Genes up- or down-regulated by high calcium medium in parathyroid tissue explants from patients with primary hyperparathyroidism.

To investigate genes modulated in the parathyroid glands by calcium, expression levels of mRNA for all genes expressed in parathyroid tissue explants (PTEs) obtained from patients with primary hyperparathyroidism (I degrees -HPT) were analyzed by oligo-DNA microarray. PTEs obtained from 4 patients with I degrees -HPT were precultured in normocalcemic medium (Ca(++) 1.0-1.1 mM) for 7 days and then cultured in hypocalcemic medium (Ca(++) 0.60 mM) or hypercalcemic (Ca(++) 1.60 mM) medium containing 4 mg/dl phosphate for an additional 7 days. As expected, expression levels of mRNA for PTH and chromogranin A were decreased to less than 50% in the hypercalcemic medium when compared with those in the hypocalcemic medium. Furthermore, oligo-DNA microarray analyses revealed that 7 genes were up-regulated by more than 2-fold and more than 30 genes were down-regulated by more than 1/2 in PTEs. Interestingly, 9 of these genes (up-regulated genes: chemokine ligand 8, multiple C2 domain and transmembrane region protein 1; down-regulated genes: matrix metallopeptidase-9, B-box and SPRY domain-containing protein, nitric oxide synthase 2A, PTH, cartilage acidic protein 1, chromogranin A, and fibrin 1) were involved in calcium metabolism or calcium-signaling pathways in the parathyroid tissue. However, the expression level of mRNA for alpha-klotho was variable, and it was not constantly decreased in hypercalcemic medium under the present experimental conditions. Although it was not possible to use normal parathyroid tissue, this is the first reported study to have investigated the expression levels of mRNA for all genes in human parathyroid adenomas that are modulated by high calcium concentration in organ culture.

Stimulating parathyroid cell proliferation and PTH release with phosphate in organ cultures obtained from patients with primary and secondary hyperparathyroidism for a prolonged period.

The pathogenesis of primary hyperparathyroidism (I degrees -HPT) and secondary hyperparathyroidism (II degrees -HPT) remains to be elucidated. To characterize their pathophysiology, we investigated the effects of calcium and phosphate on cell proliferation and PTH release in an organ culture of parathyroid tissues. Dissected parathyroid tissues obtained from patients with I degrees -HPT (adenoma) or II degrees -HPT (nodular hyperplasia) were precultured on a collagen-coated membrane for 1-4 week. After changing the medium for one containing various concentrations of phosphate, PTH release and [(3)H]thymidine incorporation were studied. In contrast to dispersed parathyroid cells cultured in a monolayer, calcium decreased PTH release in a concentration-dependent manner in parathyroid tissues. Furthermore, when parathyroid tissues obtained from II degrees -HPT were precultured for 1-4 weeks, PTH release and parathyroid cell proliferation were significantly increased in high-phosphate medium. These phosphate effects were also observed to a lesser extent in parathyroid tissues obtained from I degrees -HPT, but there was no significant difference between I degrees -HPT and II degrees -HPT. Microarray analyses revealed that mRNA levels of PTH, CaSR, and VDR were well preserved, and several growth factors (e.g. TGF-beta1-induced protein) were abundantly expressed in II degrees -HPT. Using organ cultures of hyperparathyroid tissues, in which PTH release and CaSR are well preserved for a prolonged period, we have demonstrated that phosphate stimulates parathyroid cell proliferation not only in II degrees -HPT but also in I degrees -HPT. Although the mechanism responsible for phosphate-induced cell proliferation remains to be elucidated, our in vitro findings suggest that both parathyroid tissues preserve to some extent a physiological response system to hyperphosphatemia as observed in normal parathyroid cells.

A novel missense mutation of AIRE gene in a patient with autoimmune polyendocrinopathy, candidiasis and ectodermal dystrophy (APECED), accompanied with progressive muscular atrophy: case report and review of the literature in Japan.

Autoimmune polyendocrinopathy, candidiasis, and ectodermal dystrophy (APECED) also known as autoimmune polyglandular syndrome type I, is a rare autosomal recessive disorder that results in several autoimmune diseases due to mutations in the AIRE (autoimmune regulator) gene. A 39-year-old female patient developed chronic mucocutaneous candidiasis at 3 yrs, idiopathic hypoparathyroidism at 11 yrs, chronic hepatitis at 23 yrs, Addison's disease and diabetes mellitus type I at 27 yrs. In addition, the patient developed progressive muscular atrophy of unknown etiology at the beginning of the third decade, and is bedridden at the present time. Her grandparents, parents, brother and daughter did not develop any features of APECED, but her father died of hepatoma. Direct sequencing of the AIRE gene revealed a novel missense mutation at exon 1 (R15C), which was identified to be of maternal origin. The other mutation was not found despite repeated sequencing of the whole coding regions. The R15C mutation was not detected in patients with idiopathic hypoparathyroidism (N= 10), idiopathic Addison's disease (N = 3), and normal subjects (N = 55). Although we could not analyze the father's gene, these results suggest that the patient is probably a compound heterozygote of the AIRE gene, in which the other abnormal allele could not be identified by the present analytical method. These data are compatible with the recent review that only one defective allele was detectable in some patients with clinically evident APECED. We found only six Japanese patients compatible with diagnosis of APECED, indicating that this autoimmune disease is extremely rare in our country.