Abnormal Parathyroid Cell Proliferation Precedes Biochemical Abnormalities in a Mouse Model of Primary Hyperparathyroidism

Sanjay M. Mallya, James J. Gallagher, Yvette K. Wild, Olga Kifor, Jessica Costa-Guda, Kirsten Saucier, Edward M. Brown and Andrew Arnold

Department of Oral Health and Diagnostic Sciences (S.M.M.), University of Connecticut School of Dental Medicine; Center for Molecular Medicine (S.M.M., J.J.G., Y.K.W., J.C.-G., K.S., A.A.) and Division of Endocrinology and Metabolism (A.A.), University of Connecticut School of Medicine, Farmington, Connecticut 06030-3101; and Brigham and Women’s Hospital (O.K., E.M.B.), Boston, Massachusetts 02115

Address all correspondence and requests for reprints to: Andrew Arnold, M.D., Center for Molecular Medicine, University of Connecticut Health Center, Farmington, Connecticut 06030-3101. E-mail: aarnold{at}nso2.uchc.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The properties of neoplastic proliferation and hormonal dysregulation are tightly linked in primary hyperparathyroidism (HPT). However, whether abnormal parathyroid proliferation is the cause or result of a shift in calcium-sensitive parathyroid hormonal regulation has been controversial. We addressed this issue by analyzing the temporal sequence of these fundamental abnormalities in a mouse model of primary HPT. These transgenic mice (PTH-D1) harbor a transgene that targets overexpression of the cyclin D1 oncogene to parathyroid cells, resulting in parathyroid hypercellularity with a phenotype of chronic biochemical HPT and, notably, an abnormal in vivo PTH-calcium set point. We examined parathyroid cell proliferation and biochemical alterations in PTH-D1 and control wild-type mice from ages 1–14 months. Strikingly, abnormal parathyroid proliferation regularly preceded dysregulation of the calcium-PTH axis, supporting the concept that disturbed parathyroid proliferation is the crucial primary initiator leading to the development of the biochemical phenotype of HPT. Furthermore, we observed that decreased expression of the calcium-sensing receptor in the parathyroid glands occurs several months before development of biochemical HPT, suggesting that decreased calcium-sensing receptor may not be sufficient to cause PTH dysregulation in this animal model of primary HPT.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PRIMARY HYPERPARATHYROIDISM (HPT) is a common endocrine disorder, characterized by hypersecretion of PTH and resultant hypercalcemia. Most cases of primary HPT result from a solitary adenoma in one of the parathyroid glands. Given that the overwhelming majority of parathyroid tumors are small and benign, the clinical manifestations of the tumors are almost always related to the biochemical and hormonal dysregulation they cause, rather than their tumor mass per se. The properties of neoplastic proliferation and hormonal dysregulation seem to be inexorably linked in primary HPT, a coupling not necessarily characteristic of other endocrine neoplasms such as thyroid, adrenal, or pituitary tumors. The molecular mechanisms underlying this important and consistent link between the theoretically separable processes of parathyroid cell tumorigenesis and PTH secretory dysregulation, demonstrated both in cell culture and in patients, are not fully understood, and insights into these pathways could ultimately yield new treatments for parathyroid disorders.

For parathyroid tumors in primary culture, the abnormal feedback relationship between extracellular calcium concentration and PTH secretion has been characterized through determination of the "set point," i.e. the calcium concentration that yields the half-maximal level of regulatable PTH secretion (1). Likewise, dynamic studies of patients with primary HPT have shown that, in addition to a proliferative defect leading to parathyroid hypercellularity, there is an abnormality in the feedback system through which extracellular calcium regulates PTH secretion—an apparent resetting of the set point mechanism that normally tightly couples PTH secretion with ambient calcium levels (2).

Normal parathyroid cells are able to respond to the stimulus of chronic hypocalcemia by increasing PTH secretion and also by a secondary (polyclonal) expansion of parathyroid cell mass, mediated through the calcium-sensing receptor (CaR) (3, 4). This has led to the controversial hypothesis that most parathyroid adenomas are caused by acquired mutation(s) in the genes of the set point control pathway per se, with the cell’s mistaken perception of ambient hypocalcemia being the driving force for parathyroid tumor cell proliferation (5). However, support for this hypothesis has not been forthcoming from molecular genetic studies of parathyroid tumors (6, 7, 8).

Currently, two genes are established contributors to common sporadic parathyroid adenomas, the cyclin D1 (PRAD1) oncogene (9) and the MEN1 tumor suppressor gene (10). Cyclin D1 was first discovered through its involvement in clonal activating gene rearrangements in parathyroid tumors. This rearrangement, a pericentromeric inversion of chromosome 11, juxtaposes the PTH gene’s 5'-regulatory region with the cyclin D1 gene, thereby causing overexpression of cyclin D1. Cyclin D1 is overexpressed in 20–40% of parathyroid adenomas (11, 12, 13, 14). To further study the role of cyclin D1 in parathyroid neoplasia, we recently developed a mouse model of primary HPT (15). In these mice (PTH-D1), targeted overexpression of cyclin D1 in the parathyroid glands leads to parathyroid hypercellularity and, in some cases, parathyroid adenomas, with a phenotype of chronic biochemical HPT (15). Notably, these mice develop abnormalities in the calcium set point, similar to that observed in the human disease. In the studies reported here, we have used this model to analyze the temporal sequences of proliferative and set point abnormalities that occur in parathyroid tumorigenesis, to help ascertain whether abnormal parathyroid proliferation is the cause or result of a shift in PTH-calcium set point.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Given that PTH-D1 mice are known to develop biochemical HPT by the age of 10 months (15), we first measured serum calcium and serum PTH levels in transgenic and wild-type animals ranging in age from 1–14 months (Fig. 1Go), to more clearly and precisely define the temporal characteristics of the onset and progression of biochemical abnormalities in this model of primary hyperparathyroidism. Through the age of 9 months there were no significant differences in the serum calcium and serum PTH levels in PTH-D1 mice, compared with wild-type age-matched controls. However, between the ages of 6–9 months, elevated serum calcium levels were noted in some PTH-D1 mice, although not consistently in all animals. Subsequently, at ages 10 months and 14 months (the next measured time point), both serum calcium and serum PTH levels were significantly elevated in PTH-D1 mice compared with wild-type littermates (P ≤ 0.05).



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Fig. 1. Biochemical Alterations in PTH-D1 and Wild-Type (WT) Mice

Serum calcium (A) and serum PTH (B) were measured at the indicated ages in PTH-D1 mice (closed squares) and wild-type littermates (open circles). Symbols denote means ± SD. The tabulation at the bottom of the figure indicates the numbers of animals analyzed in each age group. *, P ≤ 0.05.

 
To establish that any later-emerging phenotype in PTH-D1 mice was not merely attributable to delayed onset of expression of the cyclin D1 transgene, we examined in detail the temporal pattern of expression of cyclin D1 in the parathyroids of PTH-D1 transgenic mice and controls. At all ages examined, from the earliest analyzed time point of 1 month and extending through all subsequent ages examined, cyclin D1 expression was detected in parathyroid glands of PTH-D1 transgenics, but not wild-type mice at any age (Fig. 2Go). Histological examination of parathyroid glands from PTH-D1 mice revealed enlargement and hypercellularity. These morphological changes were first detected at 5–6 months of age and were present in most PTH-D1 mice examined.



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Fig. 2. Immunohistochemical Analysis of Cyclin D1 Expression

A, Parathyroid gland from a 7-month-old PTH-D1 transgenic mouse. Staining (dark brown) is localized to the nucleus and is distributed nonuniformly throughout the parathyroid. Adjacent thyroid tissue is negative for staining. The same pattern of expression was observed with other PTH-D1 mice. B, Parathyroid gland from a 7-month-old wild-type control mouse showing no staining. Parathyroids of wild-type mice were uniformly negative for cyclin D1 staining.

 
To determine whether this observed hypercellularity could be a result of altered parathyroid cell proliferation, we examined proliferation in the parathyroid glands of PTH-D1 mice at ages ranging from 1–14 months. Proliferation was measured by determining incorporation of bromodeoxyuridine (BrdU) by parathyroid cells, in vivo, and by immunohistochemical detection of proliferating cell nuclear antigen (PCNA) expression (Fig. 3Go). The proliferative index was determined as the ratio of the number of labeled cells per unit area of the gland (Fig. 4Go). In both PTH-D1 and wild-type mice, transient parathyroid cell proliferation was noted at the ages of 1–2 months, correlating with the timing of active skeletal growth, and subsequently decreased to low levels at 3–4 months. However, at age 5 months, a specific and dramatic increase in the rate of proliferation was noted in the parathyroid glands from PTH-D1 mice, but not in wild-type mice (P ≤ 0.05). Importantly, the PTH-D1 mice at these ages were not hypercalcemic or hyperparathyroid (Fig. 1Go). This increased parathyroid proliferation was maintained in PTH-cyclin D1 mice through the ages 10 and 14 months, by which time transgenic mice had significantly elevated serum calcium and PTH levels (Fig. 1Go). Analysis of serum calcium/PTH levels and proliferative indices from individual animals showed no significant correlation between these two parameters. In confirmation, the temporal pattern of parathyroid cell proliferation, as determined in vivo by BrdU incorporation, was similar to that determined by detection of PCNA levels (data not shown).



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Fig. 3. Analysis of Parathyroid Cell Proliferation by Immunohistochemical Detection of BrdU

A, Parathyroid gland from a 7-month-old PTH-D1 transgenic mouse, showing several darkly stained cells, indicating incorporation of BrdU. B, Parathyroid gland from a 7-month-old wild-type control mouse is negative for incorporation of BrdU.

 


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Fig. 4. Temporal Analysis of Parathyroid Cell Proliferation

Proliferating cells were identified by immunohistochemical detection with an anti-BrdU antibody. The ratio of labeled cells per unit area of the gland was determined for PTH-D1 (black bars) and wild-type (WT) littermates (open bars). Bars represent means ± SD. *, P ≤ 0.05.

 
We next analyzed expression of the CaR, an important mediator of the response of parathyroid cells to changes in circulating calcium concentration, the expression of which is reduced in human parathyroid tumors. CaR expression was determined by immunohistochemistry (IHC) in parathyroid glands of mice at ages 1, 4, 7, and 12 months. These ages represent mice that 1) have normal calcium and PTH levels, with no evidence of abnormal proliferation (ages 1 and 4 months); 2) are biochemically normal but exhibit increased proliferation (age 7 months); and 3) have biochemical HPT and demonstrate increased, abnormal parathyroid cell pro-liferation (age 12 months). CaR expression was detected by IHC, and the intensity of staining was quantitated by optical image analysis. CaR expression in PTH-D1 mice was generally decreased to approximately 50–75% of that in age-matched, control animals (Fig. 5Go). The difference in expression of CaR was statistically significant at ages 7 months (before development of biochemical HPT) and 12 months (P ≤ 0.05 and 0.005, respectively). We analyzed the relationship between serum calcium and PTH levels and expression of CaR in individual mice. There was no significant correlation between serum calcium/PTH levels and degree of CaR expression at the various age groups examined.



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Fig. 5. Immunohistochemical Detection of CaR Expression

A, Parathyroid gland from a 7-month-old PTH-D1 transgenic mouse, showing overall decreases in the staining intensity of CaR. CaR expression was usually more heterogeneous and patchy in distribution than in glands from wild-type mice. B, Parathyroid gland from a 7-month-old wild-type mouse showing rim staining of parathyroid chief cells. The gland is uniformly stained. C, Temporal analysis of CaR expression. The optical density (intensity of staining) was determined by image analysis. The staining intensity was normalized to age-matched littermate controls. Columns represent means ± SD, expressed as percent of expression in age-matched littermate controls. *, P ≤ 0.05; **, P ≤ 0.005

 
Because the vitamin D receptor (VDR) is an important regulator of parathyroid cell function, mediating the inhibitory effects of 1,25-dihydroxyvitamin D on parathyroid cell proliferation and PTH secretion, we analyzed VDR expression in parathyroid glands from PTH-D1 and control mice at selected ages. In contrast to the reduced expression of CaR, there was no difference in VDR expression in parathyroid glands from transgenic mice at ages 1, 4, and 7 months, when they were still biochemically normal, compared with age-matched controls. However, at age 14 months, when the mice had manifested biochemical HPT, expression of VDR was markedly reduced (Fig. 6Go).



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Fig. 6. Immunohistochemical Detection of VDR Expression

A, Parathyroid gland from a 14-month-old PTH-D1 transgenic mouse, showing decreased expression of VDR. B, Parathyroid gland from a 14-month-old wild-type mouse showing nuclear staining of parathyroid chief cells. The gland is uniformly stained.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Abnormal cell proliferation and dysregulation of PTH secretion are consistent and tightly intertwined features of parathyroid neoplasia. The molecular mechanisms that underlie crucial links between these properties are poorly understood. Given the low mitotic rate, slow growth, and long-term clinical/biochemical stability of most parathyroid adenomas, it has been proposed that an acquired mutation in the PTH secretory set point pathway must be the initiating primary event that leads to sporadic parathyroid neoplasia (16, 17). Although appealing, this hypothesis remains controversial. It has not, for example, been supported by results of mutational analyses of the CASR gene in parathyroid adenomas (7, 8), nor does it account for the involvement of those genes that are established contributors to parathyroid tumorigenesis (6). Moreover, we have previously shown that parathyroid-targeted overexpression of cyclin D1, an important regulator of the cell cycle, leads to a phenotype of biochemical HPT (15). Here we report on extensive characterization of this transgenic model to conclusively show that abnormal parathyroid cell proliferation precedes the development of biochemical HPT, indicating that dysregulation of parathyroid cell growth occurs before dysregulation of hormone secretion.

Further, although cyclin D1 is established as a parathyroid oncogene, the mechanism by which its gene product drives parathyroid neoplasia, often assumed to be increased proliferation (possibly caused by cell cycle dysregulation), has not been demonstrated in the context of parathyroid tissue. Our findings substantiate that the cyclin D1-induced primary disturbance of growth is a driving force for parathyroid tumorigenesis and precedes biochemical abnormalities in this model. Given that 20–40% of human parathyroid adenomas overexpress cyclin D1, it seems plausible that cyclin D1-induced hyperproliferation followed by disturbed PTH regulation is of similar importance in human parathyroid tumorigenesis. Indeed, because many parathyroid adenomas are caused by genetic abnormalities that do not involve cyclin D1, and yet maintain the same tight association between abnormal proliferation and hormonal control, we speculate that the concept of set point shift being secondary to proliferative drive constitutes a general feature of parathyroid neoplasia rather than being specific to cyclin D1-driven tumors. Analyses similar to those we have described, but performed on a mouse model of HPT manifesting inactivation of the men1 tumor suppressor (18), will be informative in this regard. Our data also allow for the possibility that biochemical HPT is a direct and independent result of the primary oncogenic defects, not mediated through their tumorigenic or proliferative actions.

Hypercalcemia due to an altered calcium set point is a central feature of primary HPT. Also, we have previously established that an abnormal calcium set point accompanies the hypercalcemia and elevated PTH levels in PTH-D1 mice (15). Heterozygous or homozygous inactivation of the CASR gene is known to cause an increased calcium set point in patients with familial hypocalciuric hypercalcemia or neonatal severe HPT (19). Because decreased CaR expression is also found in sporadic parathyroid adenomas (20, 21, 22), this decrease has been thought to be the key determinant of the altered set point in primary HPT. Here we show that a significant decrease in parathyroid CaR expression can be detected at 7 months, before the onset of increased PTH levels. Perhaps at this early stage, the degree of decrease of CaR is insufficient to cause biochemically detectable alterations of the PTH-calcium set point. Notably, mice at these ages do manifest abnormal parathyroid proliferation, further substantiating that proliferative abnormalities precede the onset of biochemical dysregulation. Previous studies using a uremic rat model of secondary HPT have also demonstrated a similar phenomenon: in this model, PTH hypersecretion and down-regulation of the CaR receptor in the parathyroid gland were reversed by dietary phosphate restriction (23). However, restoration of CaR expression lagged behind normalization of PTH levels, suggesting that the CaR does not play a major role in regulating the calcium set point in that model (23). Notably, and in marked contrast, mice that exhibit a similarly decreased level of CaR but, as a result of a germline heterozygous knockout of the casr gene, demonstrate a clear rightward shift in the set point curve with hypercalcemia and inappropriate/excessive PTH levels (24). Possibly, the stage of development at which alteration of CaR expression is imposed (embryonic/germline vs. postnatal/acquired) may lead to very different phenotypic consequences. Nevertheless, our results suggest that in human sporadic parathyroid tumors the typical decrease in CaR expression may not be the only determinant of the altered set point of the tumor cells. Furthermore, the decreased expression of CaR in the face of normal calcium and PTH levels also points to the complex nature of calcium sensing by the parathyroid cell. Perhaps factors other than receptor density, such as functional activity of the receptor or oscillations of intracellular Ca2+, may play a role in the CaR-mediated signaling mechanisms (3). It is also tempting to speculate that perhaps the primary cyclin D1 abnormality, independent of its tumorigenic role and/or putative actions as a cell cycle regulator, may have a more direct role in regulating the expression of CaR. Indeed, in vitro studies have indicated that cyclin D1 may mediate effects independent of its kinase partner cdk4 (25, 26, 27). However, the existence and functional significance of such mechanisms in parathyroid cells remain to be determined.

In addition to changes in CaR expression, decreased expression of VDR, both at the mRNA level (28) and the protein level (29), is also a consistent feature of HPT. It has been suggested that reduced expression of VDR may be an early and, perhaps, initiating event in the pathogenesis of parathyroid adenomas (29). Our data show that decreased expression of VDR follows proliferative abnormalities and occurs relatively late in the sequence to development of biochemical abnormalities. These data strongly show that reduced expression of VDR is not an early event in the development of primary HPT and that reduced expression of VDR may be a consequence, rather than a cause, of altered set point.

In summary, our data in this cyclin D1-driven transgenic model strongly suggest that a primary growth abnormality is the initiating event in primary HPT, and that deregulation of hormone secretion and resultant biochemical abnormalities arise as a secondary consequence of these proliferative abnormalities.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Animals
The development and initial phenotypic characterization of PTH-D1 transgenic mice were as described elsewhere (15). For genotyping of mice, tail DNA was analyzed by Southern blot using the human cyclin D1 cDNA as a probe, or by PCR as previously described (15). All mice were provided with the Prolab RMH 3000 regular diet containing 1.0% calcium, 0.75% phosphorus, and 2.4 IU/g of vitamin D3 (PMI Nutrition International, Richmond, IN) and water ad libitum. The studies were approved by the institutional animal care committee at the University of Connecticut Health Center.

Biochemical Analyses
At the indicated ages, control and transgenic mice were anesthetized and blood was collected by cardiac puncture and the sera promptly separated. Serum calcium levels were measured (Sigma Chemical Co., St. Louis, MO) using the o-cresolphthalein-complexone method. Serum PTH levels were measured using the mouse intact PTH ELISA (Immutopics, San Clemente, CA).

Assessment of Parathyroid Cell Proliferation
Parathyroid cell proliferation was assessed by determining incorporation of BrdU in the parathyroid glands and by detecting expression of PCNA. Mice were fed a standard diet (Prolab RMH3000) and water containing 5 mg/ml BrdU (Sigma Chemical Co.). After 5 d, the mice were euthanized; the thyroid-parathyroid complex was dissected, fixed in 4% paraformaldehyde for 1 h, and saturated overnight with 30% sucrose. The tissue was embedded in optimal cutting temperature compound medium and frozen on dry ice, and 5-µm-thick axial sections were obtained. Cell proliferation was determined by detection of BrdU incorporation, using a BrdU immunostaining kit (Zymed Laboratories, Inc., South San Francisco, CA), and by immunostaining with an anti-PCNA antibody (Zymed Laboratories, Inc.) as previously described (15).

Immunohistochemical Analysis of Cyclin D1 and VDR Expression
All immunohistochemical analyses were performed on 5-µm-thick frozen tissue sections. Cyclin D1 was detected using a biotinylated mouse monoclonal antihuman cyclin D1 antibody using the avidin-biotin peroxidase-complex method as previously described (15). Briefly, endogenous peroxidase activity in tissue was blocked with 3% hydrogen peroxide in methanol for 5 min. Tissue sections were incubated overnight at 4 C with biotinylated mouse monoclonal antihuman cyclin D1 antibody at 1:50 dilution (clone 5D4; Immuno-Biological Laboratories, Fujioka, Japan). The next day, excess antibody was washed off with PBS, and sections were then incubated with avidin-biotin peroxidase complex at room temperature for 30 min using the VectaStain Elite ABC kit (Vector Laboratories, Inc., Burlingame, CA). 3,3-Diaminobenzidine was used as chromagen, and sections were counterstained in Gill-2 hematoxylin, followed by qualitative visual assessment.

Expression of the VDR was determined, also through qualitative visual assessment, using a rat monoclonal anti-VDR antibody as previously described (30). Endogenous peroxidase activity was quenched in 3% H2O2 in PBS for 10 min. Sections were rinsed in PBS, and nonspecific binding was blocked with 20% rabbit serum in PBS for 30 min and subsequently incubated overnight with a rat monoclonal anti-VDR antibody (Clone 9A7, Affinity BioReagents, Inc., Golden, CO; diluted 1:300 in 20% rabbit serum). Excess antibody was rinsed with PBS, and sections were incubated with biotinylated antirat IgG (Vector Laboratories) and subsequently with 3,3'-diaminobenzidine (Zymed Laboratories).

Immunohistochemical Analysis and Quantitation of Calcium Receptor Expression
Expression of the CaR was detected by IHC using an affinity-purified, polyclonal anticalcium receptor (anti-CaR) antiserum 4637. The peptide against which the anti-CaR antibody was raised corresponds to amino acids 345–359 of the bovine CaR. This peptide sequence is present in all mammalian CaRs examined to date, including the mouse CaR. The detailed procedure has been described previously (15). Briefly, the thyro-parathyroid region from mice at selected ages was embedded in optimal cutting temperature compound on dry ice. Sections (5µm thick) were obtained and fixed in 4% paraformaldehyde for 10 min and washed in PBS. Sections were treated with an endogenous peroxidase inhibitor (DAKO Corp., Carpinteria, CA) and then with 1% BSA in PBS to block nonspecific binding. Sections were incubated with the primary antibody overnight at 4 C. Binding of the primary antibody was detected using peroxidase-conjugated goat anti-rabbit IgG and then with the DAKO 3-amino-9-ethylcarbazole substrate system. Specificity of the immunostaining was confirmed by incubating the anti-CaR antiserum with the peptide against which it was raised (corresponding to amino acids 345–359 of the bovine CaR) before performing immunohistochemistry as described above. Abolition of CaR immunoreactivity when the antiserum had been peptide blocked indicated that the immunostaining was specific for the immunogenic peptide sequence. Digital images of stained sections were obtained using an Olympus BX60 light microscope (Olympus Corp., Melville, NY) and the Open Lab Imaging software (Improvision Inc., Lexington, MA). Intensity of CaR staining was determined by digital image analysis. The region of the parathyroid gland was outlined to exclude areas of nonspecific background as well as any acellular areas in the parathyroid gland. The mean density in the outlined region was determined. Measurements from all regions of interest for each section were averaged. The background intensity was measured and subtracted from the measured mean value to yield a corrected density.


    FOOTNOTES
 
This work was supported in part by National Institutes of Health (NIH) Grant DE14773 from the National Institute of Dental and Craniofacial Research (to S.M.); NIH Grants DK48330, DK52005, and DK67155 (to E.M.B.); the John G. Haddad Jr. Award from The Paget Foundation (to A.A. and S.M.); and the Murray-Heilig Fund in Molecular Medicine (to A.A.).

First Published Online May 31, 2005

Abbreviations: BrdU, Bromodeoxyuridine; CaR, calcium-sensing receptor; HPT, hyperparathyroidism; IHC, immunohistochemistry; PCNA, proliferating cell nuclear antigen; VDR, vitamin D receptor.

Received for publication March 7, 2005. Accepted for publication May 26, 2005.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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