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Context: The type I membrane protein Klotho was recently shown to mediate PTH secretion in parathyroid cells in response to low extracellular calcium. In contrast, Klotho inhibits PTH secretion indirectly through the action of fibroblast growth factor-23. Abnormal Klotho expression in parathyroid disorders remains to be elucidated. Objective: The aim of the study was to determine: 1) Klotho expression in parathyroid adenomas from patients with primary hyperparathyroidism (pHPT) compared to normal tissue; and 2) its relation to the serum calcium and PTH levels. Design: Surgically removed parathyroid glands (n = 40) and four normal parathyroid tissue specimens were analyzed for Klotho mRNA and protein levels by quantitative real-time PCR and immunohistochemistry. In vitro effects of calcium on Klotho mRNA expression were studied in bovine parathyroid cells. Results: Klotho mRNA levels were significantly decreased (n = 23) or undetectable (n = 17) in parathyroid adenomas compared to normal tissues (P < 0.001). Reduced Klotho protein expression was confirmed by immunohistochemistry. Klotho mRNA levels were inversely correlated to serum calcium (r = −0.97; P < 0.0001), and calcium dose-dependently decreased Klotho mRNA expression in normal parathyroid cells in vitro (P < 0.01). Serum calcium was the only significant marker of Klotho expression in multivariate analysis with calcium, phosphate, PTH, and adenoma weight as independent variables. Conclusions: Parathyroid Klotho expression is decreased or undetectable in pHPT. We provide evidence that 1) serum calcium is strongly associated with parathyroid Klotho expression in pHPT; and 2) abnormal PTH secretion in hypercalcemic pHPT subjects is mediated by Klotho-independent mechanisms.

Primary hyperparathyroidism (pHPT) resulting from parathyroid tumors is a common endocrine disorder with incompletely understood etiology, affecting about 1% of the adult population, with an even higher prevalence for elderly individuals. In renal failure, secondary hyperparathyroidism (sHPT) occurs with multiple tumor development as a result of calcium and vitamin D regulatory disturbance.Aberrant Wnt/beta-catenin signaling with accumulation of beta-catenin in the cytoplasm/nucleus is involved in the development of a variety of neoplasms. The aim of this study was to evaluate whether the Wnt/beta-catenin signaling pathway is activated in parathyroid adenomas of pHPT and in hyperplastic glands from uremic patients with sHPT.Immunohistochemistry, Western blotting, real-time quantitative RT-PCR, and DNA sequencing were performed.beta-Catenin was accumulated in all analyzed parathyroid tumors (n = 47) from patients with pHPT and from patients with HPT secondary to uremia. The accumulation included nonphosphorylated, stabilized (transcriptionally active) beta-catenin. The overexpression was not related to increased beta-catenin mRNA levels. A protein-stabilizing mutation in exon 3 of beta-catenin (S37A) was detected in three of 20 pHPT tumors (15%). No mutation was detected in secondary hyperplastic glands (n = 20), and no evidence for truncated adenomatosis polyposis coli proteins was found in adenomas and secondary hyperplastic glands. Mutations in other Wnt signaling components leading to beta-catenin accumulation, other than in beta-catenin itself, are therefore anticipated. The beta-catenin target gene c-myc was overexpressed in a substantial fraction of the parathyroid tumors.Our results strongly suggest that modifications in the Wnt/beta-catenin signaling pathway may be involved in the development of hyperparathyroidism.

In the absence of metastases or overgrowth to adjacent organs, the lack of reliable markers for malignancy is a well-recognized problem for clinicians managing patients with endocrine tumors. Apart from inactivation of the multiple endocrine neoplasia type 1 (MEN1) gene, the molecular mechanisms involved in tumorigenesis of the endocrine organs and MEN1-associated nonendocrine lesions are vastly unknown. To try to learn more about down-stream effects on MEN1 gene inactivation, we used the BON1 cells, showing low levels of endogenous menin, and transfected them with a MEN1 gene construct. On restoring the menin expression, we recorded inhibition of cell growth. We also performed macroarray and present data on differentially expressed genes in the transfected cells, after corroboration by Northern blots and quantitative PCR. JunD was up-regulated in menin-expressing clones, whereas delta-like protein 1/preadipocyte factor-1 (involved in differentiation and growth of the pancreatic endocrine cells), proliferating cell nuclear antigen, and QM/Jif-1 (a negative regulator of c-Jun) became down-regulated. These findings might contribute to the understanding of the tissue-specific features of MEN1. We also show that homozygous inactivation of the MEN1 gene statistically correlates to higher expression of delta-like protein 1/preadipocyte factor-1, proliferating cell nuclear antigen, and QM/Jif-1, as well as lower MEN1 expression, in a limited sample of malignant endocrine pancreatic tumors.

Active vitamin D, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], plays a pivotal role in calcium homeostasis and bone metabolism. Circulating levels of 1,25(OH)(2)D(3) are thought to be dependent mainly on the activity of the renal cytochrome P450 enzyme 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-hydroxylase), which is potently induced by PTH. However, 1alpha-hydroxylase activity or expression has also been reported at several extrarenal sites, at which local synthesis of 1,25(OH)(2)D(3) appears to fulfill autocrine or paracrine functions. This includes tissues such as placenta and brain that also express LRP-2/megalin, an endocytic receptor for multiple ligands, which is involved in the renal uptake of the substrate for 1alpha -hydroxylase, 25-hydroxyvitamin D(3). We have previously demonstrated LRP-2/megalin in parathyroid cells, and here we present results from RT-PCR and immunohistochemical analyses showing coincident expression of 1alpha-hydroxylase in normal and pathological parathyroid tissue. With real-time quantitative RT-PCR analysis, the expression of 1alpha-hydroxylase mRNA was higher in the majority of parathyroid adenomas and secondary hyperplastic glands but lower in parathyroid carcinomas, compared with normal parathyroid tissue. The findings imply that in addition to feedback control by circulating 1,25(OH)(2)D(3) levels, parathyroid cells may also be influenced by local 1alpha -hydroxylase activity with possible growth regulatory and differentiating effects.

Multiple endocrine neoplasia type 1 (MEN1) is an inherited syndrome with multiple tumors of the endocrine pancreas, the parathyroid, the pituitary, and other tissues. The MEN1 gene at 11q13 is homozygously mutated in the majority of MEN1 tumors. Here we present a genome-wide loss of heterozygosity (LOH) screening of 23 pancreatic lesions, one duodenal tumor, and one thymic carcinoid from 13 MEN1 patients. Multiple allelic deletions were found. Fractional allelic loss varied from 6-75%, mean 31%. All pancreatic tumors displayed LOH on chromosome 11, whereas the frequency of losses for chromosomes 3, 6, 8, 10, 18, and 21 was over 30%. Different lesions from individual patients had discrepant patterns of LOH. Intratumoral heterogeneity was revealed, with chromosome 6 and 11 deletions in most tumor cells, whereas other chromosomal loci were deleted in portions of the analyzed tumor. Chromosome 6 deletions were mainly found in lesions from patients with malignant features. Fractional allelic loss did not correlate to malignancy or to tumor size. Our findings indicate that MEN1 pancreatic tumors fail to maintain DNA integrity and demonstrate signs of chromosomal instability.

Familial hyperparathyroidism (HPT), characterized by hypercalcemia and hypercalciuria, and familial benign hypocalciuric hypercalcemia (FHH) are the most common causes of hereditary hypercalcemia. The calcium-sensing receptor (CaR) regulates PTH secretion and renal calcium excretion. Heterozygous inactivating mutations of the gene cause FHH, whereas CaR gene mutations have not been demonstrated in HPT. In a kindred with 20 affected individuals, the hypercalcemic disorder segregated with inappropriately higher serum PTH and magnesium levels and urinary calcium levels than in unaffected members. Subtotal parathyroidectomy revealed parathyroid gland hyperplasia/adenoma and corrected the biochemical signs of the disorder in seven of nine individuals. Linkage analysis mapped the condition to markers flanking the CaR gene on chromosome 3q. Sequence analysis revealed a mutation changing phenylalanine to leucine at codon 881 of the CaR gene, representing the first identified point mutation located within the cytoplasmic tail of the CaR. A construct of the mutant receptor (F881L) was expressed in human embryonic kidney cells (HEK 293), and demonstrated a right-shifted dose-response relationship between the extracellular and intracellular calcium concentrations. The hypercalcemic disorder of the present family is caused by an inactivating point mutation in the cytoplasmic tail of the CaR and displays clinical characteristics atypical of FHH and primary HPT.

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.

Vitamin D regulates parathyroid cell proliferation and secretion of PTH. Increased prevalence of the polymorphic vitamin D receptor (VDR) alleles b, a, and T has been reported in sporadic primary hyperparathyroidism (PHPT), suggesting that these genetic variants may predispose to the disease. Recently, another polymorphism in the VDR gene was related to bone mineral density, and this VDR-FokI polymorphism causes different lengths of the VDR, implying possible functional consequences. The VDR-FokI polymorphism was studied in 182 postmenopausal women with sporadic PHPT and in matched controls. No significant differences in distribution of the VDR-FokI genotypes could be detected between the groups, although there was a tendency toward overrepresentation of the F allele in the PHPT patients (P = 0.05). There were no significant associations with age, serum calcium, serum PTH, bone mineral density, or parathyroid tumor weight. The VDR genotypes were unrelated to VDR and PTH messenger ribonucleic acid levels in the parathyroid adenomas of 42 PHPT patients. In 23 PHPT patients, the Ca2+-PTH set-points were determined in vivo and were unrelated to the VDR alleles. We suggest that the VDR-FokI polymorphism has at most a minor pathogenic importance in the development of PHPT.

Biochemical signs and severity of symptoms of primary hyperparathyroidism (pHPT) differ among patients, and little is known of any coupling of clinical characteristics of nonfamilial pHPT to genetic abnormalities in the parathyroid tumors. Mutations in the recently identified MEN1 gene at chromosome 11q13 have been found in parathyroid tumors of nonfamilial pHPT. Using microsatellite analysis for loss of heterozygosity (LOH) at 11q13 and DNA sequencing of coding exons, the MEN1 gene was studied in 49 parathyroid lesions of patients with divergent symptoms, operative findings, histopathological diagnosis, and biochemical signs of nonfamilial pHPT. Allelic loss at 11q13 was detected in 13 tumors, and 6 of them demonstrated previously unrecognized somatic missense and frameshift deletion mutations of the MEN1 gene. Many of the detected mutations would most likely result in a nonfunctional menin protein, consistent with a tumor suppressor mechanism. Clinical and biochemical characteristics of HPT were apparently unrelated to the presence or absence of LOH and the MEN1 gene mutations. However, the demonstration of LOH at 11q13 and MEN1 gene mutations in small parathyroid adenomas of patients with slight hypercalcemia and normal serum PTH levels suggest that altered MEN1 gene function may also be important for the development of mild sporadic pHPT.

Vitamin D receptor (VDR) and parathyroid hormone mRNA levels correspond to polymorphic VDR alleles in human parathyroid tumors