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Tumor-Specific Decreased Expression of Calcium Sensing Receptor Messenger Ribonucleic Acid in Sporadic Primary Hyperparathyroidism¹

Department of Molecular Medicine (F.F., M.S., C.L.), Department of Clinical Pathology (L.G.), and Department of Surgery (U.E., M.B., L.-O.F.), Karolinska Hospital, Stockholm, Sweden

Address all correspondence and requests for reprints to: Filip Farnebo, Department of Molecular Medicine, Endocrine Tumor Unit L8:01, Karolinska Hospital, S-171 76 Stockholm, Sweden.

	Abstract

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Secretion of PTH is regulated by extracellular calcium via calcium receptors (CaR) on the parathyroid cell surface. Recent studies have shown a decreased expression of CaR messenger RNA (mRNA) and CaR protein in pathological parathyroids. We studied the expression of CaR mRNA in pairs of adenoma and adenoma-associated normal gland from the same patients (n = 17) and in biopsies of normal parathyroid glands of normocalcemic subjects (n = 4) using in situ hybridization with oligonucleotide probes on frozen sections. No down-regulation of CaR mRNA caused by hypercalcemia could be demonstrated in the normal adenoma-associated parathyroids when compared with the normal parathyroids of normocalcemic subjects. In contrast, CaR mRNA in the adenomas was significantly reduced to 64% (median; range 41–98) of the corresponding normal adenoma-associated glands. No correlation was seen between CaR mRNA in the adenoma and preoperative serum calcium, PTH, or weight of the adenoma. Loss of heterozygosity studies were performed on adenomas using markers for the locus of the CaR gene on chromosome 3q. No allelic loss was demonstrated, excluding allelic loss as the cause for decreased CaR mRNA expression in the adenomas. It is concluded that the lowered levels of CaR mRNA in parathyroid adenomas may contribute to the increased set point of PTH secretion. In large adenomas the increased cell mass seems to be more important for the increased secretion of PTH.

	Introduction

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PTH is secreted from the parathyroid glands in response to lowered extracellular calcium. The signal from the exterior to the interior of the parathyroid cell is mediated by receptors in the plasma membrane. The calcium sensing receptor (CaR) has been extensively characterized during the last few years by Brown and coworkers (1, 2, 3, 4). CaR belongs to the seven transmembrane G protein-coupled receptor superfamily. Inactivating mutations in the CaR gene cause familial benign hypocalciuric hypercalcemia in heterozygous individuals and neonatal severe hyperparathyroidism in homozygous individuals, whereas activating mutations cause hypocalcemia (5, 6, 7). Changes in the CaR have been proposed to be responsible for the increase in set point of PTH secretion seen in primary hyperparathyroidism. However, it has been difficult to establish the role of CaR in sporadic hyperparathyroidism. Thus, no mutations of the CaR gene could be demonstrated in a large number of parathyroid tumors (8). Neither did two different CaR complementary DNA clones, isolated from a parathyroid adenoma, show any mutations in the coding region (3). Studies using markers flanking the CaR locus on chromosome 3q revealed loss of heterozygosity in but a few cases of sporadic parathyroid adenomas and secondary parathyroid tumors (9, 10), indicating that this is no common cause for the development parathyroid tumors.

It has been speculated that variable stability of the different messenger RNAs (mRNAs) transcribed from the CaR gene could affect the level of functional CaR expressed in the adenoma (3). An immunohistochemical study using antibodies directed against the extracellular domain of CaR (11) demonstrated a marked reduction of CaR staining in adenomas as well as hyperplastic uremic glands. In line with this, Gogusev et al. (12) recently showed a decreased expression of CaR mRNA as well as CaR protein in pathological parathyroid tissue as compared with glands from normal subjects.

We studied the expression of CaR in matched pairs of adenoma and biopsy of normal parathyroid from patients with primary hyperparathyroidism, as well as in biopsies of normal parathyroids from normocalcemic subjects using in situ hybridization of DNA oligonucleotides to mRNA. In addition, loss of heterozygosity (LOH) studies were carried out in the parathyroid adenomas using markers flanking the CaR locus on chromosome 3q.

	Materials and Methods

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Patients

Parathyroid tissue (pairs of adenoma and biopsy of adenoma-associated normal gland) from 17 patients operated on for sporadic primary hyperparathyroidism at the Karolinska Hospital were snap frozen in liquid nitrogen after removal and stored at -70 C until analysis. The patients (13 women, 4 men) had a median age of 72 yr at the time of operation (range 43–84). The preoperative median concentrations of serum calcium and intact PTH were 2.80 mmol/L (11.2 mg/dL) (2.62–2.94) and 100 ng/L (61–234), respectively. The median tumor weight was 730 mg (125–2, 900). In addition, sections of parathyroid biopsies (<10 mg) from four normocalcemic patients (all women, median age 52 yr) operated on for thyroid disorders (benign goiter 2, toxic goiter 2) were included in the study. The biopies had been taken for frozen section verification of parathyroid glands, which because of their location on the thyroid had been devascularized during dissection and therefore had to be removed for implantation in the sternocleidomastoid muscle to ascertain function (13). Biopsies of thyroid were obtained as control tissue. The study was approved by the Ethical Committee of the Karolinska Hospital. Informed consent was obtained from all patients.

Preparation of probes

Oligonucleotide probes with sequences complementary to mRNAs encoding for CaR (nt 1371–1410, GenBank/EMBL Data Bank accession number U20759 and U20760) (3) and GAPDH (nt 1149–1193, GenBank/EMBL Data Bank accession number M33197) (14) and a CaR sense probe were synthesized (Geneset, France). The oligonucleotides were labeled at the 3' end with [³⁵S]deoxycytidine ATP (NEN, Life Science Products, Boston, MA) using terminal deoxynucleotidyl transferase (Amersham Life Sciences, Japan). The labeled probes were purified through Nensorb-20 columns (DuPont, Wilmington, DE).

In situ hybridization

Cryostat sections 14 µm thick were cut at -20 C and thaw-mounted onto SuperFrostPlus (Menzel-Gläser, ) slides. Sections of adenoma and biopsy of normal parathyroid from the same patient were mounted on the same slide. Hybridization was essentially performed according to Dagerlind et al. (15). In brief, hybridization solution containing 50% formamide (Sigma, St. Louis, MO), 4 x SSC (1 x SSC: 0.15 M NaCl, 0.015 M sodium citrate), 1x Denhardt’s solution (0.02% polyvinyl-pyrrolidone (Sigma), 0.02% BSA fraction V (Sigma) and 0.02% Ficoll (Sigma), 1% N-lauroyl-sarcosine (Sigma), 0.02 M phosphate buffer (pH 7.0), 10% dextran sulphate (Sigma), 500 µg/mL heat-denaturated salmon sperm DNA (Sigma), 200 mM dithiothreitol (Sigma), and 37.5 µL/mL of the labelled probes were mixed and placed in hybridization oven for 30 min. The solution was spread out on the sections, covered with parafilm, placed in a humidified box, and incubated for 16–18 h at 42 C. After hybridization the sections were seqentially rinsed in five changes of 1 x SSC at 60 C for 60 min, put on the bench for 30 min to cool down, and then rinsed in distilled water and dehydrated in 70, 95, and 99% ethanol. After air drying, the sections were exposed to Hyperfilm beta-max x-ray film (Amersham, CEA AB, Sweden) for 4–6 days depending on the specific activity of the probes. The sections were then dipped in Kodak NTB-2 emulsion (Eastman Kodak, Rochester, NY), exposed for 1–4 weeks, developed, and finally counterstained with hematoxylin-eosin and evaluated in light and dark-field microscopy.

The x-ray films were developed with Kodak LX 24 for 5 min and fixed in Kodak AL4 for 10 min. Semiquantification of film autoradiograms was carried out by microdensitometry using a Macintosh Quadra 700 computer (Apple Computer, Cupertino, CA) equipped with a Quick Capture frame grabber board (Data Translation, Marlboro, MA) and a Northern Light precision illuminator (Imaging Research, St. Catharines, Ontario, Canada) and a CCD camera (Hamamatsu Photonics KK, Hamamatsu-City, Japan) equipped with a Nikon 55 mm lens (Nikon, Tokyo, Japan). Each image was an average of 16 video frames digitized to a 768 x 512 matrix with 256 gray levels for each picture element. Analysis was made on a Macintosh Performa 6400 computer using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/). Gray levels corresponding to eight standards (Amersham International, Buckinghamshire, England) were determined and used to construct a gray level to activity concentration function. Exposure time was chosen with regard to the specific activity of each probe to stay within the exposure range of the film. All measurements were subtracted for film background only. Care was taken to measure only homogenous areas consisting of parathyroid parenchymal cells, excluding fat cells, connective tissue, and vessels. This task was accomplished by simultaneously evaluating the sections in the microscope. At least two sections of each gland were evaluated.

LOH

High molecular weight DNA was prepared from sections of fresh frozen tumor and peripheral blood leukocytes using standard methods. The following microsatellite markers in the CaR region on chromosome 3q were selected: D3S1267, D3S1269, and D3S1316. PCRs were performed according to standard procedures, and the PCR products electrophoresed on polyacrylamide gel followed by autoradiography or digital imaging (Bio-Imaging analyzer Bas 1000, Fuji).

Statistical analysis

Data were analyzed using the software StatView 4.0 and expressed as median and range or mean ± SD. Comparisons between groups were made using the Mann-Whitney U-test. Comparisons between biopsy and adenoma from the same patients were made using the Wilcoxon signed rank test. An ANOVA table was used to evaluate correlations between variables. Probabilities of <0.05 were accepted as significant.

	Results

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Controls

No signal was seen with the sense probe in any tissue analyzed (Fig. 1).

View larger version (139K): [in this window] [in a new window]   Figure 1. A and B, Dark-field microphotographs of sections of normal parathyroid hybridized with sense (A) and antisense (B) probe against CaR mRNA. Almost no silver grains can be seen after hybridization with sense probe (A). A dense aggregation of silver grains is seen over chief cells but not over connective tissue and vessel after hybridization with antisense probe (B). x125. C and D, Microphotographs of a section of normal parathyroid remnant and adenoma hybridized with antisense probe against CaR mRNA (same section as in Fig. 2.) C, Light-field. normal remnant is mainly composed of chief cells with small micronodules of oxyphil cells, whereas adenoma is mainly composed of oxyphil cells. D, Dark field. A dense aggregation of silver grains can be seen over chief cells in normal remnant. Less grains are seen over oxyphil cells. x50. E and F, Details of right upper portion of microphotographs in C and D. E, Light field. Oxyphil nodule surrounded by chief cells. F, Dark field. Less dense aggregation of silver grains over oxyphil nodule than over chief cell areas. x250.

GAPDH mRNA could be demonstrated in all sections analyzed, parathyroid as well as thyroid. GAPDH mRNA in normal parathyroid (n = 4), adenoma-associated normal parathyroid (n = 17), and adenoma (n = 17) was 334 ± 61, 344 ± 99, and 362 ± 149 nCi/g, respectively. Although variability was great in the adenoma group, no adenoma had a GAPDH expression lower than 73% of the corresponding normal parathyroid. Thus, no specimens were excluded from the analysis because of suspicion of significant reduction of mRNA caused by RNase activity.

CaR

CaR mRNA was expressed in all parathyroid sections (Figs. 1 and 2). No expression was found in thyroid. To assess reproducibility of the semiquantification procedure, 11 sections from a biopsy of an adenoma-associated normal gland (patient No. 4) were hybridized on different slides and then exposed to x-ray film. CaR mRNA of the chief cells was 1,107 ± 154 nCi/g, yielding a variation coefficient of 14%. In the same biopsy a few oxyphil cell micronodules could be found. The CaR mRNA in these micronodules was significantly lower, 446 ± 109 nCi/g, than in the chief cells (Fig. 1).

View larger version (91K): [in this window] [in a new window]   Figure 2. A, X-ray film exposed to sections of biopsy of normal parathyroid (left) and part of adenoma (right) hybridized with antisense probe against CaR mRNA (patient No. 4). At left upper border of adenoma is a remnant of normal parathyroid. Estimated CaR mRNA of normal was 1319 nCi/g, of adenoma 815 nCi/g, and of remnant 1,313 nCi/g. (Light- and dark-field microscopy of same section of adenoma is shown in Fig. 1, C–F.) B, Schematic representation of sections in A.

The expression of CaR mRNA in the adenomas varied between 41–98% (median 64%) of that of the corresponding biopsy of normal parathyroid gland (Table 1, Fig. 3). When small nodules of oxyphilic cells were found in an adenoma consisting of predominantly chief cells, the oxyphilic cells always had a lower content of CaR mRNA. Otherwise, distribution of CaR mRNA was even within a certain adenoma, and no systematic difference in expression could be noted between adenomas mainly composed of chief cells, oxyphil cells or transitional cells (Table 1).

View larger version (31K): [in this window] [in a new window]   Figure 3. CaR mRNA in pairs of biopsy of adenoma-associated normal gland and adenoma. Estimated amount in nanocuries per gram. Three different experiments with different specific activity of probe. *, Patients 1–3; +, Patients 4–5; , Patients 6–17.

LOH studies

Markers flanking the CaR gene on chromosome 3qcen-3q21 were used to search for LOH in 11 adenomas. All tumors were informative for at least one marker, but no tumor showed LOH (Table 1).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study shows a variable decrease of CaR mRNA expression in parathyroid tumors as compared with biopsies of normal parathyroid glands from the same patients, as well as normal parathyroid glands from normocalcemic patients. The results are in agreement with recently published data by Gogusev et al. (12) that showed that the expression of CaR mRNA is decreased in parathyroid adenomas when compared with normal parathyroid glands from normocalcemic subjects. Previously Kifor et al. (11), using immunohistochemistry, showed that the CaR protein is reduced in adenomas when compared with corresponding adenoma-associated normal gland. These two studies described even lower levels of CaR mRNA/CaR protein in adenomas than our study. Kifor et al. (11) reported a CaR protein level in the adenoma of 24–65% that of the normal. Gogusev et al. (12), using a nonradioactive method for determination of hybridization signal, found a reduction of CaR mRNA expression in adenomas to approximately 29–36% of normal glands from normocalcemic subjects. No adenoma-associated normal glands were analyzed, however, and no comparable quantitative data were given for CaR protein. The level of CaR mRNA in the adenoma in the present study was in the range 41–98%, with a median of 64%. Thus, several adenomas displayed only moderate lowering of CaR mRNA expression. The discrepancies between the three studies are most probably caused by the differences in methodology. All the techniques used are semiquantative in nature, and the discrepancies should not be over interpreted.

The present investigation shows, for the first time, that there is no difference in CaR mRNA levels in parathyroid glands from normocalcemic subjects and in normal adenoma-associated glands from patients with hypercalcemia caused by hyperparathyroidism. Thus, hypercalcemia does not down-regulate the expression of CaR mRNA in normal parathyroid glands. This finding is compatible with data obtained in rats, in which neither serum calcium nor vitamin D affect the expression of CaR in the parathyroids (16).

In all instances in which oxyphils cells were found together with chief cells, the CaR mRNA of the oxyphil cells per unit area were lower. One reason for this may be the greater cell volume of the oxyphil cells. Thus, the number of transcripts per cell may not differ. No systematic difference between adenomas composed of mainly chief or oxyphils cells were seen.

It is not known what causes the lower expression of CaR mRNA in the adenomas. One possible explanation would be less stable mRNA (3). Another explanation would be loss of one of the alleles at the CaR locus on chromosome 3q. Such a loss has been demonstrated in single cases of tumors from primary as well as secondary hyperparathyroidism (9, 10). If only one functional gene was left, this would probably have affected the number of transcripts. However, the present data do not support this hypothesis, because no loss of heterozygosity could be found in the 11 tumors analyzed.

In addition to the reduced transcription of the CaR gene, further attenuation of the signal elicited by an increased extracellular calcium could result from inactivating mutations in the gene, similar to those seen in familial benign hypocalciuric hypercalcemia (5, 17). Such point mutations in the CaR gene would not be detected by the present method.

It is not clear whether the decreased expression of CaR mRNA is a primary or a secondary phenomenon. It has been speculated that mutations affecting the set point mechanism would give growth advantage to the affected parathyroid cell leading to monoclonal expansion. The growth of such a clone would be asymptotic and progressively retarded when the new secretory set point is approached (18, 19). According to this view, adenomas that have arisen because of mutations affecting set point should be comparatively small and found in patients with long-standing hypercalcemia, whereas adenomas caused by mutations in genes regulating the cell cycle (oncogenes or tumor suppressor genes) should be larger and associated with rapid progress of the disease caused by continuous cell growth. Although such a theory is attractive, it is not corroborated by the present study. In line with the findings of Kifor et al. (11), we were not able to demonstrate any correlation between the CaR mRNA and the tumor weight. An alternative explanation for adenoma formation would be that the two events follow each other. An increase of set point gives a stimulus for proliferation, which in turn increases the risk for mutations in genes regulating the cell cycle (19).

It has been suggested that in hyperparathyroidism, the increase of parenchymal cell mass is as important for the increased secretion of PTH as the increase of set point (19). That assumption is corroborated by the present results, because a positive correlation between adenoma weight and serum PTH was found, but not between CaR mRNA (as percent of corresponding normal parathyroid gland) and serum PTH. This indicates that other alterations in the control of PTH secretion are of importance in addition to the disturbance at the receptor level.

Finally, it has to be pointed out that in addition to CaR, another possible sensor of external calcium in the parathyroid cell has been described, namely gp330, which is a transmembrane protein belonging to the low density lipoprotein-receptor superfamily (20, 21). Comparisons between the expression of the two receptors have to be made to further elucidate the role of each of them.

In summary, the present results are in line with previous studies showing a reduced expression of CaR mRNA and protein in parathyroid adenomas compared with normal parathyroid glands. In addition, evidence is presented that CaR mRNA expression is not down-regulated in normal parathyroid glands in response to hypercalcemia. The findings may, to a certain extent, explain why pathological parathyroid cells are less sensitive to external calcium than normal parathyroid cells. The relative role of a reduced expression of CaR mRNA in relation to mutations affecting cell-cycle control in the development of parathyroid adenomas remains to be clarified.

	Footnotes

 
¹ This work was supported by the Swedish Medical Research Council, Swedish Cancer Foundation, the Cancer Society of Stockholm, the Magn. Bergvall Foundation, the Gustav V Jubilee Fund, the Martin Rind Foundation, and the Fredrik and Ingrid Thuring Foundation.

Received April 11, 1997.

Revised May 28, 1997.

Accepted June 18, 1997.

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