Cloning and Characterization of Two Promoters for the Human Calcium-sensing Receptor (CaSR) and Changes of CaSR Expression in Parathyroid Adenomas*

Histological analyses showed that expression of the parathyroid calcium-sensing receptor (CaSR) is decreased in parathyroid adenomas. Because reduced expression of CaSR may result in insufficient suppression of parathyroid hormone secretion, the elucidation of regulatory mechanisms of CaSR expression is indispensable for understanding the pathogenesis of parathyroid adenomas. Two cDNA clones for human CaSR with different 5'-untranslated regions have been isolated. However, the structure of the promoter region of human CaSR and the mechanisms of production of multiple CaSR mRNAs are unknown. We have cloned promoter regions of human CaSR by screening a genomic library. The human CaSR gene has two promoters and two 5'-untranslated exons (exons 1A and 1B), and alternative usage of these exons leads to production of multiple CaSR mRNAs. The upstream promoter has TATA and CAAT boxes, and the downstream promoter is GC-rich. Northern blot analysis showed that expression levels of exon 1A in parathyroid adenomas are significantly less than those in normal glands. However, expression of exon 1B was not different between adenomas and normal glands. Thus, specific reduction of the transcript driven by the upstream promoter was observed in parathyroid adenomas. Further analyses of factors that modulate the activity of the upstream promoter are necessary to clarify the pathogenesis of parathyroid adenomas.

set point of PTH secretion and serum calcium levels. In some hypercalcemic diseases, the elevation of the set point of PTH secretion has been reported. For example, inactivating mutations of CaSR result in inadequate suppression of PTH secretion and inappropriately high serum levels of PTH in patients with familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism (3)(4)(5)(6)(7). In contrast, no mutation in the CaSR gene has been reported in parathyroid adenomas from patients with primary hyperparathyroidism, the most common cause of hypercalcemia (8). However, it has been shown by immunohistochemistry and in situ hybridization that the expression of CaSR is decreased in parathyroid adenomas (9 -11). Because reduced expression of CaSR may also result in insufficient suppression of PTH secretion, the elucidation of the regulatory mechanisms of CaSR expression is indispensable for understanding the pathogenesis of parathyroid adenomas.
The coding region of human CaSR is coded by six exons. In addition, two cDNA clones for human CaSR with different 5Ј-untranslated regions have been isolated from parathyroid adenoma (2). Therefore, it is suggested that the translation start site of CaSR is in exon 2, and there is at least one 5Ј-untranslated exon in the human CaSR gene (6). However, the structure of the promoter region of human CaSR and the mechanism of the production of multiple CaSR mRNAs are unknown. In this study, we have identified and characterized the promoter regions of human CaSR by screening a genomic library. The expression levels of multiple mRNAs in normal and adenomatous parathyroid glands were also analyzed. The results indicate that the human CaSR gene has at least two promoters and that alternative usage of 5Ј-untranslated exons leads to the production of multiple CaSR mRNAs. In addition, the expression of CaSR mRNA produced by one of the two promoters of the CaSR gene is specifically reduced in parathyroid adenomas.

EXPERIMENTAL PROCEDURES
Materials-Parathyroid adenoma tissues were obtained from patients who underwent surgical treatment for primary hyperparathyroidism after informed consents were obtained. All these patients had grossly abnormal single adenomas. Normal parathyroid glands were also obtained from some of these patients because of associated thyroid diseases. The tissues were frozen in liquid nitrogen as soon as possible and stored at Ϫ80°C until use. The Megaprime DNA labeling system, [␣-32 P]dCTP, and Hybond-N were obtained from Amersham Pharmacia Biotech (Tokyo). The human genomic library was provided by the Japanese Cancer Research Resources Bank. Taq and LA Taq polymerases were purchased from TaKaRa (Otsu, Japan). The TA cloning kit and pcDNA3 were from Invitrogen (Carlsbad, CA). pBluescript II SK was from Stratagene (La Jolla, CA). The dual luciferase reporter assay system and the pGL3-basic vector were from Promega (Madison, WI).
PCR for the 5Ј-Region of CaSR Using Genomic DNA as a Template-Genomic DNA was extracted from peripheral leukocytes as described * This study was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture and a grant from the Research Society for Metabolic Bone Diseases, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBank TM /EBI Data Bank with accession number AB031325.
Screening of the Genomic Library for the Untranslated Region of the CaSR Gene-The PCR product generated by the forward primer in clone A and the reverse primer in clone B was cloned by TA cloning and sequenced as described below. The insert was cut out by EcoRI and used as a probe for screening the genomic library. The human genomic library was screened by the standard plaque hybridization method. A positive clone was isolated and purified using the polyethylene glycol precipitation method. The phage insert was mapped by restriction digestion and Southern blot analysis. A fragment of 4 kb containing the 5Ј-region of the CaSR gene was subcloned into the BamHI site of pBluescript II SK and sequenced. Sequencing indicated that the 5Јregions of the two different CaSR clones (clones A and B) are derived from two untranslated exons (exons 1A and 1B). In addition, to clarify the DNA sequence of the upstream region of a coding exon containing the translation start site (exon 2), PCR using a phage library was employed (13). A reverse primer in an exon containing the translation start site (5Ј-CCAAAATGAATAGGAAAGAGCCCCCCA-3Ј) (see Fig. 2) and a primer corresponding to the left arm of the phage (5Ј-TTATGC-CCGAGAAGATGTTGAGCAAACTTATCG-3Ј) were used. The conditions for PCR were one cycle at 94°C for 2 min, 30 cycles at 98°C for 20 s and 70°C for 5 min, and one cycle at 72°C for 7 min. The PCR product was subcloned by TA cloning and sequence by the same primers.
DNA Sequencing-The samples were sequenced using the PRISM Ready-Reaction dye deoxy terminator cycle sequencing kit and an ABI Model 373S-36 autosequencer (Perkin-Elmer, Chiba, Japan) according to the manufacturer's instructions.
RNA Extraction-Total RNA was prepared from each tissue by the acid/guanidium/phenol/chloroform method as described previously (14).
5Ј-Rapid Amplification of cDNA Ends (5Ј-RACE)-5Ј-RACE was carried out using a 5Ј-RACE system (Life Technologies, Inc.) according to the manufacturer's instruction. Briefly, total RNA (0.9 g), isolated from human parathyroid, was reverse-transcribed, and the first-strand cDNA was purified using GlassMAX DNA isolation spin cartridges. Terminal deoxynucleotidyltransferase tailing of the first-strand cDNA was carried out, and the tailed cDNA was amplified by PCR using Taq polymerase. PCR conditions were initial denaturation at 95°C for 1 min; 30 cycles at 95°C for 20 s, 57°C for 45 s, and 68°C for 30 s; and final elongation at 68°C for 5 min. The antisense primers used were 5Ј-CATACTCCTTTGGGTGGGCAAT-3Ј for exon 1A and 5Ј-AAAC-CCGGGCTCCACGAGGATGAGCTCTGGT-3Ј for exon 1B (see Fig. 2). PCR products were then cloned by TA cloning, and seven cDNA clones isolated for exon 1A and five clones for exon 1B were sequenced.
Construction of the 5Ј-Untranslated Region of CaSR/Luciferase Reporter Gene Plasmids-Sequencing and Southern blot analysis showed that there are restriction sites for KpnI and PstI in the 5Ј-flanking region and in exon 1A, respectively (see Fig. 2). Therefore, to test the promoter activity of the CaSR gene, pBluescript II containing the screened 4.0-kb fragment was digested with KpnI and PstI, and the resulting 2.0-kb fragment was subcloned into pGL3-basic cut with KpnI and PstI (A-2.0). The B-0.5 vector was also constructed by inserting a 530-bp PCR fragment corresponding to the 5Ј-upstream region of exon 1B. The PCR product was generated using two primers with restriction sites on their ends (5Ј-AAAGAGCTCTGGGCACGCGATTTGTATTTA -3Ј and 5Ј-AAACCCGGGCTCCACGAGGATGAGCTCTGGT-3Ј) (see Fig. 2), cut with HindIII and XbaI, and inserted in front of the coding sequence of luciferase in pGL3-basic.

Transient Expression of Recombinant Vectors in MCF-7 Human
Breast Cancer Cells-The MCF-7 cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (JRH Biosciences, Lenexa, KS). Cells were transiently transfected using LipofectAMINE (Life Technologies, Inc.). The DNA-liposome complex was prepared by mixing 0.25 g of DNA with 1 l of LipofectAMINE in 50 l of serum-free medium, and the mixture was incubated at room temperature for 30 min. The mixture was then diluted with 950 l of serum-free medium and added to cells plated in each well of six-well plates. After 5 h of incubation at 37°C, an equivalent amount of medium with 20% fetal bovine serum was added, and luciferase reporter assay for cell extracts was performed 29 h after the start of transfection.
Dual Luciferase Reporter Assay for the Promoter Activity-Promoter activity was measured using the dual luciferase reporter assay system and a MiniLumat LB9506 luminometer (Berthold, Osaka, Japan). Cells were transfected with 0.25 g of each reporter recombinant along with 12.5 ng of Renilla luciferase vector (pRL-TK) as an internal control. Each luciferase activity was normalized for transfection efficiency by the Renilla luciferase assay.
Northern Blot Analysis-6 g of total RNA from normal parathyroid glands and 15 g from adenoma tissues were electrophoresed on a formaldehyde-containing 1.5% agarose gel and transferred to Hybond-N. Each sample was electrophoresed twice, and two Hybond-N membranes containing the same samples were prepared. Initial hybridization was conducted using a DNA probe for the full-length coding region of CaSR generated by the Megaprime DNA labeling system (12). To examine the expression levels of exons 1A and 1B separately, PCR products corresponding to exons 1A and 1B were cloned by TA cloning. The primers used were 5Ј-GCTGCAGCCAGGAAGGACCG-3 and 5Ј-TTCTGCAAGACTCAGGTCAAGCGTTG-3Ј for exon 1A and 5Ј-CTGCT-GTGGCCGGACCCGAA-3Ј and 5Ј-CCAGGGCTCCCTCGCACAGAG-3Ј for exon 1B (see Fig. 1). After confirming the orientation of the insert by DNA sequencing, these plasmids were cut with XbaI, and T7 RNA polymerase-driven [␣-32 P]UTP-labeled antisense riboprobes were prepared using a Maxiscript kit (Ambion Inc., Austin, TX). Each membrane was hybridized with a probe for either exon 1A or 1B, respectively. The filters were washed to the high stringency of 0.1ϫ SSC at 65°C and exposed to X-Omat AR (Eastman Kodak Co.). The filters were then stripped and rehybridized with a cDNA probe for GAPDH. The amount of mRNA in each lane was quantified using a densitometer and normalized by the expression level of GAPDH.
Statistical Analyses-Statistical significance was analyzed either by one-way analysis of variance followed by Bonferroni's method for comparison of multiple means or by Student's t test. An unadjusted p value less than 0.05 was considered to be significant.

Cloning and Characterization of the 5Ј-Region of the Human
CaSR Gene-Two previously reported CaSR cDNA clones (clones A and B) have the same sequence back to Ϫ242 bp from the translation start site, but their 5Ј-upstream regions have completely different sequences (Fig. 1) (2). These results indicate that 5Ј-regions of clones A and B are derived from different exons. To clarify the relationship between these exons in the 5Ј-region of human CaSR, we conducted PCR using genomic DNA as a template. PCR using the forward primer in clone A and the reverse primer in clone B produced a PCR product of ϳ800 bp (data not shown). However, no PCR product was observed by PCR using the forward primer in clone B and the reverse primer in clone A (data not shown). These results indicate that the exon forming the 5Ј-region of clone A is in the 5Ј-position to the exon forming the 5Ј-region of clone B. To isolate the promoter region of human CaSR, we labeled the 800-bp PCR product described above and screened a human genomic library. A phage clone containing the 5Ј-flanking region of the human CaSR gene was cloned and purified using the polyethylene glycol precipitation method. A fragment of 4 kb was subcloned into pBluescript II SK. DNA sequencing of this plasmid showed that the 5Ј-regions of clones A and B are derived from two different untranslated exons (exons 1A and 1B) (Fig. 2). In addition, exon 1B was not connected to the exon containing the translation start site (exon 2). However, because exon 2 was not included in the phage clone, the length of the intron between exons 1B and 2 was not evident. The DNA sequence of the upstream region of exon 2 was determined by PCR using the phage library as s template as described under "Experimental Procedures." Sequencing indicated that the intron between exons 1B and 2 is a U2-type GT-AG intron (15). There are TATA and CAAT boxes in the upstream region of exon 1A (Fig. 2). In contrast, the region between exons 1A and 1B is GC-rich, but has no TATA box (Fig. 2). Therefore, the human CaSR gene has at least two 5Ј-untranslated exons, and alternate usage of these exons produces multiple CaSR mRNAs.
5Ј-RACE-The DNA sequences of exons 1A and 1B in Fig. 2 are the longest ones identified by 5Ј-RACE. 5Ј-RACE for clone B using mRNA from parathyroid tissue showed that clone B is at least 50 bp longer in its 5Ј-end than originally described (2). 5Ј-RACE for clone A also showed that clone A is at least 430 bp longer than the originally described clone (2) (Fig. 2).
Analysis of Promoter Activities of the Human CaSR Gene-Before examining the promoter activity of the human CaSR gene, we searched for cell lines that express CaSR. We found by Northern and Western blot analyses that MCF-7 cells express CaSR (data not shown). Therefore, we used this cell line for analyses of promoter activities. When transiently expressed in MCF-7 cells, a 4.3-fold increase in luciferase activity was observed by A-2.0 compared with that by the control vector pGL3 (Fig. 3). B-0.5 also showed a 8.2-fold increase in luciferase activity (Fig. 3). These results indicate that the human CaSR gene has at least two promoters.
Northern Blot Analysis of the Expression of CaSR mRNAs in Parathyroid Tissues-Hybridization with a probe for the fullcoding region of CaSR identified main transcripts of ϳ5.4 and ϳ4 -4.2 kb and a minor band of ϳ10 kb in both normal and adenoma tissues (Fig. 4) as already reported (2). We used riboprobes to distinguish the expression levels of exons 1A and 1B. Analyses by the exon 1A-specific riboprobe identified 5.4-, ϳ4 -4.2-, and 10-kb transcripts, suggesting the existence of alternative splicing in 3Ј-untranslated exons (Fig. 4). In contrast, the exon 1B-specific riboprobe showed only a ϳ4 -4.2-kb band (Fig. 4). Although detailed mechanisms of the production of multiple transcripts from the human CaSR gene are not clear at the moment, these results indicate that 5.4-and 10-kb transcripts are specific for exon 1A.
Expression Levels of Exons 1A and 1B in Normal Parathyroid Glands and Parathyroid Adenomas-The expression levels of exon 1A were evaluated by the exon 1A-specific riboprobe, and those of the 5.4-kb band were quantified and normalized by GAPDH expression. The expression levels of this transcript in adenomas were ϳ60% of and significantly less than those in normal parathyroid glands (p Ͻ 0.01) (Fig. 5A). In contrast, the expression levels of exon 1B were not different between nine adenomas and three normal glands (Fig. 5B). These results indicate that the reduction of the expression of CaSR in parathyroid adenomas is specific for mRNA containing exon 1A. DISCUSSION We have shown that the human CaSR gene has at least two promoters and 5Ј-untranslated exons and produces multiple mRNAs. The coding region of human CaSR is 3234 bp, and the 5Ј-untranslated region that is common to both clones A and B is 242 bp long (2). Two 3Ј-untranslated sequences of ϳ180 bp and 1300 bp have also been reported (2). We have shown here that exon 1A for human CaSR is ϳ560 bp. Therefore, using two 3Ј-untranslated regions, CaSR mRNAs containing exon 1A seem to be ϳ4.2 and 5.4 kb. The mechanism of the production of 10-kb CaSR mRNA is not clear. However, mRNA of a similar size has also been reported in a human medullary thyroid carcinoma cell line and in other species including mouse (16 -18). On the other hand, exon 1B is ϳ250 bp long. If the short 3Ј-untranslated region is used, mRNA containing exon 1B is calculated to be ϳ3.9 kb. The absence of larger transcripts by the exon 1B-specific probe indicates that only the short 3Јuntranslated sequence is used. Although the mechanism of selective usage of the 3Ј-untranslated exon is unclear at the moment, probes specific for these two 3Ј-untranslated regions may be useful to further characterize multiple CaSR mRNAs.
Human CaSR has at least two promoters. The upstream promoter has TATA and CAAT boxes, and the downstream promoter is a GC-rich promoter without a TATA box. The presence of multiple promoters is reported in many other genes, including PTH/PTH-related protein receptor and PTH-related protein (19 -22). Because the structure of the two promoter regions of human CaSR is different, it is likely that the regulation of promoter activity is promoter-specific. It is also likely that these two promoters are regulated in a tissue-dependent manner. Further study employing other tissues is necessary to address these issues. Although these two promoters are active in both normal human parathyroid glands and parathyroid adenomas, the expression of the transcript driven by the upstream promoter and containing exon 1A is decreased in adenomas, whereas that driven by the downstream promoter is not altered. These results suggest that the upstream promoter activity is reduced in parathyroid adenomas. Although normal parathyroid glands were obtained from patients with primary hyperparathyroidism in our study, the expression levels of CaSR in normal parathyroid glands from patients with primary hyperparathyroidism were shown to be the same as those from eucalcemic normal controls (11). Previous study using immunohistochemistry or in situ hybridization indicated that the expression of CaSR in parathyroid adenomas was reduced to ϳ60% of that in normal glands (10,11). Our results show that the expression level of CaSR mRNA containing exon 1A in adenomas is ϳ60% of that in normal parathyroid glands. It is difficult to precisely analyze the relative amount of CaSR mRNAs containing exons 1A and 1B because the specific activities of probes for exons 1A and 1B are not the same. However, since both transcripts containing exons 1A and 1B are evident by Northern blot analysis, our results indicate that the difference in the expression level of CaSR between adenomas and  4. Northern blot analysis of CaSR mRNA in parathyroid glands. 6 g of total RNA from normal parathyroid glands and 15 g from adenoma tissues were electrophoresed on a formaldehyde-containing 1.5% agarose gel. Each sample was electrophoresed twice, and two Hybond-N membranes containing the same samples were prepared. After initial hybridization with a probe for the full-length coding region of CaSR, membranes were stripped, and each membrane was rehybridized with a probe for either the exon 1A-or 1B-specific riboprobe. This figure is a representative result from a normal parathyroid gland. The same expression patterns of these bands were obtained from parathyroid adenomas. normal parathyroid is less than that in previous reports. This may be due to the difference of methods employed. Because the expression of CaSR is not uniform in parathyroid adenomas (23), Northern blot analysis shows the average expression level of CaSR in whole adenomas. In contrast, immunohistochemistry and in situ hybridization would be suitable for investigating the expression pattern of CaSR in different part of adenomas.
The reduced expression of CaSR may contribute to less efficient intracellular signal transduction at a given calcium level. This leads to less suppression of PTH secretion and hence higher PTH levels. Because calcimimetics suppresses proliferation of parathyroid cells (24), it is possible that the reduction of CaSR expression also has stimulatory effects on parathyroid cell growth. Thus, derangement of CaSR expression may explain the two main characteristics of parathyroid adenoma, a higher set point for PTH secretion and enhancement of cell proliferation. However, it is not clear at the moment whether the reduction of CaSR expression can explain all the abnormalities of adenoma cells because the reduction is not prominent. Since no mutation in CaSR was reported in parathyroid adenomas (8), derangement of post-receptor signal transduction is another possibility. Furthermore, it is not known whether the reduction of CaSR expression is a direct cause of parathyroid adenoma or, conversely, the results of tumorigenesis. For example, it is possible that hypercalcemia and/or high levels of 1,25-dihydroxyvitamin D in patients with primary hyperparathyroidism affect expression of CaSR. However, our preliminary experiments indicate that neither hypercalcemia nor 1,25dihydroxyvitamin D has an effect on the promoter activities of the human CaSR gene (data not shown). Further study is necessary to clarify the effect of systemic factors on CaSR expression because we cannot exclude the possibility that a region farther upstream has some effects on CaSR expression.
In conclusion, the human CaSR gene has two promoters and produces multiple mRNAs. Specific reduction of the expression levels of transcript driven by the upstream promoter underlies the pathogenesis of parathyroid adenomas. Further analyses of the factors that modulate the activity of the upstream promoter of CaSR will be necessary to clarify the mechanisms of reduced expression of CaSR in parathyroid adenomas.