Geoffrey N. Hendy

Targeted ablation of the 25-hydroxyvitamin D 1α-hydroxylase enzyme: Evidence for skeletal, reproductive, and immune dysfunction

The active form of vitamin D, 1α,25-dihydroxyvitamin D [1α,25(OH)2D], is synthesized from its precursor 25 hydroxyvitamin D [25(OH)D] via the catalytic action of the 25(OH)D-1α-hydroxylase [1α(OH)ase] enzyme. Many roles in cell growth and differentiation have been attributed to 1,25(OH)2D, including a central role in calcium homeostasis and skeletal metabolism. To investigate the in vivo functions of 1,25(OH)2D and the molecular basis of its actions, we developed a mouse model deficient in 1α(OH)ase by targeted ablation of the hormone-binding and heme-binding domains of the 1α(OH)ase gene. After weaning, mice developed hypocalcemia, secondary hyperparathyroidism, retarded growth, and the skeletal abnormalities characteristic of rickets. These abnormalities are similar to those described in humans with the genetic disorder vitamin D dependent rickets type I [VDDR-I; also known as pseudovitamin D-deficiency rickets (PDDR)]. Altered non-collagenous matrix protein expression and reduced numbers of osteoclasts were also observed in bone. Female mutant mice were infertile and exhibited uterine hypoplasia and absent corpora lutea. Furthermore, histologically enlarged lymph nodes in the vicinity of the thyroid gland and a reduction in CD4- and CD8-positive peripheral T lymphocytes were observed. Alopecia, reported in vitamin D receptor (VDR)-deficient mice and in humans with VDDR-II, was not seen. The findings establish a critical role for the 1α(OH)ase enzyme in mineral and skeletal homeostasis as well as in female reproduction and also point to an important role in regulating immune function.

Human Calcium-sensing Receptor Gene VITAMIN D RESPONSE ELEMENTS IN PROMOTERS P1 AND P2 CONFER TRANSCRIPTIONAL RESPONSIVENESS TO 1,25-DIHYDROXYVITAMIN D

The calcium-sensing receptor (CASR), expressed in parathyroid chief cells, thyroid C-cells, and cells of the kidney tubule, is essential for maintenance of calcium homeostasis. Here we show parathyroid, thyroid, and kidney CASR mRNA levels increased 2-fold at 15 h after intraperitoneal injection of 1,25-dihydroxyvitamin D3(1,25(OH)2D3) in rats. Human thyroid C-cell (TT) and kidney proximal tubule cell (HKC) CASR gene transcription increased ∼2-fold at 8 and 12 h after 1,25(OH)2D3 treatment. The human CASR gene has two promoters yielding alternative transcripts containing either exon 1A or exon 1B 5′-untranslated region sequences that splice to exon 2 some 242 bp before the ATG translation start site. Transcriptional start sites were identified in parathyroid gland and TT cells; that for promoter P1 lies 27 bp downstream of a TATA box, whereas that for promoter P2, which lacks a TATA box, lies in a GC-rich region. In HKC cells, transcriptional activity of a P1 reporter gene construct was 11-fold and of P2 was 33-fold above basal levels. 10−8 m 1,25(OH)2D3 stimulated P1 activity 2-fold and P2 activity 2.5-fold. Vitamin D response elements (VDREs), in which half-sites (6 bp) are separated by three nucleotides, were identified in both promoters and shown to confer 1,25(OH)2D3 responsiveness to a heterologous promoter. This responsiveness was lost when the VDREs were mutated. In electrophoretic mobility shift assays with either in vitrotranscribed/translated vitamin D receptor and retinoid X receptor-α, or HKC nuclear extract, specific protein-DNA complexes were formed in the presence of 1,25(OH)2D3 on oligonucleotides representing the P1 and P2 VDREs. In summary, functional VDREs have been identified in the CASR gene and provide the mechanism whereby 1,25(OH)2D up-regulates parathyroid, thyroid C-cell, and kidney CASR expression.

Inactivation of menin, a Smad3-interacting protein, blocks transforming growth factor type β signaling

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by endocrine tumors of parathyroids, pancreatic islets, and anterior pituitary. The MEN1 gene encodes a nuclear protein called menin. In MEN1 carriers inactivating mutations give rise to a truncated product consistent with menin acting as a tumor suppressor gene. However, the role of menin in tumorigenesis and its physiological functions are not known. Here, we show that menin inactivation by antisense RNA antagonizes transforming growth factor type β-mediated cell growth inhibition. Menin interacts with Smad3, and antisense menin suppresses transforming growth factor type β-induced and Smad3-induced transcriptional activity by inhibiting Smad3/4-DNA binding at specific transcriptional regulatory sites. These results implicate a mechanism of tumorigenesis by menin inactivation.

Familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Effects of mutant gene dosage on phenotype.

Neonatal severe hyperparathyroidism is a rare life-threatening disorder characterized by very high serum calcium concentrations (> 15 mg/dl). Many cases have occurred in families with familial hypocalciuric hypercalcemia, a benign condition transmitted as a dominant trait. Among several hypothesized relationships between the two syndromes is the suggestion that neonatal severe hyperparathyroidism is the homozygous form of familial hypocalciuric hypercalcemia. To test this hypothesis, we refined the map location of the gene responsible for familial hypocalciuric hypercalcemia on chromosome 3q. Analyses in 11 families defined marker loci closely linked to the gene responsible for familial hypocalciuric hypercalcemia. These loci were then analyzed in four families with parental consanguinity and offspring with neonatal severe hyperparathyroidism. Each individual who was homozygous for loci that are closely linked to the gene responsible for familial hypocalciuric hypercalcemia had neonatal severe hyperparathyroidism. The calculated odds of linkage between these disorders of > 350,000:1 (lod score = 5.56). We conclude that dosage of the gene defect accounts for these widely disparate clinical phenotypes; a single defective allele causes familial hypocalciuric hypercalcemia, while two defective alleles causes neonatal severe hyperparathyroidism.

Mutations of the calcium-sensing receptor (CASR) in familial hypocalciuric hypercalcemia, neonatal severe hyperparathyroidism, and autosomal dominant hypocalcemia.

The calcium‐sensing receptor (CASR) is a plasma membrane G protein coupled receptor that is expressed in the parathyroid hormone (PTH) producing chief cells of the parathyroid gland and the cells lining the kidney tubule. By virtue of its ability to sense small changes in circulating calcium concentration ([Ca2+]o) and to couple this information to intracellular signaling pathways that modify PTH secretion or renal cation handling, the CASR plays an essential role in maintaining mineral ion homeostasis. Inherited abnormalities of the CASR gene located on chromosome 3p13.3‐21 can cause either hypercalcemia or hypocalcemia depending upon whether they are inactivating or activating, respectively. Heterozygous loss‐of‐function mutations give rise to familial (benign) hypocalciuric hypercalcemia (FHH) in which the lifelong hypercalcemia is asymptomatic. The homozygous condition manifests itself as neonatal severe hyperparathyroidism (NSHPT), a rare disorder characterized by extreme hypercalcemia and the bony changes of hyperparathyroidism which occur in infancy. The disorder autosomal dominant hypocalcemia (ADH) is due to gain‐of‐function mutations in the CASR gene. ADH may be asymptomatic or present with neonatal or childhood seizures. A common polymorphism in the intracellular tail of the CASR, Ala to Ser at position 986, has a modest effect on the serum calcium concentration in healthy individuals. Hum Mutat 16:281–296, 2000.

Familial isolated hyperparathyroidism: clinical and genetic characteristics of 36 kindreds.

Familial hyperparathyroidism (HPT) encompasses a clinically and genetically heterogeneous group of disorders. Syndromes with familial HPT include multiple endocrine neoplasia type 1 (MEN1) (Mendelian Inheritance in Man [MIM] 1311001) (63, 87), multiple endocrine neoplasia type 2A (MEN2A) (MIM 171400)(42, 79, 86), familial hypocalciuric hypercalcemia (FHH) (MIM 145980, 145981, 600740) also known as familial benign hypercalcemia (38, 64), and the hyperparathyroidism-jaw tumor syndrome (HPT-JT; HRPT2) (MIM 145001) (45). Familial isolated hyperparathyroidism2 (FIH; HRPT1) (MIM 145000) is a subgroup of familial HPT that can result from the incomplete expression of a syndromic form of familial HPT or from full expression of other entities (Figure 1). It is unknown how many as yet unrecognized clinical entities, including mutant genotypes, can also present as FIH. MEN1 is an autosomal dominant disorder characterized by endocrine and nonendocrine tumors, most strikingly involving the parathyroids, enteropancreatic endocrine system, and pituitary. Because FIH is seen less frequently than full expressions of MEN1, because HPT is the earliest and most frequent endocrinopathy in MEN1, and because even some large families with an apparent phenotype of FIH ultimately express MEN1, we (59, 67) and others (2, 61) previously speculated that most kindreds with FIH were occult expressions of MEN1. The gene responsible for MEN1 has been cloned (15), leading to powerful gene sequencing methods applicable to MEN1, FIH, and other conditions (63). MEN2A, unlike MEN1, is not typically a consideration in the differential diagnosis of FIH, because the higher penetrance of medullary thyroid carcinoma and pheochromocytoma than of HPT in MEN2A dominates the clinical presentation in a family (42, 79, 86). FHH is an autosomal dominant trait usually causing mild HPT (62) with relative hypocalciuria; hypercalcemia in FHH is highly penetrant at all ages, even in the perinatal period (64). Mild hypermagnesemia is sometimes seen in FHH but is unusual in other forms of primary HPT (53, 64). FHH cases almost always remain hypercalcemic following standard subtotal parathyroidectomy (PTX) (64). FHH always presents 0025-7974/02/8101-0001/0 MEDICINE® 81: 1-26, 2002 Vol. 81, No. 1 Copyright

Autosomal dominant pseudohypoparathyroidism type Ib is associated with a heterozygous microdeletion that likely disrupts a putative imprinting control element of GNAS

Patients with pseudohypoparathyroidism type Ib (PHP-Ib) have hypocalcemia and hyperphosphatemia due to renal parathyroid hormone (PTH) resistance, but lack physical features of Albright hereditary osteodystrophy. PHP-Ib is thus distinct from PHP-Ia, which is caused by mutations in the GNAS exons encoding the G protein alpha subunit. However, an imprinted autosomal dominant form of PHP-Ib (AD-PHP-Ib) has been mapped to a region of chromosome 20q13.3 containing GNAS. Furthermore, loss of methylation at a differentially methylated region (DMR) of this locus, exon A/B, has been observed thus far in all investigated sporadic PHP-Ib cases and the affected members of multiple AD-PHP-Ib kindreds. We now report that affected members and obligate gene carriers of 12 unrelated AD-PHP-Ib kindreds and four apparently sporadic PHP-Ib patients, but not healthy controls, have a heterozygous approximately 3-kb microdeletion located approximately 220 kb centromeric of GNAS exon A/B. The deleted region, which is flanked by two direct repeats, includes three exons of STX16, the gene encoding syntaxin-16, for which no evidence of imprinting could be found. Affected individuals carrying the microdeletion show loss of exon A/B methylation but no epigenetic abnormalities at other GNAS DMRs. We therefore postulate that this microdeletion disrupts a putative cis-acting element required for methylation at exon A/B, and that this genetic defect underlies the renal PTH resistance in AD-PHP-Ib.

A986S polymorphism of the calcium-sensing receptor and circulating calcium concentrations

BACKGROUND The regulation of extracellular calcium concentration by parathyroid hormone is mediated by a calcium-sensing, G-protein-coupled cell-surface receptor (CASR). Mutations of the CASR gene alter the set-point for extracellular ionised calcium [Ca2+]o and cause familial hypercalcaemia or hypocalcaemia. The CASR missense polymorphism, A986S, is common in the general population and is, therefore, a prime candidate as a genetic determinant of extracellular calcium concentration. METHODS We genotyped the CASR A986S variant (S allele frequency of 16.3%) in 163 healthy adult women and tested samples of their serum for total calcium, albumin, total protein, creatinine, phosphate, pH, and parathyroid hormone. A prospectively generated, random subset of 84 of these women provided a whole blood sample for assay of [Ca2+]o. FINDINGS The A986S genotype showed no association with total serum concentration of calcium, until corrected for albumin. In a multivariate regression model, biochemical and genetic variables accounted for 74% of the total variation in calcium. The significant predictors of serum calcium were: albumin (p<0.001), phosphate (p=0.02), parathyroid hormone (p=0.007), pH (p=0.001), and A986S genotype (p=0.009). Fasting whole-blood [Ca2+]o also showed an independent positive association with the 986S variant (p=0.013). INTERPRETATION The CASR A986S variant has a significant effect on extracellular calcium. The CASR A986S polymorphism is a likely candidate locus for genetic predisposition to various bone and mineral disorders in which extracellular calcium concentrations have a prominent part.

Mapping of the calcium-sensing receptor gene (CASR) to human chromosome 3q13.3-21 by fluorescence in situ hybridization, and localization to rat chromosome 11 and mouse chromosome 16.

The calcium-sensing receptor (CASR), a member of the G-protein coupled receptor family, is expressed in both parathyroid and kidney, and aids these organs in sensing extracellular calcium levels. Inactivating mutations in the CASR gene have been described in familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT). Activating mutations in the CASR gene have been described in autosomal dominant hypoparathyroidism and familial hypocalcemia. The human CASR gene was mapped to Chromosome (Chr) 3q13.3-21 by fluorescence in situ hybridization (FISH). By somatic cell hybrid analysis, the gene was localized to human Chr 3 (hybridization to other chromosomes was not observed) and rat Chr 11. By interspecific backcross analysis, the Casr gene segregated with D16Mit4 on mouse Chr 16. These findings extend our knowledge of the synteny conservation of human Chr 3, rat Chr 11, and mouse Chr 16.

Abundant expression of parathyroid hormone-related protein in primary rat aortic smooth muscle cells accompanies serum-induced proliferation.

Parathyroid hormone-related protein (PTHrP), which is responsible for producing hypercalcemia in patients with humoral hypercalcemia of malignancy, has recently been identified in several normal tissues. Because PTHrP, like parathyroid hormone (PTH), is known to exhibit vasodilatory properties, we investigated the expression and regulation of PTHrP mRNA in cultured rat aortic smooth muscle cells (SMC). We report here that PTHrP mRNA is expressed in SMC and is markedly induced by serum in a time- and concentration-dependent fashion. Addition of 10% fetal calf serum to serum-deprived, confluent cells, resulted in a marked induction of PTHrP mRNA by 2 h with a peak at 4-6 h. PTHrP was detected in SMC by immunocytochemistry and radioimmunoassay of conditioned medium, and was shown to be up-regulated within 24 h after the addition of serum. The serum induction of PTHrP mRNA was blocked by actinomycin D and by cycloheximide indicating the need for protein synthesis to evoke the serum effect on PTHrP gene transcription. In addition, treatment with dexamethasone, which has been previously shown to reduce the constitutive expression of PTHrP in human cancer cells, also blunted the serum induction of PTHrP mRNA in SMC. Treatment of quiescent cells with the serum mitogens platelet-derived growth factor or insulin-like growth factor-I had no effect on PTHrP, whereas the vasoactive peptides endothelin, norepinephrine and thrombin stimulated PTHrP expression. Exogenous addition of recombinant PTHrP-(1-141) had no significant effect on SMC DNA synthesis as measured by [3H]thymidine incorporation. In summary, the abundance of PTHrP mRNA and the characteristics of its regulation in SMC suggest a major role for PTHrP as a local modulator in vascular smooth muscle.