Publications

This manuscript was withdrawn at the request of the authors.

Considerable progress has been made in the development and testing of agents to treat osteoporosis. Most impressive are reports on new antiresorptive agents--both bisphosphonates (ibandronate and zoledronic acid) and monoclonal antibodies (MAbs) (denosumab) directed against receptor activator of nuclear factor kappaB-ligand, a key molecule in the control of commitment and activation of osteoclasts. Bisphosphonates promise convenience and potency at slowing bone loss, whereas denosumab offers powerful suppression of resorption and rapid offset of action. Attention is also shifting from the osteoclast as a target for new therapies to the osteoblast and the osteocyte, with its complex network within the depths of bone. Wnt signaling through the frizzled receptor and its coreceptor, the low-density lipoprotein receptor related protein-5, appears from both molecular and in vivo evidence to be a pivotal pathway for modulating osteoblastic activity, bone formation, and bone strength. The recently identified product of the SOST gene or sclerostin has also been shown to block Wnt signaling. Sclerostin is produced by the osteocytes buried in the bone and is a new target to treat bone loss. Clinical trial reports indicate that the calcimimetic cinacalcet can effectively treat PTH hypersecretion due to primary and secondary hyperparathyroidism and parathyroid carcinoma. Lastly, it is now recognized that the matrix protein dentin matrix protein-1 enhances the release of the phosphate-regulating factor fibroblast growth factor 23 and that mutations in dentin matrix protein-1 play a causative role in a form of hypophosphatemic rickets.

Changes in extracellular [Ca2+] modulate the function of bone cells in vitro via the extracellular Ca2+-sensing receptor (CaR). Within bone microenvironments, resorption increases extracellular [Ca2+] locally. To determine whether enhanced CaR signaling could modulate remodeling and thereby bone mass in vivo, we generated transgenic mice with a constitutively active mutant CaR (Act-CaR) targeted to their mature osteoblasts by the 3.5 kb osteocalcin promoter. Longitudinal microcomputed tomography of cancellous bone revealed reduced bone volume and density, accompanied by a diminished trabecular network, in the Act-CaR mice. The bone loss was secondary to an increased number and activity of osteoclasts, demonstrated by histomorphometry of secondary spongiosa. Histomorphometry, conversely, indicates that bone formation rates were unchanged in the transgenic mice. Constitutive signaling of the CaR in mature osteoblasts resulted in increased expression of RANK-L (receptor activator of nuclear factor-kappaB ligand), the major stimulator of osteoclast differentiation and activation, which is the likely underlying mechanism for the bone loss. The phenotype of Act-CaR mice is not attributable to systemic changes in serum [Ca2+] or PTH levels. We provide the first in vivo evidence that increased signaling by the CaR in mature osteoblasts can enhance bone resorption and further propose that fluctuations in the [Ca2+] within the bone microenvironment may modulate remodeling via the CaR.

We co-immunoprecipitated the Ca(2+)-sensing receptor (CaR) and type B gamma-aminobutyric acid receptor (GABA-B-R) from human embryonic kidney (HEK)-293 cells expressing these receptors and from brain lysates where both receptors are present. CaRs extensively co-localized with the two subunits of the GABA-B-R (R1 and R2) in HEK-293 cell membranes and intracellular organelles. Coexpressing CaRs and GABA-B-R1s in HEK-293 cells suppressed the total cellular and cell surface expression of CaRs and inhibited phospholipase C activation in response to high extracellular [Ca(2+)] ([Ca(2+)](e)). In contrast, coexpressing CaRs and GABA-B-R2s enhanced CaR expression and signaling responses to raising [Ca(2+)](e). The latter effects of the GABA-B-R2 on the CaR were blunted by coexpressing the GABA-B-R1. Coexpressing the CaR with GABA-B-R1 or R2 enhanced the total cellular and cell surface expression of the GABA-B-R1 or R2, respectively. Studies with truncated CaRs indicated that the N-terminal extracellular domain of the CaR participated in the interaction of the CaR with the GABA-B-R1 and R2. In cultured mouse hippocampal neurons, CaRs co-localized with the GABA-B-R1 and R2. CaRs and GABA-B-R1s also co-immunoprecipitated from brain lysates. The expression of the CaR was increased in lysates from GABA-B-R1 knock-out mouse brains and in cultured hippocampal neurons with their GABA-B-R1 genes deleted in vitro. Thus, CaRs and GABA-B-R subunits can form heteromeric complexes in cells, and their interactions affect cell surface expression and signaling of CaR, which may contribute to extracellular Ca(2+)-dependent receptor activation in target tissues.

Extracellular calcium-sensing receptors (CaRs) and metabotropic or type B gamma-aminobutyric acid receptors (GABA-B-Rs), two closely related members of family C of the G protein-coupled receptor superfamily, dimerize in the formation of signaling and membrane-anchored receptor complexes. We tested whether CaRs and two GABA-B-R subunits (R1 and R2) are expressed in mouse growth plate chondrocytes (GPCs) by PCR and immunocytochemistry and whether interactions between these receptors influence the expression and function of the CaR and extracellular Ca(2+)-mediated cell differentiation. Both CaRs and the GABA-B-R1 and -R2 were expressed in the same zones of the growth plate and extensively colocalized in intracellular compartments and on the membranes of cultured GPCs. The GABA-B-R1 co-immunoprecipitated with the CaR, confirming a physical interaction between the two receptors in GPCs. In vitro knockout of GABA-B-R1 genes, using a Cre-lox recombination strategy, blunted the ability of high extracellular Ca(2+) concentration to activate phospholipase C and ERK1/2, suppressed cell proliferation, and enhanced apoptosis in cultured GPCs. In GPCs, in which the GABA-B-R1 was acutely knocked down, there was reduced expression of early chondrocyte markers, aggrecan and type II collagen, and increased expression of the late differentiation markers, type X collagen and osteopontin. These results support the idea that physical interactions between CaRs and GABA-B-R1s modulate the growth and differentiation of GPCs, potentially by altering the function of CaRs.

The extracellular Ca(2+)-sensing receptor (CaSR) plays a nonredundant role in the functions of the parathyroid gland (PTG) and the kidney. Severe hyperparathyroidism, premature death, and incomplete gene excision in Casr(-/-) mice have precluded the assessment of CaSR function in other tissues. We generated mice with tissue-specific deletion of Casr in the PTG, bone, or cartilage. Deletion of Casr in the PTG or bone resulted in profound bone defects, whereas deletion of Casr in chondrocytes (cartilage-producing cells) resulted in death before embryonic day 13 (E13). Mice in which chondrocyte-specific deletion of Casr was induced between E16 and E18 were viable but showed delayed growth plate development. Our data show a critical role for the CaSR in early embryogenesis and skeletal development.

BACKGROUND

Primary hyperparathyroidism (PHPT) is a common endocrine disorder that is frequently asymptomatic. The 2002 International Workshop on Asymptomatic PHPT addressed medical management of asymptomatic PHPT and summarized the data on nonsurgical approaches to this disease. At the Third International Workshop on Asymptomatic PHPT held in May 2008, this subject was reviewed again in light of data that have since become available. We present the results of a literature review of advances in the medical management of PHPT.

METHODS

A series of questions was developed by the International Task Force on PHPT. A comprehensive literature search for relevant studies evaluating the management of PHPT with bisphosphonates, hormone replacement therapy, raloxifene, and calcimimetics was conducted. Existing guidelines and recent unpublished data were also reviewed. All selected relevant articles were reviewed, and the questions developed by the International Task Force were addressed by the Consensus Panel.

RESULTS

Bisphosphonates and hormone replacement therapy are effective in decreasing bone turnover in patients with PHPT and improving bone mineral density (BMD). Fracture data are not available with either treatment. Raloxifene also lowers bone turnover in patients with PHPT. None of these agents, however, significantly lowers serum calcium or PTH levels. The calcimimetic cinacalcet reduces both serum calcium and PTH levels and raises serum phosphorus. Cinacalcet does not, however, reduce bone turnover or improve BMD.

CONCLUSIONS

Bisphosphonates and hormone replacement therapy provide skeletal protection in patients with PHPT. Limited data are available regarding skeletal protection in patients with PHPT treated with raloxifene. Calcimimetics favorably alter serum calcium and PTH in PHPT but do not significantly affect either bone turnover or BMD. Medical management of asymptomatic PHPT is a promising option for those who are not candidates for parathyroidectomy.

CONTEXT

Patients with persistent primary hyperparathyroidism (PHPT) after parathyroidectomy or with contraindications to parathyroidectomy often require chronic treatment for hypercalcemia.

OBJECTIVE

The objective of the study was to assess the ability of the calcimimetic, cinacalcet, to reduce serum calcium in patients with intractable PHPT.

DESIGN

This was an open-label, single-arm study comprising a titration phase of variable duration (2-16 wk) and a maintenance phase of up to 136 wk.

SETTING

The study was conducted at 23 centers in Europe, the United States, and Canada.

PATIENTS

The study included 17 patients with intractable PHPT and serum calcium greater than 12.5 mg/dl (3.1 mmol/liter).

INTERVENTION

During the titration phase, cinacalcet dosages were titrated every 2 wk (30 mg twice daily to 90 mg four times daily) for 16 wk until serum calcium was 10 mg/dl or less (2.5 mmol/liter). If serum calcium increased during the maintenance phase, additional increases in the cinacalcet dose were permitted.

MAIN OUTCOME MEASURE

The primary end point was the proportion of patients experiencing a reduction in serum calcium of 1 mg/dl or greater (0.25 mmol/liter) at the end of the titration phase.

RESULTS

Mean +/- sd baseline serum calcium was 12.7 +/- 0.8 mg/dl (3.2 +/- 0.2 mmol/liter). At the end of titration, a 1 mg/dl or greater reduction in serum calcium was achieved in 15 patients (88%). Fifteen patients (88%) experienced treatment-related adverse events, none of which were serious. The most common adverse events were nausea, vomiting, and paresthesias.

CONCLUSIONS

In patients with intractable PHPT, cinacalcet reduces serum calcium, is generally well tolerated, and has the potential to fulfill an unmet medical need.

Nephrogenic systemic fibrosis (NSF) is a disease of thickened, hard, hyperpigmented skin lesions with or without systemic fibrosis occurring in patients with renal insufficiency and associated with the administration of gadolinium-containing contrast. The pathogenesis of this disease is unclear, and there is no definitive treatment. We describe a 71-yr-old patient with stable chronic lymphocytic leukemia (CLL), end-stage renal disease (ESRD), and NSF who presented with hypercalcemia in 2006. Before onset of renal insufficiency in 2002, serum calcium, phosphorus, and PTH levels were normal. In 2004, the patient began hemodialysis, and he was diagnosed with NSF in 2005, shortly after undergoing an MRI with gadolinium contrast administration. Over the next 6 mo, albumin-corrected serum total calcium levels rose from 9.9 to 13.1 mg/dl (normal range, 8.5-10.5 mg/dl) with normal serum phosphorus levels. On admission in September 2006, 1,25-dihydroxyvitamin D [1,25(OH)(2)D] levels were elevated at 130.7 pg/ml (normal range, 25.1-66.1 pg/ml). Biopsy of an NSF lesion showed increased 25-hydroxyvitamin D(3)-1-alpha hydroxylase (CYP27B1) immunostaining compared with the biopsy from a normal control. This is the first reported association of NSF with hypercalcemia caused by elevated 1,25(OH)(2)D levels. This metabolic disturbance should be sought in future cases to determine a connection between NSF, 1,25(OH)(2)D metabolism, and CYP27B1 activation in the skin, which may shed light on the pathogenesis of this unusual local and systemic fibrosing disorder.