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Endocrinology Vol. 147, No. 10 4608-4617
Copyright © 2006 by The Endocrine Society

Ca2+-Dependent Regulation of Calcitonin Gene Expression by the Transcriptional Repressor DREAM

Miho Matsuda, Tada-aki Yamamoto and Masato Hirata

Laboratory of Molecular and Cellular Biochemistry (M.M., M.H.), and Department of Orthodontics (T.Y.), Faculty of Dental Science, and Station for Collaborative Research, Kyushu University, Fukuoka 812-8582, Japan

Address all correspondence and requests for reprints to: Masato Hirata, Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, Kyushu University, Fukuoka 812-8582, Japan. E-mail: hirata1{at}dent.kyushu-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Calcitonin (CT), whose secretion from thyroid glands is regulated by increases in the concentration of extracellular Ca2+, is a well-known hormone that regulates calcium homeostasis. However, the molecular mechanisms underlying the gene expression dependent on Ca2+ have not been clarified. The downstream regulatory element (DRE) antagonist modulator (DREAM) was recently identified as a Ca2+-dependent transcriptional repressor. In the present study, we investigated the possible involvement of DREAM in the regulation of CT gene expression and secretion. A luciferase assay using TT cells, a thyroid carcinoma cell line, showed that a particular region in the CT gene promoter repressed the promoter activity under basal conditions but induced the activity when the Ca2+ concentration was increased. We found two DRE sequences in a region located upstream from the transcription start site. Gel retardation assay confirmed that DREAM bound to the CT-DRE and also indicated that DREAM bound to the DRE in a Ca2+-dependent manner. We generated stable transfectants of TT cells with wild-type or mutant DREAM, which lacked the responsiveness to Ca2+ changes. In contrast to the wild type, overexpression of the mutant DREAM inhibited the increase in CT secretion induced by a calcium ionophore. The addition of forskolin to increase cAMP activated the CT promoter, probably by the interaction of DREAM with cAMP-responsive element binding proteins, independent on the activation by Ca2+. Together, these results suggest that DREAM plays an important role in human CT gene expression in a Ca2+- and cAMP-dependent manner.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CALCITONIN (CT), A 32-AMINO ACID polypeptide, is secreted from the thyroid gland and regulates calcium homeostasis through its effects on target tissue such as bone. Since the first identification of CT by Copp et al. (1), many studies have revealed that CT functions as an inhibitor of bone resorption and thus decreases serum calcium levels, in contrast to activation of osteoclastic functions by PTH and 1{alpha},25-dihydroxy vitamin D3. Hoff et al. (2) found the osteosclerotic phenotype in CT gene knockout mice. The knockout mice exhibited no apparent developmental defects at birth, and their baseline calcium-related chemistry values were not different from the wild type. However, the mutant animals showed a drastic increase in bone formation, developing greater bone mass as they grew, and maintaining bone mass during estrogen deficiency by increasing bone formation, indicating that CT gene products play dual roles in bone biology: prevention of bone resorption in hypercalcemic states and regulation of bone formation.

The CT gene also expresses CT gene-related peptide-{alpha} (CGRP-{alpha}; 37 amino acids) by alternative splicing (3, 4, 5, 6). Alternative splicing in thyroid C cells results in the processing of the primary transcript to CT mRNA, which is composed of exons 1 to 4, whereas in neuronal cells, it is processed to CGRP mRNA consisting of exons 1 to 3, and 5 plus 6. As a result, CT expression is predominant in thyroid C cells, whereas CGRP is produced throughout the central and peripheral nervous system. This process is well known as CT/CGRP gene expression, and the molecular mechanism by which the mRNA is alternatively spliced in a tissue-specific manner, has been investigated by several groups (7, 8, 9, 10, 11).

In contrast, there have been only a few reports regarding the molecular mechanisms underlying the transcriptional regulation of the CT/CGRP gene. The Ras-responsive transcriptional element in the upstream region of the gene binds a zinc finger transcription factor to mediate an increase in CT/CGRP gene expression during the cell differentiation process (12). An 18-bp enhancer controls the cell-specific expression of the rat CT/CGRP gene by binding to the basic helix-loop-helix-Zip protein upstream stimulatory factor-1 and 2, and the forkhead protein (13). However, nothing is known about the mechanisms as to how Ca2+ concentration regulates CT gene transcription despite the evidence that CT is a hormone for calcium homeostasis and its production is regulated by the serum calcium level, as well as TSH (1).

DREAM (downstream regulatory element antagonist modulator) was first identified as a Ca2+-dependent transcriptional repressor of the prodynorphin gene (14). The prodynorphin gene contains a consensus DNA sequence called downstream regulatory element (DRE), and its transcription requires direct association with DREAM. This molecule has a mass of 29 kDa, four EF hand motifs, and is the first known Ca2+-binding protein to function as a DNA-binding transcriptional regulator; the Ca2+-unbound DREAM binds to the DRE site as a tetramer to repress the transcription of target genes, whereas an increase in the intracellular Ca2+ concentration causes the dissociation of the Ca2+-bound DREAM from the DRE site, resulting in transcriptional derepression. Functional studies on DREAM have been carried out mainly using neuronal tissues because prodynorphin, of which DREAM was first identified as a transcription factor, is expressed and functions in the central nervous system. However, endogenous DREAM is highly expressed not only in the brain but also in the thyroid glands in human tissues (14). Furthermore, the DRE motif has also been found in c-fos genes (14), indicating that DREAM acts on genes other than prodynorphin.

The discovery of DREAM encouraged us to examine the possible involvement of DREAM in the Ca2+-dependent CT gene expression. In the CT gene promoter sequences, we identified two DRE motifs that were the targets of DREAM, but not the mutant DREAM lacking the Ca2+ responsiveness, for the Ca2+-dependent dissociation. Luciferase assays using TT cells, a thyroid carcinoma cell line, showed that a region containing the DRE motifs repressed the promoter activity but induced the activity when the Ca2+ concentration was increased. Further cellular assay using stable transfectant of TT cells showed that overexpression of the mutant DREAM inhibited the secretion of CT induced by a calcium ionophore. These results suggest that DREAM plays an important role in human CT gene expression in a Ca2+-dependent manner. Involvement of DREAM in a cAMP-regulated CT gene expression was also examined.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and transfection
The human medullary thyroid carcinoma cell line, TT, was purchased from the American Type Culture Collection (ATCC, Manassas, VA) and grown in RPMI 1640 medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. NB69, the human neuroblastoma cell line, was grown in RPMI 1640 medium supplemented with 15% fetal bovine serum. HeLa cells were cultured in DMEM (Sigma) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Lipofectoamine2000 (Invitrogen, Carlsbad, CA) was used for the production of transient or stable transfectants of TT cells in accordance with the manufacturer’s instructions. Briefly, for transient transfection for the luciferase assay, 3 µg of each plasmid construct and 30 ng pRL-TK vector (Promega, Madison, WI) as an internal control were introduced into TT cells in each well of six-well plates with Lipofectoamine2000. After 4 h, the medium was changed to a fresh one containing 10% fetal bovine serum, with or without ionomycin, forskolin, or both, and the cells were cultured at 37 C for 12 h, followed by analysis for luciferase activity. For the construction of stable transfectants, each green fluorescent protein (GFP) construct (GFP-alone, GFP-DREAM-wild-type, GFP-DREAM-mutant) was transfected into TT cells, followed by cultivation in medium containing 0.2 mg/ml G418 (Invitrogen). Stable transfectants were established by selection with G418 and cloned. The transfection into HeLa cells was carried out by Lipofectamine and Plus Reagent (Invitrogen), according to the manufacturer’s protocols.

Nuclear protein extraction
Nuclear extracts from TT, HeLa, and NB69 cells were prepared according to the method of Adachi et al. (15). Cultured cells were incubated on ice in buffer A [10 mM HEPES (pH 7.8), 10 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 2 mg/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, and 0.1% Nonidet P-40]. Nuclei from the cell lysates were pelleted (12,000 x g for 1 min), lysed with buffer C [50 mM HEPES (pH 7.9), 420 mM KCl, 0.1 mM EDTA, 5 mM MgCl2, 10% glycerol, 1 mM dithiothreitol, 2 mg/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride and phosphatase inhibitors consisting of 5 mM NaF, 1 mM ß-glycerophosphate, 2.5 mM pyrophosphate, and 0.2 mM sodium orthovanadate] and incubated on ice for 1 h. After centrifugation (12,000 x g for 10 min), the nuclear proteins extracted in the supernatant were used.

Western blotting
Nuclear extracts from TT, HeLa, and NB69 cells were separated by SDS-PAGE, followed by transfer to polyvinyldifluoride membrane and immunoblotting with the specific antibody of interest. Antibodies used were as follows: DREAM (Upstate, Charlottesville, VA), GST (glutathione S-transferase; Santa Cruz Biotechnology, Santa Cruz, CA), GFP (MBL, Nagoya, Japan), CREB [cAMP-responsive element (CRE) binding protein; Cell Signaling Technology, Beverly, MA], phospho(serine at 133) CREB (Cell Signaling Technology) and CREM (cAMP-responsive element modulator protein; Santa Cruz Biotechnology). Blots were developed with horseradish peroxidase-coupled secondary antibodies and visualized using the ECL system (Amersham Biosciences, Uppsala, Sweden).

Plasmid construction
The full-length DREAM fusion protein with GST was constructed as follows: full-length DREAM cDNA was prepared using Moloney murine leukemia virus reverse transcriptase (TOYOBO, Tokyo, Japan) and then total RNA from human brain was used to amplify full-length DREAM by PCR. The synthetic oligonucleotide primers used were: forward primer 5'-GAATTCGGCAAACATGAGGCAGCTGC-3' and reverse primer 5'-GTCGACTCGAGCCCCTTCTTGGTG-3' (EcoRI and SalI sites underlined). The PCR product was subcloned into pGEM-T Easy vector (Promega). The plasmid was cleaved at the EcoRI/SalI sites followed by treatment of the insert with Klenow fragment for end-filling and insertion of the DNA fragment into the SmaI site of the pGEX vector (Amersham). The sequences and orientation of the insert were confirmed by sequencing and restriction endonuclease mapping. The DREAM mutant, with Asp at 223 to Ala (D223A) and Asn at 225 to Ala (N225A) replacement within the fourth EF hand in DREAM (14), was generated using the MutanR-K kit (TaKaRa, Kyoto) according to manufacturer’s instructions, using full-length DREAM cDNA and the oligonucleotide 5'-CTTCGAGAAAATGGCCCGGGCCCAGGATGGGGTAG-3'. The substitutions were confirmed by sequencing and the mutant DREAM cDNA was subcloned into the pGEX vector as described above. For the construction of luciferase reporter plasmids containing various 5'-flanking regions of the CT gene, a 1.9-kb fragment (–1757 to +133) including the regulatory region of the human CT gene was amplified by PCR with human genomic DNA as a template and the specific primers 5'-ATCGAGCTCGAAGAGATGTAGCGCGAG-3' and 5'-TCGCTCGAGACCTGAGCCAGGATCTCG-3' (SacI and XhoI sites underlined). The PCR product was cloned with SacI and XhoI into the pGL3-Basic reporter plasmid (Promega). The construct was digested with BglII to create an 860-bp segment (–732 to +133), which was then cloned with BglII into the same plasmid vector. The orientation of the insert was confirmed by sequencing. A 700-bp fragment (–567 to +133) was prepared by PCR with the specific primers 5'-AGCGGTACCACACAACCAGGAATTGG-3' and 5'-TCGCTCGAGACCTGAGCCAGGATCTCG-3' (KpnI and XhoI sites underlined) and inserted with KpnI and XhoI into the vector. A 570-bp fragment (–435 to +133) was created by digestion of the 860-bp luciferase construct with SmaI and ApaI. The fragment was treated with Mung Bean Nuclease (TOYOBO) to create blunt ends and cloned with SmaI into the vector. The direction was confirmed by restriction enzyme digestion. A 370-bp segment (–234 to +133) was amplified by PCR with the specific primers 5'-AGCGCTAGCGCTCACTTTAAGGGCG-3' and 5'-TCGCTCGAGACCTGAGCCAGGATCTCG-3' (NheI and XhoI sites underlined) and inserted with NheI and XhoI into the vector. The constructs of the DREAM (wild type and mutant) with GFP were produced as follows; pGEM-T Easy vector inserted DREAM cDNA was digested with EcoRI/SalI sites and the fragment containing DREAM cDNA was subcloned into pBluescriptRII SK–. The plasmid was cleaved at the HindIII/SalI sites and the DNA fragment of interest was inserted into the phGFP105-C1 vector, which was modified from the phGFP-S65T vector by changing several amino acids of GFP to enhance the fluorescent intensity (16). For the construction of expression plasmids for DREAM sense and antisense, the oligonucleotide of DREAM antisense was inserted into phGFP105-C1 digested with XhoI/EcoRI. The oligonucleotide of DREAM sense was also inserted into phGFP105-C1 using SalI and BamHI restriction sites. The sequences of the antisense oligonucleotides were 5'-GAATTCGTCGACCGCTGCCAGCCGGCCTGGG-3' (underlined, EcoRI; italic, SalI) and 5'-CTCGAGGATCCCCACTTCCTTAGCCGGCTG-3' (underlined, XhoI; italic, BamHI). These oligonucleotides were also used for the DREAM sense construct.

Luciferase assays
Luciferase activity was measured using cell extracts prepared using a Dual Luciferase Reporter Assay System (Promega), according to the manufacturer’s protocol. Briefly, transfected cells were lyzed in a lysis buffer, followed by three freezing-thawing cycles. After centrifugation, aliquots of the supernatants were mixed with the luciferase substrate and measured using a luminometer (Berthold Lumat; Wallac, Gaithersburg, MD). Luciferase values were normalized to the Rluc activity, derived from pRL-TK vector cording for Renilla luciferase, which was cotransfected as an internal reference.

Expression and purification of GST fusion proteins
GST-DREAM (wild type and mutant) fusion proteins were expressed in Escherichia coli BL 21 and purified on glutathione-Sepharose 4B (Pharmacia, Piscataway, NJ), according to routine procedures. Purity was more than 90%, as assessed by SDS-PAGE and Coomassie brilliant blue staining.

Gel retardation assay
Double-stranded oligonucleotides used as probes were as follows: prodynorphin (PD)-DRE (5'-GATCGAAGCCGGAGTCAAGGAGGCCCCTG-3') containing the DRE motif of the human prodynorphin gene (14) and CT-DRE (5'-CCAGCTACTGGAGTCAGATTTCTTG-3') containing the DRE consensus sequences at around position –450 of the upstream region of the human CT gene. Underlined sequences indicate the DRE consensus sequence: [G(G/A/T/C)A(A/G)(C/T)(C/T)(A/G)AG] (14). The binding reactions were performed using a modification of a procedure described previously (17). Complementary synthetic oligonucleotides were annealed and end-labeled with [{gamma}-32P]ATP using T4 polynucleotide kinase (TOYOBO), before incubation with 4–10 µg of nuclear extracts from TT cells or 50 ng of each GST-DREAM fusion protein in reaction buffer [25 mM HEPES-KOH (pH 7.9), 50 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, 10% glycerol, and phosphatase inhibitors described above]. For competition experiments, up to 200-fold molar excess of unlabeled oligonucleotides were incubated with nuclear extracts and labeled probes. For the experiment with the change of Ca2+ concentration, CaCl2 (0, 5, or 10 µM) was added to the reaction mixture with wild-type or mutant GST-DREAM protein and labeled probes. Electrophoresis of DNA-protein complexes was performed at room temperature on a 4% native polyacrylamide gel, and the gels were dried and autoradiographed.

45Ca2+ binding overlay assay
45Ca2+ overlay assay was performed according to the method of Maruyama et al. (18). Briefly, GST-fusion proteins of DREAM (wild type and mutant) were resolved by 15% SDS-PAGE and electrophoretically transferred to a polyvinyldifluoride membrane, followed by incubation with 45Ca2+ for 10 min, with or without excess unlabeled CaCl2 (50 µM). Unbound 45Ca2+ was removed by washing, and bound radioactivity was visualized by autoradiography.

Measurement of CT secretion
The cells were incubated in medium containing dimethylsulfoxide, 10 µM forskolin (CALBIOCHEM, San Diego, CA), or 1 µM A23187 (Sigma) for 3 h, followed by an ELISA (Peninsula Laboratories, San Carlos, CA) for CT released into the medium, according to the manufacturer’s specifications. The OD was measured at 450 nm using a microplate reader. The detection limit of these assays was as low as 2–3 pg/well. The standards range was 0–25 ng/ml. Mean values of triplicate determinations are given in ng/ml.

RT-PCR and Southern blotting
Total RNA was extracted from TT cells using the RNeasyR Mini kit (QIAGEN, Chatsworth, CA), and cDNA was synthesized and amplified using the GeneAmp RNA PCR Core kit (PerkinElmer, Boston, MA) according to the manufacturers’ instructions. The DNA templates were replaced with water and DREAM cDNA inserted into pBluescriptRII SK– (Stratagene, La Jolla, CA), as negative and positive controls for the PCR, respectively. The nucleotide sequences of synthetic oligonucleotide primers used in the reactions were 5'-ACCAAAGAGGAGATGCTGGC-3' and 5-GTCGACTCGAGCCCCTTCTTGGTG-3', which are located in the 5'- and 3'-untranslated regions of the DREAM transcript, respectively. The expected 568-bp PCR product was assessed by Southern blotting using a 590-bp fragment containing the coding region of DREAM as a probe.

Immunoprecipitation assay
TT cells (1 x 107 cells) incubated with 1 µM ionomycin, 10 µM forskolin, or both for 30 min, were treated for extraction of the nuclear fraction as described above. Anti-DREAM antibody-Protein G Sepharose (Amersham Biosciences) complexes were mixed with the nuclear extract (0.5 mg) and incubated at 4 C for 2 h. Immunoprecipitates were washed twice with buffer [12.5 mM HEPES-KOH (pH 7.8), 100 mM KCl, 0.1 mM EDTA, 2% glycerol, 1.25 mM MgCl2 and 1 mM dithiothreitol], and finally suspended in a SDS buffer, followed by SDS-PAGE and immunoblotting for CREB, phospho (serine at 133) CREB, CREM, and DREAM. Immunoprecipitation by anti-CREB or anti-CREM antibody was also performed in the same manner.

Statistics
Data are presented as mean ± SEM. Student’s t test was used and significance was represented by * or ** for P < 0.05 or P < 0.01, respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of DREAM in TT cells
We first examined whether DREAM is expressed in a cell line, TT, which is a human thyroid carcinoma cell line, and has been widely used for the investigation of CT gene expression (10, 12). Figure 1AGo shows the result of Western blotting using an anti-DREAM antibody, indicating the presence of endogenous DREAM in TT cells as well as in NB69 cells, which are of neuronal origin and known to express DREAM and prodynorphin (19, 20), and rat brain but not in HeLa cells. The result justifies the usage of TT cells in the following experiments to explore the possible involvement of DREAM in CT expression with special reference to the Ca2+ concentration dependence.


Figure 1
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  		FIG. 1. DREAM expression in TT cells and deletion analysis of the transcription regulatory region in the upstream region of the human CT gene. A, Expression of DREAM. Nuclear proteins were extracted from HeLa, TT and NB69 cells, respectively, and used for the expression analysis. Western blotting with anti-DREAM antibody (200-fold dilution) shows the expression of DREAM in each cell line (50 µg protein of nuclear extract). Rat brain extract (10 µg) was also analyzed as a positive control. B, Schematic representation of the 5' deletions (pCT1–4) in the promoter region of the human CT gene and the empty reporter vector pGL3-basic (pGL3). +1 shows the transcription start site of the gene. C and D, Transient transfections in TT (C) and HeLa cells (D) with the reporter constructs (pCT1 to 4 and pGL3) shown in B. Cells were stimulated with none or 1 µM ionomycin for 30 min. The results are represented as luciferase activity relative to that of the pCT1 construct under basal conditions in each cell line. The pRL-TK vector was cotransfected as an internal reference, and luciferase values were normalized to the Rluc activity. Values are mean ± SEM from at least three independent experiments. **, Significant difference (P < 0.01) from the corresponding untreated cells (Student’s t test).

 
Transcriptional activity of the human CT gene
To analyze the regulatory regions of human CT gene expression, we cloned the upstream region containing the first exon and prepared several deletion constructs linked to a luciferase reporter gene as shown in Fig. 1BGo. Transient transfection was performed with HeLa cells, a cell line that does not express endogenous DREAM or CT (see Fig. 1AGo and Refs.21 and 22), and TT cells, followed by a luciferase assay in the presence or absence of ionomycin. In TT cells, the activity of the pCT3 construct was about 2.7 times higher than that of the pCT1 or pCT2 construct at basal conditions, but stimulation with the Ca2+ ionophore, ionomycin at 1 µM, increased the activity of pCT1 and 2 to a level comparable with that of pCT3. The ionophore effect was not observed with pCT3 and pCT4 (Fig. 1CGo). The stimulatory effect of the ionophore was abolished when cells were preincubated with O,O'-bis(2-aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid/acetoxymethylester, a membrane permeable Ca2+-specific chelator, indicating that the effect is mediated by an increased intracellular Ca2+ concentration (data not shown). In contrast, HeLa cells apparently exhibited little change in the activity, in all conditions (Fig. 1DGo). The results suggest that the 132-bp region (–567 to –436) in pCT1 and 2 contains cis elements for the repression of gene expression, which respond to a change in cellular Ca2+ concentration in TT cells.

Downstream regulatory elements in the upstream region of the CT gene
We searched the binding sites for transcriptional repressors in the 132-bp region, as determined by luciferase analysis, and identified two DRE core sequences (GTCA) (Fig. 2AGo). They are located at around –450 bp from the transcriptional start site and the flanking sequences of one of them are similar to the DRE found first in human prodynorphine, that is, the original consensus DRE, G(G/A/T/C)A(A/G)(C/T)(C/T)(A/G)AG (14), suggesting that these are functional DRE for CT expression.


Figure 2
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  		FIG. 2. Recombinant DREAM. A, The sequence of DRE and their flanking region upstream of the human CT gene. The region containing two DRE core sequences (underlined) was found at around –450 bp upstream from the transcription start site. Vertical lines represent sequences identical with the prodynorphin (PD)-DRE (underlined). B, Schematic representation of the DREAM structure. Gray boxes indicate EF hands. The mutations are introduced at 223 and 225 residues in the fourth EF-hand motif. C, The expression of the wild-type and mutant constructs (10 µg of protein) was confirmed by Western blotting with anti-GST antibody (1000-fold dilution). D, Binding of the recombinant wild type and the mutant of DREAM to the PD-DRE. The probe sequence used is shown as PD-DRE in A, which is derived from the 5' untranslated region of the human prodynorphin gene. The probe and 50 ng of each protein were used for the gel retardation assay. E, 45Ca2+ overlay assay. GST alone (left lane), GST-DREAM wild-type (WT, middle lane) and the mutant (MT, right lane) were electrophoresed (5 µg of protein in each lane), followed by transfer to the membrane and incubation with 45Ca2+ only (10 µCi 45Ca2+/5 ml nominal Ca2+-free saline solution, left panel) or 45Ca2+ plus 50 µM cold Ca2+ (right panel).

 
To investigate the Ca2+-dependent binding of DREAM to the region including these DRE, wild-type or mutant type recombinant DREAM protein was constructed and purified in a bacterial expression system, as shown in Fig. 2Go, B and C. Two mutations, which make the recombinant protein insensitive to calcium, were introduced into the fourth EF hand, according to the report by Carrion et al. (14). Their binding to the DRE was confirmed by gel retardation assay, using a synthetic oligonucleotide containing the DRE of the human prodynorphin gene that had been shown to bind endogenous DREAM (Fig. 2DGo). As expected, the wild type, but not the mutant type, DREAM showed Ca2+ binding ability (Fig. 2EGo). The results indicate that the wild-type and mutant type recombinant DREAM behave as expected.

Using the recombinant DREAM, gel retardation experiments were performed using the oligonucleotide probe containing the DRE sequence that we identified in the human CT promoter in the present study (Fig. 3AGo). A retarded band, indicating binding with DREAM, was observed with both wild-type and mutant DREAM, although the latter exhibited less binding, in the absence of exogenous Ca2+ and in the presence of 2 mM EGTA (Fig. 3BGo). Increasing the Ca2+ concentration decreased the amount of the retarded band in a concentration-dependent manner with the wild-type DREAM, whereas no decrease was observed with the mutant, indicating that the CT-DRE binds to DREAM in a Ca2+-dependent manner. Endogenous DREAM binding to the CT-DRE was then analyzed by gel retardation assay using nuclear extracts from TT cells. The CT-DRE probe bound to endogenous DREAM in TT cells as well as to the PD-DRE, and the binding was specifically reduced upon competition with an excess of cold DRE fragment (Fig. 3CGo). Supershift assay using anti-DREAM antibody was further performed, indicating a specific binding of DREAM to DRE fragment (Fig. 3DGo). From these results, we tentatively concluded that the putative DRE elements in the upstream region (located at about –450 bp) of the CT gene are functional for specific Ca2+-dependent binding to DREAM in TT cells. Together with the results shown in Fig. 1CGo, the results suggest that Ca2+-dependent regulation of CT gene expression is mediated by DRE-DREAM interaction.


Figure 3
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  		FIG. 3. Ca2+-dependent binding of DREAM to CT-DRE. A, The sequence shows the CT-DRE oligonucleotide probe used in the gel retardation assay. B, Gel retardation assay was performed with 50 ng GST fused-recombinant DREAM wild type (WT) and mutant (MT). Each protein and the probe were incubated with less than 0.01, 0.5, or 10 µM CaCl2. Ca2+ concentration was determined in the presence of 2 mM EGTA plus required CaCl2. Graph shows the summary of four independent experiments. Density was represented relative to that observed with mutant DREAM in the absence of Ca2+. **, Significant difference (P < 0.01) from the corresponding untreated cells (Student’s t test). C, The specific binding of endogenous DREAM in TT cells to the CT-DRE. Lanes 2–7 indicate a self competition assay with 0- to 200-fold molar excess of the unlabeled probe. The oligonucleotide probe containing the DRE motif in the human PD gene (PD-DRE, the sequences were shown in Fig. 2AGo) was used as a positive control (lane 1). D, Supershift assay. Signal (arrowhead) for binding of DREAM and the DRE was shifted upper by the addition of anti-DREAM antibody (indicated by an arrow), but not by control IgG.

 
Effect of DREAM on the secretion of CT
To analyze the effect of the gene expression regulated by DREAM on the secretion of CT in TT cells, GFP-tagged wild-type and mutant DREAM molecules were constructed, followed by transfection into TT cells and selection of stable transfectants by G418 as described in Materials and Methods. The expression was confirmed by Western blotting using anti-GFP and DREAM antibodies (Fig. 4AGo). These transfectants were then analyzed for secreted CT in response to stimulation with either forskolin or A23187. Forskolin is an adenylate cyclase activator and thus leads to activation of cAMP-dependent protein kinase (PKA), which phosphorylates cAMP-responsive element (CRE) binding proteins, resulting in the enhancement of CT secretion (23, 24, 25). A23187 is a calcium ionophore and thus leads to an increase in the cellular concentration of Ca2+, resulting in the dissociation of DREAM from the DRE motif. As shown in Fig. 4BGo, all transfectants examined similarly secreted CT in response to forskolin stimulation, whereas CT secretion in response to A23187 stimulation varied; such a robust increase was not observed in TT cells transfected with the mutant DREAM.


Figure 4
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  		FIG. 4. Effect of DREAM on CT secretion. A, The expression of GFP fusion proteins of DREAM in TT cells was analyzed by Western blotting using anti-GFP (1000-fold dilution) and anti-DREAM (1000-fold dilution) antibodies. GFP only (left lane), GFP-DREAM-WT (middle lane) and GFP-DREAM-MT (right lane) are shown. Ten micrograms of cellular extracts were used in the blots shown in which endogenous DREAM in the left lane was not detected. When 200 µg of the extracts were used, a trace of endogenous DREAM was detected in the blot by anti-DREAM antibody (data not shown). GFP-mutant DREAM was expressed about 50~80-fold excess of endogenous DREAM. B, CT secretion from stable transfectants was analyzed by ELISA after stimulation with 10 µM forskolin or 1 µM A23187. Values are represented by the mean ± SEM of three independent experiments. **, Significant difference (P < 0.01) from the corresponding untreated cells (Student’s t test).

 
Interaction of DREAM with CREM or CREB for the expression of the human CT gene
It has been reported that CRE and CRE-like motifs are present in the 5' flanking region of the CT gene and are involved in transcriptional activation (23, 24, 25). This was confirmed in the present study by showing the stimulatory effect of forskolin on CT secretion. However, the cAMP pathway might function in CT gene expression not only alone but also collaboratively with the Ca2+ pathway because the prodynorphin gene in which DRE was first identified was recently shown to be also comprised of CRE, and the transcription is thought to be determined by interactions between DREAM and CRE-binding proteins including CREM and CREB (20, 26, 27); DREAM was also dissociated from the DRE to derepress gene expression by its interaction with CREM, which was strengthened by PKA-mediated phosphorylation. Also, binding of CREB and CREM to CRE was blocked by the presence of DREAM, which was relieved by an increase in cellular Ca2+ concentration (20, 27).

Thus, we questioned whether interactions between these transcription factors, including CREM and CREB, are also involved in CT gene expression in relation to the function of DREAM. To investigate whether any interactions between DREAM, CREM, and CREB occur in vivo and the transcriptional activity when such interactions occur, we performed immunoprecipitation analysis using nuclear extracts from TT cells and reporter gene assay. Neither CREB nor CREM was immunoprecipitated by an anti-DREAM antibody from nuclear extracts of unstimulated TT cells (Fig. 5AGo, lane 1). Similarly, a little CREB or CREM was immunoprecipitated under conditions of increased cellular Ca2+ concentration (Fig. 5AGo, lane 2). However, when TT cells were stimulated with forskolin to increase cellular cAMP, both CREM and CREB were immunoprecipitated with anti-DREAM antibody, indicating the association of DREAM with CREB and CREM (Fig. 5AGo, lane 3). Phosphorylation of CREB under such conditions was confirmed by immunoblotting using an antibody to phospho (serine at 133)-specific CREB (Fig. 5AGo). In the case of stimulation with both forskolin and ionomycin, the immunoprecipitate of CREM by anti-DREAM antibody was slightly reduced, whereas that of CREB was almost null (Fig. 5AGo, lane 4 and a graph). Because the immunoprecipitation using an antibody to phospho (serine at 133)-specific CREB was unsuccessful for unidentified reasons, we further performed the immunoprecipitation using an anti-CREB or anti-CREM antibody to confirm the interaction with DREAM. DREAM in the nuclear extracts were immunoprecipitated with CREB and CREM when TT cells were stimulated with forskolin (Fig. 5BGo), consistent with those shown in Fig. 5AGo. Gel retardation experiment was then performed using nuclear extracts from TT cells stimulated with forskolin or ionomycin, and the DRE sequence. As shown in Fig. 5CGo, the amount of DREAM bound to the DRE from nuclear extracts of TT cells stimulated with forskolin or ionomycin were less than that of the control cells. These results indicate that cAMP not only acts on the CRE independently of Ca2+ and DREAM but also triggers the dissociation of DREAM from the DRE motif by its interaction with the phosphorylated forms of CREB and CREM for CT gene expression. Figure 5DGo shows the results of luciferase analysis in TT cells. Stimulation with forskolin or ionomycin increased the transcriptional activity of the CT gene by 2- to 3-fold, and that with both stimuli increased further.


Figure 5
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  		FIG. 5. Interaction of DREAM, CREM and CREB, and promoter assay. A, Immunoprecipitation of nuclear extracts from TT cells. Nuclear extract was prepared before (lane 1) and after stimulation for 30 min with 1 µM ionomycin (lane 2), 10 µM forskolin (lane 3) or ionomycin plus forskolin (lane 4), followed by immunoprecipitation with anti-DREAM antibody, and immunoblotting with anti-CREB (1000-fold dilution), anti-CREM (1000-fold dilution), anti-DREAM (1,000-fold dilution) and antiphospho (serine at 133)-specific CREB antibody (1000-fold dilution). Graph summarizes three independent blots. Densities of CREB and CREM were represented relative to that of DREAM. **, P < 0.01 by Student’s t test. B, Immunoprecipitation by anti-CREM or anti-CREM antibody. Nuclear extracts were immunoprecipitated by anti-CREB or anti-CREM antibody, followed by immunoblotting by anti-DREAM antibody. Another blot gave a similar result. C, Binding of endogenous DREAM in TT cells to the DRE. Nuclear extracts were subjected to the binding to the DRE, followed by an electrophoresis and an autoradiography. For this experiment, nuclear extracts were incubated with DRE in the presence of Ca2+ in addition to phosphatase inhibitors. D, Transient transfection in TT cells was performed with the luciferase construct with the CT gene promoter containing the DRE (pCT2, shown in Fig. 1BGo). After stimulation with 1 µM ionomycin, 10 µM forskolin or both, cell extracts were assayed for luciferase activity. The pRL-TK vector was cotransfected as an internal reference, and luciferase values were normalized to the Rluc activity. Values are mean ± SEM of at least three independent experiments. * and **, P < 0.05 and P < 0.01 by Student’s t test, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
CT inhibits bone resorption, thus resulting in a decrease of serum calcium level (for review, see Refs. 28 and 29), which is derived from the inhibitory effects on osteoclasts such as a rapid loss of the ruffled border (30), a rapid cessation of the motility (31), and an inhibition of the secretory activities of tartrate-resistant acid phosphatase (32) and proton (33). On the other hand, there are apparent paradox; a hypocalcaemia (but not hypercalcaemia) even when circulating CT is absent such as after thyroidectomy (34), or the absence of osteopetrosis under conditions of high circulating CT in medullary thyroid carcinoma (29), raising a question whether CT is a physiologically relevant hormone to regulate serum calcium level. However, recent studies have reported surprising additional actions which might provide an explanation to the paradox. CT, at physiological concentrations, blocked the inhibitory effect of high extracellular Ca2+ on osteoclasts by interfering extracellular Ca2+-sensing system, thus causing activation of osteoclasts under high extracellular Ca2+ conditions (35). CT interacts with osteoblasts despite no convincing evidence for CT receptors on any osteoblast-like cells. Thus, CT exerts an effect on osteoblasts that enhances osteoinduction by bone morphogenetic protein-2 (36). It also increases the concentration of IGF in serum-free cultures of human osteoblast-like cells (37). Furthermore, CT also prevents osteoblasts and osteocytes from apoptosis (38). Moreover, CT increases mRNA expression of 25-hydroxyvitamin D3 1{alpha}-hydroxylase, which catalyzes biosynthesis of 1{alpha},25-dihydroxyvitamin D3 in renal proximal tubules (39). These results indicate that CT is still an important hormone involved in bone biology, although the effects are expanded beyond those on osteoclasts originally identified. Therefore, the molecular mechanisms underlying the regulation of CT gene expression are subjects to be investigated.

CT has been clinically introduced for the treatment of disorder of bone and mineral metabolism including osteoporosis, Paget’s disease and bone loss after renal transplantation (29, 40), principally owing to its inhibitory effect on osteoclastic bone resorption. Clinical usage of CT is limited, due almost entirely to its bioavailabilty (29). Several new approaches are currently being used or invented, including novel allosteric activators of the CT receptors to make them more sensitive (41), modulator of Ca2+ sensing system on the thyroid C-cell surface to release more endogenous CT (29), and application methodology (CT formulation for oral, pulmonary, or transdermal application) (29). Furthermore, there are new gene therapy vectors available that would allow osteoclast precursors to carry the CT gene to specific bone sites of osteolysis (29). Therefore, understanding of the mechanisms underlying CT gene expression would provide novel clues for CT to be an important therapeutic agent for disorder of bone and mineral metabolism.

The present study was undertaken to examine human CT gene expression mechanisms with a special reference to the possible involvement of DREAM in the Ca2+-dependent regulation. We first determined that TT cells contain DREAM and identified two DRE core sequences in the 5'-flanking region of the human CT gene. DRE sites were first identified in the 5'-untranslated regions of the prodynorphin and c-fos genes, and in the 3'-untranslated region of the hrk gene (14, 42, 43), suggesting either that the DRE is functional only when located downstream of the transcription start site, or that it is a position-dependent regulatory element (26, 44). However, a functional DRE was identified recently upstream from the TATA box of the thyroglobulin-promoter region and in the upstream region of the p21cip1 gene (21, 45). The present study showed that CT-DRE, containing two DRE core sequences, one of which is in an inverted direction, is also located upstream from the TATA box of the gene. The inverted DRE sequence was shown to be functional, exhibiting a similar or greater affinity for DREAM binding (46). Therefore, it is likely that the DRE is functional as not only an orientation- but also a position-independent regulatory element, and each gene may have the best position of DRE for transcriptional regulation.

An increase in plasma Ca2+ levels leads to the secretion of CT from thyroid C cells to reduce the level of Ca2+. The increase in plasma Ca2+ probably triggers the activation of Ca2+-sensing receptors on the surface membrane of thyroid cells (47), followed by the activation of the Gq-coupled signaling pathway to increase cellular Ca2+ (48). This then causes a conformational change in DREAM (49), which dissociates from DRE resulting in derepression as shown in the present study. CT secretion was increased in TT cells stimulated with forskolin and A23187, which was probably mediated by an increase in cAMP levels followed by PKA activation and in the intracellular Ca2+ level, respectively. Transfection of wild-type DREAM did not modify the secretion, indicating that endogenous DREAM is sufficient to regulate Ca2+-dependent CT gene expression. In contrast, mutant DREAM transfected into TT cells behaved as a dominant-negative in response to A23187 stimulation. Although the apparent affinity of mutant DREAM for CT-DRE is 10~15-fold weaker (see Fig. 3BGo), the expression was roughly 50~80-fold excess over the endogenous level (see the legend of Fig. 4Go), thus causing the replacement of endogenous DREAM with the mutant molecule.

We also examined the activation of CT gene expression by PKA stimulation, and the relationship between a Ca2+/DREAM system and cAMP/CRE-regulatory system. The interaction of DREAM with CREM and CREB was detected in cells stimulated with forskolin, indicating that DREAM is released from the DRE site by its interaction with CREM and CREB phosphorylated by PKA. This was consistent with the result that the interaction strengthened by PKA phosphorylation prevents binding of DREAM to the DRE, resulting in the derepression of the prodynorphin gene in a cAMP-dependent manner (20). However, the interaction was attenuated when stimulated together with ionomycin, which was also consistent with the results for the prodynorphin gene expression system (20, 27). These results indicate that increased cAMP levels also involve the DREAM system in CT gene expression in addition to the previously identified CRE system (23, 24, 25). However, the physiological significance of the latter system is questionable because phosphorylated CREB, which binds to DREAM preventing its binding to other proteins, requires an association with the CREB-binding protein, CBP, to bind to the CRE site for activation of target gene transcription (50, 51, 52). Conversely, the system involving cAMP/CREB/CBP must be functional for CRE if Ca2+ is increased together with cAMP in cells. The functional difference between CREB and CREM was not discriminated in the present study, although the results exhibited difference in the binding to DREAM when cells were stimulated with forskolin and ionophore. Further studies are apparently needed to fully understand the function of the DREAM-CREB/CREM interaction in TT cells. Taken together, it can be concluded that DREAM is involved in the Ca2+- and cAMP-modulated CT gene expression influencing each other, as in the prodynorphin gene expression system (53).


   Acknowledgments
 
We thank Drs. Takashi Kanematsu and Hiroshi Takeuchi of this laboratory for their assistance in the generation of mutant DREAM and CT release assay, respectively.


   Footnotes
 
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to M.M. and M.H.).

Disclosure Statement: M.M., T.Y., and M.H. have nothing to declare.

First Published Online July 13, 2006

Abbreviations: CGRP, CT gene-related peptide; CT, calcitonin; CRE, cAMP-responsive element; CREB, CRE binding protein; CREM, CRE modulatory protein; DRE, downstream regulatory element; DREAM, DRE antagonist modulator; GFP, green fluorescent protein; GST, glutathione S-transferase; PD, prodynorphin; PKA, cAMP-dependent protein kinase.

Received February 24, 2006.

Accepted for publication July 6, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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