Many pathogenic factors, including hypocalcemia, phosphate retention, vitamin D deficiency, reduction in the density of both vitamin D receptor (VDR) and Ca2+-sensing receptor (CaR) in the parathyroid cells, have been reported to participate in the development of secondary hyperparathyroidism (2HPT) in patients with chronic renal failure1,2,3. Histopathologically, the pattern of parathyroid hyperplasia in patients with chronic renal failure has been classified as two major patterns: diffuse and nodular. In the former, the diffuse parenchymal cell proliferation with normal lobular structures is the characteristic finding, while the latter is recognized as the glands exhibiting at least one well-circumscribed, encapsulated nodule with virtually fat cell free accumulation of parenchymal cells. Based on the investigations of the relationship between glandular weight and the pattern of hyperplasia, it has been reported that nodular hyperplasia is usually heavier than hyperplastic parathyroid glands with a diffuse pattern. It has been considered that the hyperplasia may change its growth pattern from diffuse to nodular as the parathyroid gland becomes heavier4,5.
By using image cytometric DNA analyses, a greater growth is more obvious in the cells of the nodules than in the surrounding parenchymal cells6. Using clonal analysis with a random inactivation of the X-chromosome-linked gene, Tominaga et al found a polyclonal pattern of cell proliferation in the diffuse hyperplastic parathyroid glands, whereas there was a distinct monoclonality in the nodules of nodular hyperplasia7. Arnold et al also reported that 64% of patients who underwent parathyroidectomy for advanced renal hyperparathyroidism had at least one monoclonal parathyroid gland8.
The inhibitory effects of calcitriol on 2HPT have been shown during calcitriol pulse therapy by either oral or intravenous administration9,10,11, which successfully decreased the serum parathyroid hormone (PTH) level. Furthermore, the antiproliferative effect of calcitriol on parathyroid glands was shown during oral calcitriol pulse therapy, which successfully decreased the size of hyperplastic parathyroid glands9. Kremer et al showed that calcitriol abolished the expression of a protooncogene, c-myc, and delayed the subsequent proliferation of bovine parathyroid cells in primary culture12. However, the response to calcitriol therapy in nodular hyperplastic glands, which express less VDR density than diffuse hyperplasia, was reported to be poor as compared to that in diffuse hyperplasia2; the mechanism by which calcitriol regulates parathyroid cell proliferation via VDR remains unknown.
Liu et al found that p21, a cyclin-dependent kinase inhibitor (CDKI), is transcriptionally induced by 1,25-dihydroxyvitamin D3 (1,25D) in a VDR-dependent manner, but not a p53-dependent manner, and that p27 also is induced by 1,25D in the myelomonocytic cell line, U93713. It is known that the p21 and p27 gene codes for CDKI regulate the progression from the G1 to the S phase of the cell cycle by inhibiting cyclin-dependent kinase14,15,16. Moreover, a recent publication by Cozzolino and collaborators demonstrated that, in uremic rats, the efficacy of 1,25D and its less calcemic analog, 19-nor-1,25D, to prevent high phosphorus-induced parathyroid hyperplasia, could be partially attributed to the induction of parathyroid p21 expression17.
We hypothesized that the reduced p21 and p27 production via the decreased nuclear VDR expression leads to parathyroid cell proliferation in hyperplastic parathyroid glands, especially nodular hyperplasia. The present study examined the expressions of VDR, p21, p27, p53, and Ki67 antigen as a proliferation marker to elucidate the pathogenic effects of the CDKIs, p21 and p27, on parathyroid cell proliferation in hemodialysis patients with advanced 2HPT requiring parathyroidectomy.
METHODS
Parathyroid gland tissues
The tissue samples of parathyroid glands were obtained from the patients who underwent total parathyroidectomy with simultaneous autotransplantation for severe 2HPT, parathyroidectomy for primary hyperparathyroidism with single adenoma, and thyroidectomy for goiter, at Kyushu University between 1995 and 1999. Those included 20 normal parathyroid glands as a control (C), 29 hyperplastic parathyroid glands in 8 patients with 2HPT, and 16 adenomas (Ad) with primary hyperparathyroidism. Histologically, 29 hyperplastic glands were diagnosed as having a diffuse pattern (Df) of hyperplasia in 6 and a nodular pattern (Nd) of hyperplasia in 23. Hematoxylin and eosin (H&E)-stained sections were reviewed independently by two different pathologists (K.T. and H.H.) for the diagnoses.
Immunohistochemistry
All specimens were fixed in 10% formalin and were routinely processed to paraffin. Formalin-fixed, paraffin-embedded tissue sections were serially cut at 4 
m and mounted on aminopropyltriethoxysilane-coated glass slides. These sections were deparaffinized in xylene and were rehydrated through an ethanol series. The sections were treated with 0.1 mol/L citrate, pH 6.0, in an 800-W microwave oven for 15 minutes for antigen retrieval before immunohistochemical staining. The sections were pretreated with 5% skim milk at room temperature for one hour to block nonspecific binding of the primary antibodies. The serial sections were then incubated with the primary antibodies at 4°C overnight. Antibodies included anti-p21 antibody (Santa Cruz Biotechnology, Inc., CA, USA) at a 1/100 dilution, anti-p27 antibody (Santa Cruz Biotechnology) at a 1/1000 dilution, anti-p53 antibody (Novocastra Laboratories Ltd., Newcastle, UK) at a 1/800 dilution, anti-VDR antibody (Biomeda Laboratories Ltd., Foster City, CA, USA) at a 1/200 dilution and anti-Ki67 antibody (Novocastra Laboratories) at a 1/100 dilution. Ki67 antigen is a nuclear protein that is expressed in proliferating cells and may be required for maintaining cell proliferation. It is expressed in the granular components of the nucleolus during the late G1, S, G2 and M phases of cell cycle, and has been used as a marker for cell proliferation of solid tumors. Immunostaining was performed with the Elite avitin biotin peroxidase kit (Nichirei, Tokyo, Japan) according to manufacturer's specifications. Slides were counterstained with hematoxylin for two minutes. To demonstrate the specificities of primary antibodies used for immunohistochemistry, immunohistochemical staining was performed with a replacement of the first antibody with an equivalent dilution of pre-immune immunoglobulin G or by pre-absorption of the primary antibodies with the peptide against which it was raised. For quantitation, all of the immunohistochemistry was performed with serial sections.
Quantitation
Only distinct nuclear immunoreactivity was deemed positive for VDR, p21, p27, p53, and Ki67 antigen. The distribution of immunoreactivity was analyzed by quantifying nuclear staining in randomly selected areas on each specimen without knowledge of either diagnosis or outcome. The number of cells expressing VDR, p21, p27, p53, and Ki67 antigen were determined by counting a minimum of 1000 cells per slide using the NIH image program (NIH, Bethesda, MD, USA). Color images of 10 appropriate locations in each specimen were obtained at magnification of 20 
10, covering an area of 717 558 m2 using the Fujix digital camera C-300i (Nikon, Tokyo, USA). The images were then digitized and stored using Photograb 300 version 1.0 (Fuji Photo Film Co., Tokyo, Japan) as PICT files. The brightness and contrast of each image were uniformly enhanced or subtracted by Adobe Photoshop version 4.0 followed by analysis using the NIH images freeware (version 1.60; available on the Internet: http://rsb.info.nih.gov/nih-image/download.html). The PICT image files were opened in a gray scale mode by the NIH image software. The plot area, mean density and plot number corresponding to positive cell number were determined using the "Analyze Particles" command after setting a proper threshold, followed by analysis using Excel v98 (Microsoft Corp., Redmond, WA, USA). The threshold was defined in our study as 100 using the "Density Slice" command in the NIH image software. To avoid the influence of non-specific positive staining, plots of more than 50 pixels were excluded. The number of positive immunoreactive nuclei per 1000 parenchymal cells was expressed as the labeling index (LI). In nodular hyperplasia, cells were counted only in the nodular area of nodular hyperplasia and did not include the diffuse area. VDR, p21, p27, p53, and Ki67 antigen immunoreactive cells were randomly counted over a minimum of 10 fields. When 10% of parathyroid glands were blindly recounted for p21, p27, p53, VDR, and Ki67 antigen, the LIs did not vary more than 
5% from the original count.
Statistical analysis
Data are expressed as the mean SD. All statistics were analyzed by using the StatView program (Abacus Concepts, Berkeley, CA, USA). One-way analysis of variance (ANOVA) followed by the Student t test with Bonferroni correction were used when indicated for comparison of data among the four groups according to the histological findings. A linear regression analysis also was used for the relationship between p21 or p27 LI and either VDR LI, Ki67 LI, gland weight, or p53 LI. A P value of less than 0.05 was considered statistically significant.
RESULTS
Clinical characteristics of the patients
Clinical history was recorded and the follow-up information for each patient carefully measured, including age, preoperative serum level of calcium, inorganic phosphorus, alkaline phosphatase and intact parathyroid hormone (iPTH) levels; the glandular weight was determined when either the hyperplastic glands or primary adenomas were resected. Indeed, preoperative corrected serum calcium was highest in the patients with primary hyperparathyroidism (11.8 1.2 mg/dL), followed by those with 2HPT (10.7 0.8 mg/dL) and normal parathyroid function (9.1 0.4 mg/dL). Serum inorganic phosphorus was highest in the patients with 2HPT (5.7 1.0 mg/dL), followed by those with normal parathyroid function (3.7 0.5 mg/dL), primary hyperparathyroidism (2.3 0.4 mg/dL). Serum alkaline phosphatase and iPTH values were higher in patients with 2HPT (834 448 IU/L and 1101 515 pg/mL, respectively) than those with primary hyperparathyroidism (404 222 IU/L, 282 211 pg/mL, respectively; Table 1). In 2HPT patients, the average dose of oral alfacalcidol was 0.42 0.23 g/day.
Weight and microscopic findings of the resected glands
The single glandular weight of resected parathyroid glands in nodular hyperplasia (1.4 1.6 g) was comparable with that of primary adenomas (1.5 1.4 g), but it was smaller in diffuse hyperplasia (0.3 0.2 g).
Microscopically, normal parathyroid glands showed the constitution of parenchymal cells, such as chief and oxiphil cells, with numerous stromal fat cells Figure 1a. Ad revealed a remarkably enlarged single nodule with compressed rim of normal parathyroid tissue Figure 1b. In Df, the number of parenchymal cells diffusely increased with normal lobular structure and usually stromal fat cells co-existed Figure 1c. Nd exhibited well-circumscribed, encapsulated nodules with virtually fat-free accumulation of parenchymal cells Figure 1d.
Figure 1.
Representative microscopic findings of parathyroid gland tissues in each group (H&E stain, 200). (A) Normal parathyroid gland is composed of chief cells, oxyphilic cells, and stromal fat cells. (B) Parathyroid adenoma is surrounded with compressed rim of normal parathyroid containing numerous stromal fat cells. (C) Diffuse parathyroid hyperplasia is characterized by cell proliferation with normal lobular constitution. (D) Nodular parathyroid hyperplasia is comprised of several nodules surrounded by fibrous capsules.
Expression of VDR and p21 protein
Immunohistochemical staining of VDR protein revealed mainly nuclear localization and, to a lesser degree, cytoplasmic localization in each histological type. The representative tissues are shown in Figure 2. Among the four groups, VDR LI in Nd (162 194) was significantly lower than that in Df (495 337), Ad (383 262), and C (659 234), respectively (P < 0.01; Figure 3).
Figure 2.
Representative immunohistochemical findings of vitamin D receptor (VDR) protein expression in parathyroid gland tissues with diaminobenzidine chromogen and hematoxylin counterstain (400). (A) Normal parathyroid gland with nearly all nuclei showing brown staining for VDR protein. (B) Parathyroid adenoma with some nuclei staining for VDR. (C) Diffuse parathyroid hyperplasia with the majority of nuclei staining for VDR. (D) Nodular parathyroid hyperplasia with few nuclei staining for VDR.
Figure 3.
Difference in the mean labeling index (LI) of vitamin D receptor (VDR) protein expression among each histological type of parathyroid glands. Nodular parathyroid hyperplasia (HP) shows the lowest VDR LI. *P < 0.01 vs. normal; **P < 0.01 vs. diffuse HP; ***P < 0.01 vs. adenoma.
Full figure and legend (35K)p21 protein expression revealed nuclear localization, as shown in Figure 4. Semiquantitative analyses showed that p21 LI in both Nd (85 110) and Ad (136 122) were significantly lower as compared to those in C (359 228) and Df (360 191; P < 0.01), as shown in Figure 5.
Figure 4.
Representative immunohistochemical findings of p21 protein expression in parathyroid gland tissues with diaminobenzidine chromogen and hematoxylin counterstain (400). (A) Normal parathyroid gland. Nearly all nuclei show brown staining for p21 protein. (B) Parathyroid adenoma. Some nuclei show positive staining for p21. (C) Diffuse parathyroid hyperplasia. The majority of nuclei show positive staining for p21. (D) Nodular parathyroid hyperplasia. Only few nuclei show positive staining for p21.
Figure 5.
Difference in the mean labeling index (LI) of p21 protein expression among each histological type of parathyroid glands. Nodular hyperplasia (HP) and adenoma show the lowest p21 LI. *P < 0.01 vs. normal; **P < 0.01 vs. diffuse HP.
Full figure and legend (29K)The distributions of VDR and p21 were examined with the serial sections. Parathyroid sections with high nuclear VDR expression elicited high p21 expression, whereas the areas that lacked VDR had no detectable p21 Figure 6. A significant positive correlation between VDR LI and p21 LI was found only in Nd (r = 0.92, P < 0.01; Figure 7). In Nd, the weight of the resected glands inversely correlated with p21 LI (r = -0.44, P < 0.05; Figure 8).
Figure 6.
Representative immunohistochemical findings of expression of p21 and VDR in serial sections of diffuse hyperplastic parathyroid gland tissues showing early nodular formation with diaminobenzidine chromogen and hematoxylin counterstain (200). (A) Nuclear VDR expression. The majority of nuclei are stained strongly with VDR protein in the left side, but only a small number of nuclei show positive staining in the right side. (B) p21 expression. Immunohistochemistry for p21 expression show positive staining in the nuclei of the left side, which is the same as the VDR expression.
Figure 7.
Correlation between the labeling index of VDR expression (VDR LI) and that of p21 (p21 LI) only in nodular hyperplasia. A significant positive correlation is found between VDR LI and p21 LI in nodular hyperplasia: y = 1.2 + 0.52x; r = 0.915, P < 0.01.
Full figure and legend (8K)Figure 8.
Correlation between the labeling index of p21 expression (p21 LI) and the weight of parathyroid gland only in nodular hyperplasia. A significant negative correlation was found between p21 LI and the weight of parathyroid gland in nodular hyperplasia: y = 2.0 - 0.007x; r = 0.442, P < 0.05.
Full figure and legend (6K)Expression of VDR and p27 protein
p27 protein expression revealed nuclear localization, as shown in Figure 9. Semiquantitative analyses revealed that p27 LI in both Nd (97 156) and Ad (187 196) were significantly lower as compared to those in C (631 170) and Df (532 146; P < 0.01), as shown in Figure 10.
Figure 9.
Representative immunohistochemical findings of p27 protein expression in parathyroid gland tissues with diaminobenzidine chromogen and hematoxylin counterstain (400). (A) Normal parathyroid gland. Nearly all nuclei show brown staining for p27 protein. (B) Parathyroid adenoma. Some nuclei show positive staining for p27. (C) Diffuse parathyroid hyperplasia. The majority of nuclei show positive staining for p27. (D) Nodular parathyroid hyperplasia. Only few nuclei show positive staining for p27.
Figure 10.
Differences in the mean labeling index (LI) of p27 protein expression among each histological type of parathyroid glands. Nodular hyperplasia (HP) and adenoma show the lowest p21 LI. *P < 0.01 vs. normal; **P < 0.01 vs. diffuse HP.
Full figure and legend (29K)The distributions of VDR and p27 were examined on the serial sections. Parathyroid sections with high nuclear VDR expression elicited high p27 expression, whereas areas that lacked VDR had no detectable p27 Figure 11. A significant positive correlation between VDR LI and p27 LI was found only in Nd (r = 0.76, P < 0.01), as shown in Figure 12. In Nd, the weight of the resected glands inversely correlated with p27 LI (r = -0.41, P < 0.05; Figure 13).
Figure 11.
Representative immunohistochemical findings p27 and VDR expression in the serial sections of diffuse hyperplastic parathyroid gland tissues, which show early nodular formation with diaminobenzidine chromogen and hematoxylin counterstain (200). (A) Nuclear VDR expression. The majority of nuclei are stained strongly with VDR protein expression on the lower side, but only a small number of nuclei show positive staining in the upper side (200). (B) p27 expression shows positive staining in the nuclei of the lower side, the same as VDR expression.
Figure 12.
Correlation between the labeling index of VDR expression (VDR LI) and that of p27 (p27 LI) only in nodular hyperplasia. A significant positive correlation was found between VDR LI and p27 LI in nodular hyperplasia: y = 15.4 + 0.65x; r = 0.76, P < 0.01.
Full figure and legend (8K)Figure 13.
Correlation between the labeling index of p27 expression (p27 LI) and the weight of parathyroid gland only in nodular hyperplasia. A significant negative correlation was found between p27 LI and the weight of parathyroid gland in nodular hyperplasia: y = 1.99 - 0.005x; r = 0.41, P < 0.05.
Full figure and legend (7K)Expression of Ki67 antigen
A remarkable increase in Ki67 expression was observed in both Ad and Nd, as depicted in Figure 14. Semiquantitatively, Ki67 LI was significantly higher in Ad (33 27) and Nd (20 18) than in Df (4 4) and C (1 1; P < 0.01; Figure 15). Although the correlation between Ki67 LI and either p21 or p27 LI was examined with serial sections, there was no significant correlation in any histological type of parathyroid glands.
Figure 14.
Immunohistochemical findings of Ki67 antigen in parathyroid gland tissues with diaminobenzidine chromogen and hematoxylin counterstain (400). (A) Normal parathyroid gland: A few nuclei showed brown staining for Ki67 antigen. (B) Parathyroid adenoma: A majority of the nuclei showed positive staining for Ki67. (C) Diffuse parathyroid hyperplasia: In this figure, only 2 nuclei showed positive staining for Ki67. (D) Nodular parathyroid hyperplasia: Some nuclei showed positive staining for Ki67, but the incidence was less as compared to adenoma.
Figure 15.
Difference in the mean labeling index of Ki67 expression (Ki67 LI) among each histological type of parathyroid glands. Both adenoma and nodular hyperplasia (HP) showed a significantly higher ki67 LI. *P < 0.01 vs. normal; **P < 0.01 vs. diffuse HP.
Full figure and legend (19K)Expression of p53 protein
Immunohistochemical analyses of p53, as a principal transcriptional regulator of p21, was performed in each histological type Figure 16. NIH image analyses revealed that p53 LI in Ad of 77 63 was the highest value among the four groups (P < 0.01), and it was comparable between other three groups, as shown in Figure 17. Although the correlation between p53 LI and p21 LI was examined with serial sections, there was no significant correlation in any histological type of parathyroid glands.
Figure 16.
Representative immunohistochemical findings of p53 expression in parathyroid gland tissues with diaminobenzidine chromogen and hematoxylin counterstain. (A) Normal parathyroid gland where a few nuclei are stained brown for p53 protein. (B) Parathyroid adenoma; the majority of nuclei show positive staining for p53. (C) Diffuse parathyroid hyperplasia, showing a few nuclei that have a positive staining for p53. (D) Nodular parathyroid hyperplasia where a few nuclei show positive staining for p53, the same as that in the diffuse hyperplasia.
Full figure and legend (322K)Figure 17.
Difference in the mean labeling index of p53 expression (p53 LI) among each histological type of parathyroid glands. Parathyroid adenoma shows the highest p53 LI. Interestingly, no up-regulation of p53 is found in any of the hyperplastic glands. *P < 0.01 vs. normal; **P < 0.01 vs. diffuse hyperplasia (HP); ***P < 0.01 vs. nodular HP.
Full figure and legend (20K)DISCUSSION
The present study demonstrates that the reduced expression of p21 and p27, in association with the decreased density of VDR, is a major pathogenic feature of the nodular hyperplastic parathyroid gland in patients with advanced 2HPT.
Calcitriol reduces parathyroid cell proliferation by decreasing the expression of the early gene, c-myc12, the gene that modulates the progression from G1 to S phase in the cell cycle. A decrease in plasma calcitriol and/or a disturbance of its action at the level of the parathyroid cell, which are both frequently observed in uremic patients, may cause a lack of inhibition of c-myc expression and lead to progression of the cell cycle. Calcitriol activates the p21 gene through a VDR-dependent and p53-independent manner, as well as the p27 gene in the myelomonocytic cell line, U93713. Moreover, a recent study by Cozzolino and colleagues demonstrates that, in uremic rats, the efficacy of 1,25D and its less calcemic analog, 19-nor-1,25D, to prevent high phosphorus-induced parathyroid hyperplasia, could partially be attributed to the induction of parathyroid p21 expression, and the increases in p21 correlate inversely with reduced Ki6717. This finding indicates another possible mechanism by which calcitriol may regulate the proliferation of parathyroid cells.
In patients with severe 2HPT, hyperplastic parathyroid glands often show a resistance to the physiological concentration of serum 1,25D10,18,19,20. The reason for this resistance is partly explained by VDR deficiency in the hyperplastic glands21,22,23. In the present study, nodular hyperplasia showed a significantly lower VDR density than diffuse hyperplasia, which is compatible with previous reports2. Therefore, the effects of 1,25D through its VDR might be limited in nodular hyperplasia. Parathyroid sections with high nuclear VDR expression elicited high both p21 and p27 expression, whereas the areas that lacked VDR had no detectable p21 and p27. The semiquantitative LI of p21 and p27 significantly correlated with that of nuclear VDR, but not with p53, indicating that p21 and p27 expression in the hyperplastic parathyroid glands due to 2HPT may be regulated in a VDR-dependent manner, as reported in the myelomonocytic cell line, U93713. It was reported that p21 and p27 participate in regulating the transition from the G1 to S phase of the cell cycle14,15, and a functional vitamin D responsive element has been identified in the p21 promoter area13. Therefore, the suppressive action of 1,25D on the cell proliferation also might be diminished in nodular hyperplasia.
Recently, Dusso et al demonstrated the direct effect of phosphorus on parathyroid cell proliferation in the early phase of uremia by elegantly designed experiments done in 5/6 nephrectomized rats24. Dietary phosphorus regulated p21 expression in association with growth promoter, transforming growth factor-
(TGF-). Low dietary-phosphorus induced p21 expression, whereas high phosphorus intake enhanced TGF- with a subsequent stimulation of parathyroid cell proliferation, independent of changes in serum 1,25D. In their study, there was a significant correlation between the decreased p21 expression and the enhanced PCNA expression, showing the direct relationship of p21 with parathyroid cell proliferation. Our present study found an important role for p21 and p27 in the parathyroid cell proliferation of patients with advanced 2HPT. Although the expression of Ki67 did not correlate with p21 and p27 expression in the present study, Ki67 LI was significantly higher in both primary adenomas and nodular hyperplasia, showing a decreased p21 and p27 expression, than in diffuse hyperplasia and normal parathyroid gland. Also, in nodular hyperplasia, the weight of the resected parathyroid glands inversely correlated with p21 and p27 LI. Therefore, our results support a hypothesis that decreased p21 and p27 expression may lead to parathyroid cell proliferation in advanced 2HPT. Thus, completion of the cell cycle, induced by the reduced p21 and p27 expression in a VDR-dependent manner, is suggested to be a major factor for parathyroid cell proliferation.
Stimuli for parathyroid cell proliferation include long-standing hypocalcemia, hyperphosphatemia, and vitamin D deficiency. In the present study, parathyroid sections with high nuclear VDR expression elicited a high expression of both p21 and p27, whereas areas lacking VDR had no detectable p21 and p27. Therefore, among these factors, decreased VDR expression accompanied by vitamin D deficiency may play a major role in parathyroid cell proliferation and continuous over-secretion of PTH, even after achieving a correction of calcium-phosphorus imbalance.
In the prevention and management of 2HPT, which results in increased synthesis and secretion of PTH, it is crucial to understand the mechanisms of regulating cell growth. Induction of p21 was shown to be sufficient to induce growth arrest in monocyte-macrophages13, keratinocytes25, and human cancer cells26, as well as to suppress tumorigenesis in vivo27. Induction of p27 also was demonstrated to be sufficient to induce growth arrest in monocyte-macrophages13. In the hyperplastic parathyroid glands of patients with 2HPT, obtained most likely by needle biopsy, in situ immunohistochemical analysis of p21 and p27 expression may be a useful parameter to estimate the response to active vitamin D therapy, and help assess the decision of surgical treatment. Up-regulation of parathyroid VDR content through 1,25D administration may correct parathyroid VDR and therefore normalize p21 and p27 levels.
We focused on the CDKIs, p21 and p27, and VDR as potential modulators, however, recent studies also have demonstrated that the several other key regulators including c-myc, PRAD1/cyclin D1 and retinoblastoma protein (Rb), might be involved in the regulation of parathyroid cell growth. Based on recent studies, it is suggested that cell cycle progression is important for induction of parathyroid cell proliferation. In nodular hyperplastic parathyroid glands, Tominaga et al demonstrated that expression of PRAD1/cyclin D1, pRB and Ki67 antigen was significantly higher than in diffuse hyperplastic parathyroid glands28. Vasef et al also showed an overexpression of cyclin D1 protein in human hyperplastic parathyroid glands29. On the other hand, Bianchi et al reported that hypercalcemia suppressed expression of PRAD1/cyclin D1 in a rat parathyroid cell line30. These studies suggest that the higher expression of PRAD1/cyclin D1 may correlate with a decreased expression of the calcium sensing receptor (CaR) in nodular hyperplasia. The lack of a cause and effect relationship for the temporal association between increased proliferation and reduced expression of CaR has been documented. The studies by Imanishi et al demonstrated that the reduction in CaR follows rather than determines the increase in proliferating activity by targeted over-expression of cyclin D1 in parathyroid glands31. Moreover, similar findings were reported for parathyroid hyperplasia in an experimental model of renal failure32. Thus, it is conceivable that cyclin D1 may play a major role in parathyroid cell proliferation in uremia. Regarding the control of cyclin D1, in addition to the impact of 1,25D therapy on parathyroid-p21 expression, Cozzolino et al's study demonstrated evidence of an additional mechanism for both 1,25D therapy and high dietary calcium to suppress parathyroid growth17. These authors showed that both 1,25D therapy and high dietary calcium prevent the increases in parathyroid-TGF- induced by renal failure and high dietary phosphorus. Induction of cyclin D1 is one of the mechanisms downstream from TGF- binding to its receptor, the epidermal growth factor receptor, to induce growth33. Therefore, the reduction of parathyroid TGF- caused by two effective maneuvers to arrest parathyroid gland enlargement in renal failure could be an important mechanism to down-regulate parathyroid-cyclin D1. Furthermore, it has been reported that calcitriol reduced parathyroid cell proliferation by decreasing the expression of the early gene, c-myc12. Thus, the vitamin D transcriptional up-regulation of the p21 and p27 gene may be one of the mechanisms for the antiproliferative effects of the calcitriol/VDR complex in blocking the G1-S transition.
In our present study, VDR expression also was substantially reduced in parathyroid adenoma, but there was no significant correlation between either VDR or glandular weight, and either p21 or p27 expression. Parathyroid adenomas have substantiated the derangement in genes thought to be involved primarily in the regulation of parathyroid growth, such as the PRAD1/cyclin D1 oncogene34,35,36 and the MEN1 tumor suppressor gene37,38,39. Thus, the mechanisms for the decreased p21 and p27 expression in primary adenomas might be different from those in renal hyperplasia. These studies and our data suggest that factors other than the reduced expression of p21 and p27 contribute to parathyroid cell proliferation in primary adenomas.
In summary, this study demonstrates the higher mitogenic properties in nodular hyperplasia in patients with advanced 2HPT. It could be attributed to down-regulation of the expression of the CDKIs, p21 and p27, in association with the down-regulated expression of VDR in the nucleus of parathyroid gland cells. Based on these results and the evidence in the literature on the mechanisms of action of p21 and p27, we conclude that the decreased expression of both p21 and p27, in a VDR-dependent manner, is a major factor for nodular hyperplasia, the severest case of 2HPT.
References
| 1. | Llach F. Secondary hyperparathyroidism in renal failure: The trade-off hypothesis revisited. Am J Kidney Dis 1995; 25: 663−679. | PubMed | ISI | ChemPort | |
| 2. | Fukuda N, Tanaka H & Tominaga Y et al. Decreased 1,25-dihydroxyvitamin D3 receptor density is associated with a more severe form of parathyroid hyperplasia in chronic uremic patients. J Clin Invest 1993; 92: 1436−1443. | PubMed | ISI | ChemPort | |
| 3. | Kifor O, Moore FJ & Wang P et al. Reduced immunostaining for the extracellular Ca2+-sensing receptor in primary and uremic secondary hyperparathyroidism. J Clin Endocrinol Metab 1996; 81: 1598−1606 10.1210/jc.81.4.1598. | Article | PubMed | ISI | ChemPort | |
| 4. | Tominaga Y, Sato K & Tanaka Y et al. Histopathology and pathophysiology of secondary hyperparathyroidism due to chronic renal failure. Clin Nephrol 1995; 144 Suppl: S42−S47. |
| 5. | Tominaga Y, Tanaka Y & Sato K et al. Histopathology, pathophysiology, and indications for surgical treatment of renal hyperparathyroidism. Semin Surgl Oncol 1997; 13: 78−86. | ChemPort | |
| 6. | Tominaga Y, Tanaka Y & Sato K et al. Recurrent renal hyperparathyroidism and DNA analysis of autografted parathyroid tissue. World J Surg 1992; 16: 595−602. | Article | PubMed | ISI | ChemPort | |
| 7. | Tominaga Y, Kohara S & Namii Y et al. Clonal analysis of nodular parathyroid hyperplasia in renal hyperparathyroidism. World J Surg 1996; 20: 744−750 10.1007/s002689900113. | Article | PubMed | ISI | ChemPort | |
| 8. | Arnold A, Brown MF & Urena P et al. Monoclonality of parathyroid tumors in chronic renal failure and in primary parathyroid hyperplasia. J Clin Invest 1995; 95: 2047−2053. | PubMed | ISI | ChemPort | |
| 9. | Fukagawa M, Okazaki R & Takano K et al. Regression of parathyroid hyperplasia by calcitriol-pulse therapy in patients on long-term dialysis. N Engl J Med 1990; 323: 421−422. | PubMed | ISI | ChemPort | |
| 10. | Slatopolsky E, Weerts C & Thielan J et al. Marked suppression of secondary hyperparathyroidism by intravenous administration of 1,25-dihydroxy-cholecalciferol in uremic patients. J Clin Invest 1984; 74: 2136−2143. | PubMed | ISI | ChemPort | |
| 11. | Cannella G, Bonucci E & Rolla D et al. Evidence of healing of secondary hyperparathyroidism in chronically hemodialyzed uremic patients treated with long-term intravenous calcitriol. Kidney Int 1994; 46: 1124−1132. | PubMed | ChemPort | |
| 12. | Kremer R, Bolivar I & Goltzman D et al. Influence of calcium and 1,25-dihydroxycholecalciferol on proliferation and proto-oncogene expression in primary cultures of bovine parathyroid cells. Endocrinology 1989; 125: 935−941. | PubMed | ISI | ChemPort | |
| 13. | Liu M, Lee MH & Cohen M et al. Transcriptional activation of the Cdk inhibitor p21 by vitamin D3 leads to the induced differentiation of the myelomonocytic cell line U937. Genes Dev 1996; 10: 142−153. | PubMed | ISI | ChemPort | |
| 14. | Reed SI, Bailly E & Dulic V et al. G1 control in mammalian cells. J Cell Sci 1994; 18 Suppl: 69−73. | ChemPort | |
| 15. | Sherr CJ & Roberts JM. Inhibitors of mammalian G1 cyclin-dependent kinases. Gene Dev 1995; 9: 1149−1163. | PubMed | ISI | ChemPort | |
| 16. | Niculescu AB, 3rd, Chen X & Smeets M et al. Effects of p21 (Cip1/Waf1) at both the G1/S and the G2/M cell cycle transitions: pRb is a critical determinant in blocking DNA replication and in preventing endoreduplication. Mol Cell Biol 1998; 18: 629−643. | PubMed | ISI | ChemPort | |
| 17. | Cozzolino M, Lu Y & Finch J et al. p21WAF1 and TGF- mediate parathyroid growth arrest by vitamin D and high calcium. Kidney Int 2001; 60: 2109−2117. | Article | PubMed | ISI | ChemPort | |
| 18. | Chan YL, McKay C & Dye E et al. The effect of 1,25 dihydroxycholecalciferol on parathyroid hormone secretion by monolayer cultures of bovine parathyroid cells. Calcified Tissue Int 1986; 38: 27−32. | ISI | ChemPort | |
| 19. | Delmez JA, Tindira C & Grooms P et al. Parathyroid hormone suppression by intravenous 1,25-dihydroxyvitamin D. A role for increased sensitivity to calcium. J Clin Invest 1989; 83: 1349−1355. | PubMed | ISI | ChemPort | |
| 20. | Dunlay R, Rodriguez M & Felsenfeld AJ et al. Direct inhibitory effect of calcitriol on parathyroid function (sigmoidal curve) in dialysis. Kidney Int 1989; 36: 1093−1098. | PubMed | ISI | ChemPort | |
| 21. | Korkor AB. Reduced binding of [3H]1,25-dihydroxyvitamin D3 in the parathyroid glands of patients with renal failure. N Engl J Med 1987; 316: 1573−1577. | PubMed | ISI | ChemPort | |
| 22. | Merke J, Hugel U & Zlotkowski A et al. Diminished parathyroid 1,25(OH)2D3 receptors in experimental uremia. Kidney Int 1987; 32: 350−353. | PubMed | ISI | ChemPort | |
| 23. | Brown AJ, Dusso A & Lopez HS et al. 1,25-(OH)2D receptors are decreased in parathyroid glands from chronically uremic dogs. Kidney Int 1989; 35: 19−23. | PubMed | ISI | ChemPort | |
| 24. | Dusso AS, Pavlopoulos TP & Naumovich L et al. p21WAF1 and transforming growth factor- mediate dietary phosphate regulation of parathyroid cell growth. Kidney Int 2001; 59: 855−865 10.1046/j.1523-1755.2001.00568.x. | Article | PubMed | ISI | ChemPort | |
| 25. | DiCunto F, Topley G & Calautti E et al. Inhibitory function of p21Cip1/WAF1 in differentiation of primary mouse keratinocytes independent of cell cycle control. Science 1998; 280: 1069−1072 10.1126/science.280.5366.1069. | Article | PubMed | ISI | ChemPort | |
| 26. | Bonfanti M, Taverna S & Salmona M et al. p21WAF-1-derived peptides linked to an internalization peptide inhibit human cancer cell growth. Cancer Res 1997; 57: 1442−1446. | PubMed | ISI | ChemPort | |
| 27. | Yang ZY, Perkins ND & Ohno T et al. The p21 cyclin-dependent kinase inhibitor suppresses tumorigenesis in vivo. Nat Med 1995; 1: 1052−1056 10.1038/nm1095-1052. | Article | PubMed | ISI | ChemPort | |
| 28. | Tominaga Y, Tsuzuki T & Uchida K et al. Expression of PRAD1/cyclin D1, retinoblastoma gene products, and Ki67 in parathyroid hyperplasia caused by chronic renal failure versus primary adenoma. Kidney Int 1999; 55: 1375−1383 10.1046/j.1523-1755.1999.00396.x. | Article | PubMed | ISI | ChemPort | |
| 29. | Vasef MA, Brynes RK & Sturm M et al. Expression of cyclin D1 in parathyroid carcinomas, adenomas, and hyperplasia: A paraffin immunohistochemical study. Mod Pathol 1999; 12: 412−416. | PubMed | ISI | ChemPort | |
| 30. | Bianchi S, Fabiani S & Muratori M et al. Calcium modulates the cyclin D1 expression in a rat parathyroid cell line. Biochem Biophys Res Commun 1994; 204: 691−700. | Article | PubMed | ISI | ChemPort | |
| 31. | Imanishi Y, Hosokawa Y & Yoshimoto K et al. Primary hyperparathyroidism caused by parathyroid-targeted overexpression of cyclin D1 in transgenic mice. J Clin Invest 2001; 107: 1093−1102. | PubMed | ISI | ChemPort | |
| 32. | Ritter CS, Finch JL & Slatopolsky EU et al. Parathyroid hyperplasia in uremic rats precedes down-regulation of the calcium receptor. Kidney Int 2001; 60: 1737−1744 10.1046/j.1523-1755.2001.00027.x. | Article | PubMed | ISI | ChemPort | |
| 33. | Lin SY, Makino K & Xia W et al. Nuclear localization of EGF receptor and its potential new role as a transcription factor. Nat Cell Biol 2001; 3: 802−808 10.1038/ncb0901-802. | Article | PubMed | ISI | ChemPort | |
| 34. | Arnold A, Staunton CE & Kim HG et al. Monoclonality and abnormal parathyroid hormone gene in parathyroid adenomas. N Engl J Med 1988; 318: 658−662. | PubMed | ISI | ChemPort | |
| 35. | Arnold A, Kim HG & Gaz RD et al. Molecular cloning and chromosomal mapping of DNA rearranged with the parathyroid hormone gene in a parathyroid adenoma. J Clin Invest 1989; 3: 2034−2040. |
| 36. | Motokura T, Bloom T & Kim HG et al. A novel cyclin encoded by a bcl1-linked candidate oncogene. Nature 1991; 350: 512−515 10.1038/350512a0. | Article | PubMed | ISI | ChemPort | |
| 37. | Heppner C, Kester MB & Agarwal SK et al. Somatic mutations of the MEN1 gene in parathyroid tumors. Nat Genet 1997; 16: 375−378 10.1038/ng0897-375. | Article | PubMed | ISI | ChemPort | |
| 38. | Carling T, Correa P & Hedberg J et al. Parathyroid MEN1 gene mutations in relation to clinical characteristics of non-familial primary hyperparathyroidism. J Clin Endocrinol Metab 1998; 83: 2960−2963. | Article | PubMed | ISI | ChemPort | |
| 39. | Farnebo F, Teh B-T & Kytola S et al. Alternations of the MEN1 gene in sporadic parathyroid tumors. J Clin Endocrinol Metab 1998; 83: 2627−2630 10.1210/jc.83.8.2627. | Article | PubMed | ISI | ChemPort | |
Acknowledgments
A part of the study was presented at the 36th Annual Meeting of American Society of Nephrology at Tronto, in 2000. We thank Miss Hideko Noguchi for her technical assistance with the experiments.
