Reduced parathyroid vitamin D receptor messenger ribonucleic acid levels in primary and secondary hyperparathyroidism

Vitamin D, via its receptor (VDR), inhibits the hormone secretion and proliferation of parathyroid cells. Vitamin D deficiency and reduced parathyroid VDR expression has been associated with development of hyperparathyroidism (HPT) secondary to uremia. VDR polymorphisms may influence VDR messenger RNA (mRNA) levels and have been coupled to an increased risk of parathyroid adenoma of primary HPT. VDR mRNA relative to glyceraldehyde-3-phosphate dehydrogenase mRNA levels were determined by RNase protection assay in 42 single parathyroid adenomas of patients with primary HPT, 23 hyperplastic glands of eight patients with uremic HPT, and 15 normal human parathyroid glands. The adenomas and hyperplasias demonstrated similar VDR mRNA levels, which were reduced (42 +/- 2.8% and 44 +/- 4.0%) compared with the normal glands (P < 0.0001). Comparison of parathyroid adenoma with a normal-sized parathyroid gland of the same individual (n = 3 pairs) showed a 20-58% reduction in the tumor. Nodularly enlarged glands represent a more advanced form of secondary HPT and showed greater reduction in the VDR mRNA levels than the diffusely enlarged glands (P < 0.005). The reduced VDR expression is likely to impair the 1,25(OH)2D3-mediated control of parathyroid functions, and to be of importance for the pathogenesis of not only uremic but also primary HPT. Circulating factors like calcium, PTH, and 1,25(OH)2D3 seem to be less likely candidates mediating the decreased VDR gene expression in HPT.     

Inactivating calcium‐sensing receptor mutations in patients with primary hyperparathyroidism

Objective Primary hyperparathyroidism (HPT) is characterised by autonomous secretion of PTH from enlarged parathyroid glands leading, in most patients, to asymptomatic hypercalcaemia. Familial hypocalciuric hypercalcaemia (FHH) is an autosomal dominant disorder caused by inactivating mutations in the calcium‐sensing receptor (CaSR) gene; it is characterised by lifelong and usually asymptomatic hypercalcaemia. Establishing the correct diagnosis is important because surgery can be curative in HPT, but ineffective in FHH. There is overlap in the diagnostic criteria for the two disorders and some patients carrying inactivating mutations in the CaSR gene, which is suggestive of FHH, also have HPT with hyperplastic parathyroid glands or adenomas. Design and patients CaSR gene mutations were analysed and clinical and biochemical parameters evaluated in 139 consecutive outpatients presenting with hypercalcaemia and suspected of having HPT. Results Six different mutations of the CaSR gene were found in eight patients. In four patients, classical FHH was suspected based on clinical and biochemical results and was confirmed by the CaSR mutations. In the other four patients, HPT was diagnosed based on the biochemical profile or symptoms; in these four patients, the parathyroids were operated on and single adenomas were histologically confirmed. In all four patients, serum calcium decreased postoperatively; and in three patients, serum calcium normalised postoperatively. The CaSR mutations in these patients were R25X, E250K and Q926R. Conclusion The coexistence of HPT and FHH in four of 139 patients suggests a pathogenetic role of CaSR mutations in HPT. Despite also having a CaSR mutation, these patients benefited from parathyroid surgery.     

Parathyroid cell growth in patients with advanced secondary hyperparathyroidism: vitamin D receptor, calcium sensing receptor, and cell cycle regulating factors

The parathyroid gland (PTG) is a unique endocrine organ in which the quiescent glandular cells begin to proliferate in response to the demand for maintaining calcium (Ca) homeostasis in the progressive course of renal failure, leading to secondary hypereparathyroidism (SHPT). SHPT is characterized with continuous over-secretion of parathyroid hormone (PTH) and high turn-over bone disease, osteitis fibrosa, and the major factors include a deficiency of active vitamin D, hypocalcemia, and phosphate retention. With long-term end-stage renal failure, SHPT becomes resistant to conventional medical treatment such as phosphate binders and active vitamin D supplementation, and the growth of the PTG accelerates with the pattern of hyperplasia changing from diffuse to nodular type. In this process, the sigmoid curve between extracellular Ca concentration (exCa) and the plasma level of PTH shifts to the upper-rightward, indicating both an absolute increase in PTH secretion and the resistance of PT cells to exCa. Many experimental and human studies have revealed down-regulation of vitamin D receptor (VDR), calcium-sensing receptor (CaSR), and retinoid X receptor (RXR) in PT cells. The sustained proliferation of PT cells after obtaining autonomicity is another characteristic feature of SHPT. In this context, it has been demonstrated that the cell cycle is markedly progressed, where the expression of cyclin-dependent kinase inhibitor (CDKI), p21 and p27, is depressed in a VDR-dependent manner. These pathological features are most evident in nodular hyperplasia, in which monoclonal proliferation is obvious, indicating the phenotypic changes have occured in PT cells. It has been observed by Fukagawa and colleagues that pharmacologically high dose of active vitamin D administered orally can cause small-size PTG hyperplasia to regress in patients with advanced SHPT. Successful renal transplantation may also restore VDR and CaSR expressions in the diffuse type, in association with increasing TUNEL-positive cells. Thus, it is important to vigorously treat SHPT when the PT cell proliferation is in the reversible stage of diffuse hyperplasia.     

Calcium-sensing receptor expression and signalling in human parathyroid adenomas and primary hyperplasia

OBJECTIVE: Both in vivo and in vitro evidence indicates that primary hyperparathyroidism is characterized by a reduced sensitivity to extracellular calcium ([Ca2+]o). The existence of alterations in the expression and signalling of calcium sensing receptor (CaSR) in parathyroid neoplasia is still uncertain. In order to clarify the role of CaSR in the reduced [Ca2+]o sensing of parathyroid neoplasia we investigated PTH secretion and intracellular effectors triggered by CaSR activation as well as the levels of expression of CaSR and CaSR coupled G proteins (Gq/G11) in parathyroid adenomas and primary hyperplasia. MATERIALS AND METHODS: The study included 27 parathyroid adenomas, 4 cases of primary hyperplasia and pools of normal parathyroid biopsies. Tissues were either snap frozen in liquid nitrogen or placed in sterile medium for cell dispersion. The effects of increasing [Ca2+]o on in vitro PTH release, intracellular cAMP levels and intracellular calcium ([Ca2+]i) in cells loaded with the Ca2 + indicator fura-2 were evaluated. CaSR mRNA levels were assessed by semiquantitative RT-PCR analysis, using GAPDH as internal standard, while CaSR protein was detected by western blot analysis using a specific polyclonal antibody. Purified antisera selective for G11alpha and Gqalpha were used to detect this class of proteins. RESULTS: In basal conditions (at 0.5 mM [Ca2+]o) in vitro PTH released ranged from 29.4 to 1186 pg/well/60 minutes. Increasing [Ca2+]o from 0.5 to 1, 2.5 and 5 mM caused a variable effect. One group (n = 7) showed a significant but partial reduction of PTH release (of 17 to 60% of basal levels) that occurred at physiological [Ca2+]o concentrations (1 mM) while the remainder showed either inhibition detectable only at 2.5 mM (n = 15) or total (n = 9) resistance to [Ca2+]o. In the responsive cells, [Ca2+]o (1-5 mM) caused a pertussis toxin-insensitive [Ca2+]i rise (ranging from 10% to 260%), due to Ca2+ release from intracellular stores, and an inhibition of forskolin-stimulated cAMP levels. By RT-PCR almost all tumours tested showed a substantial reduction in CaSR mRNA levels when compared to the normal tissue (CaSR/GAPDH ratio: 3.1 +/- 0.5 vs. 15.5 +/- 3.1; P < 0.001), which was confirmed by immunoblotting analysis demonstrating low levels of CaSR protein in tumour tissues. Moreover, low amounts of G11alpha and Gqalpha, the G proteins involved in CaSR coupling, were observed in the majority of pathological tissues. CONCLUSIONS: The study shows that the activation of the calcium sensing receptors expressed in adenomatous parathyroid glands modulates intracellular effectors in a similar way to those operating in the normal parathyroid. Although a reduction of calcium sensing receptor expression is probably involved in the poor inhibition of PTH release induced by [Ca2+]o, this is not the only factor altering [Ca2+]o sensing in parathyroid adenomas, since tumours characterized by different in vitro sensitivity to [Ca2+]o showed similar CaSR levels. The low content of G proteins of the Gq subfamily might represent an additional alteration leading to a defective [Ca2+]o sensing.     

Expression of calcium-sensing receptor and characterization of intracellular signaling in human pituitary adenomas.

Extracellular Ca(2+)-sensing receptor (CaSR) has been recently identified in rat and mouse pituitary and in AtT-20 cells. The aim of the study was to investigate the presence of CaSR in the human pituitary and its signaling pathway. Normal parathyroid biopsies, autoptic normal pituitaries, and seven nonfunctioning and six GH-secreting adenomas were studied. Southern blot analysis of the RT-PCR products from pituitary adenomas indicated that the PCR fragments obtained were products of specific amplification of CaSR messenger ribonucleic acid. Sequence analysis showed nucleotide identity of these products with the available human parathyroid CaSR. By immunoblotting analysis CaSR, was detected in normal and adenomatous pituitary tissues. In all tumors studied, extracellular Ca2+ (2.5 mmol/L) induced a significant increase in intracellular Ca2+, mainly due to Ca2+ mobilization (from 82.7+/-11 to 148+/-36 nmol/L; P < 0.001). Similar results were obtained with the CaSR activators gadolinium and neomycin. Moreover, CaSR activators significantly increased cAMP levels; this effect was not mimicked by other agents able to increase intracellular Ca2+, such as TRH. CaSR agonists did not increase resting GH secretion in any GH-secreting adenomas, but amplified the GH response to GHRH. In this study we first demonstrate CaSR expression in the human pituitary and provides evidence for an additional mechanism by which calcium might regulate pituitary cell function.     

Vitamin D receptor, oestrogen receptor-alpha and calcium-sensing receptor genotypes, bone mineral density and biochemical markers in Paget's disease of bone

OBJECTIVES: The significance of genetic polymorphisms in the development of Paget's disease of bone is unclear at present. METHODS: We analysed the BsmI polymorphism of the vitamin D receptor (VDR) gene, the PvuII and XbaI polymorphisms of the oestrogen receptor-alpha (ER alpha) gene, and the A986S polymorphism of the calcium-sensing receptor (CaSR) gene in 69 pagetic patients and 120 healthy subjects. We also examined the relationship of these polymorphisms with lumbar spine and femoral neck BMD as well as with biochemical parameters (serum alkaline phosphatase, osteocalcin and parathyroid hormone) in Paget's disease. RESULTS: The XbaI and PvuII genotype distributions of the ER alpha gene were significantly different between patients with Paget's disease and control subjects (P<0.001). Also, the CaSR A986S genotype frequency was significantly different between pagetic patients and controls (P<0.01). No significant effect of gene polymorphisms on BMD or biochemical parameters of bone turnover was observed. CONCLUSION: Our results suggest that the ER alpha PvuII/XbaI and CaSR A986S polymorphisms may contribute to genetic susceptibility to Paget's disease. However, further studies are required to investigate the underlying pathomechanism and to replicate the associations.     

Ki‐67 expression as a prognostic factor in diffuse large B‐cell lymphoma patients treated with rituximab plus CHOP

Background: Assessment of tumor cell proliferation based on Ki‐67 expression yielded conflicting prognostic predictions of patients with diffuse large B‐cell lymphoma (DLBCL). The introduction of rituximab to the DLBCL treatment regime has led to alterations in the significance of previous prognostic factors. Methods: We analyzed Ki‐67 expression and its correlation with prognosis in 144 patients with DLBCL treated with rituximab plus CHOP (R‐CHOP) between July 2003 and January 2008. Results: The complete response (CR) rates following R‐CHOP administration were not significantly different, based on Ki‐67 expression status (P = 0.104). However, higher rates of relapse were observed in the high Ki‐67 expression group (Ki‐67 ≥ 85%, n = 46) with 25.0%, compared to 10.0% in the low Ki‐67 expression group (Ki‐67 < 85%, n = 88) (P = 0.040). The 2‐yr event‐free survival (EFS) rates were 44.3% and 74.1% in the high and low Ki‐67 expression groups, respectively (P = 0.011). The 2‐yr overall survival (OS) rate was 66.4% in the high Ki‐67 expression group and 82.2% in the low Ki‐67 expression group (P = 0.016). In multivariate analysis, Ki‐67 expression was a significant prognostic factor for EFS [hazard ratio (HR) = 2.909; 95% confidence interval (CI) 1.261–6.708; P = 0.012]. Ki‐67 was associated with OS but with borderline significance (HR = 2.876; 95% CI, 0.972–8.508; P = 0.056). Conclusion: Elevated Ki‐67 expression seems to be associated with higher relapse after CR and inferior EFS in patients with DLBCL treated with R‐CHOP.     

The influence of the progression of secondary hyperparathyroidism on the set point of the parathyroid hormone-calcium curve

The influence of secondary hyperparathyroidism (2 HPT) on the set point of the parathyroid hormone (PTH)-Ca(2+) curve is controversial. In vitro experiments have shown an increase in the set point. However, clinical studies with hemodialysis patients have provided a variety of results (increases, decreases and no changes in the set point have been reported). The present study was designed to investigate the influence of the progression of 2 HPT on the set point of the PTH-Ca(2+) curve. The PTH-Ca(2+) curve and the expression of parathyroid calcium receptor (CaR mRNA) and vitamin D receptor (VDR mRNA) have been studied in normal rabbits (group I, n=9) and in nephrectomized rabbits (group II, n=18) at two stages after inducing 2 HPT: 2-3 weeks (group IIA) and 5-6 weeks (group IIB). In group I, the set point of the PTH-Ca(2+) curve was 1.63+/-0.03 mM. A progressive hypocalcemia was detected during the evolution of 2 HPT (groups IIA and IIB). Rabbits from group IIA had a significant (P<0.001) decrease in the set point to values of 1.45+/-0.02 mM. However, the set point increased significantly in group IIB (P<0.001) to 1.56+/-0.03 mM. CaR mRNA was similarly decreased in groups IIA (39+/-12%) and IIB (48+/-7%). No changes were detected in VDR mRNA. In conclusion, a reduction in the set point of the PTH-Ca(2+) curve in response to decreased extracellular Ca(2+) was detected in the early phases of 2 HPT. However, with the progression of 2 HPT the set point tended to increase even though extracellular Ca(2+) was markedly decreased. The increase in the set point in the course of 2 HPT seems to be a complex process that cannot be fully explained by changes in parathyroid CaR mRNA or VDR mRNA.     

Immunohistochemical investigation of angiogenic factors in parathyroid proliferative lesions

OBJECTIVE: The pathological distinction between parathyroid neoplasms and hyperplasias remains difficult in several cases. Endoglin (CD105) is a proliferation-associated and hypoxia-inducible protein abundantly expressed in angiogenic endothelial cells. Vascular endothelial growth factor (VEGF) induces angiogenesis and VEGF-R2 is a tyrosine kinase receptor expressed early in development by endothelial cell precursors. We attempted to examine whether immunohistochemical expression of CD105, VEGF and VEGF-R2 may be useful in distinguishing between parathyroid hyperplasia and neoplasia as well as to elucidate, to some extent, the mechanism of neovascularization in proliferative lesions of the parathyroid gland. DESIGN: Tissue specimens were taken from 38 patients with primary hyperparathyroidism (HPT) (17 adenomas and 21 primary hyperplasias) and from 30 patients with secondary HPT. Normal glands served as controls. METHODS: In a standard immunohistochemical procedure, monoclonal antibodies to endoglin, VEGF and VEGF-R2 were applied to detect angiogenic endothelial cells. Immunostaining was estimated by image analysis and statistical analysis was subsequently performed. RESULTS: Positive CD105 immunoreaction was significantly increased in parathyroid adenomas by comparison with primary hyperplasias (P = 0.033) and with secondary hyperplasias (P = 0.033). When parathyroid adenomas, primary hyperplasia and secondary hyperplasia specimens were comparatively evaluated, VEGF immunoreaction was much more common in adenomas (P = 0.018). In addition, in samples with secondary hyperplasia, VEGF-R2 immunoreactivity was positively linked with VEGF expression as well as with the apoptotic index of parathyroid cells (P = 0.038 and 0.010 respectively). In secondary hyperplasia specimens, an inverse correlation between cyclin D1 immunoexpression and angiogenic indexes, such as CD105 and VEGF, was noticed (P = 0.007 and 0.0017 respectively). CONCLUSIONS: This study shows increased angiogenesis in parathyroid adenomas compared with parathyroid proliferative lesions. In secondarily hyperplastic glands increased angiogenesis and increased apoptosis occur simultaneously; in the latter glands, the overexpression of cyclin D1 does not appear to be the genetic abnormality responsible for increased angiogenesis.     

The distribution of two different vitamin D receptor polymorphisms (BsmI and start codon) in primary hyperparathyroidism

INTRODUCTION: The bb genotype of the BsmI polymorphism of the vitamin D receptor (VDR) is more common in primary hyperparathyroidism (HPT) than in the general population in Swedish and German women. However, little is known about the association of HPT with the start codon polymorphism of the VDR (defined by FokI). OBJECTIVE: To study the distribution of the VDR genotypes in a group of women with HPT compared with a control group. The bone mineral density (BMD) of different genotypes was also investigated. METHODS: VDR alleles were typed by polymerase chain reaction (PCR) assay around the polymorphic BsmI or FokI restriction sites in 67 control women (48.5 +/- 10 years) and 53 women with HPT (61.4 +/- 11 years). They were all Caucasian and born in the Canary Islands. Lumbar and proximal femur BMDs were measured by dual X-ray absorptiometry (DXA) and quantitative computed tomography (QCT). RESULTS: The 'bb' genotype was equally frequent in controls and HPT subjects (46.3 and 45.3%, respectively). There was a trend towards a lower prevalence of the FF genotype amongst women with HPT as compared with controls (41.5 vs. 57.1%; P = 0.09). BMD was lower in patients with HPT compared with controls in the lumbar spine and the proximal femur. CONCLUSIONS: The association of the BsmI polymorphism of the VDR gene with HPT is not applicable to all geographical areas. In Canarian postmenopausal women suffering from HPT, VDR genotype distribution is similar to that found in controls. A possible association of HPT with the FokI polymorphism deserves further investigation.     

Regulation of glucagon-like peptide-1 receptor and calcium-sensing receptor signaling by L-histidine

Receptor-specific agonists of the extracellular calcium-sensing receptor (CaSR) potentiate glucose-induced insulin secretion, an effect similar to that of glucagon-like peptide-1 (GLP-1). We have sequenced the full open reading frame of the CaSR from rat insulinoma (INS-1) cells and find that the predicted amino acid sequence of the receptor is identical with that of the receptor from the parathyroid gland. This receptor couples to both Gq/11 and Gi/o, and this dual coupling may partly explain the varying effects of nonspecific agonists on secretion reported previously. L-Histidine (L-His) increases the sensitivity of the CaSR to extracellular Ca2+ and potentiates glucose-dependent insulin secretion from INS-1 cells. This potentiation is partially inhibited at low extracellular [Ca2+] where the CaSR is ineffective. Coexpression of the CaSR and GLP-1 receptor (GLP-1R) produces a pertussis toxin-sensitive inhibition of GLP-1-induced cAMP production in response to elevated extracellular [Ca2+]. However, l-His potentiates cAMP response element reporter activity in INS-1 cells and in human embryonic kidney-293 cells expressing either the GLP-1R alone or the CaSR and GLP-1R. INS-1 cells express the RNA for the CaSR at a lower level than that for the GLP-1R. This difference in expression level of the receptors may explain the potentiation of insulin secretion by L-His despite coupling of the CaSR to Gi/o. In conclusion, L-His can potentiate both GLP-1R- and CaSR-activated signaling pathways, and these effects may play a role in the potentiation of glucose-induced insulin secretion in response to meals containing protein in addition to carbohydrates and fat.     

Reduced p18INK4c, p21CIP1/WAF1 and p27KIP1 mRNA levels in tumours of primary and secondary hyperparathyroidism

OBJECTIVE: Hyperparathyroidism (HPT) refers to states of excessive production of parathyroid hormone (PTH). The eukaryotic cell cycle is driven forward by cyclins and their cyclin-dependent kinase (CDK) partners. Cyclin-dependent kinase inhibitors (CKIs), which generally inhibit cell cycle progression, modulate the activity of cyclin-CDK complexes. DESIGN: In order to quantify the expression of the CKI genes p18, p21, and p27 semiquantitative RT-PCR with mRNA specific-primers was performed on four normal parathyroid biopsies, 31 parathyroid adenomas of primary HPT and 13 hyperplastic glands from uraemic patients with secondary HPT. PATIENTS: Parathyroid adenomas and secondary hyperplastic glands were obtained from 31 and 13 randomly selected patients undergoing parathyroidectomy in the clinical routine, respectively. Four normal parathyroid gland biopsies were obtained at surgery for pHPT or from normocalcemic patients undergoing thyroidectomy for goitre. RESULTS: The relative p27 expression (p27/GAPDH) was significantly reduced in parathyroid adenomas compared to normal parathyroid gland biopsies. Furthermore, 42% and 53% of the parathyroid adenomas displayed undetectable p18 and p21 expression levels, respectively. All 13 adenomas that lacked p18 expression showed undetectable p21 expression. The p18 expression was significantly lower in tumours of uraemic sHPT as compared to normal parathyroids and an undetectable expression level was observed for p21 and p27 in 61% and 53%, respectively. CONCLUSION: Parathyroid adenomas and secondary hyperplastic glands exhibit aberrant reduced expression of the CKIs p18, p21, and p27. This suggests that deranged collaboration of different CKIs may contribute to the development of both primary and secondary HPT.     

Immunohistochemical detection of cell cycle regulators,Fhit protein and apoptotic cells in parathyroid lesions

OBJECTIVE: The pathological distinction between parathyroid neoplasms and hyperplasias remains difficult. Changes in cell cycle control may lead to clonal proliferation and precede tumorigenesis. The parathyroid adenoma 1 oncogene, subsequently identified as the gene encoding cyclin D1, has been shown to be important to parathyroid tumour development. In addition to cell proliferation, the mechanisms of parathyroid cell turnover include apoptosis. The tumour-suppressor activity of the fragile histidine triad gene (FHIT) is linked to its proapoptotic function and cell cycle control. We attempted to evaluate the cellular proliferative kinetics and apoptotic function of the parathyroid glands in patients with non-familial hyperparathyroidism (HPT). DESIGN: TIssue specimens were taken from 40 patients with primary HPT (17 adenomas, two carcinomas and 21 primary hyperplasias) and from 30 patients with secondary HPT. Normal glands served as controls. METHODS: In a standard immunohistochemical procedure, monoclonal antibodies to Ki-67 antigen and single-stranded DNA were applied to detect cycling and apoptotic cells respectively; polyclonal antibodies to cyclin D1 and Fhit protein were used. Immunostaining was estimated by image analysis and statistical analysis was subsequently performed. RESULTS: Significantly higher proliferative and apoptotic indexes were detected in the diseased glands in comparison with normal controls. In neoplastic and secondarily hyperplastic glands, apoptotic indexes were higher than in primarily hyperplastic glands; the difference between neoplastic and primarily hyperplastic glands was statistically significant (P=0.034). Cyclin D1 was overexpressed in a considerable proportion of tumours (68.4%). A reduction of Fhit protein immunoreactivity was selectively noticed in carcinomas. CONCLUSIONS: In primary hyperplasia, the remarkable proliferation of parathyroid glands may be due to the reduction of the apoptotic process. FHIT gene abnormalities are worthy of investigation in parathyroid carcinogenesis.