      • Karas H.
      • Wingender E.
      • Werner T.
      ) with consensus VDR/RXR half-sites (see Table II in Ref.
      • Haussler M.R.
      • Whitfield G.R.
      • Haussler C.A.
      • Hsieh J.-C.
      • Thompson P.D.
      • Selznick S.H.
      • Dominguez C.E.
      • Jurutka P.U.
      ) revealed potential VDREs in both P1 and P2 promoters. Promoter-luciferase reporter constructs were prepared in which the VDREs were mutated (see Fig. 8). When these constructs were transfected into HKC cells, the 1,25(OH)2D3 -stimulated component of the transcriptional activity was lost (Fig. 10A).
      Figure thumbnail gr10
      FIG. 10Human CASR P1 and P2 promoters both have VDREs. HKC cells were transfected with the indicated constructs (see ) in the absence (−) or presence (+) of 10−8m 1,25(OH)2D3. A, a comparison of the ability of P1 and P2 wild type and mutant VDREs to confer transcriptional responsiveness to 1,25(OH)2D3. B, a comparison of the ability of P1 and P2 wild type and mutant VDREs to confer transcriptional responsiveness to 1,25(OH)2D3to a heterologous (SV40) promoter.

      PI and P2 VDREs Confer 1,25(OH)2D3Responsiveness to a Heterologous Promoter

      Portions of the P1 and P2 promoters containing their respective VDREs (either wild type or mutated) were cloned upstream of the SV40 promoter driving the luciferase 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 transiently transfected into HKC cells (Fig.10B). The fold stimulation with the vitamin D metabolite corresponds exactly to that obtained with the P1 and P2 VDREs in the context of their natural promoters (Fig. 10, compare A and B). Constructs with mutated VDREs had lost the ability to confer 1,25(OH)2D3 responsiveness to the heterologous promoter (Fig. 10B).

      Similar Protein-DNA Complexes Form on the P1, P2, and Mouse Osteopontin VDREs with VDR and RXR

      EMSAs were conducted with oligonucleotides representing the well characterized mOP VDRE, the P1 VDRE, or the P2 VDRE and VDR and RXRα prepared by in vitrotranscription/translation (Fig. 11). The protein-DNA complexes formed with the P1, P2, and mOP VDREs had similar electrophoretic mobilities, their formation was stimulated by ligand, and they were shifted in a similar fashion by addition of antibodies against either the VDR or RXRα (Fig. 11). The VDR antibody N-20 reduced the intensity of the complex, whereas the VDR antibody C-20 supershifted it. The RXRα antibody Δ197 reduced the intensity of the complex, whereas the RXRα antibody D-20 supershifted it (Fig.11). Addition of unlabelled oligonucleotides reduced the intensity of the labeled mOP, P1, and P2 VDRE-protein complexes in a similar manner (data not shown). When the EMSAs were conducted with labeled mutated mOP, P1, and P2 VDREs, the normal DNA-protein complexes failed to form (data not shown).
      Figure thumbnail gr11
      FIG. 11Comparison of protein-DNA complexes formed in gel retardation assays with oligonucleotides representing mOP , the CASR P2 and P1 VDREs, and in vitrotranscribed/translated VDR and RXR α.Electrophoretic mobility shift assays were conducted as described under “Experimental Procedures” and antibodies against VDR or RXRα were added as shown. The VDR/RXRα containing complex is indicated by an arrow (→).
      When EMSAs were conducted with HKC nuclear extract, similar ligand-induced protein-DNA complexes were formed with the mOP and P2 VDRE oligonucleotides, which were shifted by addition of VDR or RXRα antibodies (Fig. 12). Similar findings were obtained with the P1 VDRE oligonucleotide and the HKC nuclear extract (data not shown).
      Figure thumbnail gr12
      FIG. 12Comparison of protein-DNA complexes formed in gel retardation assays with oligonucleotides representing mOP and the CASR P2 VDREs and HKC nuclear extract. Electrophoretic mobility shift assays were conducted as described under “Experimental Procedures” and antibodies against VDR or RXRα were added as indicated. The VDR/RXRα containing-complexes formed on the mOP and P2 oligonucleotides is indicated by an arrow (→).

      DISCUSSION

      We 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 (
      • Brown A.J.
      • Zhang M.
      • Finch J.
      • Ritter C.
      • McCracken R.
      • Morrissey J.
      • Slatopolsky E.
      ,
      • Yao J.
      • Karnauskas A.J.
      • Bushinsky D.A.
      • Favus M.J.
      ) 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 (
      • Brown A.J.
      • Zhang M.
      • Finch J.
      • Ritter C.
      • McCracken R.
      • Morrissey J.
      • Slatopolsky E.
      ,
      • Yao J.
      • Karnauskas A.J.
      • Bushinsky D.A.
      • Favus M.J.
      ). 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 (
      • Haussler M.R.
      • Whitfield G.R.
      • Haussler C.A.
      • Hsieh J.-C.
      • Thompson P.D.
      • Selznick S.H.
      • Dominguez C.E.
      • Jurutka P.U.
      ,
      • Toell A.
      • Polly P.
      • Carlberg C.
      ). 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 (
      • Ohyama Y.
      • Ozono K.
      • Uchida M.
      • Shinki T.
      • Kato S.
      • Suda T.
      • Yamamoto O.
      • Noshiro M.
      • Kato Y.
      ,
      • Hahn C.N.
      • Kerry D.M.
      • Omdahl J.L.
      • May B.K.
      ,
      • Zierold C.
      • Darwish H.M.
      • DeLuca H.F.
      ).
      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 (
      • Silver J.
      • Naveh-Many T.
      • Mayer H.
      • Schmelzer H.J.
      • Popovtzer M.M.
      ,
      • Demay M.B.
      • Kiernan M.S.
      • DeLuca H.F.
      • Kronenberg H.M.
      ). 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 (
      • Delmez J.A.
      • Tindira C.
      • Grooms P.
      • Dusso A.
      • Windus D.W.
      • Slatopolsky E.
      ,
      • Dunlay R.
      • Rodriguez M.
      • Felsenfeld A.J.
      • Llach F.
      ,
      • Kwan J.T.C.
      • Almond M.K.
      • Beer J.C.
      • Noonan K.
      • Evans S.J.W.
      • Cunningham J.
      ,
      • Malberti F.
      • Surian M.
      • Cosci P.
      ,
      • Ledger G.A.
      • Burritt M.F.
      • Kao P.C.
      • Fallon W.M.
      • Riggs B.L.
      • Khosla S.
      ). 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 (
      • Shiraishi K.
      • Tsuchida M.
      • Wada T.
      • Yoshihiro S.
      • Takai K.
      • Suga A.
      • Kaneda Y.
      • Naito K.
      ). 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 (
      • Trechsel V.
      • Eisman J.A.
      • Fischer J.A.
      • Bonjour J.-P.
      • Fleisch H.
      ,
      • Matsumoto T.
      • Ikeda K.
      • Morita K.
      • Fukomoto S.
      • Takahashi H.
      • Ogata E.
      ,
      • Weisinger J.R.
      • Favus M.J.
      • Langman C.B.
      • Bushinsky D.A.
      ). Calcium directly regulates 1,25(OH)2D3production in the human proximal tubular (HKC) cell line (
      • Bland R.
      • Walker E.A.
      • Hughes S.V.
      • Stewart P.M.
      • Hewison M.
      ).
      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.
      • Brown E.M.
      • Pollak M.
      • Hebert S.C.
      ). 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 (
      • Bapty B.W.
      • Dai L-J.
      • Ritchie G.
      • Jirik F.
      • Canaff L.
      • Hendy G.N.
      • Quamme G.A.
      ,
      • Bapty B.W.
      • Ritchie G.
      • Canaff L.
      • Hendy G.N.
      • Quamme G.A.
      ). 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 (
      • Pearce 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.
      ,
      • Lienhardt A.
      • Bai M.
      • Lagarde J.-P.
      • Rigaud M.
      • Zhang Z.
      • Jiang Y.
      • Kottler M.-L.
      • Brown E.M.
      • Garabedian M.
      ). 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 (
      • Sands J.M.
      • Flores F.X.
      • Kato A.
      • Baum M.A.
      • Brown E.M.
      • Ward D.T.
      • Hebert S.C.
      • Harris H.W.
      ). However, in autosomal dominant hypocalcemia patients with activating CASR gene mutations, the normal counter-regulatory mechanisms are clearly often insufficient to protect against the vitamin D-stimulated hypercalciuria leading to nephrocalcinosis (
      • Pearce 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.
      ,
      • Lienhardt A.
      • Bai M.
      • Lagarde J.-P.
      • Rigaud M.
      • Zhang Z.
      • Jiang Y.
      • Kottler M.-L.
      • Brown E.M.
      • Garabedian M.
      ).
      Altered regulation of CASR expression by vitamin D may be critical in genetic hypercalciuria contributing to stone formation. In kindreds predisposed to idiopathic hypercalciuria and calcium nephrolithiasis, linkage of the trait to chromosome 12q12–14 markers near the VDR locus was found (
      • Scott P.
      • Ouimet D.
      • Valiquette L.
      • Guay G.
      • Proulx Y.
      • Trouvé M.L.
      • Gagnon B.
      • Bonnardeux A.
      ). The same investigators found that markers flanking and within the CASR locus on chromosome 3q13.3–21 were not linked to idiopathic hypercalciuria (
      • Petrucci M.
      • Scott P.
      • Ouimet D.
      • Trouvé M.L.
      • Proulx Y.
      • Valiquette L.
      • Guay G.
      • Bonnardeaux A.
      ). Linkage studies in a genetic hypercalciuric stone-forming rat model, which demonstrates many of the features of human hypercalciuric nephrolithiasis, suggested a quantitative trait locus on chromosome 7q in a part that encodes the VDR (
      • Hoopes R.Jr.
      • Reid R.
      • Thakker R.
      • Szpirer C.
      • Bushinsky D.
      • Scheinman S.
      ). The hypercalciuric rat demonstrates increased sensitivity of the VDR to 1,25(OH)2D3, leading to a defect in renal calcium absorption (
      • Yao J.
      • Kathpalia P.
      • Bushinsky D.A.
      • Favus M.J.
      ). From the findings of the present study, it would be predicted that elevated levels of CASR expression, secondary to enhanced vitamin 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 CASR mRNA levels were found in the hypercalciuric rats relative to normal rats (
      • Yao J.
      • Karnauskas A.J.
      • Bushinsky D.A.
      • Favus M.J.
      ).
      Loss of CASR function, as occurs in the inherited disorder neonatal severe hyperparathyroidism because of homozygous inactivation of the CASR gene (
      • Pollak M.R.
      • Chou Y.-H., W.
      • Marx S.J.
      • Steinman B.
      • Cole D.E.C.
      • Brandi M.L.
      • Papapoulos S.E.
      • Menko F.H.
      • Hendy G.N.
      • Brown E.M.
      • Seidman C.E.
      • Seidman J.G.
      ) or in the cases in which heterozygous inactivation of the CASR gene causes familial hypocalciuric hypercalcemia with atypical hyperparathyroidism (
      • Soei Y.L.
      • Karperien M.
      • Bakker B.
      • Breuning M.H.
      • Hendy G.N.
      • Papapoulos S.E.
      ,
      • Carling T.
      • Szabo E.
      • Bai M.
      • Ridefelt P.
      • Westin G.
      • Gustavsson P.
      • Trivedi S.
      • Hellman P.
      • Brown E.M.
      • Dahl B.
      • Rastad J.
      ) or familial isolated hyperparathyroidism (
      • Simonds W.F.
      • James-Newton L.A.
      • Agarwal S.K.
      • Yang B.
      • Skarulis M.C.
      • Hendy G.N.
      • Marx S.J.
      ), have established the link between impaired parathyroid calcium sensing and dysregulated proliferation. Although somatic mutation of the CASR gene is not a significant factor in parathyroid tumorigenesis (
      • Hosokawa Y.
      • Pollak M.R.
      • Brown E.M.
      • Arnold A.
      ,
      • Thompson D.B.
      • Samowitz W.S.
      • Odelberg S.
      • Davis R.K.
      • Szabo J.
      • Heath III, H.
      ,
      • Degenhardt S.
      • Toell A.
      • Weidmann W.
      • Dotzenrath C.
      • Spindler K.D.
      ,
      • Cetani F.
      • Pinchera A.
      • Pardi E.
      • Cianferotti L.
      • Vignali E.
      • Picone E.
      • Miccoli P.
      • Viacava P.
      • Marcocci C.
      ), more than half of the parathyroid glands of patients with primary and severe uremic secondary hyperparathyroidism show reduced CASR expression (
      • Chikatsu N.
      • Fukumoto S.
      • Takeuchi Y.
      • Suzawa M.
      • Obara T.
      • Matsumoto T.
      • Fujita T.
      ,
      • Kifor O.
      • Moore F.D.
      • Wang P.
      • Goldstein M.
      • Vassilev P.
      • Kifor I.
      • Hebert S.
      • Brown E.M.
      ,
      • Farnebo F.
      • Enberg U.
      • Grimelius L.
      • Backdahl M.
      • Schalling M.
      • Larsson C.
      • Farnebo L.O.
      ,
      • Farnebo F.
      • Hoog A.
      • Sandelin K.
      • Larsson C.
      • Farnebo L.O.
      ,
      • Gogusev J.
      • Duchambon P.
      • Hory B.
      • Giovannini M.
      • Goureau Y.
      • Sarfati E.
      • Drueke T.
      ). Thus, mutations in growth-regulating genes may secondarily alter the calcium set-point by decreasing expression of the CASR (
      • Cetani F.
      • Picone A.
      • Cerrai P.
      • Vignali E.
      • Borsari S.
      • Pardi E.
      • Viacava P.
      • Naccarato A.G.
      • Miccoli P.
      • Kifor O.
      • Brown E.M.
      • Pinchera A.
      • Marcocci C.
      ,
      • Hendy G.N.
      • Arnold A.
      ). Evidence for this comes from a mouse model in which a cyclin D1 transgene is under the control of the PTH gene regulatory region (
      • Imanishi Y.
      • Hosokawa Y.
      • Yoshimoto K.
      • Schipani E.
      • Mallya S.
      • Papanikolaou A.
      • Kifor O.
      • Tokura T.
      • Sablosky M.
      • Ledgard F.
      • Gronowicz G.
      • Wang T.C.
      • Schmidt E.V.
      • Hall C.
      • Brown E.M.
      • Bronson R.
      • Arnold A.
      ). Parathyroid gland CASR expression is reduced, the calcium set-point is shifted to the right, parathyroid enlargement occurs and serum calcium and PTH levels are increased. However, the specific genes and precise mechanisms involved in down-regulation of parathyroid CASR expression are not known.
      The antiproliferative effects of vitamin D metabolites on the parathyroid gland are well documented. Patients with inherited disorders in which there is homozygous inactivation of the VDR or its ligand (and the corresponding mouse models of these disorders) manifest marked parathyroid hyperplasia (see Ref.
      • Panda D.K.
      • Miao D.
      • Tremblay M.L.
      • Sirois J.
      • Farookhi R.
      • Hendy G.N.
      • Goltzman D.
      and references therein). However, like the CASR gene, somatic mutation of the VDR gene does not contribute to parathyroid tumorigenesis (
      • Wu H.I.
      • Arnold A.
      ,
      • Brown S.B.
      • Brierley T.T.
      • Palanisamy N.
      • Salusky I.B.
      • Goodman W.
      • Brandi M.L.
      • Drueke T.B.
      • Sarfati E.
      • Urena P.
      • Chaganti R.S.K.
      • Pike J.W.
      • Arnold A.
      ), but parathyroid VDR expression is reduced in both primary and secondary hyperparathyroid patients (
      • Carling T.
      • Rastad J.
      • Szabo E.
      • Westin G.
      • Akerstrom G.
      ,
      • Rao D.S.
      • Han Z.-H.
      • Phillips E.R.
      • Palnitkar S.
      • Parfitt A.M.
      ). Thus the reduced CASR expression may, in part, be secondary to decreased VDR expression. Additional evidence for the involvement of both the VDR and CASR in controlling parathyroid function and/or growth comes from studies showing association of VDR and CASR gene polymorphisms with primary and/or secondary uremic hyperparathyroidism (
      • Carling T.
      • Kindmark A.
      • Hellman P.
      • Lundgren E.
      • Ljunghall S.
      • Rastad J.
      • Akerstrom G.
      • Melhus H.
      ,
      • Carling T.
      ,
      • Yamauchi M.
      • Sugimoto T.
      • Yamguchi T.
      • Yano S.
      • Kanzawa M.
      • Kobayashi A.
      • Chihara K.
      ) and the parathyroid responsiveness to extracellular calcium in end stage renal disease (
      • Yokoyama K.
      • Shigematsu T.
      • Kagami S.
      • Tsukada T.
      • Arai T.
      • Hara S.
      • Yamada A.
      • Kawaguchi Y.
      • Hosoya T.
      ,
      • Yokoyama K.
      • Shigematsu T.
      • Tsukada T.
      • Hara S.
      • Yamada A.
      • Kawaguchi Y.
      • Hosoya T.
      ).
      In summary, we have identified the transcriptional start sites of promoters P1 and P2 of the human CASR gene and demonstrated functional VDREs in both promoters. Thus, 1,25(OH)2D acting via the VDR is important for the tonic expression of parathyroid and kidney CASR and helps to maintain the normal functions of these tissues. Alterations in the vitamin D regulation of CASR expression are likely to contribute to the development of primary and secondary hyperparathyroidism. Our studies provide the basis for further work investigating the link between vitamin D action and altered CASR expression in the development of hyperparathyroid and hypercalciuric states.

      ACKNOWLEDGEMENTS

      We thank Drs. Bernard Turcotte and John H. White for insights into aspects of these studies and critical review of the manuscript. We thank Dr. Lise Binderup (Leo Pharmaceutical Products, Ballerup, Denmark) and Dr. Milan Uskokovic (Hoffman LaRoche Inc., Nutley, NJ) for providing 1,25(OH)2D3, Dr. Martin Hewison (University of Birmingham, Birmingham, UK) for the HKC cells, Dr. Gregor Sutcliffe (The Scripps Research Institute, La Jolla, CA) for the cyclophilin plasmid, and Martine Girard for technical assistance with the animal studies.

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